Characterization of the telomere binding protein 
ZNF524 
Dissertation 
Zur Erlangung des Grades 
Doktor der Naturwissenschaften 
am Fachbereich Biologie 
der Johannes Gutenberg-Universität Mainz 
Hanna Braun 
geb. am 19.05.1990 in Simmerath 
Mainz, 2022 
Dekan: Prof. Dr. Eckhard Thines  
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 14.11.2022
Table of Contents 
Preface ......................................................................................................................................................1 
Summary ..................................................................................................................................................2 
Zusammenfassung ....................................................................................................................................3 
Abbreviations ...........................................................................................................................................4 
Introduction ..............................................................................................................................................4 
The structure of telomeres ...................................................................................................................4 
The shelterin complex ..........................................................................................................................5 
Direct telomere binders beyond shelterin ...........................................................................................8 
The end replication problem ................................................................................................................8 
3’-overhang processing ....................................................................................................................9 
Telomeres as fragile sites .............................................................................................................. 10 
Telomere transcripts and their function ........................................................................................... 12 
Telomere length maintenance .......................................................................................................... 13 
Telomerase .................................................................................................................................... 13 
Alternative lengthening of telomeres ........................................................................................... 14 
Upper length limitations ............................................................................................................... 16 
The end protection ............................................................................................................................ 16 
Telomeres in aging and disease ........................................................................................................ 18 
Telomeres in senescence and cancer ............................................................................................ 18 
Telomere biology disorders ........................................................................................................... 20 
Rationale ............................................................................................................................................ 22 
Results ................................................................................................................................................... 23 
ZNF524 localizes to telomeres .......................................................................................................... 23 
ZNF524 binds to telomeric repeats via its zinc fingers .................................................................. 23 
ZNF524 localizes to telomeres in vivo ........................................................................................... 24 
Functional analysis of ZNF524 ........................................................................................................... 29 
Global effects of ZNF524 ............................................................................................................... 32 
The function of ZNF524 at telomeres ........................................................................................... 34 
Synthetic lethality with ZNF524 .................................................................................................... 43 
Discussion .............................................................................................................................................. 48 
Emergence of novel telomere binders .............................................................................................. 48 
Direct interaction of ZNF524 and other zinc finger proteins with telomeric sequences .................. 49 
ZNF524 as a telomeric protein .......................................................................................................... 51 
Proliferation and cell cycle progression are not impaired by the removal of ZNF524 ..................... 53 
ZNF524 is not essential for telomere length homeostasis ................................................................ 53 
i 
TRF2/RAP1 localization to telomeres is influenced by ZNF524......................................................... 54 
Stoichiometry of shelterin and expression levels of its members allow for subcomplex 
formation ....................................................................................................................................... 55 
Binding patterns of shelterin members to telomeric sequences allow for differential regulation of 
subcomplexes ................................................................................................................................ 55 
ZNF524-depleted telomeres resemble intermediate-state telomeres ............................................. 56 
Potential involvement of ZNF524 in telomeric chromatin organization ........................................... 58 
Conclusion ......................................................................................................................................... 62 
Appendix ................................................................................................................................................ 63 
Materials and Methods ......................................................................................................................... 73 
Materials ............................................................................................................................................ 73 
Methods ............................................................................................................................................ 89 
References ........................................................................................................................................... 104 
Acknowledgements ................................................................................................................................. iv 
ii 
Preface 
The work presented in this thesis was conducted by me and other members of the laboratory and 
the study was supported by a research collaboration. I was involved in the conceptualization of the 
project and planned the research. I also performed and analyzed most experiments with the 
support in the following experiments and techniques: 
• the staining and imaging of samples for the telomere colocalization, telomere dysfunction 
induced foci and the TRF2 signal intensity studies.as well as the counting of telomere 
dysfunction induced foci.  
• the preparation of metaphase spreads of U2OS WT and ZNF524 KO clones for 
chromosome orientation FISH, as well as the staining and imaging and of these samples and 
support in the scoring for chromosomal aberrations. 
• performance and analysis of the C-circle assay. 
• preparation of samples for ChIP-seq. 
• analysis of the sequencing results of the synthetic lethality screen and help with the 
visualization of the data. 
• analysis of the ChIP-seq results and visualization of the data. 
• analysis of the RNA-seq results and visualization of the data.  
Parts of the text and figures included in the Results, Discussion as well as Materials and Methods 
sections were used to prepare the following scientific manuscript: 
Braun et al. (2022). “ZNF524 directly interacts with telomeric DNA and supports telomere integrity.” 
In submission. 
1 
Summary 
Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes. They are generally 
organized as double-stranded tandem repeats (TTAGGGn in vertebrates), terminating in a G-rich 
single-stranded overhang. The telomeric sequence is recognized and bound by dedicated proteins that 
support the chromatin structure, regulate telomere length and block unwanted DNA damage repair. 
The well-described shelterin complex (composed of TRF1, TRF2, RAP1, TIN2, POT1 and TPP1) plays a 
crucial role in telomere protection and is constitutively present at telomeres. For instance, TRF2 is 
essential in the prevention of non-homologous end joining, which would otherwise lead to telomere 
fusions followed by extensive genomic alterations, including chromothripsis and kataegis. In addition 
to the shelterin complex, several direct telomere binders have been described more recently, including 
HOT1 and the zinc finger proteins ZBTB10 and ZBTB48/TZAP.  
In this study, I describe ZNF524, an uncharacterized protein that harbors four C2H2-type zinc finger 
domains towards the C-terminus. We show that binding of ZNF524 to telomeric sequences depends 
on the second zinc finger domain and a point mutation disrupting the zinc finger structure results in 
abrogated binding. Furthermore, we validate the localization of ZNF524 to telomeres within the cell. 
During functional analysis using ZNF524 KO cell lines, we found a reduced localization of TRF2 and 
RAP1 to telomeres in the absence of ZNF524 whereas total TRF2/RAP1 protein levels remained 
unchanged. Interestingly, other shelterin members were unperturbed, indicating a unique influence of 
ZNF524 on the TRF2/RAP1 subcomplex. In agreement with reduced TRF2/RAP1 at telomeres, we 
detected a slightly increased DNA damage signaling at ZNF524-depleted telomeres as well as a higher 
recombination rate defined by telomeric sister chromatid exchanges.  
Thus, ZNF524 localizes to telomeres and safeguards their integrity. To gain deeper insight into the 
mechanism, we conducted a genome-wide synthetic lethality screen and identified a number of 
genetic interactors with ZNF524 which will aid in future investigations. 
2 
Zusammenfassung 
Telomere sind Nukleoprotein-Strukturen an den Enden linearer Chromosome. Sie bestehen allgemein 
aus doppelsträngigen Wiederholungen (TTAGGGn in Vertebraten), welche in einem einzelsträngigen, 
G-reichen Überhang enden. Die Telomersequenz wird von bestimmten Proteinen erkannt und
gebunden. Diese Proteine unterstützen die Chromatinstruktur und die Regulierung der Telomerlaenge
und verhindern außerdem unerwünschte Reparatur der DNS. Der gut beschriebene Shelterin-Komplex
(bestehend aus TRF1, TRF2, RAP1, TIN2, POT1 und TPP1) ist konstitutiv an Telomeren vorhanden und
spielt eine wichtige Rolle für den Schutz der Telomere. Zum Beispiel: TRF2 ist essentiell in der
Vorbeugung von nicht-homologer DNS Reparatur, welche anderenfalls zu Telomerfusionen, gefolgt
von extensiven Genomveränderungen, wie Chromothripsis und Kataegis, führen würde. Zusätzlich zu
dem Shelterin-Komplex wurden unlängst andere Proteine als direkte Telomerbinder beschrieben, wie
HOT1 und die Zinkfingerproteine ZBTB10/TZAP.
In dieser Abhandlung berichte ich von ZNF524, einem nicht charakterisierten Protein, das im C-
Terminus vier Zinkfingerdomänen vom Typ C2H2 beinhaltet. Wir konnten zeigen, dass die Bindung von 
ZNF524 an Telomersequenzen von der zweiten Zinkfingerdomaene abhängt und dass eine 
Punktmutation, welche die Zinkfingerstruktur unterbricht, zu einem Bindeverlust führt. Des Weiteren 
validierten wir die Lokalisierung von ZNF524 an Telomere innerhalb der Zelle. Während funktioneller 
Untersuchungen mit ZNF524 KO Zelllinien fanden wir, in Abwesenheit von ZNF524, eine reduzierte 
Lokalisierung von TRF2 und RAP1 zu Telomeren obwohl die totale Proteinmenge von TRF2/RAP1 
unverändert blieb. Interessanterweise war die Lokalisierung anderer Proteine des Shelterin-Komplexes 
unvermindert. Dies weist auf einen einzigartigen Einfluss von ZNF524 auf den TRF2/RAP1 Teilkomplex 
hin. Passend zu reduzierten TRF2/RAP1 Mengen an Telomeren, detektierten wir einen leichten Anstieg 
an DNS Schäden sowie eine erhöhte Rekombinationsrate, definiert durch telomerische 
Schwesterchromatidaustausche, an ZNF524-freien Telomeren. 
Zusammenfassend lässt sich festhalten, dass ZNF524 an Telomere bindet und ihre Integrität bewahrt. 
Um tiefere Einsicht in den Mechanismus zu erhalten, führten wir einen genomweiten Screen für 
Synthetische Letalität durch und identifizierten einige genetische Interaktoren von ZNF524, welche für 
zukünftige Forschung hilfreich sein werden. 
3 
Abbreviations 
9-1-1 RAD9/RAD1/HUS1 DNA DNA deoxyribonucleic acid 
damage response checkpoint DNA2 DNA replication ATP-
53BP1 p53 binding protein dependent helicase/nuclease 
°C degrees celsius 2 
α anti DNA-PKcs DNA-dependent protein 
A adenine kinase catalytic subunit 
ALT alternative lengthening of DNase unspecific DNA cleaving 
telomeres endonuclease 
alt-NHEJ alternative non-homologous DNMT DNA methyl transferase 
end joining ds double-strand 
APB ALT-associated promyelocytic DSB double-strand break 
leukemia body E. coli Escherichia coli 
ASF1 Anti-Silencing Factor 1 ECTR extrachromosomal telomeric 
ATM Ataxia telangiectasia mutated repeats 
ATP adenosine tri-phosphate EMSA electrophoretic mobility shift 
ATR ATM and RAD3-related assay 
ATRX Transcriptional regulator EXO1 exonuclease 1 
ATRX FANC fanconi anemia 
BIR Break induced replication complementation group 
BLM Bloom syndrome RecQ-like FITC fluorescin isothiocyanate 
helicase FPC fork protection complex 
BMF bone marrow failure G guanine 
bp base pairs G1 G1 cell cycle phase 
C cytosine G2 G2 cell cycle phase 
C2H2 Cys2-His2-type zinc finger g grams 
C-circle extrachromosomal circular G4 G (guanine) quadruplex 
DNA GAL4 galactose metabolism 4 
CDK cyclin dependent kinase γH2AX S139 phosphorylated histone 
CoIP Co-immunoprecipitation H2A.X 
CRISPR/Cas9 Clustered regularly GFP green fluorescent protein 
interspaced short palindromic G-strand telomeric guanine-rich strand 
repeats/ CRISPR-associated HDAC histone deacetylase 
protein 9 HDR Homology directed repair 
CST CDC1, STN1, TEN1 complex HGPS Hutchinson-Gilford Progeria 
C-strand telomeric cytosine-rich strand Syndrome 
CTC1 Conserved telomere HH Hoyeraal-Hreidarsson 
maintenance component 1 syndrome 
CTCF CCCTC-binding factor HMG-5 High-Mobility-Group-Protein 
DAPI 4′,6-Diamidin-2-phenylindol 5 
dATP deoxyadenosine tri- HMT histone methyl transferase 
phosphate hnRNP Heterogeneous nuclear 
DAXX Death domain-associated ribonucleoprotein 
protein 6 HOT1/HMBOX1 Homeobox 1 
DC dyskeratosis congenita  HP1 heterochromatin protein 1  
DDR DNA damage response HR homologous recombination 
DEG differentially expressed gene hTR Telomerase RNA component 
DKC1 Dyskerin IP immunoprecipitation 
D-loop displacement loop kb kilobase pairs 
4 
 
Kd equilibrium dissociation RNAi RNA interference 
constant RPA Replication factor A 
KD Knock down rpm rounds per minute 
kDa kilodalton RT room temperature 
KO Knock-out RTEL1 Regulator Of Telomere length 
L liters 1 
LFQ label-free quantitation S DNA synthesis phase of cell 
Lig ligase cycle (between G1 and G2) 
M molar SASP Senescence associated 
mg milligrams secretory phenotype  
S. cerevisiae Saccharomyces cerevisiae, 
MiDAS mitotic DNA synthesis 
budding yeast 
mL milliliters 
S. pombe Schizosaccharomyces pombe, 
mM millimolar fission yeast 
MRE11 Meiotic recombination 11 sgRNA single-guide RNA 
homolog 1 SLX Synthetic Lethal of unknown 
MRN MRE11, RAD50, NBS1 (X) function 
complex Sm nuclease Serratia marcescens nuclease 
MS mass spectrometry 
ss single-strand 
MTS multiple telomeric signals 
STN1 suppressor of CDC13 
Myb Myeloblastosis gene 
SUV39-H1 Histone-lysine N-
NHEJ non-homologous end joining methyltransferase SUV39H1 
nM nanomolar T thymine 
NOR Nuclear orphan receptor TBP telomere biology disorders  
NR2C2 nuclear receptor subfamily 2, T. thermophila Tetrahymena thermophila 
group C, member 2 T-circles telomeric circles 
NR2F2 nuclear receptor subfamily 2, 
TEN1 Telomere length regulation 
group F, member 2 
protein TEN1 homolog 
nt nucleotides 
TERC Telomerase RNA component 
NuRD nucleosome remodeling and 
TERRA telomeric repeat-containing 
histone deacetylase 
RNA 
OB-fold oligonucleotide binding-fold 
TERT Telomerase reverse 
ORC origin recognition complex transcriptase 
PARP1 poly(ADP-ribose) polymerase TIF telomere dyfunction induced 
1 foci 
PCNA Proliferating-Cell-Nuclear- TIN2 TINF2, TRF-interacting nuclear 
Antigen factor 2 
PD population doubling TLC1 telomerase component 
PML promyelocytic leukemia t-loop telomeric loop 
PNK polynucleotid kinase TOR target of rapamycin 
POL polymerase TPE telomere position effect 
POT1 protection of telomeres 1 TPP1 TINT1, PTOP, PIP1, interactor 
pRB p16 retinoblastoma protein of POT1 and TIN2 
qFISH quantitative fluorescence in- TRF Telomere restriction 
situ hybridization fragment 
RAD Radiation sensitive TRF1/2 Telomere repeat binding 
RAP1 Repressor/Activator protein 1 factor 1/2 
RFP red fluorescent protein TRFH TRF homology dimerization 
R-loop RNA:DNA hybrid structure domain 
RNA ribonucleic acid 
2 
 
T-SCE telomeric sister-chromatid ZBTB zinc-finger and BTB-domain 
exchange containing protein 
TZAP Telomeric zinc finger- ZNF zinc-finger protein 
Associated Protein μL microliter 
v/v volume per volume μm micrometer 
w/v weight per volume MEF mouse embryonic fibroblast 
WRN Werner Syndrome RecQ-like MTS multiple telomere signals 
helicase ZF zinc finger 
WT wild-type 
g times gravity 
3 
Introduction 
The ends of linear chromosomes have been a source of fascination for researchers over decades. From 
the very beginning on, the importance of protective end structures to safeguard chromosome integrity 
was a central dogma that gave rise to the field of telomere biology (from Greek: τέλος (telos) = end, 
μέρος (méros) = part) (Muller, 1938; McClintock, 1941). The definition of the so-called end replication 
problem further fueled the interest in these capping structures and with the discovery of telomeric 
repetitive sequences and the reverse transcriptase telomerase, telomere biology reached substantial 
significance in both life sciences and medical research (Watson, 1972; Blackburn and Gall, 1978; 
Greider and Blackburn, 1985). Over the years, a plethora of discoveries have advanced our 
understanding of major functions and mechanisms at the telomeres. 
The structure of telomeres 
Telomeres are nucleoprotein structures at the ends of linear chromosomes. While in most species, 
telomeres end in repetitive sequences they vary greatly in length and sequence. In vertebrate cells, 
TTAGGG repeats are the dominant sequence, while we find (TTAGGC)n in C. elegans, (C1-3A/TG1-3)n in S. 
cerevisiae, (G2-6TTAC[A])n in S. pombe, (TTTAGGG)n in Arabidopsis thaliana, and (TTGGGG)n in 
Tetrahymena thermophila (Greider and Blackburn, 1985; Richards and Ausubel, 1988; Wicky et al., 
1996; Dehé and Cooper, 2010; Wellinger and Zakian, 2012). Despite identical telomere sequences 
across mammalian species, differences in length span from 2 kb to 50 kb. Human telomeres display a 
length of 10-15 kb at birth, with a G-rich 3’-overhang of less than 300 nt (Makarov, Hirose and 
Langmore, 1997; McElligott and Wellinger, 1997; Wright et al., 1997; Palm and De Lange, 2008). In 
contrast, mouse telomeres range from 20 kb to 65 kb and can even reach up to 150 kb in length (Kipling 
and Cooke, 1990). However, this length is restricted to extensively bred laboratory mice, while wild-
derived mice have an average telomere length closer to 20 kb (Hemann and Greider, 2000).  
Adjacent to the canonical repeats is a stretch of segmentally duplicated DNA tracts known as 
subtelomeric region (Riethman, Ambrosini and Paul, 2005). In this region additional hexameric repeats 
could be identified that resemble the canonical T-type TTAGGG repeats but differ in one nucleotide: 
the N-type TTGGGG, G-type TGAGGG (Allshire, Dempster and Hastie, 1989) and C-type TCAGGG (Baird, 
Jeffreys and Royle, 1995) repeats. In alternative lengthening of telomeres (ALT) cells, these repetitive 
elements are interspersed with the telomeric sequence probably due to the recombinogenic nature of 
the pathway (Conomos et al., 2012). 
The double-stranded telomeric region eventually culminates in a single-stranded 3’-overhang of the 
G-rich strand. It is found at mammalian telomeres throughout the cell cycle and forms independent of 
the telomere length maintenance status. Therefore, this overhang is a result of active telomere 
processing after replication, potentially by C-strand resection, and it is not synthetized by telomerase 
(Makarov, Hirose and Langmore, 1997; McElligott and Wellinger, 1997). The overhang is essential for 
telomere integrity as it allows for the formation of a lariat structure called the t-loop (Griffith et al., 
1999): functional telomeres fold back onto themselves allowing for the single-stranded region to 
invade the TTAGGG double-strand thereby creating a displacement loop (D-loop). The formation of 
these t-loops strongly relies on the interaction with a dedicated telomeric protein, the shelterin 
member TRF2 (de Lange, 2018). 
4 
 
The shelterin complex 
Many accessory proteins are present at telomeres. Some constitutively coat telomeres throughout the 
cell cycle while others perform regulatory functions that only require transient interactions. The most 
dominant binder of telomeres is the shelterin complex. This complex composes of six different 
proteins: TRF1 (telomeric repeat binding factor 1), TRF2 (telomeric repeat binding factor 2), RAP1 (the 
human orthologue of yeast repressor/activator protein 1; gene name TERF2IP), TIN2 (TRF interacting 
protein; gene name TINF2), POT1 (protection of telomeres 1) and TPP1 (TINT1, PTOP, PIP1; gene name 
ACD).  
Both TRF1 and TRF2 bind telomeric DNA as homodimers specifically at the double-stranded repeats. 
They were the first proteins described as telomere binders: by performing an electrophoretic mobility 
shift assay (EMSA) using TTAGGG repeats in HeLa lysate, a novel protein was detected that recognized 
TTAGGG repeats very specifically as it could not be competed off by similar sequences (namely 
TTGGGG and TTAGGC) (Zhong et al., 1992; Chong et al., 1995). Simultaneously, the same protein was 
described as a homologue to Tbf1, a known TTAGGG binder in fission yeast (Bilaud et al., 1996). Here, 
also an EMSA was applied followed by immunoblotting. By purifying antibodies against different 
sections of the novel protein, the DNA binding domain with homology to Tbf1 could be mapped (Bilaud 
et al., 1996). This first telomere binder was termed TRF1. By sequence comparison and 
immunoblotting, TRF2 was discovered soon after (Bilaud et al., 1997; Broccoli et al., 1997). Both 
proteins recognize and bind telomeres in a similar fashion through their homeobox domains. At the 
time of discovery, parallels to other myb-domain containing proteins were drawn by sequence 
alignment. However, while the known myb-domain containing proteins would bind via two to three 
myb domains, both TRF1 and TRF2 only harbor one domain per protein (Broccoli et al., 1997). 
Interestingly though, both TRF1 and TRF2 are able to form homodimers, but not heterodimers, via 
their TRF homology dimerization domains (TRFH) (Fairall et al., 2001). Following this line of thought, 
experimental data confirmed that by formation of these homodimers, the binding of TRF1 and TRF2 
to TTAGGG also relies on two myb domains and that dimerization with a Δmyb mutant diminished 
telomere binding (Bianchi et al., 1997; König, Fairall and Rhodes, 1998). Crystallization of the DNA 
binding domains revealed an additional hook that is located C-terminal to the myb-like domain and 
critical for binding, thereby defining the domain as a homeobox (Court et al., 2005). While TRF1 and 
TRF2 bind telomeres in an almost identical manner, their functions diverge (Nishikawa et al., 2001; 
Court et al., 2005; Hanaoka, Nagadoi and Nishimura, 2009). Both have implications as negative 
telomere length regulators but only TRF2 is essential to the prevention of non-homologous end joining 
at telomeres (Van Steensel and De Lange, 1997; Smogorzewska et al., 2000; Karlseder, Smogorzewska 
and De Lange, 2002). Indeed, a strong increase in telomere fusions was observed when expressing a 
dominant-negative form of TRF2  that lacks only the DNA-binding homeobox domain and thereby 
sequesters functional TRF2 from telomeres via homodimerization (Van Steensel, Smogorzewska and 
De Lange, 1998).  
Since its discovery, TRF2 has been the focus of ample research unravelling a plethora of diverse 
functions at mammalian telomeres. For instance, TRF2 is involved in the topology of telomeric 
chromatin as it localizes to branched DNA as well as Holliday junctions and is additionally able to wrap 
DNA around its homodimerization domain thereby inducing telomere winding (Poulet et al., 2009; 
Doksani et al., 2013; Benarroch-Popivker et al., 2016; Schmutz et al., 2017). As previously mentioned, 
the formation of the protective t-loop depends on TRF2 and is potentially mediated by these effects 
on chromatin structure. Most strikingly, TRF2 protects telomeres from ATM-mediated DNA damage 
response (DDR) and thereby prevents telomeric end-to-end fusions that would subsequently lead to 
genomic instability, break-fusion-bridge cycles, chromothripsis and kataegis (Van Steensel, 
5 
 
Smogorzewska and De Lange, 1998; Denchi and De Lange, 2007; Maciejowski et al., 2015). 
Furthermore, TRF2 is responsible for recruitment of the fellow shelterin member RAP1 (Li, Oestreich 
and De Lange, 2000). Not only does TRF2 perform these constitutively required functions but it also 
recruits factors to the telomere that are only transiently needed and subject to regulatory 
mechanisms. For example, the nuclease Apollo aids with both 3’-overhang processing and replication 
and the helicase RTEL1 is only bound during S-phase to facilitate replication (Wu et al., 2010; Ye et al., 
2010; Mendez-Bermudez et al., 2018; Sarek et al., 2019).  
Shelterin member RAP1 associates with telomeres indirectly (Li, Oestreich and De Lange, 2000), while 
its yeast homologue harbors two myb domains and locates to telomeres independent of additional 
factors (Wellinger and Zakian, 2012). Even though human RAP1 also contains a myb like domain, its 
affinity to DNA is low due to its a lack of positive surface charge that could interact with the negatively 
charged phosphate backbone of the DNA (Hanaoka et al., 2001). In addition to the known interaction 
with TRF2, these findings indicate that localization of RAP1 towards telomeres is mediated by TRF2. 
Upon closer examination, this protein-protein interaction was mapped to the RCT domain of RAP1 and 
the hinge domain of TRF2 (Li, Oestreich and De Lange, 2000; Chen et al., 2011). While some in vitro 
data suggests that RAP1 is able to bind telomeric DNA and preferentially associates with junctions 
between ss- and ds-DNA, this was not yet confirmed in vivo (Arat and Griffith, 2012). Further in vitro 
studies also showed that RAP1 increases the specificity of telomere recognition by TRF2 albeit at the 
cost of affinity. In this setting, RAP1 also maintains the necessary susceptibility to D-loop unwinding 
thereby regulating the function of TRF2 (Janoušková et al., 2015; Nečasová et al., 2017). Early 
investigations of RAP1’s function showed an involvement in TRF2-mediated inhibition of NHEJ in vitro 
(Bae and Baumann, 2007) and tethering of RAP1 to TRF2-depleted telomeres is able to reduce fusion 
events despite persistent DNA damage signaling (Sarthy et al., 2009). To further assess the relevance 
of RAP1 in human cell lines, knock-outs were generated and tested for growth effects, DNA damage 
signaling, recombination events and telomere position but a change in comparison to WT was not 
observed (Kabir, Hockemeyer and de Lange, 2014). While overexpression of RAP1 leads to telomere 
elongation (Li and De Lange, 2003), the KO does not affect telomere length (Kabir, Hockemeyer and de 
Lange, 2014). In mouse cells, RAP1 protects telomeres from recombination as shown by an increase in 
t-SCE in RAP1 deficient cells. Again, neither NHEJ nor DNA damage signaling were enhanced, indicating 
that TRF2’s protective function does not rely on the recruitment of RAP1 (Martinez et al., 2010; Sfeir 
et al., 2010). While a removal of RAP1 at functioning telomeres is not detrimental, RAP1’s importance 
surfaces at challenged telomeres: if the basic domain of TRF2 is deleted, the interaction with RAP1 is 
able to partially rescue the telomere attrition and concomitant fusion phenotype (Rai et al., 2016). In 
a telomerase-deficient background, RAP1 prevents telomere fusions and eases telomere shortening in 
human cell lines and mice (Martínez et al., 2016; Lototska et al., 2020). In conclusion, RAP1 supports 
TRF2 function and becomes essential at challenged or shortened telomeres. 
In contrast to TRF2, TRF1 does not have a strong implication in the protection of telomeres but is rather 
involved in the facilitation of telomere replication, for example through the recruitment of helicases 
(Martínez et al., 2009; Sfeir et al., 2009; Zimmermann et al., 2014). Telomeres pose obstacles for the 
replisome that are detrimental if not properly resolved. As a result, the removal of TRF1 in mouse cells 
leads to growth defects and onset of senescence (Karlseder et al., 2003; Iwano et al., 2004; Sfeir et al., 
2009). The functional divergence of TRF1 and TRF2 is potentially due to their differing N-terminal 
domain, which is acidic in TRF1 and basic in TRF2 (Chong et al., 1995; Bilaud et al., 1997; Broccoli et al., 
1997). 
A third member of the shelterin complex with DNA binding capabilities is POT1. Unlike TRF1 and TRF2, 
POT1 binds to the single-stranded part of telomeres via two OB fold domains located at the N-terminus 
6 
 
of the protein (Lei, Podell and Cech, 2004). While the majority of mammals has only one POT1 protein, 
a gene duplication occurred in the rodent lineage leading to POT1a and POT1b (Hockemeyer et al., 
2006). Deletion of POT1 in mouse cells activates ATR-mediated DNA damage signaling, leading to 
aberrant homologous recombination (HR) and onset of senescence, thereby marking the significance 
of POT1 in telomere protection (Wu et al., 2006; Denchi and De Lange, 2007). Recently, these findings 
were strengthened by Glousker et al. who reported an enrichment of the DDR complexes MRN 
(MRE11, Rad50, NBS1) and 9-1-1 (Rad9-Hus1-Rad1) at POT1 deficient telomeres in human cell lines 
(Glousker et al., 2020). This was accompanied by an increase of TIFs, telomere aberrations, and other 
phenotypes connected to increased HR activity (Glousker et al., 2020; Pinzaru et al., 2020). While POT1 
limits recombinations, it also facilitates telomere replication: POT1 mutations occurring in cutaneous 
T cell lymphoma additionally cause replication defects (Pinzaru et al., 2016). Also, POT1 loss-of-binding 
mutants lacking the OB domains  resulted in replication stress at telomeres, manifesting in mitotic DNA 
synthesis and ultrafine anaphase bridges (Pinzaru et al., 2020). At functional telomeres, POT1 forms a 
complex with TPP1 and in vitro studies confirmed that multiple POT1-TPP1 dimers can coat single-
stranded telomeric repeats where they aid with compaction of the DNA (Taylor et al., 2011; Corriveau 
et al., 2013). By binding to the ss 3’-overhang, the POT1-TPP1 heterodimer protects the telomeres 
from RPA-mediated DDR. As RPA and POT1-TPP1 have similar affinities for telomeric sequences and 
RPA is more abundant than the complex, it seems unlikely that RPA is simply outcompeted. Instead, 
the formation of G4 in the 3’-overhang seems advantageous for POT1-TPP1 binding (Ray et al., 2014) 
Furthermore, an involvement of TERRA and hnRNPA1 in the regulation of RPA displacement was 
suggested (Flynn et al., 2011) but the exact mechanism is still under investigation. Research in mouse 
cells showed that TPP1 also supports telomere protection by suppressing ATM-mediated DDR as 
opposed to ATR-mediated suppression by POT1 (Guo et al., 2007). Additionally, POT1-TPP1 is involved 
in processivity of telomerase, its recruitment to telomeres, translocation during telomere synthesis 
and thereby telomere elongation and maintenance (Latrick and Cech, 2010; Zaug et al., 2010; Rajavel, 
Mullins and Taylor, 2014; Pike et al., 2019; Sekne et al., 2022). Strikingly, TPP1 promotes telomerase 
processivity also when tethered to the telomere independently of POT1 (Lim et al., 2017). Indeed, TPP1 
harbors an OB fold that directly interacts with telomerase and recruits it to the telomere (Nandakumar 
et al., 2012; Zhong et al., 2012). Different isoforms of TPP1 have been reported that differ in their 
ability to drive telomere elongation, thereby suggesting an additional layer of telomerase regulation 
by TPP1 (Grill et al., 2019; Boyle et al., 2020). The promotion of telomerase activity is further 
strengthened by the interaction of TPP1 with shelterin member TIN2 (Abreu et al., 2010; Zhong et al., 
2012; Pike et al., 2019). TIN2 bridges the members of the shelterin complex through interactions with 
TPP1, TRF2 and TRF1 (Ye et al., 2004; Hu et al., 2017; Lim et al., 2017). The disruption of these 
interactions leads to DDR activation indicating the necessity of the fully assembled shelterin complex 
at functional telomeres (Hu et al., 2017). Especially in mouse cells, the deletion of TIN2 results in 
decreased binding of TRF1, TRF2 and POT1-TPP1. Subsequently, recombination events increase and 
both ATM and ATR are activated (Takai et al., 2011). 
The fully assembled shelterin complex is assumed to consist of a TRF2 homodimer bound by two RAP1 
proteins per dimer, the TRF1 homodimer, POT1, TPP1 and TIN2 (de Lange, 2018). The six member 
complex exists both in solution and chromatin-bound (Takai et al., 2010). Additionally, subcomplexes 
consisting only of a subset of shelterin members can form in vitro. For example, TRF2-TIN2-TPP1-POT1, 
TIN2-TPP1-POT1 and TRF2-TIN2-TPP1 exhibit a strong affinity to junctions between the single- and 
double-stranded DNA (Lim et al., 2017). The POT1-TPP1 heterodimer can also form independently but 
is less abundant than the other shelterin members (Takai et al., 2010). Biochemical data shows that, 
with regard to plain telomeric ds-DNA, the TRF2/RAP1 subcomplex has similar recognition and binding 
properties as the fully assembled shelterin complex: both are able to form in solution and then 
associate to telomeric sequences mainly by diffusive 3D search in a non-cooperative manner (Erdel et 
7 
 
al., 2017). While the in vitro data strongly suggest the existence of these subcomplexes in vivo, 
evidence thereof is still lacking.  
Direct telomere binders beyond shelterin 
Over the last decade, additional proteins have been identified that associate with telomeres, some of 
them even binding directly (Déjardin and Kingston, 2009; Grolimund et al., 2013; Kappei et al., 2017). 
Homeobox telomere-binding protein 1 (HOT1) was discovered in a mass spectrometry-based DNA-
protein interaction screen (Déjardin and Kingston, 2009; Kappei et al., 2013). HOT1 binds to telomeres 
via a homeobox domain, similar to TRF1 and TRF2, and functions as a positive telomere length 
regulator potentially mediated by interactions with components of the telomerase holoenzyme and 
localization to Cajal bodies (Kappei et al., 2013). Another recently identified telomere length regulator 
is ZBTB48 or TZAP (Jahn et al., 2017; Li et al., 2017). In contrast to HOT1, ZBTB48 promotes telomere 
shortening potentially through trimming. Interestingly, ZBTB48 does not harbor a homeobox or OB 
domain but binds to telomeres via a zinc finger (Jahn et al., 2017; Li et al., 2017; Zhao et al., 2018). Zinc 
finger proteins often function as transcription factors, which also holds true for ZBTB48 in addition to 
its telomeric function (Jahn et al., 2017). Another protein whose binding to telomeres is mediated by 
zinc fingers is ZBTB10. ZBTB10 interacts with TRF2 and prefers the subtelomeric sequence TTGGGG 
over TTAGGG repeats but its exact function remains elusive (Bluhm et al., 2019). As previously 
mentioned, variant repeats are interspersed through ALT positive telomeres. Especially the TCAGGG 
variant repeats are recognized and bound by the nuclear receptors NR2C2 (TR4) and NR2F2 (COUP 
TF2) (Déjardin and Kingston, 2009; Conomos et al., 2012). While the recruitment of the nuclear 
receptors to the variant repeats is assumed to drive ALT, recent data in several NR2F2 depleted ALT 
positive cell lines suggests a fine-tuned cell line dependent mechanism (Marzec et al., 2015; Alhendi 
and Royle, 2020). Still, the nuclear receptors NR2C2 and NR2F2 influence the association of ZNF827 to 
ALT telomeres, which in turn recruits the NuRD chromatin remodeler complex thereby promoting ALT 
activity (Conomos, Reddel and Pickett, 2014). Given that zinc finger proteins are emerging as direct 
telomere binders, it is tempting to speculate that ZNF827, which was also identified in a 
phylointeractomics screen with telomeric sequences, does not necessarily rely on the nuclear 
receptors for interaction with telomeres but might bind independently. Even though shelterin is the 
most prominent telomeric protein complex, additional direct interactors of telomeres perform crucial 
functions for telomere integrity. 
The end replication problem 
In the 1960’s Leonard Hayflick made an important observation while establishing cell lines from human 
tissue samples: human cells from healthy tissue do not divide indefinitely while maintaining a diploid 
state. After a certain number of population doublings, that is now known as the Hayflick limit, the 
division time increases and eventually cells enter senescence (Hayflick and Moorhead, 1961; Hayflick, 
1965). In 1972, Watson postulated the concept of the end replication problem (Olovnikov, 1971, 1996; 
Watson, 1972) giving a potential explanation for the phenomenon: In semi-conservative replication, 
DNA polymerases require the 3’-OH for the extension of the newly synthesized strand. Leading strand 
synthesis therefore results in a blunt end while lagging strand synthesis can never reach the terminus 
of the parental strand because RNA primers are needed as starting points for the DNA polymerase. 
The removal of the last primer will always generate a 3’-overhang and lead to a gradual shortening of 
linear DNA with every replication round. While the exact number of nucleotides is still under 
discussion, telomeres seem to shorten at a higher rate than what is expected if the final primer is 
placed at the very end of the telomere. Indeed, the processing of telomeres after replication 
constitutes more complex mechanisms. As mentioned previously, functional telomeres require a 3’-
8 
 
overhang for the formation of the crucial t-loop. Since the leading strand synthesis culminates in a 
blunt end, resection of the C-strand is necessary which continuously shortens the leading end telomere 
with every cell division.  
3’-overhang processing 
Post-replicative processing of telomeres reinstates the 3’-overhang. While the exact mechanism is not 
yet understood in humans, it is assumed that it might have similarities to the processing in mice: To 
generate a first overhang at the leading end telomere, which is presumably blunt ended after 
replication, the 5’-3’ exonuclease Apollo is recruited to the telomere. Apollo interacts with TRF2 
through a C-terminal YxLxP motif and is not found at telomeres upon disruption of this interaction sites 
(Lenain et al., 2006; Chen et al., 2008; Lam et al., 2010). In humans, Apollo is known to interact with 
TRF2, too. Depletion of Apollo results in a senescence like phenotype, fragile telomeres and TIFs (van 
Overbeek and de Lange, 2006). Furthermore, the removal of Apollo only shows an increase of leading 
end telomere chromatid type fusions while the lagging end telomere is not effected, strengthening the 
hypothesis of an Apollo-mediated initiating excision (Lam et al., 2010; Wu et al., 2010). This first 
resection by Apollo could also provide the template for telomere elongation in telomerase-positive 
cells. While these mechanisms seem to mainly take place at the blunt ended leading telomere, the 
lagging telomere obtains a short overhang due to incomplete replication. In humans, the last primer is 
placed 50-100 nt distant from the very end of the strand where it remains for ~1h after replication 
(Chow et al., 2012). This resembles a previously suggested amount of telomere loss per cell division 
(Huffman et al., 2000). Exo1 continues the resection of the C-strand at both leading and lagging end 
telomeres. As a longer 3’-overhang was observed in S/G2 phase in comparison to G1 phase it seems 
that the C-strand is temporarily hyper-resected. For compensation, a fill-in reaction takes place that is 
mediated by the CST (CTC1, STN1, TEN1) complex and relies on the interaction of POT1b, CST and the 
Polα/primase complex (Casteel et al., 2009; Wu, Takai and De Lange, 2012). While POT1b recruits the 
CST complex to telomeres, it also limits the resection (Hockemeyer et al., 2008). In humans, the 
function of POT1b could be taken over by POT1 and TPP1 as both proteins have implications to interact 
with Polα/primase complex (Diotti et al., 2015). Also, STN1 interacts with TPP1 which could lead to its 
recruitment to telomeres, specifically the G-rich strand (Wan et al., 2009). In humans, STN1/CST seems 
to fulfill the same function as in mice since the depletion of STN1 results in a delayed processing of the 
elongated 3’-overhang on both leading and lagging telomeres (Huang, Dai and Chai, 2012; Wang et al., 
2012). Furthermore, the CST-mediated fill-in reaction is uncoupled from telomerase-dependent 
telomere maintenance in a timely manner and CST seems to additionally partake in the termination of 
telomerase activity (Zhao et al., 2009; Chen, Redon and Lingner, 2012). While the mechanism of 3’-
overhang processing is not fully understood yet, it can be assumed that it is tightly regulated and highly 
defined in humans as 80% of the C-strands end in 3’-CCAATC-5’ (Sfeir et al., 2005).  
9 
Figure 1. Telomere shortening and processing of the G-rich 3’ overhang. 
After semi-conservative replication of telomeres, the 3’ overhang is lost and needs to be reconstituted to allow 
for telomere protection. For an initial resection of the blunt ended leading strand telomere, Apollo is recruited. 
Subsequently, Exo1 resects the leading strand of both daughter telomeres thereby elongating the 3’ overhang. 
An exacerbated loss of telomeric sequence is prevented by subsequent strand fill-in synthesis, which putatively 
involves the CST complex and Pol α. Despite these tightly regulated processes, a reduction of telomeric repeats 
cannot be prevented. As a result, average telomere length shortens with every population doubling thereby 
shaping the end replication problem [modified after (Palm and De Lange, 2008; Wu, Takai and De Lange, 2012; 
Kappei, 2013)]. 
Telomeres as fragile sites 
Telomeres are considered hard-to-replicate regions due to their repetitive G-rich sequence and their 
dense heterochromatic structure (Maestroni, Matmati and Coulon, 2017). The repetitive G-rich 
sequence of the lagging telomere allows for the formation of a four-stranded DNA structure termed 
G-quadruplexes (G4). While spontaneous folding of telomeric sequences into G4 structures was
initially demonstrated in vitro (Sundquist and Klug, 1989; Williamson, Raghuraman and Cech, 1989;
Tang et al., 2008), they were later also identified at telomeres in vivo using structure specific antibodies
(Lam et al., 2013) or ligand molecules (Biffi et al., 2013). As such, they pose an obstacle to telomerase-
10 
dependent telomere length maintenance and the replication machinery leading to replication fork 
stalling or even collapse if not properly dissolved (Zahler et al., 1991). Regulated and reliable resolution 
of G4 during telomere elongation and replication is therefore essential to ensure telomere 
homeostasis. A very prominent mechanism includes RECQ helicases, namely Bloom’s syndrome 
helicase (BLM) and Werner syndrome ATP-dependent helicase (WRN) (Opresko et al., 2002; Lillard-
Wetherell et al., 2004; Barefield and Karlseder, 2012). BLM unwinds G4 in vitro and has higher affinity 
for G4 than for DNA duplex substrates (Sun et al., 1998) or Holiday Junctions (Huber, 2002) indicating 
preferred involvement in smoothening DNA for replication rather than resolution of recombination 
intermediates. While G4 on the leading strand are removed by both BLM and WRN (Drosopoulos, 
Kosiyatrakul and Schildkraut, 2015), the lagging strand mainly relies on WRN (Crabbe et al., 2004). 
Similarly, regulator of telomere length (RTEL1) and Fanconi anemia complementation group J (FANCJ) 
resolve G4 in vitro (London et al., 2008; Wu, Shin-ya and Brosh, 2008) and also in vivo as shown recently 
by fluorescence lifetime imaging microscopy (Lansdorp and van Wietmarschen, 2019; Summers et al., 
2021). 
Telomeres’ ability to form G4 structures is one of the factors contributing to them resembling so called 
fragile sites that are prone to replication stress. At telomeres, this fragility manifests as multiple 
telomeric signals (MTS) or elongated foci in metaphase spreads. This phenotype is strongly connected 
to the loss of shelterin member TRF1. Conditional deletion of TRF1 establishes the formation of MTS, 
which is even exacerbated by treatment with aphidicolin, a DNA polymerase α inhibitor (Martínez et 
al., 2009; Sfeir et al., 2009). In combination with diminished replication as determined by Single 
molecule analysis of replicated DNA (SMARD), this further strengthens the link between TRF1 and 
telomere replication (Sfeir et al., 2009). Additionally, an increase in DDR signaling is observed at TRF1-
depleted telomeres (Martínez et al., 2009; Sfeir et al., 2009). The mechanism by which TRF1 facilitates 
telomere replication involves the recruitment of the helicase BLM, which could for example resolve 
G4, and is also connected to TIN2- and TPP1/POT1-mediated repression of ATR activation and 
subsequent DDR (Zimmermann et al., 2014). 
Another key player in the prevention of telomere fragility is RTEL1, a DNA helicase that was first 
discovered as a positive regulator of telomere length in mice (Ding et al., 2004). Similar to TRF1, an 
increase in MTS was observed upon deletion of RTEL1 in mouse embryonic fibroblasts (MEFs). This 
effect was further exacerbated by induction of replication stress via treatment with aphidicolin, G4 
stabilizers or BLM knock-out (Uringa et al., 2012; Vannier et al., 2012). Additionally, the removal of 
RTEL1 causes telomere loss and an accumulation of telomeric circles, which is rescued by depletion of 
the SLX4 endonuclease or inhibition of replication (Vannier et al., 2012). These findings suggest that, 
in the absence of RTEL1, t-loops are not dissolved and replicated but excised by SLX4 leading to 
telomere free ends and extrachromosomal t-circles. RTEL1 is recruited to telomeres during S-phase by 
TRF2 (Sarek et al., 2015). This recruitment is regulated by a phospho-switch on Ser365 of TRF2, 
meaning that phosphorylation by cyclin-dependent kinases (CDK) prevents the interaction with RTEL1 
and only during S-phase, when the site is dephosphorylated, can RTEL1 be bound and recruited 
(Vannier et al., 2013; Sarek et al., 2015, 2019). The re-phosphorylation of TRF2 is crucial for the timely 
release of RTEL1, which is then again available for the association with proliferating cell nuclear antigen 
(PCNA) and genome-wide replisome activity (Vannier et al., 2013; Sarek et al., 2019). TRF2 not only 
supports telomere replication by cell cycle-dependent recruitment of RTEL1 but also releases 
topological stress by interaction with Apollo thereby complementing the topoisomerase TOP2α (Ye et 
al., 2010). 
11 
 
Telomere transcripts and their function 
Initially, chromosomes were believed to be transcriptionally silenced. Yet, over the past two decades, 
evidence emerged that telomeres are transcribed into long noncoding RNAs, called telomeric repeat-
containing RNA (TERRA). Transcription by RNA polymerase II starts in the subtelomeric region and 
moves towards the chromosome end using the C-rich strand as template. In mammalian cells, the 
resulting transcripts are of heterogenous length, ranging from 100 nt to 9 kb (Azzalin et al., 2007; 
Schoeftner and Blasco, 2008). Typical for RNA polymerase II transcription, TERRA is protected by a 5’ 
7-methyguanosine cap yet only a subset of TERRA harbor a 3’-polyadenylation tail (Azzalin and Lingner,
2008; Oliva-Rico and Herrera, 2017). TERRA promoter regions are characterized by CpG islands in the
subtelomeres which present high affinity binding sites for Nuclear Respiratory Factor 1 (NRF1) and
CTCF, a driver of TERRA transcription (Nergadze et al., 2009; Deng et al., 2012; Diman et al., 2016;
Beishline et al., 2017). While studies suggest that several chromosomes could be the origin of TERRA
transcription (Azzalin et al., 2007; Schoeftner and Blasco, 2008; Nergadze et al., 2009; Deng et al.,
2012), the findings regarding only one telomere as the main source remain controversial (De Silanes
et al., 2014; Montero et al., 2016; Diman and Decottignies, 2018). In telomerase-positive cells, TERRA
levels peak during G1 and are lowest during S-phase indicating a cell cycle-dependent regulation of
transcription (Porro et al., 2010) that would aim at the timely separation of transcription and
replication of telomeres. This aspect is especially interesting as TERRA can hybridize to complementary
DNA regions thereby forming displacement loops and potentially blocking the replisome. These
DNA:RNA hybrid structures are referred to as R-loops and their existence at human telomeres is
indisputable, especially at telomeres using the ALT pathway (Azzalin et al., 2007; Arora et al., 2014;
Diman and Decottignies, 2018). While R-loops can occur due to recruitment to telomeres and
subsequent strand displacement by TERRA, (Oliva-Rico and Herrera, 2017) they can also form co-
transcriptionally (Arora et al., 2014). More and more evidence is emerging about the role of TERRA
and its R-loops in the replication, length maintenance, chromatin structure and DDR signaling at
telomeres. Strong heterochromatic marks like H3K9me3 and heterochromatin protein 1 α (HP1α) have
been shown to repress the transcription of TERRA (Arnoult, Van Beneden and Decottignies, 2012). In
contrast, TERRA itself also promotes H3K9me3, localizes to heterochromatin and interacts with the
histone methyltransferase SUV39H1, indicating a contribution of TERRA to heterochromatin formation
and maintenance, also at telomeres (Deng et al., 2009; Nergadze et al., 2009; Arnoult, Van Beneden
and Decottignies, 2012; Porro et al., 2014; Montero et al., 2018). These findings could indicate a
negative feedback-loop where TERRA represses its own transcription. Additionally, TERRA has
implications in telomere replication. Intuitively, it is tempting to speculate that TERRA and the
formation of R-loops at telomeres only hinder the replisome thereby causing replication stress
(Feuerhahn et al., 2010; Doksani, 2019). Interestingly, Beishline et al. showed that disruption of CTCF-
driven TERRA transcription in cells exposed to replication stress leads to an increase in ultrafine
anaphase bridges and micronuclei, which were rescued by a reintroduction of TERRA (Beishline et al.,
2017). This mechanism suggests that TERRA works in cis to facilitate replication of telomeres and
cannot be reduced to a mere obstacle. The idea of TERRA promoting replication is further supported
by the TERRA-TRF2-mediated recruitment of the origin recognition complex (ORC), which initiates
replisome assembly (Deng et al., 2009).
Of note, TERRA also promotes DDR at telomeres when TRF2 is reduced. Extended zeocin treatment of 
HeLa cells causes a prolonged mitotic arrest that results in loss of TRF2 at telomeres and a subsequent 
increase in γH2AX DDR signaling and end-to-end fusions. Interestingly, this reduction of TRF2 also led 
to enhanced TERRA transcription (Porro et al., 2014). Similarly, in the absence of DNA 
methyltransferases DNMT1 and DNMT3b, TRF2 removal leads to an increase in TERRA-mediated 
recruitment of lysine-specific demethylase 1 (LSD1) and MRE11 which stimulates the nucleolytic 
12 
processing of the 3’ overhang (Porro, Feuerhahn and Lingner, 2014). In agreement with the previously 
mentioned data, this could indicate that TERRA also aids with fusions of unprotected telomeres 
(Cusanelli and Chartrand, 2015). In contrast, intact telomeres rely on TERRA and hnRNPA1 to facilitate 
the replacement of RPA with POT1 on single-stranded telomere sequences which supports the 
protection of telomeres (De Silanes, D’Alcontres and Blasco, 2010; Flynn et al., 2011). Especially at 
telomeres maintained by the ALT pathway, TERRA and R-loops promote recombination events (Arora 
et al., 2014; Cusanelli and Chartrand, 2015; Flynn et al., 2015). The chromatin remodeler ATRX 
functionally antagonizes TERRA genome-wide and the loss of ATRX, as often observed in ALT positive 
cells, is linked to the loss of cell cycle-dependent regulation of TERRA (Flynn et al., 2015; Chu et al., 
2017). Subsequently, RPA persistently binds to telomeres creating a recombinogenic environment. 
While this might not be desired in healthy cells, it is crucial for telomere homeostasis in ALT positive 
cells and a repression of ATR activation in response to RPA results in apoptosis (Flynn et al., 2015). 
While ATRX and TERRA regulate each other via competition, the RNA endonuclease RNaseH1 actively 
degrades R-loops at telomeres (Arora et al., 2014). A depletion of RNaseH1 leads to accumulation of 
TERRA hybrids and subsequent telomere excision while a surplus of RNaseH1 weakens the 
recombinogenic potential of ALT dependent cells resulting in telomere shortening (Arora et al., 2014). 
These finding again demonstrate the importance of a tight regulation of TERRA at telomeres (Cusanelli 
and Chartrand, 2015).  
Telomere length maintenance 
In somatic cells, telomeres shorten with each cell division until they reach a critical length. About 5 
dysfunctional telomeres suffice to induce cellular senescence, a state in which cells metabolize but do 
not replicate (Kaul et al., 2012). The onset of senescence naturally prevents tumorigenesis. However, 
if this coincides with the loss of p53 and RB, cells can bypass senescence. As a result, more critically 
short telomeres (<13 TTAGGG repeats) accumulate in the cell leading to fusions, telomeric crisis, 
chromothripsis and genomic rearrangements (Capper et al., 2007; Shay and Wright, 2011). At this 
point, cells will undergo apoptosis unless they re-elongate their telomeres by one of two mechanisms: 
1.) the reactivation of the enzyme telomerase or 2.) the alternative lengthening of telomeres (ALT) 
mechanism. As a result, cells become immortal and give rise to cancer. While telomerase- and ALT-
mediated telomere elongation are not exclusive, a recent study found that a subset of tumors shows 
neither telomerase expression nor hallmarks of ALT (namely alterations in ATRX or DAXX) (Barthel et 
al., 2017; Viceconte et al., 2017; De Vitis, Berardinelli and Sgura, 2018). Indeed, the majority of cancers 
re-activate telomerase, predominantly by TERT promoter mutations, followed by ALT positive cancers 
comprising the second largest group, with few cancer types displaying both pathways and rare cases 
that lack telomere elongation but instead rely on a telomere pool of sufficient length. Also in contrast 
to somatic cells, germline and embryonic stem cells continuously maintain their telomeres to ensure 
their proliferative potential and genomic stability. They do so by regulated telomerase expression. 
Telomerase 
Telomerase is a ribonucleoprotein enzyme that synthesizes telomeres de novo using an internal RNA 
template. It was first discovered and characterized in the ciliate Tetrahymena thermophila (Greider 
and Blackburn, 1987) and shortly after also in the human cell line HeLa (Morin, 1989). In humans, 
telomerase is active in germline cells and embryonic stem cells but is silenced upon differentiation in 
most tissues with reduced activity in the majority of adult stem cells (Kim et al., 1994; Hiyama and 
Hiyama, 2007). In about 85-90% of cancers, telomerase is reactivated and guarantees their 
immortality. The enzymatically active subunit (telomerase reverse transcriptase, TERT) and the RNA 
13 
subunit (telomerase RNA, hTR) add the canonical TTAGGG telomeric repeats with high precision to the 
G-rich 3’-overhang by reverse transcriptase activity. 
In more detail, the RNA component hTR consists of three structurally and functionally distinct motifs 
(Chen, Blasco and Greider, 2000). The pseudoknot domain with the adjacent template sequence (t/PK) 
and the conserved regions 4 and 5 (CR4/5) are bound by TERT and crucial for catalytic activity. This 
core unit is sufficient for telomerase activity in vitro. However, in vivo, the telomerase holoenzyme 
undergoes extensive biogenesis. Proper assembly of telomerase requires the box H/ACA domain of 
hTR which interacts with DKC1 (dyskerin), NOP10, NHP2 and GAR1. This composition is also shared by 
small nucleolar ribonucleoproteins and is mediated by localization to Cajal bodies (CBs) (Jády, Bertrand 
and Kiss, 2004; Theimer et al., 2007). Additionally, the CB protein TCAB1 associates with the H/ACA 
lobe of hTR (Nguyen et al., 2018). TCAB1 is essential for telomerase localization to CBs and if mutated 
or lacking, telomerase biogenesis and reverse transcriptase activity are hampered (Venteicher et al., 
2009). Even though localization of telomerase to CBs and their importance for processivity is 
undisputed, recent data suggests that the actual process of telomere elongation does not take place 
within the CBs (Laprade et al., 2020). Recently, the structural interplay of the members of the human 
telomerase holoenzyme was finally solved using Cryo-electron microscopy (Nguyen et al., 2018). It 
revealed the formation of two RNA-tethered lobes. The H/ACA lobe is bound by two tetramers 
composed of NOP10, NHP2, GAR1 and DKC1 each and one additional TCAB1. The second lobe contains 
the catalytic core where t/PK and CR4/5 encircle TERT. In turn, TERT surrounds the template sequence 
and forms the active site (Nguyen et al., 2018). Within the catalytic core, the RNA sequence 3’-
CAAUCCCAAUC-5’ serves as template for the elongation of the telomeric 3’ overhang. The overhang 
itself binds as primer to the template and starts the reverse transcription. In one round of elongation, 
50-60 nts are added to a single telomere (Zhao et al., 2009). Telomeric repeats are added in a stepwise 
manner while telomerase activity is additionally influenced by the presence of POT1-TPP1 and the 
immediate formation of G4 (Hwang, Opresko and Myong, 2014; Jansson et al., 2019). 
The amount of fully assembled telomerase per cell is limited suggesting an active recruitment to 
telomeres rather than random diffusion (Xi and Cech, 2014). This notion is supported by findings that 
telomerase does not act on every telomere but only selectively elongates a subset (Ouellette et al., 
2000; Hemann et al., 2001), for example, telomerase is primarily active on short telomeres in yeast 
(Chang, Arneric and Lingner, 2007). Also, TERT transcription and activity spike during S-phase indicating 
that telomere elongation by telomerase takes place after replication (Xi and Cech, 2014). While the 
exact trigger and mechanism of telomerase recruitment to telomeres is not fully understood, there is 
ample of evidence that TPP1 interacts with telomerase through its OB-fold and thereby facilitates 
telomerase activity (Xin et al., 2007; Abreu et al., 2010; Tejera et al., 2010). This might be further 
regulated by HOT1 which interacts with members of the telomerase holoenzyme and promotes TERT-
chromatin binding (Kappei et al., 2013). Interestingly, the POT1-TPP1 heterodimer also recruits the CST 
complex to telomeres where it counteracts telomerase, adding another layer of telomere elongation 
control (Chen, Redon and Lingner, 2012). As elaborated previously, the CST complex is involved in 
strand fill-in and potentially finalizes the replication at telomeres. It will be interesting to see in the 
future, what exact mechanism prompts telomerase to act at specific telomeres, how this is timed with 
regular replication and what decides the termination of telomere elongation. 
Alternative lengthening of telomeres 
While the majority of cancers depend on telomerase for telomere elongation, about 10-15% of cancers 
rely on ALT. This mechanism is especially dominant in sarcomas and astrocytomas (Henson and Reddel, 
2010; De Vitis, Berardinelli and Sgura, 2018). Interestingly, details of several pathways in connection 
14 
 
to ALT have emerged over the recent years raising the question if ALT is not a single mechanism but in 
fact a collection of many that seem to have homologous recombination (HR) as a common 
denominator. 
Indeed, several specific characteristics of ALT positive cells have been well-defined and these hallmarks 
of ALT argue for an HR-driven mechanism: an increased level of telomere recombination events as 
determined by telomeric sister chromatid exchanges (t-SCEs) was observe (Dunham et al., 2000; 
Londoño-Vallejo et al., 2004), as well as elevated levels of extrachromosomal telomeric repeats (ECTR) 
including t-circles, C-circles and G-circles (Cesare and Griffith, 2004; Henson et al., 2009; Nabetani and 
Ishikawa, 2009). In comparison to telomeres elongated by telomerase are ALT dependent telomeres 
more heterogeneous in both length and sequence (Bryan et al., 1995). They can become as long as 
50 kb, while telomerase-maintained telomeres are typically about 10 kb in length. In addition to the 
canonical TTAGGG repeats, the aforementioned variant repeats (N-, G-, and T-type) that are usually 
inherent to the subtelomeric regions are interspersed throughout the telomeres (Allshire, Dempster 
and Hastie, 1989; Baird, Jeffreys and Royle, 1995; Conomos et al., 2012). Both characteristics could be 
explained by the homology driven nature of the ALT pathway. Another indication arguing for HR at ALT 
telomeres was the discovery that telomeres localize to promyelocytic leukemia (PML) bodies, then 
termed ALT-associated PML bodies (APBs) (Henson and Reddel, 2010). These nuclear structures 
contain not only the name-giving PML protein but also telomeric DNA, TRF1/2 and factors involved in 
DNA synthesis and recombination, namely RAD51, RAD52, RPA and the SMC5/6 complex (Yeager et 
al., 1999; Fasching et al., 2007; Potts and Yu, 2007; Draskovic et al., 2009). APBs are believed to serve 
as hubs for HR of telomeres as their disruption correlates with a decrease in t-SCEs and telomere 
shortening in ALT cells. The clustering of telomeres during ALT is RAD51 dependent and reminiscent of 
homology searches during homology directed repair (HDR) (Cho et al., 2014). As template for 
recombination-mediated DNA synthesis one could imagine the t-loop, ECTR (linear or circular), sister 
telomeres, and distant telomeres after long range movement (Cesare and Reddel, 2010). 
Although we do not fully understand what determines the onset of ALT as opposed to the reactivation 
of telomerase, several potential drivers of ALT have been postulated. For example, TERRA not only 
localizes to telomeres (Azzalin et al., 2007) but also APBs (Arora et al., 2014). In human ALT cells, TERRA 
transcription is upregulated allowing for an increase in R-loop formation, a DNA:RNA hybrid structure 
that is regulated by RNaseH1. These TERRA R-loops can transform telomeric chromatin into a suitable 
substrate for HR and thereby promote ALT (Arora et al., 2014; Arora and Azzalin, 2015). In addition to 
the canonical factors of homology directed repair, several chromatin remodelers and epigenetic 
effectors modulate the nucleoprotein architecture and thereby influence the ALT pathway. Most 
notably, the mutation of the chromatin remodeling complex, alpha thalassemia/mental retardation 
syndrome X-linked (ATRX) and death-domain associated protein (DAXX), has been identified as a very 
common and predictive feature of ALT tumors (Heaphy et al., 2011). Furthermore, an orchestrated 
recruitment of the NuRD complex via ZNF827 and the nuclear receptors promotes HR at ALT telomeres, 
putatively through remodeling of the telomeric chromatin (Conomos, Reddel and Pickett, 2014). 
Another factor is the Anti-Silencing Factor 1 (ASF1), a histone chaperone that assist DNA replication. 
Reduction of its paralogs ASF1a and ASF1b by RNAi leads to an increase in ALT hallmarks like t-SCEs, 
ECTR and APBs in previously ALT negative cells. This artificial induction of ALT phenotypes clearly links 
ALT to replication stress, chromatin remodeling and chromatin dysfunction (O’Sullivan et al., 2014). 
Following the link to replication stress, it was proposed that break-induced replication (BIR) could 
trigger telomere elongation by ALT. This mechanism is effective at collapsed replication forks and has 
been mainly studied in yeast where it promotes telomere recombination. Now, there is emerging 
evidence in human cell lines arguing for a related mode of action at ALT telomeres, like the involvement 
15 
 
of polymerase δ subunits POLD3 and POLD4, RAD52 and the occurrence of conservative replication 
(Dilley et al., 2016; Roumelioti et al., 2016; Min, Wright and Shay, 2017; Zhang et al., 2019).  
Upper length limitations 
While telomere elongation is essential for a cell’s proliferative potential, an upper limit is provided by 
negative regulation of telomere length. Interestingly, soon after their discovery it was reported that 
the overexpression of TRF1 or TRF2 results in telomere shortening (Van Steensel and De Lange, 1997; 
Smogorzewska et al., 2000). It was then suggested that negative regulation of telomere length is based 
on trimming of telomeric DNA from the chromosome ends, a mechanism that involves t-loop excision 
mediated by HR- and DDR sensing- factors (Li and Lustig, 1996; Pickett et al., 2009, 2011). While this 
mode of action is reminiscent of the ALT mechanism, trimming is not restricted to ALT dependent cells 
(Pickett et al., 2011; Rivera et al., 2017). This is further supported by the identification of ZBTB48/TZAP, 
a negative telomere length regulator with implications in telomere trimming (Jahn et al., 2017; Li et 
al., 2017). Indeed, this additional layer of telomere length control is more rapid than the gradual 
shortening by cell divisions and could counteract over-elongation of telomeres.  
The end protection 
As a consequence of linear chromosomes, telomeres resemble double-strand breaks (DSBs) that could 
be recognized by the DNA damage surveillance machinery of the cell and consequently induce repair 
mechanisms. Thus, telomere structures need to be protected in order to prevent unwanted end-to-
end fusions or unscheduled recombination events.  
Genome-wide, DSBs occur as a consequence of internal or external DNA damage agents. The majority 
of DSBs is repaired by Non-homologous end joining (NHEJ) or Homology directed repair (HDR). While 
HDR is the pathway of choice during S-/G2 phase when a homologous sister chromatid is available, 
NHEJ can occur throughout the entire cell cycle (Tacconi and Tarsounas, 2015). NHEJ is a quick and 
promiscuous repair mechanism and therefore widely applicable. However, this pathway is more error-
prone than HDR and can result in insertions or deletions of nucleotides with unknown and potentially 
detrimental outcome (Tacconi and Tarsounas, 2015). Upon recognition of a DSB, the Ku70-Ku80 
heterodimer (Ku) binds to the open ends and, together with DNA-dependent protein kinase catalytic 
subunit (DNA-PKcs), recruits additional factors required for canonical NHEJ (cNHEJ), like  the nuclease 
Artemis for minor end resection and finally DNA ligase 4 (Lig4) (Ciccia and Elledge, 2010; Chang et al., 
2017). If the NHEJ pathway is impaired, the alternative NHEJ (aNHEJ) pathway takes place which 
depends on poly (adenosine diphosphate ribose) polymerase 1 (PARP1) as well as DNA ligases 3 (Lig3) 
and 1 (Lig1) instead of Lig4. This pathway is microhomology-driven and relies on more extensive end 
resection by MRN and CtIP, resulting in overhangs of 15-100 bp (Chang et al., 2017). Of note, many of 
the factors known to be activated upon DSB throughout the genome perform the same function at 
unprotected or defected telomeres (Arnoult and Karlseder, 2015). In mammalian cells, cNHEJ at 
telomeres is repressed by TRF2/RAP1 (Bae and Baumann, 2007; Sarthy et al., 2009). Upon knock-out 
of TRF2 in dividing cells, uncontrolled telomere fusions and chromosome concatenations take place; 
an observation that was not made for any of the other shelterin members, pointing out the importance 
of TRF2 for telomere protection (Van Steensel, Smogorzewska and De Lange, 1998; Denchi and De 
Lange, 2007; Sfeir and De Lange, 2012; de Lange, 2018). The removal of Lig4 in the same scenario 
drastically reduces the amount of fusion events, indicating the involvement of the cNHEJ pathway. 
However, a small number of telomere fusions was still observed upon double knock-out of TRF1 and 
TRF2 in MEFs, despite the absence of Lig4. This observation suggests that the Lig4-independent aNHEJ 
can also function at telomeres. Upon knock-out of the Ku complex, a repressor of aNHEJ, this effect 
16 
 
was exacerbated and then rescued by removal of Lig3 (Bombarde et al., 2010; Sfeir and De Lange, 
2012). Similar as in genome-wide DDR, aNHEJ might be a rescue pathway that is prominently active if 
cNHEJ is compromised and therefore, in the first instance, also repressed in presence of TRF2 at 
telomeres. While HDR is involved in telomere length maintenance in ALT positive cancer cells and 
partakes in telomere replication, spontaneous HDR is undesired at telomeres in functional cells as 
unequal strand exchanges and recombination events lead to telomere loss (de Lange, 2018; Doksani, 
2019). This telomere loss can be quite extreme if long fragments are cleaved off as circular or linear 
ECTRs due to resolvase activity at Holiday junctions. At telomeres, HDR activity is repressed by 
TRF2/RAP1 and POT1 as their removal results in an increase in t-SCEs (Sfeir et al., 2010; Rai et al., 2016; 
Doksani, 2019). Additionally, the Ku complex is known to repress HDR at DSBs. However, it is difficult 
to differentiate this activity at telomeres as Ku deletion is lethal to mammalian cells (Celli, Denchi and 
de Lange, 2006). 
The DNA damage response signaling is controlled by kinases including ataxia telangiectasia mutated 
(ATM) and ataxia telangiectasia and Rad3 related (ATR). Upon recognition of the DSB by the MRN 
complex (MRE11, RAD50 and NBS1), ATM is recruited which starts a phosphorylation cascade involving 
γH2AX, MDC1, RNF8 and RNF168 (Blackford and Jackson, 2017). Recruitment and phosphorylation of 
these factors induces additional recruitment cycles thereby spreading and amplifying the DDR signal. 
The ubiquitin ligases RNF8 and RNF168 facilitate recruitment of 53BP1 to DNA damage sites by 
ubiquitylation of histones. At telomeres, this signaling cascade is blocked by TRF2 (Denchi and De 
Lange, 2007; Arnoult and Karlseder, 2015). In MEFs, it was shown that the iDDR sequence within the 
hinge domain of TRF2 is responsible for this repression of 53BP1 signaling by inhibition of RNF168 
(Okamoto et al., 2013). In general, ATR mediated DNA damage signaling occurs in the presence of 
single-stranded DNA. Replication protein A (RPA) binds to the ssDNA and ATR is recruited to the 
damage site by its interaction partner ATRIP. Subsequently, Rad17 loads the 9-1-1 complex (Rad9, Rad1 
and Hus1) to the ss-/ds-junction of the lesion and ATR is finally activated by TopBP1 or ETAA1 
(Blackford and Jackson, 2017). At telomeres, ATM and ATR signaling are repressed by two distinct 
mechanisms: while TRF2 inhibits ATM, POT1-TPP1 block the ATR pathway (Denchi and De Lange, 2007). 
As RPA and POT1 both recognize and bind to ssDNA, it has been proposed that POT1 outcompetes RPA 
at the telomeric 3’-overhang thereby preventing the initiation of DDR signaling. Also, research in MEFs 
showed a supportive function of TPP1 and TIN2 in POT1-mediated telomere protection that could 
explain how POT1 displaces RPA despite similar binding affinities for ss-telomeric DNA and a higher 
abundance of RPA (Takai et al., 2011). Interestingly, in vitro data showed that POT1 prevents RPA-
mediated unfolding of telomeric G4 suggesting an additional mode of action for telomere protection 
(Ray et al., 2014). 
An additional mechanisms assumed to hide the chromosome ends from any DDR machinery is the 
formation of t-loops, as the ss-overhang might not be as accessible to DNA damage signaling factors 
anymore once it is part of the D-loop structure. First, the formation of t-loop structures was verified 
biochemically and then in 2013 Doksani et al. demonstrated the presence of t-loops in mouse cells 
using stochastic optical reconstruction microscopy (STORM)(Stansel, De Lange and Griffith, 2001; 
Doksani et al., 2013). With this method they also verified the importance of TRF2 for both the 
formation and maintenance of the t-loop. Upon deletion of TRF2, the number of t-loops was reduced 
even when end-to-end fusions were repressed by removal of ATM. Neither the deletion of RAP1 nor 
POT1 could recapitulate this effect indicating the specificity of TRF2 for this function (Doksani et al., 
2013). The mechanisms by which TRF2 promotes and stabilizes t-loop formation are starting to surface: 
TRF2 locates to the junction between the duplex repeats and the ss-overhang (Stansel, De Lange and 
Griffith, 2001) protecting the t-loop from branch migration and Holiday Junction resolution (Schmutz 
et al., 2017). In addition, the hybridization domain of TRF2 (TRFH) can wrap ~90 bp DNA around itself. 
17 
A loss of function mutant, termed Top-less, cannot support t-loop formation anymore (Benarroch-
Popivker et al., 2016), further pinpointing the unique involvement of TRF2.  
Telomeres in aging and disease 
While telomere length on a cellular level is regarded as an indicator for the replicative potential, its 
possible indication for longevity of an organism remains controversial. Similar to proliferating cells, the 
average telomere length decreases with age directly linking telomere state to the process of aging in 
humans (Canela et al., 2007; Turner, Vasu and Griffin, 2019). Also, if telomere maintenance goes awry, 
this can result in premature aging syndromes (Holohan, Wright and Shay, 2014). Still, studies have 
shown that telomere length varies greatly between individuals of the same age and therefore, the 
absolute length itself does not serve as a sufficient indicator for lifespan in humans. A high-throughput 
telomere length quantification by FISH resulted in the proposal to determine the rate of telomere 
shortening or the rate of increase of short telomeres per nucleus to predict longevity (Canela et al., 
2007; Whittemore et al., 2019). Studies in mice came to the same conclusion further strengthening 
the need for dynamic observation of telomere length changes over time (Vera et al., 2012). In addition 
to age, telomere length was suggested to be susceptible to genetic factors, psychological stress, 
obesity, smoking and alcohol consumption while physical fitness has beneficial effects (Turner, Vasu 
and Griffin, 2019). Interestingly, it was demonstrated that induced pluripotency in senescent cells was 
not only possible but resulted in rejuvenation and reset telomere length, again strengthening the link 
between telomere biology and aging (Lapasset et al., 2011; McHugh and Gil, 2018). 
Telomeres in senescence and cancer 
Senescent cells are characterized by a ceased replicative potential. While these cells continue to 
metabolize, they are unable to undergo cell division. In addition, their morphology changes as well as 
their biochemical and functional properties. Even a unique secretome profile was observed for 
senescent cells, called the Senescence associated secretory phenotype (SASP) (Coppé et al., 2008). 
SASP consists of proinflammatory cytokines, chemokines, growth factors and proteases that stimulate 
immune cells and facilitate the clearance of senescent cells (Coppé et al., 2008; Turner, Vasu and 
Griffin, 2019). Another potential function of SASP is the stimulation of nearby progenitor cells thereby 
promoting tissue regeneration. This mode of action contributes to wound healing and tissue repair and 
is associated with the so-called acute senescence (Krizhanovsky et al., 2008; Demaria et al., 2014; Van 
Deursen, 2014; Ritschka et al., 2017). As opposed to acute senescence, the chronic senescence is 
proposed to be linked to organismal aging (Victorelli and Passos, 2017; McHugh and Gil, 2018). Here, 
senescent cells are not sufficiently cleared from the tissue anymore, leading to accumulations that 
impair the regeneration and function of organs and subsequently evoke aging phenotypes (Van 
Deursen, 2014; Turner, Vasu and Griffin, 2019). It is not clear how exactly telomeres contribute to the 
different forms of senescence and aging in healthy organisms (Van Deursen, 2014). 
The onset of cellular senescence is strongly linked to the state of individual telomeres which often 
leads to the comparison of telomeres with a cellular clock (Bernadotte, Mikhelson and Spivak, 2016). 
Indeed, due to the end replication problem, telomeres of somatic cells shorten with each cell division. 
Once a critical length is reached, DDR triggers the activation of p16(INK4a) or p53 and subsequently 
p21 and RB to induce cell cycle arrest (D’Adda Di Fagagna et al., 2003; Jacobs and De Lange, 2004; 
Maciejowski and De Lange, 2017). Interestingly, the initiation of this cascade depends on the shortest 
telomeres rather than the average telomere length and as few as five dysfunctional telomeres suffice 
for induction (Hemann et al., 2001; Zou et al., 2004; Kaul et al., 2012). Short telomeres cannot be 
adequately bound by the protective shelterin complex anymore and it was shown that the loss of 
18 
 
shelterin members leads to t-loop unwinding followed by telomere deprotection thereby locking the 
cells in p53-mediated cell cycle arrest (D’Adda Di Fagagna et al., 2003; Victorelli and Passos, 2017; Van 
Ly et al., 2018; Turner, Vasu and Griffin, 2019). This telomere related growth arrest can also occur 
independently of telomere length but when telomeres are damaged or dysfunctional due to loss of  
individual shelterin members or exogenous DNA damage induction, likewise arresting the cells by 
checkpoint activation (Karlseder, Smogorzewska and De Lange, 2002; Wu et al., 2006; Fumagalli et al., 
2012; Hewitt et al., 2012; Victorelli and Passos, 2017). If the cause of telomere failure cannot be 
rectified, the cells become senescent or apoptotic (Fumagalli et al., 2012; Hewitt et al., 2012; Turner, 
Vasu and Griffin, 2019).  
While the effects of senescence and aging seem unfavorable, they naturally prevent tumorigenesis. If 
critically short or dysfunctional telomeres concur with a loss or mutation of the tumor suppressors p53 
or RB, cells are able to bypass senescence, continue to divide and thereby pass on genomic defects to 
the daughter cells (Brown, Wei and Sedivy, 1997; Beauséjour et al., 2003; Jacobs and De Lange, 2004). 
This bypass is referred to as lifespan elongation. With deactivated or insufficient cell cycle check points, 
short and dysfunctional telomeres accumulate over cell divisions and eventually the so-called 
telomeric crisis is reached (D’Adda Di Fagagna et al., 2003; Zou et al., 2004; Hayashi et al., 2015). During 
crisis, end-to-end fusions of the telomeres lead to breakage-fusion-bridge cycles, chromothrypsis, 
kataegis and genomic instability (Shay and Wright, 2011; Maciejowski et al., 2015; Maciejowski and De 
Lange, 2017; Voronina et al., 2020). The resulting genomic rearrangements, aneuploidy and prolonged 
mitotic arrest cause most cells to undergo cell death by apoptosis or autophagy (Davoli and de Lange, 
2012; Hayashi et al., 2015; Nassour et al., 2019). Recent data has shown that telomere-dysfunction 
induced autophagy plays a major role in the prevention of crisis-escape and thereby inhibits 
tumorigenesis (Nassour et al., 2019). However, a concomitant reactivation of telomerase or the 
induction of the ALT pathway renders cells immortal and they enter a cancerous state (Maciejowski 
and De Lange, 2017). Therefore, telomere maintenance is central in cancer development.  
 
Figure 2. Telomeres and senescence act as cancer suppressors.  
In somatic cells, telomeres shorten with each cell division. A few short or dysfunctional telomeres suffice to 
trigger check point activation followed by cell cycle arrest and senescence. In the absence of p53 or RB, cells 
continue to cycle and accumulate very short and dysfunctional telomeres, which eventually leads to telomere 
crisis. Cells that escape cell death during crisis and manage to reinstate telomere length maintenance, undergo 
genomic rearrangements and become cancerous. 
19 
 
Telomere biology disorders 
An array of genetic diseases has been linked to abnormally short telomeres and telomere dysfunctions 
that has therefore been clustered under the terms telomeropathies or telomere biology disorders 
(TBD). Given the heterogeneity of clinical manifestations and genetic alterations, telomeropathies 
have been proposed as a spectrum disorder rather than distinct diseases (Holohan, Wright and Shay, 
2014).  
The most prominent genetic disorder is dyskeratosis congenita (DC). The X-linked form of DC is caused 
by mutations in the DKC1 gene that derives its name from the very same circumstance (Heiss et al., 
1998). Patients suffering from DC often present with the mucocutaneous triad of nail dysplasia, lacy 
skin and oral leukoplakia (Vulliamy et al., 2006). Furthermore, the risk of developing progressive bone 
marrow failure (BMF), pulmonary fibrosis and a subset of cancers is highly elevated (Ballew and Savage, 
2013). In addition to DKC1, mutations in hTR, NOP10, NHP2 and WRAP53 (encoding for TCAB1) have 
been reported in DC patients (Vulliamy et al., 2006; AlSabbagh, 2020). As these genes express crucial 
components of the telomerase holoenzyme, the direct link between DC and telomere length was 
further strengthened. Furthermore, growth defects as well as an increase in TIFs were observed in 
patient-derived T-lymphocytes (Kirwan et al., 2011).  In combination with reports about alterations in 
shelterin member TIN2, which is linked to telomerase recruitment by its interaction with TPP1 and also 
protects telomeres from DDR, the data suggests that differing mechanistic details can lead to similar 
telomere dysfunctions and clinical symptoms. Clinically severe variants of DC have been reported, like 
the Revesz syndrome and the Hoyeraal-Hreidarsson syndrome (HH). While these variants are difficult 
to distinguish by clinical criteria, the Revesz syndrome has been mainly linked to de novo mutations in 
exon 6 of TIN2 (Karremann et al., 2020). In addition to the classical triad of DC, HH patients also present 
with cerebellar hypoplasia and pancytopenia (Høyeraal, Lamvik and Moe, 1970; Hreidarsson et al., 
1988). Here, telomeres of patient-derived lymphocytes are even shorter than those of age-matched 
classical DC patients and it could be argued that the disease severity correlates with telomere length 
(Alter et al., 2012). Interestingly, ectopic expression of RTEL1 was able to rescue the loss of telomere 
(Deng et al., 2013). Similar as in classical DC, mutations were found in DKC1, TINF2 and TERT but also 
in ACD and RTEL1 (LeGuen et al., 2013; Glousker et al., 2015). Additionally, genomic rearrangements 
and telomeric aberrations are hallmarks of HH (Touzot et al., 2012; LeGuen et al., 2013). 
Shortened telomeres and mutations in TERT and hTR have also been reported in pulmonary fibrosis, 
liver cirrhosis or aplastic anaemia (Barbaro, Ziegler and Reddel, 2016). As shortened telomeres can be 
inherited, a genetic anticipation of telomere biology disorders was observed. While older generations 
with shortened telomeres presented with idiopathic pulmonary fibrosis or aplastic aneamia as well as 
an onset during adulthood, younger generations suffered from the more severe DC, and also 
experienced an earlier onset of the disorders (Armanios, 2012; Holohan, Wright and Shay, 2014). 
Independent of the previously mentioned DC, Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare 
progeroid disease with a characteristic mutation in LMNA, the gene for Lamin A (Ahmed et al., 2018). 
While HGPS does not present with any known mutations in telomere- or DDR-related genes, the 
premature aging phenotype could be implicative for an altered telomere state. Indeed, a shortening 
of telomeres was observed in HGPS fibroblast but not in hematopoietic cells that do not express Lamin 
A (Decker et al., 2009). Stable expression of the characteristic Lamin A mutant, termed progerin, leads 
to dysfunctional telomeres, proliferative defects and premature replicative senescence, an effect that 
could be reduced by overexpression of TERT (Kudlow et al., 2008; Benson, Lee and Aaronson, 2010). 
These findings argue for a link between the Lamin A mutation and telomeres, possibly mediated by 
TRF2 (Wood et al., 2014), but the exact mechanism remains elusive. Aguado et al. were able to show 
20 
that dysfunctional telomeres in HGPS cells induce transcription of telomeric non-coding RNAs. 
Counteraction of these RNAs with telomeric antisense oligonucleotides inhibits DDR and premature 
senescence in patient fibroblast. In the HGPS mouse model it even increases lifespan (Rossiello et al., 
2017; Aguado et al., 2019). These findings are steps towards not only understanding telomeropathies 
but also treating them and thereby improving patients’ lifes.  
21 
Rationale 
As described above, telomeres are indispensable for genomic stability and human health. Functional 
telomeres act as natural tumor suppressors and ensure proper aging while maintaining self-renewal 
capabilities in germline and embryonic stem cells. The open ends are protected from the DDR 
machinery of the cell by t-loop formation and coating with the shelterin complex. Thereby, telomeres 
prevent ATM- and ATR-mediated DNA damage signaling, unscheduled HDR and end-to-end fusions by 
NHEJ (de Lange, 2018). Beyond the shelterin complex, additional telomere binders like HOT1, ZBTB48 
and transiently associated factors have functions in telomere replication, elongation and maintenance 
(Kappei et al., 2013; Jahn et al., 2017; Li et al., 2017; de Lange, 2018). These known telomere binders 
were recapitulated by Kappei et al. in a mass spectrometry-based interactomics screen utilizing 
telomeric sequences which also identified additional, uncharacterized candidates for telomere 
association (Kappei et al., 2017).  
In this work, I aim to characterize one of these novel telomere-associated proteins, namely the zinc 
finger protein ZNF524. To date, there is no published data on the telomeric function of ZNF524. 
However, annotation of ZNF524 revealed four zinc finger domains towards the C-terminus of the 
protein that could be responsible for sequence recognition and telomere binding. Therefore, it is 
important to validate the direct interaction of ZNF524 with telomeres and determine the DNA binding 
domain. During the course of this thesis, I have applied a plethora of molecular biology techniques as 
well as classic telomere biology assays to gain a deeper understanding of the function of ZNF524 at 
telomeres and the potential influence on cancer progression and telomeropathies.  
  
22 
 
Results 
ZNF524 localizes to telomeres 
ZNF524 is a 29 kDa protein that harbors four zinc fingers towards its C-terminus (Figure 3 A). While 
structure predictions suggested that ZNF524 interacts with DNA via these zinc fingers, experimental 
evidence was missing until the identification of ZNF524 in a pull down screen with telomeric DNA bait 
followed by mass spectrometry-based quantification of interactors. 
ZNF524 binds to telomeric repeats via its zinc fingers 
In a previous mass spectrometry based DNA-protein interaction screen (Kappei et al., 2017), ZNF524 
was identified as a binder to the telomeric repeat sequence TTAGGG in human fibroblasts (IMR90). To 
verify this association, a DNA-pulldown was performed using whole cell lysate of telomerase positive 
HeLa and ALT dependent U2OS cells. Binding to TTAGGG repeats was reproduced with extracts from 
both cell lines. Additionally, we observed interaction with the telomeric variant repeats TCAGGG, 
TGAGGG and TTGGGG but not the scrambled control sequence GTGAGT (Figure 3 B). For detection of 
ZNF524 by Western Blot, we raised a polyclonal antibody against ZNF524 (Figure 7). Using bacterially 
expressed His-ZNF524 in an identical setup, we demonstrated a similar association as observed for the 
endogenous protein. As bacterial lysate can be assumed to lack putative assisting factors, these 
findings strongly argue for a direct interaction of ZNF524 with the telomeric sequences (Figure 3 C). 
 
Figure 3. ZNF524 binds to telomeric repeats via its four zinc fingers 
(A) Schematic overview of ZNF524 with its four C-terminal zinc fingers. (B) DNA pulldowns with canonical TTAGGG 
and variant telomeric repeats using HeLa and U2OS lysate. After supplementing the lysate with the indicated 
biotinylated oligonucleotides, interactors were separated using streptavidin coupled magnetic beads. The 
scrambled GTGAGT repeat sequence served as control bait. (C) DNA pulldowns with canonical and variant 
repeats with bacterially expressed His-ZNF524 WT, His-ZNF524 MD (minimal domain) and His-ZNF524 ZF2 
mutant. (D) FLAG-ZNF524 ZF mutants were overexpressed in HEK293 and subjected to the same DNA pulldown 
using the canonical and the scrambled control sequence. 
ZNF524 harbors four Cys2His2-type (C2H2) zinc fingers at the C-terminal region of the protein, 
presenting putative DNA binding domains (Figure 3 A). We therefore created a minimal domain (MD) 
limited to these four annotated zinc fingers (aa 109-239)(Figure 3 A). The bacterially expressed MD 
23 
 
construct bound to telomeric and variant repeat sequences in a comparable manner as the full length 
ZNF524 construct, supporting the hypothesis that the four ZF MD indeed comprises the DNA binding 
domain (DBD)(Figure 3 C). The typical C2H2-type ZF structure composes of two antiparallel β-sheets 
and an α-helix. Two cysteine and two histidine residues of the canonical CX2–4CX12HX2–6H motif interact 
with a zinc ion to maintain structural integrity. To examine the interaction of ZNF524 with telomeric 
sequences in more detail, point mutations were introduced into each individual zinc finger thereby 
replacing the first Cys of the C2H2 binding motif with an Ala and ensuring sufficient disruption of the 
structure. The FLAG-ZNF524 mutant constructs were then overexpressed in HEK293 cells and 
subjected to the same telomeric DNA pulldown. Strikingly, ZNF524 does not enrich at TTAGGG repeats 
anymore upon disruption of the second zinc finger (Figure 3 D). This was verified with the bacterially 
expressed ZNF524 ZF2 mutant as well (Figure 3 C). Single mutations of the other three ZFs do not result 
in a loss of binding (Figure 3 D). Interestingly, double mutations of ZF1+ZF3, ZF1+ZF4 or ZF3+ZF4 lead 
to strongly reduced or abrogated DNA-protein interaction, which is also true for the ZF1+ZF3+ZF4 triple 
mutant (Figure 3 D). These findings indicate that ZF2 is essential yet not sufficient for recognition and 
binding of TTAGGG repeats.  
ZNF524 localizes to telomeres in vivo 
After having established telomere binding in vitro, we wanted to examine whether ZNF524 also 
localizes to telomeres in vivo. Therefore, we introduced doxycycline inducible C-terminally GFP-tagged 
constructs into U2OS cells. In addition to ZNF524-GFP WT, we also tested ZNF524-GFP ZF2 mut 
expecting the loss of binding that we observed in the previously mentioned DNA pulldowns.  
24 
 
Figure 4. ZNF524 binds to telomeres in the cell 
(A) Fluorescence microscopy of co-localization between TRF2 (red) and ZNF524-GFP (green). Representative 
images of doxycycline induced ZNF524-GFP WT and ZNF524-GFP ZF2 mutant in U2OS cells are shown (scale bar 
10 µm). Nuclei were counterstained with DAPI (blue). (B) Quantification of co-localization events of ZNF524-GFP 
WT with TRF2 (n=99 nuclei). Telomeric foci and the overlap with GFP foci were scored on maximum intensity 
projections of the acquired z-stacks. (C) Representative slot blot after ChIP using GFPtrap beads to enrich for 
ZNF524-GFP WT and ZNF524-GFP ZF2 mut. Co-precipitated chromatin was visualized using either a telomeric 
probe (left) and or an Alu control probe (right). (D) Quantification of telomeric probe signal in ChIP experiments. 
The enrichment over input was normalized against the NLS-GFP negative control (n=3; error bars represent SD; 
* p<0.05, Welch’s test). (E) Quantification of ChIP-seq experiments comparing ZNF524-GFP WT to ZNF524-GFP 
ZF2 mut and NLS-GFP negative controls. A minimum of 7 and a maximum of 25 hexameric repeats were 
considered (n=3; error bars represent SD; * p<0.001, one-way ANOVA followed by Dunnett’s multiple comparison 
tests). 
25 
 
Indeed, in immunofluorescence microscopy we saw the formation of nuclear foci upon induction of 
ZNF524-GFP WT expression. An additional staining against TRF2 indicated the position of telomeres 
within the nucleus (Figure 4 A). Quantification of 99 nuclei revealed that on average 66% of telomeres 
are occupied by ZNF524. Strikingly, in 20% of cells almost all telomeres (90-100%) are bound by 
ZNF524-GFP (Figure 4 B). While not every telomere also displays a GFP signal, it is striking that every 
ZNF524-GFP foci colocalizes with a TRF2 signal. A subset of cells (~15%) present with a strong GFP 
signal throughout the entire nucleus, potentially masking GFP foci. For these cells, only 0-10 GFP foci 
could be distinguished from the pan-nuclear GFP signal. These cells were included in the quantification 
but might cause an underestimation of ZNF524-GFP foci and colocalization events. In parallel, the assay 
was performed with the ZNF524 ZF2 mut that lost binding to TTAGGG repeats in our in vitro assays. 
Despite the evident expression of the mutant construct, we could not observe any foci formation thus 
validating the previously observed loss of binding also in a cellular context (Figure 4 A). Additionally, 
we performed chromatin immunoprecipitation using GFPtrap beads against the GFP-tagged constructs 
expressed in U2OS. In addition to ZNF524-GFP WT, we also tested ZNF524-GFP ZF2 mut for which we 
did not expect telomeric enrichment. A GFP-tagged nuclear localization sequence (NLS) that does not 
contain a DNA binding domain served as negative control. Probing for telomeric sequences after slot 
blotting revealed an enriched binding of ZNF524 WT to telomeric DNA when compared to both the ZF2 
mut and the NLS (Figure 4 C, D). ZNF524 WT retrieved around 2.6-fold more telomeric DNA than 
ZNF524 ZF2 mut (Figure 4 D). In comparison to the NLS construct, we could not observe an increased 
telomere recovery for ZNF524-GFP ZF2 mut thereby again validating the loss of binding upon ZF2 
disruption. As expected, ZNF524-GFP WT did not enrich for the Alu control sequence (Figure 4 C, D). 
Using the same experimental setup for ChIP followed by next generation sequencing (NGS) (ChIP-seq), 
we recapitulated an enrichment of TTAGGG stretches of 7 to 25 repeats (Figure 4 E), which are here 
considered as chromatin fragments derived from telomeres.  
As U2OS cells are ALT dependent and harbor unusually long telomeres, we tested whether ZNF524 
also localizes to telomeres in other cell lines. Thus, we chose three telomerase positive cell lines (HeLa, 
HeLa 1.3 and HT1080ST) and three additional ALT cell lines (GM847, Saos2 and WI-38 VA-13) for 
doxycycline-induced overexpression of ZNF524-GFP WT and ZNF524-GFP ZF2 mut followed by 
telomeric fluorescent in situ hybridization (FISH) and IF. 
26 
 
27 
Figure 5. ZNF524 localizes to telomeres in ALT cell lines 
Fluorescence microscopy of co-localization between telomeric FISH (red) and GFP-ZNF524 (green). 
Representative images of doxycycline induced ZNF524-GFP WT and the ZF2 mutant in HeLa (A), HeLa1.3 (B), 
HT1080ST (C), GM847 (D), Saos-2 (E), and WI-38 VA-13 (F) cells are shown (scale bar 10 µm). Nuclei were 
counterstained with DAPI (blue). Quantification of co-localization events of ZNF524-GFP WT with telomeric PNA 
in GM847 (D), Saos-2 (E), and WI-38 VA-13 (F) (n=148, 86 or 101 nuclei respectively). Telomeric foci and the 
overlap with GFP foci were scored on maximum intensity projections of the acquired z-stacks.  
In the telomerase positive cell lines, we did not detect foci formation for either ZNF524-GFP WT or 
ZNF524-GFP ZF2 mut, despite successful overexpression (Figure 5 A, B, C). In contrast, each of the ALT 
cell lines displayed colocalization between ZNF524-GFP WT (Figure 5 D, E, F). While fewer telomeres 
coincided with ZNF524-GFP when compared to U2OS cells, it is remarkable that, again, each detected 
ZNF524-GFP dot corresponds to a telomeric signal. Furthermore, ZNF524-GFP WT signals and 
telomeres overlapped mainly in very large foci, reminiscent of the ALT-specific APBs. Again, we did not 
see colocalization events in ZNF524-GFP ZF2 mut overexpressing cells. 
To reach a better understanding of ZNF524’s direct protein environment at the telomere, we 
performed a BioID assay. Therefore, ZNF524 WT and ZNF524 ZF2 mut were N-terminally tagged with 
the biotin ligase BirA*, stably integrated in U2OS and overexpressed by doxycycline induction. Of note, 
comparing ZNF524 WT to ZNF524 ZF2 mut specifically targets proximity partners of ZNF524 at the 
telomeres. In contrast, proteins that associate with ZNF524 independently of DNA-binding might also 
interact with ZNF524 ZF2 mut and are therefore not enriched in this set-up.  
 
Figure 6. ZNF524 localizes proximal to telomeric factors 
Volcano plot of BioID assay comparing proximity partners of ZNF524 WT as opposed to ZNF524 ZF2 mut in U2OS 
cells. BirA*-ZNF524 WT and ZF2 mut were induced with 100 ng/ml doxycycline. Specifically-enriched proteins 
(red circles) are distinguished from background binders by a two-dimensional cut-off of >4-fold enrichment and 
p<0.01. Two-dimensional error bars represent the standard deviation after iterative imputation cycles during the 
label-free analysis with substituted zero values (e.g. no detection in the ZF2 mut samples). Detection of ZNF524 
is highlighted in blue. 
Measurement of biotinylated proteins on the mass spectrometer showed an enrichment of the known 
telomere binders NR2C2, NR2C1, TRF2 and TRF1 for BirA*-ZNF524 WT (Figure 6). While TRF2 and TRF1 
constitutively bind to telomeres, NR2C2 and NR2C1 have a stronger affinity to the variant repeat 
TCAGGG than to the canonical sequence (Conomos et al., 2012). As these four proteins are abundantly 
present at U2OS (ALT dependent) telomeres, the data further supports the association of ZNF524 with 
telomeres in vivo.  
28 
 
Functional analysis of ZNF524 
Generation of an α-ZNF524 polyclonal antibody 
To validate the both localization of ZNF524 to telomeres (Figure 3 B) and the generation of ZNF254 KO 
cell lines, I tested multiple commercially available α-ZNF524 antibodies that neither detected the 
denatured protein (as confirmed by Western Blot (WB)) nor the native protein (as confirmed by 
Immunofluorescence (IF) and Immunoprecipitation (IP)). We thus raised our own α-ZNF524 antibody 
(Figure 7 A). To this end, I first bacterially expressed a His-MBP-ZNF524 fusion protein. The His-tag 
allowed for Ni-NTA purification of the recombinant protein while the MBP-tag improved the solubility 
and the immunogenic potential of the recombinant protein. After Immobilized metal affinity 
chromatography (IMAC), fractions of at least 75% purity (E22 – E25) were dialyzed and used for 
immunization of rabbits by the external company (Figure 7 B). To prepare the purification of antibodies 
specific for ZNF524 from animal serum, I subsequently expressed His-ZNF524, which was first purified 
against Ni-NTA (Figure 7 C) and then against Heparin (Figure 7 D). The negatively charged Heparin 
resembles the DNA phosphate backbone thereby promoting interaction with DNA binding proteins. 
Indeed, fractions E44 – E46 were sufficiently pure after this Ion exchange chromatography (Figure 7 
D). To pack the α-ZNF524-specific column, His-ZNF524 was covalently bound to iodoacetyl-groups of 
coupling resin via reduced thiol side chains of cysteine residues. The rabbit serum was applied to the 
ZNF524 coupled columns which retained specific antibodies. Subsequently, bound antibodies were 
eluted under acidic conditions, followed by immediate neutralization, and the resulting elution 
fractions tested for specificity in WB, IF and IP. While the α-ZNF524-containing fractions did not 
recognize ZNF524 in IF or IP, they were successfully validated in WB (Figure 7 E). A strong signal was 
observed for both WT HeLa and U2OS lysate while the ZNF524 KO in HeLa and U2OS clones as well as 
RNAi treatment resulted in a loss or a reduction of signal respectively. Therefore, our antibody is 
suitable for detection of the denatured protein but not for native or formaldehyde-treated ZNF524.  
 
29 
 
 
Figure 7. ZNF524 antibody generation and validation 
(A) Schematic overview of the workflow for the generation and validation of the ZNF524 polyclonal antibody. (B) 
His-MBP-ZNF524 was bacterially expressed and purified by IMAC for immunization of rabbits. The recombinant 
protein is highlighted by a black box. For purification of specific α-ZNF524 antibodies from rabbit serum, His-
ZNF524 was expressed and purified against NiNTA (C) and Heparin (D) and subsequently coupled to Sulfo-Link 
columns. Recombinant His-ZNF524 is indicated by black boxes. (E) After α-ZNF524 purification, the antibody was 
validated in WB using HeLa and U2OS lysate from WT and ZNF524 KO clones as well as lysate from U2OS cells 
treated with control esiRNA or ZNF524 esiRNA. 
30 
 
Generation of ZNF524 knock-out clones  
To facilitate functional analyses, we created ZNF524 KO clones. We therefore used the widely 
applicable CRISPR/Cas9 system and designed three guide RNAs to target different regions in the first 
coding exon of ZNF524. To represent the two major telomere length maintenance mechanisms, we 
opted for the telomerase positive HeLa cell line and the ALT dependent U2OS cell line. After single cell 
sorting and clonal expansion, the ZNF524 KO was validated on a protein level by WB and on a genomic 
level by next generation sequencing (Figure 8). We identified 5 KO clones per cell line that all differ in 
their genomic alterations (Figure 8 A, B) and result in abrogated protein expression (Figure 8 C). 
 
Figure 8. Generation of ZNF524 KO clones in HeLa and U2OS cells 
(A) Next generation sequencing analysis of genomic modifications in ZNF524 KO clones as induced by three 
different sgRNAs. The region targeted by the three sgRNAs was amplified from the genome (indicated by the red 
box) and sequenced by Illumina MiSeq. The sequences of the different variants are plotted in comparison to the 
31 
 
reference sequences (A: green, C: blue, G: gray, T: red). Deletions are represented by dotted lines and insertions 
indicated by shape- and color-coded symbols. The respective sequences are listed below the plot. On the right, 
the calculated allele frequencies of each KO clone are listed and color-coded by percentage. (B) Absolute counts 
obtained for each clone by next generation sequencing range from 25,900 to 87,106 with a minimum of 11,070 
counts per variant. The color code indicates the percentage of each variant per clone. (C) The U2OS and HeLa 
ZNF524 KO clones were verified on a protein level by WB using our self-produced α-ZNF524 antibody. Tubulin 
served as loading control. 
Global effects of ZNF524 
Proliferation and cell cycle distribution are not effected by ZNF524 depletion 
Removal of telomeric proteins can have detrimental effects on the cell, like growth defects, senescence 
onset, cell cycle arrest and even cell death. Therefore, we first investigated the effect of ZNF524 
depletion on proliferation and cell cycle distribution. Population doublings of five WT clones and five 
ZNF524 KO clones were monitored over a period of five weeks, for both U2OS (Figure 9 A) and HeLa 
cell lines (Figure 9 B). However, a significant difference in proliferation was not observed. Telomeric 
defects can trigger checkpoint activation, thereby effecting duration of cell cycle phases or even 
leading to cell cycle arrest. Thus, we determined the cell cycle stages by flow cytometry analysis of 
U2OS and HeLa clones that had been fixated in ethanol and the cellular DNA stained with propidium 
iodide (PI). The unsynchronized cells were assigned to the respective cell cycle stage based on the DNA 
content/PI intensity. Again, we did not detect ZNF524-dependent effects on cell cycle distribution for 
either cell line (Figure 9 C, D). These findings suggest that ZNF524 depletion itself does not have 
detrimental effects on overall cell proliferation. 
 
Figure 9. Proliferation and cell cycle distribution are not impaired in ZNF524 KO cells 
(A) Growth curves of U2OS WT and ZNF524 KO cells. Cells were cultured at sub-confluent densities and the 
accumulated population doublings determined. (B) Growth curves of HeLa WT and ZNF524 KO cells. Cells were 
cultured at sub confluent densities and the accumulated population doublings determined. (C) Cell cycle 
distribution in U2OS WT and ZNF524 KO cells. The cell cycle stage of at least 10,000 cells per clone was 
32 
 
determined by flow cytometry of propidium iodide- stained cells. (D) Cell cycle distribution in HeLa WT and 
ZNF524 KO cells. The cell cycle stage of at least 10,000 cells per clone was measured by propidium iodide staining 
and flow cytometry. Each experiment was performed with five WT and five ZNF524 KO clones for robustness and 
statistically analyzed by Student’s t-test (not significant). 
Transcription factor activity 
As C2H2 type Zinc finger proteins form the largest family of transcription factors in humans, we 
hypothesized that the depletion of ZNF524 could have effects on the transcriptome. We performed 
RNA sequencing (RNA-seq) of the five WT and five ZNF524 KO clones to determine up- or 
downregulated genes in response to the lack of ZNF524. We used both U2OS and HeLa clones to allow 
for comparison of ALT- or telomerase-dependent changes. 
  
 
Figure 10. Limited effects of ZNF524 KO on differential gene expression 
(A, B) Volcano plots showing differentially expressed genes (DEGs) as determined by RNA-seq in U2OS (A) and 
HeLa (B) WT and ZNF524 KO clones. ZNF524 is highlighted in red. The negative log10 (adjusted p-value) is plotted 
against the log2 fold change. Grey dots indicate DEGs as defined by an FDR<0.01 cut-off. Genes with a higher 
FDR are depicted as black dots. (C, D) Visualization of the first two principal components of WT (grey dots) and 
ZNF524 KO clones (black dots) in U2OS (C) and HeLa (D) transcriptomes. 
We did not identify any differentially expressed genes (DEGs) in U2OS ZNF524 KO clones as compared 
to the WT (Figure 10 A). In HeLa clones, we identified 156 DEGs, a much smaller number than what is 
expected for a potential transcription factor (Figure 10 B, Table 1), and mostly moderate fold changes. 
Interestingly, ZNF524 was not upregulated in the WT clones as compared to ZNF524 KO clones. Even 
though the protein is not present upon disruption of ZNF524 (Figure 8 C), a comparable amount of 
transcript is still present within the cell, suggesting that the genetic deletion does not subject the mRNA 
to nonsense-mediated decay. When looking at sample relatedness by comparing principal components 
1 and 2, we do not see a clustering of the clones according to their ZNF524-dependent genetic 
33 
 
background (Figure 10 C, D). Instead, the mixed distribution of WT and ZNF524 KO clones indicates 
that the variance between clones of the same genetic background resembles the variance between 
the WT and the KO group. A high heterogeneity was also observed between ZBTB48 KO clones, but 
DEGs were still reproducibly detected by RNA-seq and subsequently validated (Jahn et al., 2017). 
Therefore, the data suggest that ZNF524 does not widely act as a transcription factor, a notion that is 
further supported by the lack of unique binding sites identified during ChIP-seq. 
The function of ZNF524 at telomeres 
ZNF524 does not play a major role in telomere length maintenance 
Telomere length maintenance is influenced by several factors localizing to telomeres, for example 
HOT1, ZBTB48 or the CST complex (Kappei et al., 2013; Jahn et al., 2017; Li et al., 2017). To determine 
whether ZNF524 ranks among those, we examined a potential involvement of ZNF524 in telomere 
length maintenance related mechanisms both in U2OS (ALT dependent) and HeLa (telomerase 
positive) cells. In U2OS WT and ZNF524 KO clones, we studied telomere length by quantitative FISH 
(qFISH) and telomere restriction fragment (TRF) analysis (Figure 11 A, B, C). Both approaches did not 
reveal a change in telomere length in the absence of ZNF524, even after 3 months of continuous 
culture. Additionally, we probed for ALT activity by C-circle assay. C-circles are a form of ECTR that 
commonly occurs in ALT positive cancer cells as a byproduct of HR mediated telomere elongation. 
Amidst the heterogeneity between the clonal lines, we did not observe a ZNF524-dependent effect 
(Figure 11 D).  
 
  
 
34 
 
 
Figure 11. Telomere length maintenance by ALT remains intact in ZNF524 KO U2OS 
(A) Representative images of FISH staining in U2OS WT and ZNF524 KO clones. The TAMRA-labeled C-rich 
telomere probe (red) marks the telomeres. Nuclei were counterstained with DAPI (blue). Scale bars represent 
10 µm. (B) qFISH analysis of the TAMRA-labeled C-rich telomere probe. The bean plot shows the individual data 
points as densities with the solid line indicating the mean. The experiment was performed with 3 WT and 3 KO 
clones. n is the number of quantified foci and Student’s t-test compares the mean values of the individual clone 
(n. s.). (C) Telomere restriction fragment analysis of U2OS WT and ZNF524 KO clones. Five clones per condition 
were cultured for three months after ZNF524 removal by CRISPR/Cas9. Student’s t-test analysis was not 
significant. (D) C-circle assay of U2OS WT and ZNF524 KO clones. The slot blot shows the C-circle amplification 
products of five WT and five KO clones with 7.5 ng DNA template and the no φ29 polymerase negative control. 
The bar plot shows the signal intensity quantification of the C-circle assay depicting the mean intensities ± SD 
with the intensity value of each individual clone depicted as a black dot. Statistical analysis was done by Welch-
test (n. s.).  
As HeLa cells elongate their telomeres by telomerase instead of the ALT mechanism in U2OS cells, we 
repeated the TRF analysis in HeLa WT and ZNF524 KO clones (Figure 12 A). While the average telomere 
length decreased from 4.4 kb to 2.7 kb upon ZNF524 depletion, this difference was not significant 
(p=0.098). Additionally, we performed the qPCR-based telomeric repeat amplification protocol (TRAP) 
analysis, which focuses on telomerase activity rather than absolute telomere length. The repeat 
amplification was analyzed based on cycle threshold (Ct) values as well as repeat amplicon separation 
by gel electrophoresis (Figure 12 B). Both read-outs showed equal telomere amplification activity in 
WT and ZNF524 KO clones suggesting that ZNF524 is likely not involved in telomerase processivity and 
activity. This notion was further strengthened when we challenged the cells with the telomerase 
35 
 
inhibitor BIBR1532. While treatment with sufficient concentrations of BIBR1532 lead to the expected 
decrease in cell viability and arrest in S-phase, we did not see a ZNF524-dependent effect (Figure 12 C, 
D). 
 
 
Figure 12. Telomere length maintenance by telomerase remains intact in ZNF524 KO HeLa 
(A) Telomere restriction fragment analysis of HeLa WT and ZNF524 KO clones. Five clones per condition were 
cultured for 3 months after CRISPR/Cas9 treatment and analyzed. Student’s t-test was used for statistical analysis 
(n.s.) (B) Telomeric repeat amplification protocol of HeLa WT and ZNF524 KO clones. The product of 30 
amplification cycles is shown. Four clones per condition were loaded. Heat inactivated lysate and water input 
served as negative controls. (C) Viability assay of HeLa WT and ZNF524 KO clones treated with telomerase 
inhibitor BIBR1532. Cells were treated with 10 μM and 50 μM BIBR1532. The amount of viable cells was 
determined by Alamar blue staining, subsequent measurement of absorbance and normalization against an 
untreated control population. The individual values of the five WT and five KO clones are indicated by black dots. 
(D) Cell cycle distribution of HeLa WT and ZNF524 KO clones comparing untreated and 1 μM BIBR1532 treated 
cells. Cell cycle stage of five WT and five KO clones was determined by flow cytometry after PI staining. 
In conclusion, neither ALT-mediated telomere elongation nor telomere synthesis by telomerase seem 
to depend on ZNF524 in steady-state cancer cells. 
The localization of TRF2 and RAP1 to telomeres is mediated by ZNF524 
Studies have shown that telomere associated factors can influence each other’s recruitment to or 
abundance at telomeres. We therefore asked if members of the shelterin complex are affected by the 
presence or lack of ZNF524 at telomeres.  
36 
 
To this end, we examined the presence of the double-strand binders TRF1 and TRF2 by IF. To reduce 
technical effects to a minimum, we co-stained for TRF1 and TRF2, stained the five WT and the five 
ZNF524 KO clones in parallel and imaged all samples in one session. 
 
Figure 13. ZNF524 positively effects localization of TRF2 and RAP1 to telomeres 
 (A) Representative immunofluorescence images of U2OS WT and ZNF524 KO cells stained for TRF1 (green) and 
TRF2 (red). Scale bars represent 10 µm. Nuclei were counterstained with DAPI (blue). For better visualization of 
signal intensities, TRF2 staining is additionally shown in Red Hot. (B) Quantification of TRF1 and TRF2 IF signals in 
WT and KO clones. The violin plot shows the individual data points as densities. 1487-4576 telomeres per clone 
37 
 
were analyzed for TRF1 and 2300-5290 telomeres per clone were analyzed for TRF2; the mean is indicated by a 
solid line; p-values are determined by Student’s t-test and indicated above the plot; * p<0.05. For statistical 
comparison of the KO clone-based conditions (KO, KO + ZNF524 WT, KO + ZNF524 ZF2 mut) a paired Student’s t-
test was chosen.   
Indeed, we found that TRF2 occurrence at telomeres is reduced in the absence of ZNF524 (Figure 13 
A, B). Interestingly, this effect was not apparent for TRF1 (Figure 13 C). Next, we wondered if the 
reduction of TRF2 at ZNF524-depleted telomeres could be rescued by reintroducing ZNF524 in our KO 
clones. Therefore, we lentivirally transduced the five ZNF524 KO clones with DOX-inducible ZNF524-
HA WT or ZNF524-HA ZF2 mut. Strikingly, overexpression of ZNF524-HA WT restored TRF2 levels at 
telomeres while ZNF524-HA ZF2 mut did not (Figure 13 B). These findings directly link telomere binding 
of ZNF524 to TRF2 occurrence at telomeres. Exogenous expression of ZNF524-HA WT or ZNF524-HA 
ZF2 mut did not alter TRF1 signal intensity (Figure 13 C). 
As ZNF524 influences telomere association of TRF2 but not of TRF1, we additionally repeated the IF 
staining for the shelterin members RAP1 and POT1.  
Figure 14. ZNF524 does not influence telomere abundance of the single-strand binder POT1 
(A) Representative IF pictures of U2OS WT and ZNF524 KO cells stained for RAP1 (red). (B) Quantification of the
RAP1 IF signal in WT and KO clones depicted as violin plots. 2245-5290 telomeres per clone were analyzed; the
mean is indicated by a solid line; p-values are determined by Student’s t-test and indicated above the plot;
* p<0.05.  (C) Representative images of IF staining for POT1 (red) coupled to telomeric FISH staining (green). Both
U2OS WT and ZNF524 KO are shown. (D) Quantification of POT1 IF signal. The violin plot depicts the intensity
values of 5 WT and 5 KO clones. 329-790 telomeres per clone were analyzed. The mean is indicated by a solid
line and significance determined by Student’s t-test.
38 
RAP1 relies on its interaction partner TRF2 for localization to telomeres. Indeed, similar to TRF2, IF-
based analysis of RAP1 occurrence at telomeres revealed a reduction in ZNF524 KO cells (Figure 14 A, 
B). Similar to TRF1, IF analysis also showed that the abundance of the single-strand binder POT1 
remained unchanged upon ZNF524 depletion (Figure 14 C, D). Taken together, these data indicate a 
positive effect of ZNF524 on the localization of the TRF2/RAP1 subcomplex to telomeres while the 
shelterin complex as a whole is unaffected. 
Next, we wondered if this reduction of TRF2 and RAP1 at telomeres was a result of decreased protein 
levels and therefore determined the protein amounts of TRF2 and RAP1 by quantitative WB (Figure 15 
A, B). Additionally, we measured the proteome and re-analyzed RNA-seq data of the U2OS WT and 
ZNF524 KO clones, to specifically look at the shelterin members and the telomere binders 
HMBOX1/HOT1 and ZBTB48/TZAP (Figure 15 C, D, E).  
 
Figure 15. ZNF524 does not influence the expression of other telomere binders 
(A) Quantitative Western blot showing total TRF2 and RAP1 protein levels in U2OS WT and ZNF524 KO clones 
with GAPDH as loading control. (B) Quantification of TRF2 and RAP1 signal normalized to GAPDH. The bar plot 
shows the mean intensities ± SD. The intensity values of the individual clones are depicted as black dots. 
Statistical comparison by Welch-test (n.s.). (C) Volcano plot of proteome measurements in U2OS WT and ZNF524 
KO clones. Members of the shelterin complex are highlighted (orange) among the background cloud proteins. 
(D) Volcano plot of RNA-seq results in U2OS WT and ZNF524 KO clones. Members of the shelterin complex are 
highlighted (orange) in the non-differentially regulated genes. 5 WT and 5 KO clones were measured. (E) 
Heatmap of telomere binders and the shelterin complex members identified by RNA-seq in the individual U2OS 
WT and ZNF524 KO clones. 
As the protein and transcript levels are not significantly different in WT and ZNF524 KO clones with 
regard to the shelterin complex, it seems that ZNF524 rather functions as a mediator of TRF2 binding 
to telomeres than a transcription factor for the telomeric proteins. Interestingly, we identified 76 up- 
or down-regulated proteins when comparing the proteome of U2OS WT and ZNF524 KO clones (Table 
2), which were not reflected in the transcriptome of the U2OS clones. These findings hint at ZNF524-
39 
 
dependent post-transcriptional processes that do not influence mRNA levels and will be subject of 
future investigations. 
Since TRF2 occurrence at telomeres is higher in the presence of ZNF524, we wondered whether 
ZNF524 physically interacts with TRF2. To test this hypothesis, we performed a Co-
immunoprecipitation experiment with overexpressed ZNF524-GFP and FLAG-TRF2. 
 
Figure 16. ZNF524 does not constitutively bind to TRF2 
Co-Immunoprecipitation of ZNF524-GFP and TRF2-FLAG overexpression. IPs and input containing both 
overexpression constructs are indicated by the black box. NLS-GFP overexpression served as negative control. 
While the α-FLAG IP successfully enriched FLAG-TRF2 and endogenous RAP1, it did not interact with 
ZNF524-GFP. Vice versa, targeting ZNF524-GFP did not enrich for FLAG-TRF2 (Figure 16). A very weak 
signal for endogenous RAP1 was detected but this was also present in the NLS-GFP negative control 
indicating unspecific binding rather than ZNF524-RAP1 interaction. Taken together, the lack of ZNF524-
dependent differential expression and direct interaction suggests an indirect mechanism by which 
ZNF524 regulates TRF2/RAP1 occurrence at telomeres. 
 
DNA damage and telomeric aberrations occur at telomeres lacking ZNF524 
As TRF2 and RAP1 are known to mediate telomere protection from the DNA damage response 
machinery, we next looked for telomere dysfunction induced foci (TIF) as indicated by an overlap of 
telomere FISH signal and staining against a DDR protein. Using 53BP1 as marker, we indeed showed 
increased DNA damage signaling at telomeres lacking ZNF524 (Figure 17 A, C). In U2OS WT clones, we 
on average observed 0.7 TIFs that increased to an average of 1.1 TIFs in cells lacking ZNF524 (Figure 17 
B). In HeLa WT and ZNF524 KO clones, we even detected an average increase from 1.4 to 2.9 TIFs per 
cell (Figure 17 D).  
40 
 
 
Figure 17. DNA damage signaling increases in cells lacking ZNF524 
53BP1 immunofluorescence staining (green) coupled with telomeric FISH (red) indicates telomere dysfunction 
induced foci (TIFs) in U2OS (A) and HeLa (C) WT and ZNF524 KO clones, scale bars represent 10 µm. Nuclei were 
counterstained with DAPI (blue). (B, D) Quantification of TIFs per cell in U2OS (B) and HeLa (D) clones; 5 WT and 
5 KO clones were counted with at least 35 nuclei per clone; upper plot: Frequency of cells with the indicated 
number of TIFs; error bars represent SD; lower plot: the vertical lines (red) represent the fitted expected number 
of TIFs (GLMM for negative binomially distributed data). Error bars represent 95% confidence intervals for the 
mean number of TIFs. The p-value was calculated using a Likelihood Ratio Test; ** p<0.01. 
As mentioned previously, unprotected telomeres are recognized by the DDR machinery leading to 
telomere fusions. In the absence of TRF2, these fusion events are caused by ATM-mediated DDR repair. 
With a reduction of TRF2 at ZNF524-depleted telomeres and an increase in telomeric DNA damage 
signaling, we tested for an upregulated phosphorylation and thereby activation of ATM and its 
downstream partner CHK2 by probing for pATM in quantitative WB (Figure 18 A, B) and for pCHK2 in 
WB (Figure 18 C). However, a lack of ZNF524 does not induce an increase in overall ATM 
phosphorylation. As expected, phosphorylation levels of its downstream effector CHK2 in consequence 
remain unchanged as well. 
 
 
41 
 
Figure 18. Loss of ZNF524 does not upregulate pATM or pCHK2 
(A) Quantitative Western blot showing total pATM protein levels in U2OS WT and ZNF524 KO clones with tubulin
as loading control. U2OS cells treated with 40 mJ UV served as positive control. (B) Quantification of pATM signal
normalized to tubulin. The bar plot shows the mean intensities ± SD. The intensity values of the individual clones
are depicted as black dots. Statistical comparison by Welch-test. (C) Western blot showing total pCHK2 protein
levels in U2OS WT and ZNF524 KO clones with GAPDH as loading control. U2OS cells treated with 40 mJ UV served
as positive control.
With a reduction of TRF2/RAP1 and an increase in DNA damage signaling, we looked for additional 
chromosome aberrations as a result of ZNF524 removal. Therefore, we examined mitotic telomeres by 
chromosome orientation fluorescence in situ hybridization (CO-FISH) which specifically stains the 
parental C- and G-rich telomeric strands, allowing to trace telomeric sister chromatid exchanges (t-
SCEs). This set-up can also be used to score end-to-end fusions. The experiment was performed in 
U2OS cells, where we had previously observed the reduction of TRF2/RAP1 at telomeres in addition to 
the increase in TIFs.  
Figure 19. Increased telomeric sister chromatid exchanges at ZNF524 depleted telomeres 
(A) CO-FISH with Cy3-labeled G-rich telomere probe (red) and FITC-labelled C-rich telomere probe (green) in
U2OS cells. White boxes and blue boxes indicate telomeres with telomeric sister chromatid exchanges (t-SCEs)
or fragility phenotype respectively. Scale bars represent 10 µM. Metaphases were counterstained with DAPI
(blue). (B) Quantification of t-SCEs events per metaphase; 5 WT and 4 KO clones were counted with at least 10
metaphase spreads per clone; upper plot: Frequency of cells with the indicated number of t-SCEs; error bars
represent SD; lower plot: the vertical lines (red) represent the fitted expected number of t-SCEs (GLMM for
negative binomially distributed data). Error bars represent 95% confidence intervals for the mean number of t-
SCEs. The p-value was calculated using a Likelihood Ratio Test; *** p<0.001.
As already implicated by the lack of upregulated ATM phosphorylation, we did not see telomere 
fusions despite the reduction in TRF2 localization to telomeres in ZNF524 KO clones (Figure 19 A). 
These results are reasonable because TRF2 is still present at ZNF524 KO telomeres and minimal levels 
of TRF2 have been shown to suffice for prevention of NHEJ (Cesare et al., 2013). Similarly, the amount 
42 
of sister chromatid fusions remained equally low in both WT and ZNF524 KO clones. We did however 
observe a significant increase in t-SCEs upon ZNF524 depletion (Figure 19 A, B). ALT positive cells have 
a basal level of t-SCEs that is necessary for homologous recombination (HR)-mediated telomere 
maintenance. Elevated t-SCE levels are indicative for an increase in recombination events which has 
previously been linked to deficiencies in RAP1 mediated HR prevention (Sfeir et al., 2010; Rai et al., 
2016). This is in agreement with our findings of reduced telomeric RAP1 in ZNF524 KO cells.   
Synthetic lethality with ZNF524 
So far, we can conclude that the removal of ZNF524 by KO does not influence the viability or 
proliferation of cells. As for telomeric phenotypes, TRF2 and RAP1 are reduced at telomeres lacking 
ZNF524 while DNA damage signaling and homologous recombination are significantly increased. While 
these are fascinating findings, the overall fitness of the cell is not impaired. This could for example be 
the case if the loss of ZNF524 was compensated for by a pathway of similar function. Also, ZNF524 
could be fairly redundant under ideal culturing conditions but become essential under exposure to 
stress. We thus wondered if genetically challenging the cells could give us additional information on 
ZNF524’s function, especially in the context of telomeres, DDR and HR. To this end, we performed a 
genome-wide negative synthetic lethality screen. Synthetic lethality occurs if the removal of two or 
more genes renders a cell nonviable while the deletion of these genes individually would not affect the 
proliferative behavior of the cell. 
Therefore, we chose a CRISPR/Cas9-based approach for our U2OS WT and ZNF524 KO clones (Figure 
20). To examine genome-wide involvement of ZNF524 we applied a pooled sgRNA library. It comprises 
187,536 gRNAs that target 18,543 genes (Park et al., 2017). The pooled library was introduced into 
both the WT and the KO clones where the sgRNAs stably integrate into the genome. Over time, the 
gRNAs that evoke a synthetic lethality are depleted from the KO cell population while they steadily 
remain in the WT population. Determination of the gRNAs that are more abundant in the WT samples 
in comparison to the KO samples therefore indicate genes and proteins that are important co-factors 
of ZNF524’s function. 
43 
Figure 20. Schematic depiction of negative synthetic lethality screen workflow 
U2OS WT and ZNF524 KO clones were transduced to stably express S. pyogene Cas9. After selection and 
verification of Cas9 activity, the clones were lentivirally infected with the pooled sgRNA library comprising 
187,536 gRNAs targeting a total of 18,543 genes. We aimed for a multiplicity of infection of 1. The clones were 
selected for successful transduction and cultured for 1 month. Cells carrying gRNAs against genes evoking a 
synthetic lethality with ZNF524 would deplete from the pool in ZNF524 KO clones during this time. The surviving 
cells were harvested and genomic DNA extracted. The gRNA sequences integrated into the genome were 
amplified by PCR and marked with a 6-nucleotide barcode indicating the clone. The amplicons were sequenced 
by Illumina NextSeq 500 for high output, the reads were aligned to the sgRNA library sequences and reads per 
sgRNA entity were counted. A significant reduction in reads in KO clones as compared to WT clones indicates a 
genetic link. 
After transduction of the clonal lines with Cas9, the activity was determined using a GFP reporter assay 
(Doench et al., 2014). In short, a vector carrying both the sequence for GFP expression as well as a GFP 
targeting gRNA sequence was introduced into the Cas9 positive cells. In case of active Cas9, the GFP 
sequence would be altered at the target site leading to a reduced GFP signal while cells without active 
Cas9 would continue to express functional GFP. The GFP signal was determined by flow cytometry and 
Cas9 activity was successfully validated in all U2OS WT and ZNF524 KO clones. 
Five days post-transduction with the sgRNA library, a first timepoint was collected to define the initial 
situation. Principal component analysis (PCA) showed that at this early timepoint the WT and KO clones 
did not deviate from each other in terms of sgRNA distribution. However, after four weeks of culture, 
the U2OS WT and ZNF524 KO clones cluster away from the initial samples and away from each other, 
indicating both a timepoint- and a ZNF524-dependent effect (Figure 21 A).  
44 
Figure 21. Synthetic lethality screen reveals genetic interactions with ZNF524 
(A) Visualization of the first two principal components of WT and ZNF524 KO clones in the synthetic lethality
screen. PCA was done on scaled sgRNA counts. The center of each distribution is marked by a large dot. The initial
situation is shown in blue (WT) and red (KO) while the situation after four weeks is represented in green (WT SL)
and tile (KO SL) with smaller dots for the individual clones. (B) Volcano plot depicting genes based on sgRNA
identification. The -log10(p-value) is plotted against the log2(Fold change of WT/KO). Significant depletion as
defined by FDR<0.1 is indicated in red. The nuclear receptors NR2C2 and NR2F2 are highlighted in blue. (C) Gene
onthology terms of genes inducing synthetic lethality with ZNF524.
Indeed, we identified 264 genes as putative genetic interactors of ZNF524 by significant depletion of 
their respective sgRNAs (Figure 21 B). Gene onthology analysis of these 264 synthetically lethal genes 
showed an enrichment of biological processes like positive regulation of transcription, DNA damage 
response and also negative regulation of telomerase as defined by PINX, POT1 and p53 (Figure 21 C). 
Interestingly, the nuclear receptors NR2C2 and NR2F2 were also identified (Figure 21 B). NR2C2 and 
NR2F2 are so-called orphan receptors as their function is still being uncovered, however there have 
been implications in promotion of HR. As these factors also locate to telomeres in ALT positive cell 
lines, like the U2OS cell line used in this screen, we verified their synthetic lethality with ZNF524. To 
this end, we designed a competitive proliferation assay (Figure 22 A). U2OS WT clones 2 and 3 were 
45 
transduced with a vector for constitutive expression of EGFP while ZNF524 KO clones 1 and 2 were 
transduced with an identical vector for iRFP expression. WT clone 2 and KO clone 1 were mixed in 
identical ratios (Mix 1  replicate 1) and so were WT clone 3 and KO clone 2 (Mix 2  replicate 2) for 
biological replicates. Subsequently, replicate 1 and 2 were transduced for expression of Cas9 and 
sgRNAs against NR2C2 or NR2F2. In case of a verified synthetic lethality, we expect the WT cells to 
eventually outcompete the impeded ZNF524 KO cells leading to decreasing ratios of ZNF524 KO:WT 
(Figure 22 D).  
Figure 22. Genetic link between ZNF524 and nuclear receptors 
(A) Set up of the SL validation experiment. (B) Western blot of replicates transduced with sgRNA targeting NR2C2
or Gal4 negative control. Replicate 1 corresponds to a mix of WT2 and KO1 while replicate 2 corresponds to a
mix of WT3 and KO2. (C) T7endonuclease 1 assay to validate genetic modifications upon transduction with sgRNA
targeting NR2F2 or Gal4 negative control. Replicate 1 corresponds to a mix of WT2 and KO1 while replicate 2
corresponds to a mix of WT3 and KO2. (D) Exemplary density plot of mixed population in SL validation by flow
cytometry. KO (iRFP) signal is plotted against WT (GFP) signal. Q1: iRFP positive subpopulation, Q2:
Subpopulation positive for both GFP and iRFP, Q3: GFP-positive population, Q4: Population without specific
signal. (E) Quantification of ZNF524 KO over WT ratios in NR2C2 KO replicates. The ratios were normalized to the
46 
Gal4 negative control of the respective replicate. The experiment was done in duplicates. (F) Quantification of 
ZNF524 KO over WT ratios in NR2F2 KO replicates. The ratios were normalized to the Gal4 negative control of 
the respective replicate. The experiment was done in duplicates. 
After treatment with sgRNA targeting NR2C2 or Gal4 as negative control, the protein depletion was 
monitored by WB (Figure 22 B). The reduction in NR2C2 was sufficient to continue. As a suitable 
antibody against NR2F2 was not available, genomic alterations were determined by T7 endonuclease 1 
assay that visualizes sequence modifications by restriction fragments (Figure 22 C). Both replicates 
showed sgRNA-mediated genome alterations and were used for SL validation. The composition of the 
replicates was determined by flow cytometry at 3-4 day intervals over the course of a month (Figure 
22 D). Mixes transfected with sgRNA against Gal4 served as negative control and for normalization. 
And indeed, upon KO of NR2C2 the ratio of ZNF524 KO to WT clones decreased to ~0.7 in comparison 
to the Gal4 sgRNA transduced mixes (Figure 22 E). This was true for both replicate 1 and replicate 2. 
Similarly, the ratio for NR2F2 sgRNA transduced cells decreased to ~0.8 for both replicates (Figure 22 
F). These data suggest that the combined removal of ZNF524 and NR2C2 or NR2F2 does indeed 
negatively affect the fitness of the cell.  
  
47 
 
Discussion 
Emergence of novel telomere binders 
Over the past three decades researchers have been looking for telomere associated proteins, with 
TRF1 and TRF2 being among the first to be identified as direct telomere binders. After the discovery of 
the entire shelterin complex by the mid 2000’s, this endeavor has become increasingly difficult and 
only with the emergence of different screening approaches more candidates have entered the stage. 
In 2009, Déjardin and Kingston developed a protocol for proteomics of isolated chromatin segments 
and purified telomeric chromatin (PICh) (Déjardin and Kingston, 2009). This mass spectrometry-based 
analysis of associated proteins identified the shelterin complex as well as several known transient 
telomere binders like the MRN complex, Apollo or Ku70. Additionally, telomerase associated proteins, 
like NHP2, and ALT specific proteins, like PML, were found in the respective cell lines indicating the 
comprehensiveness of the screen. The screen also for the first time brought orphan nuclear receptors 
in context with ALT and identified HMBOX1. In comparison to the >200 proteins found by the PICh 
approach, a quantitative telomeric chromatin isolation protocol (QTIP) identified fewer novel 
candidates, like the THO complex, LRIF1 or SMCHD1 but also included many known telomere 
associated proteins (Grolimund et al., 2013). While PICh relies on hybridization of a sequence specific 
probe to crosslinked chromatin, QTIP utilizes α-TRF1 and α-TRF2 antibodies to isolate telomeric 
chromatin potentially accounting for the difference in identified proteins. Another mass spectrometry-
based screen used telomeric oligonucleotides to isolate potential telomere binders from nuclear 
extract (Kappei et al., 2013). Here, HMBOX1/homeobox telomere-binding protein 1 (HOT1) was again 
identified and this time characterized in more detail revealing its direct binding to telomeres and its 
function as a positive regulator of telomere length maintenance (Kappei et al., 2013). During these 
years, more screens identified additional candidates, yet the overlap between screens was mostly 
limited to already known telomere binders (Giannone et al., 2010; Nittis et al., 2010; Lee et al., 2011). 
To get an evolutionary perspective of telomeric proteins, Kappei et al. conducted a phylointeractomics 
screen across 16 vertebrate species which identified 25 proteins additionally to the shelterin complex 
members (Kappei et al., 2017). Proteins of known telomeric function include the orphan nuclear 
receptors NR2C1 and NR2C2, which have mainly been linked to ALT (Déjardin and Kingston, 2009; 
Conomos et al., 2012; Marzec et al., 2015), the nuclease Apollo which is involved in telomere end 
processing (Wu, Takai and De Lange, 2012), the helicase RECQL1 that promotes telomere maintenance 
(Popuri et al., 2014) and HOT1, which contributes to telomere elongation (Kappei et al., 2013). Among 
the identified proteins was also a group of zinc finger proteins that up to this point had not been 
described as telomere binders. In contrast, the known telomere binders TRF1, TRF2 and HOT1 harbor 
homeobox domains responsible for binding to double-stranded TTAGGG while POT1 carries an OB-fold 
domain to attach to the single-stranded overhang of the telomeres. Yet, subsequently to the screen, 
the zinc finger protein ZBTB48, also known as TZAP, was characterized as a telomere length regulator 
that directly interacts with telomeres via a zinc finger domain (Jahn et al., 2017; Li et al., 2017; Zhao et 
al., 2018). Also, the zinc finger protein ZBTB10 directly interacts with both telomeric and variant 
repeats and localizes to a subset of telomeres (Bluhm et al., 2019). In this thesis work, we showed that 
ZNF524, a zinc finger protein discovered in the previously mentioned interactomics screen by Kappei 
et al, is also a direct binder of telomere sequences. 
48 
Direct interaction of ZNF524 and other zinc finger proteins with telomeric 
sequences 
In telo pulldowns with U2OS (ALT dependent) or HeLa (telomerase positive) lysates, we verified in vitro 
binding of endogenous ZNF524 to telomeric repeats. Additionally, the enrichment of bacterially 
expressed His-ZNF524 to telomeric sequences indicated a direct interaction between ZNF524 and the 
respective DNA. This was also true for subtelomeric variant repeat sequences, yet with reduced 
affinity. In parallel, our collaborators in Fudong Li’s lab (MOE Key Laboratory for Cellular Dynamics, 
School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of 
China; Hefei, China) performed isothermal titration calorimetry with a ZNF524 minimal domain 
construct composing of only the ZF motifs. Similar to both the full-length ZNF524 and our ZNF524 MD, 
this construct bound the canonical TTAGGG repeats with high affinity (KD = 0.09 μM). They also 
recapitulated the decrease in affinity for the variant repeats TCAGGG (KD = 0.28 μM), TGAGGG (KD = 
0.27 μM), and TTGGGG (KD = 0.33 μM) by ITC (Figure 23 A). These results are similar to affinities 
determined for the telomeric zinc finger protein ZBTB48. As measured by fluorescent polarization, 
ZBTB48’s DNA binding domain displays a KD of 0.17 μM for TTAGGG while the affinities to TCAGGG (KD 
= 0.37 μM), TGAGGG (KD = 0.53 μM), and TTGGGG (KD = 0.38 μM) are reduced. As for ZBTB48, this 
suggests a preference of ZNF524 for canonical telomeric repeats while also allowing for potential 
interaction with subtelomeric regions, as already reported for ZBTB10 (Bluhm et al., 2019). In contrast 
though, ZBTB10 DBD prefers the variant TTGGGG (KD = 0.106 μM) over the canonical TTAGGG (KD = 
0.218 μM) repeats, hinting at ZBTB10’s preferred localization to subtelomeres, while ZNF524 and 
ZBTB48 are predominantly found at the telomere (Jahn et al., 2017; Li et al., 2017; Zhao et al., 2018; 
Bluhm et al., 2019). While it seems that ZNF524, ZBTB48 and ZBTB10 have similar affinities for TTAGGG 
or TTGGGG respectively, we need to keep in mind that we are comparing ITC data (ZNF524) to FP data 
(ZBTB48 and ZBTB10) and KD values of telomere binding proteins can vary greatly depending on the 
methodology, as we have seen for TRF2 (Hanaoka, Nagadoi and Nishimura, 2009; Erdel et al., 2017; 
Veverka, Janovič and Hofr, 2019).   
49 
 
 
Figure 23. Isothermal titration calorimetry reveals optimal binding of ZNF524’s four ZFs to TTAGGG 
repeats 
(A) Isothermal titration calorimetry results using a ZNF524 minimal domain containing only the four zinc fingers 
(110-223 aa) with the telomeric 12-bp ds(TTAGGG)2 and the telomeric variants ds(TCAGGG)2, ds(TGAGGG)2 and 
ds(TTGGGG)2; ds(GTGAGT)2 serves as negative control sequence;. KD values with standard deviations are noted 
in the lower right corner. (B) Isothermal titration calorimetry results for different combinations of ZNF524 ZFs 
with a 12-bp ds(TTAGGG)2;. KD values with standard deviations are noted in the lower right corner. Data was 
collected and analyzed by Ziyan Xu and Fudong Li (MOE Key Laboratory for Cellular Dynamics, School of Life 
Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China; Hefei, China). 
In telo pull-downs with overexpressed ZF point mutants, ZF2 was determined as the only domain 
essential for DNA binding as its disruption abrogated telomere recognition. Yet, mutation of any two 
ZFs displayed reduced binding or even loss of binding even if ZF2 was intact. We therefore concluded 
that ZF2 is essential yet not sufficient for telomere binding. These findings were independently 
confirmed by ITC measurements by the Li lab: As compared to the 90 nM affinity of the ZNF524 
minimal domain comprising all four ZFs, ZNF524 constructs composing of only three or two ZFs showed 
at least 6-fold reduced affinities. Similar to our ZF2 point mutant, the complete removal of ZF2 
abrogated binding (Figure 23 B). These findings demonstrate that all four ZF are necessary for maximal 
affinity binding thereby putting ZNF524 in contrast to ZBTB48: Despite harboring 11 zinc fingers, only 
one is responsible for telomere recognition and binding by ZBTB48 (Zhao et al., 2018). To further 
characterize the DBD of ZNF524, the Li lab solved the crystal structure of ZNF524 MD when bound to 
TTAGGG repeats (Figure 24 A). 
50 
 
Figure 24. ZNF524 employs all four zinc fingers for base-specific recognition of telomeric sequences 
(A) Overall structure of the four zinc fingers (ZF1 (blue), ZF2 (violet), ZF3 (green) and ZF4 (salmon) in complex
with duplex telomeric DNA (G-strand (orange), C-strand (cyan)), (B) Schematic representation of base-specific
contacts of the ZFs with telomeric sequences. Hydrogen bonds (blue) and Van der Waals contacts (black) are
highlighted. (C) Sequences of ZF1, ZF2, ZF3 and ZF4 of ZNF524 are aligned to ZF2 of ZBTB10 and ZF11 of ZBTB48.
The four zinc-coordinating residues of each finger are indicated by blue background. The first zinc-coordinating
histidine in each finger serves as reference position 0 for the RxxHxxR motif (bold). Data were collected and
analyzed by Ziyan Xu and Fudong Li (MOE Key Laboratory for Cellular Dynamics, School of Life Sciences, Division
of Life Sciences and Medicine, University of Science and Technology of China; Hefei, China).
Indeed, all four zinc fingers adopt the classical β-sheet – β-sheet – α-helix conformation, with the α-
helix inserting into the major groove of the double helix, and, in addition to unspecific interaction with 
the phosphate backbone, make base-specific contacts through either hydrogen bonds or Van der Walls 
contacts (Figure 24 B). This puts ZNF524 in contrast to ZBTB10 and ZBTB48, where only two or one ZF, 
respectively, are sufficient for telomere recognition. For both proteins, an adjacent C-terminal arm is 
involved but while it has a supportive function for ZBTB10, it is indispensable for ZBTB48 (Zhao et al., 
2018; Bluhm et al., 2019). Such an arm-structure was not detected for ZNF524, indicating its sole 
reliance on the ZF domains. It is remarkable that each of ZNF524’s ZFs contributes to the base-specific 
recognition of the TTAGGG repeats, yet ZF2 harbors the majority of DNA-interacting residues. This is 
in agreement with our previous conclusion that ZF2 is central to telomere binding. Interestingly, when 
comparing ZNF524 ZF2, ZBTB10 ZF2 and ZBTB48 ZF11, we found a common RxxHxxR motif (Figure 24 
C). The crystal structures of ZNF524 and ZBTB48 demonstrate the importance of these three residues 
for base-specific contact (Zhao et al., 2018). The RxxHxxR motif might therefore be a common feature 
of telomeric zinc finger proteins, even with otherwise differing DNA binding domains. 
ZNF524 as a telomeric protein 
Not only does ZNF524 bind to telomeric sequences in biochemical assays but it also localizes to 
telomeres within the cell. IF staining of U2OS cells overexpressing ZNF524-GFP WT showed 
colocalization with TRF2. The percentage of telomeres occupied by ZNF524-GFP WT per cell varied 
mostly between 60% and 100%, only a minority of cells had less than 10% of its telomeres bound by 
ZNF524. Therefore, ZNF524-GFP colocalizes with more telomeres than ZBTB10-GFP OE (on average 6 
51 
events per cell in G1 phase) but is comparable to FLAG-ZBTB48 OE (~80%). For ZBTB48, telomere 
localization was dose-dependent as FLAG-ZBTB48 OE displayed more colocalization events with TRF2 
than endogenous ZBTB48 (~50%). As our homemade α-ZNF524 antibody only recognized denatured 
ZNF524, we unfortunately could not quantify endogenous ZNF524 at telomeres. However, ZNF524 
resembles ZBTB48 in the sense that both apply ZF domains for TTAGGG binding and promiscuously 
recognizes subtelomeric variants repeats. Keeping these parallels in mind and given that a strong OE 
of ZNF524-GFP WT in comparison to endogenous levels was required for foci formation, it is plausible 
that ZNF524 colocalization to telomeres might also be dose-dependent. In turn, this could also explain 
the lack of ZNF524-GFP signal at telomeres in telomerase positive cell lines, which, on average, have 
shorter telomeres than ALT dependent cell lines. A telomeric occupancy sufficient for detection by IF 
might simply not be reached on shorter telomeres. Alternatively, ZNF524 might not localize to 
telomeres in telomerase positive cell lines. However, as the increase in TIFs in HeLa ZNF524 KO clones 
indicates a telomeric function of ZNF524 in telomerase positive cell lines, the latter explanation seems 
less likely. Using the OE construct, we also observed pan-nuclear GFP signal in some cells which 
impeded quantification of foci. As a result, these cells had low scores on ZNF524-GFP WT localization 
to telomeres (0-10% colocalization). At this point, we cannot differentiate if we are seeing an artefact 
of protein overexpression or if ZNF524 indeed distributes throughout the nucleus in a subset of cells, 
potentially linked to cell cycle phases. Overall, these data suggest that, in contrast to TRF1 and TRF2, 
ZNF524 is not constitutively present at telomeres and, potentially, only a subset of telomeres is 
occupied by ZNF524. Following this line of thought, it is possible that, similar to HOT1 and ZBTB48 
primarily localizing to telomeres that are in need of elongation or limitation thereof, ZNF524 only binds 
when necessary in a dynamically regulated manner. Further investigations into ZNF524 localization to 
telomeres for example throughout the cell cycle or upon DNA damage induction are obvious future 
steps. Interestingly, when investigating the colocalization of ZNF524-GFP with FISH signal in additional 
ALT cell lines, we observed a strong overlap with extraordinarily large telomere foci. While this would 
need to be confirmed with additional PML staining, one could speculate that these large foci are indeed 
APBs and that ZNF524 is either recruited to or involved in formation of APBs, potentially linking ZNF524 
to HR, telomere homeostasis and conformational changes in chromatin.  
Having established ZF2 mut as a non-binding control by IF and by ChIP, the BioID assay became 
especially appealing. Using BirA*-ZNF524 WT in comparison to BirA*-ZNF524 ZF2 mut allowed us to 
specifically target proximity partners at the telomeres. Indeed, we identified the direct telomere 
binders TRF2, TRF1, NR2C2 and NR2C1 further underscoring the presence of ZNF524 at telomeres. 
While TRF1 and TRF2 are constitutively present at all telomeres, NR2C1 and NR2C2 are mainly linked 
to subtelomeric variant repeats and telomeres in ALT positive cells. As previously mentioned, it is 
tempting to speculate that ZNF524 is involved with APBs and the identification of these nuclear 
receptors which have been shown to promote ALT adds to this perception. Of note, biotinylation did 
not extend to any of the known TRF1/2 interaction partners like RAP1 or TIN2, potentially due to steric 
hindrance. Alternatively, one could imagine a scenario where TRF1 and TRF2 homodimers bind to 
telomeres independently of the fully assembled shelterin or other interaction partners and that these 
sites are preferred by ZNF524. Interestingly, ZNF524 was also slightly enriched. With equal expression 
levels of BirA*-ZNF52 WT and BirA*-ZNF524 ZF2 mut, equal self-biotinylation of the constructs would 
render ZNF524 in the non-specific background. It is tempting to speculate that BirA*-ZNF524 WT 
localizes close to telomere-bound endogenous ZNF524 thereby leading to the observed enrichment. 
Yet, a potential telomere-mediated proximity or interaction between ZNF524 proteins still need to be 
confirmed. 
Surprisingly, DPY30 was also found by BioID. DPY30 is a core subunit of the SET1/MLL complex, a 
methyltransferase modulating H3K4, and depletion of DPY30 results in reduced H3K4 methylation, 
52 
hampered proliferation and a senescence phenotype (Ernst and Vakoc, 2012; Simboeck et al., 2013). 
The core unit of the SET1/MLL complex consists in addition to DPY30 of WDR5, RBBP5 and ASH2L 
(WRAD). GFP-DPY30 does not co-purify ZNF524 suggesting an indirect or transient interaction (van 
Nuland et al., 2013). While stoichiometric analysis of the complex revealed a potential formation of 
the WRAD complex independently of SET1 or MLL, it is noteworthy that none of the other complex 
members were enriched in our BioID assay (van Nuland et al., 2013). Especially since DPY30 associates 
with nucleosomes via ASH2L, enrichment of ASH2L could have been expected (Tremblay et al., 2014). 
This could be explained by the discovery that not all DPY30 is bound by the SET1/MLL complex: DPY30 
is expressed more abundantly than the other SET1/MLL complex members and was furthermore linked 
to the NURF complex, indicating SET1/MLL independent functions (van Nuland et al., 2013). Other 
explanation for the lack of WRAD and NURF proteins as interactors of DPY30 address the limitations 
of the assay, for example steric hindrance: We also did not identify interaction partners of TRF1 and 
TRF2, namely RAP1 and TIN2, arguing for a limited biotinylation radius of the ZNF524 fusion protein. 
Alternatively, ZNF524 might be in close proximity with the WRAD and NURF complexes independently 
of telomere binding and would therefore not be enriched in comparison to ZNF524 ZF2 mut. Overall, 
proximity to DPY30 could implicate ZNF524 in epigenetic pathways but speculations in this direction 
need further investigations.  
Proliferation and cell cycle progression are not impaired by the removal of ZNF524 
For functional analysis of ZNF524, we created U2OS and HeLa ZNF524 KO clones. Despite the disruption 
of the gene and a lack of protein expression, the cells were viable. In contrast, TRF2 and TRF1 are 
essential for cell survival (Van Steensel, Smogorzewska and De Lange, 1998; Karlseder et al., 2003; 
Iwano et al., 2004; Celli and de Lange, 2005; Sfeir et al., 2009). Yet, in comparison to other telomere 
binders, non-lethal phenotypes have been reported before: despite their undisputable role in telomere 
biology, cell death was also not observed in RAP1-, HOT1- or ZBTB48-deficient cells (Kappei et al., 2013; 
Kabir, Hockemeyer and de Lange, 2014; Jahn et al., 2017; Li et al., 2017). Telomeric defects can also 
lead to early onset of cellular senescence or trigger checkpoint activation leading to accumulation of 
cells in a certain cell cycle phase. Sometimes, these effects can even be observed in cells with 
compromised checkpoints, for example reduced growth rates in ZNF827-depleted ALT cells or G2/M 
arrests in cells with TRF1-deficient or damaged telomeres (Cho et al., 2014; Conomos, Reddel and 
Pickett, 2014; García‐Beccaria et al., 2015). However, when determining the cell cycle stages by flow 
cytometry and measuring the population doublings over a period of 34 days, we did not detect 
differences between WT and ZNF524 KO clones for either U2OS or HeLa clones. There are a couple of 
possible explanations for these findings: The effects of ZNF524 KO might not be as detrimental in 
cancer cell lines with defective checkpoint activation or, alternatively, a synergistic pathway rescues 
the function of ZNF524. The later, we have started to address by a synthetic lethality/synthetic sick 
screen where we indeed identified an array of potential genetic interactors that could function in 
tandem with ZNF524. Additionally, it would be interesting to expand our efforts to primary cells as well 
as organismal studies thereby gaining insight into ZNF524’s function in unperturbed genetic 
backgrounds. 
ZNF524 is not essential for telomere length homeostasis 
When it comes to telomere elongation in cancer cells, two pathways have been described: 1.) 
reactivation of telomerase or 2.) alternative lengthening of telomeres (ALT). To cover both pathways, 
we included the telomerase positive HeLa cell line as well as the ALT positive U2OS cell line in our 
investigations. In U2OS cells, we did not observe ZNF524-dependent effects on bulk telomere length. 
Typically, telomeres of ALT positive cancer cell lines are longer than those of telomerase positive cells 
53 
 
and display a higher heterogeneity. Among this heterogeneity, TRF analysis and qFISH might not have 
been able to detect minor changes. Additionally, we probed for the formation of C-circles, a form of 
ECTR that occurs as a byproduct of ALT activity. Again, we observed a high heterogeneity among the 
clones and the depletion of ZNF524 did not lead to an apparent effect. The occurrence of C-circles is 
assumed to correlate to ALT activity and changes have been observed for other telomeric zinc finger 
proteins. For example, ZNF827 promotes HR at telomeres by recruitment of the NuRD complex and C-
circle levels decreased upon knock down of ZNF827 (Conomos, Reddel and Pickett, 2014). Additionally, 
increased C-circle levels can be indicative for telomere trimming, which was observed for ZBTB48, a 
zinc finger protein shown to negatively regulate telomere length homeostasis (Jahn et al., 2017; Li et 
al., 2017). Another telomere ds binder, HOT1, is a positive regulator of telomere elongation by 
association with telomerase (Kappei et al., 2013). Thus, we wondered if ZNF524 might be involved in 
telomerase dependent telomere elongation but did not see a significant ZNF524-dependent change in 
telomere length or in telomerase activity as measured by TRAP assay. In addition, the inhibition of 
telomerase by treatment with BIBR1532 did not reveal ZNF524-dependent effects, arguing against 
synthetic lethality between ZNF524 and the inhibitor and therefore a direct involvement of ZNF524 in 
telomerase activity. Indeed, ZBTB10 also lacks implication in telomere length control (Bluhm et al., 
2019), indicating more diverse roles of zinc finger proteins at telomeres. 
Nevertheless, the absence of ZNF524 results in a tendency towards shorter telomeres. Interestingly, 
we identified three proteins related to telomerase regulation in our synthetic lethality screen: PINX1, 
POT1 and p53. PINX1 is an interactor of TRF1 and promotes its localization to nucleoli as well as 
telomeres (Yoo, Oh and Park, 2009). While initially described as an inhibitor of telomerase, it was later 
on shown to be important for telomere elongation, potentially through interactions with POT1 and 
telomerase itself (Zhou and Lu, 2001; Zhang et al., 2009; Cheung et al., 2012; Yoo, Park and Oh, 2014; 
Ho et al., 2019). As subject to p53 regulation, PINX1 creates a link between p53 inactivation and 
telomerase reactivation in immortalized cell lines (Wu et al., 2014). Given the tendency towards 
shorter telomeres in HeLa ZNF524 KO clones, a connection between ZNF524 and telomere 
homeostasis should be considered. However, the previously published data were rather specific to 
telomerase positive cells and might not necessarily transfer to ALT dependent cells. As the screen was 
conducted in U2OS cells that do not rely on telomerase for telomere length maintenance, these 
candidates could also be indicative for ZNF524’s involvement in other pathways. For example, POT1 is 
additionally involved in telomere protection from DDR and there is a plethora of p53-dependent 
pathways that could lead to the synthetic lethality/ sickness observed in our screen. Therefore, careful 
validation of the SL candidates and further investigations are crucial to a better understanding of these 
ambiguous results. 
TRF2/RAP1 localization to telomeres is influenced by ZNF524 
Shelterin and its member proteins are known to shape the telomeric landscape, for example by 
recruitment of transient factors needed throughout the cell cycle, including RTEL1 and BLM during 
replication or Apollo and telomerase in late S-phase for telomere elongation (van Overbeek and de 
Lange, 2006; Sfeir et al., 2009; Xi and Cech, 2014; Drosopoulos, Kosiyatrakul and Schildkraut, 2015; 
Sarek et al., 2015). Interestingly, it was also demonstrated that increased abundance of TRF2 at 
telomeres prevented ZBTB48 localization to telomeres while long telomeres with putatively diluted 
shelterin occupancy displayed enhanced ZBTB48 binding (Li et al., 2017). Clearly, telomere binding 
proteins influence each other through diverse modes of actions. We hence enquired whether the 
presence or absence of ZNF524 would have an effect on shelterin. To this end, we quantified the IF 
signal of TRF1, TRF2, RAP1 and POT1 in U2OS WT and ZNF524 KO cells. Only TRF2 and RAP1 abundance 
at telomeres showed ZNF524 dependencies: in the absence of ZNF524, we observed a 40-50% 
54 
 
reduction of TRF2 and RAP1 at telomeres while TRF1 and POT1 remained unchanged. These findings 
suggest an influence of ZNF524 specifically on the TRF2/RAP1 subcomplex as opposed to the fully 
assembled shelterin complex in vivo. While the functionalities of the individual shelterin members lead 
to assumptions about the involvement of potential subcomplexes, evidence is only emerging. The fact 
that ZNF524 removal specifically effects TRF2 and RAP1 introduces a functional relevance to the 
TRF2/RAP1 subcomplex. 
Stoichiometry of shelterin and expression levels of its members allow for subcomplex formation  
While there are reports about the formation of subcomplexes in vitro, evidence for their existence in 
vivo is difficult to obtain and so far rather indirect through stoichiometry or functional observations. 
Stoichiometry based approaches have addressed the question which shelterin members have the 
potential to form subcomplexes. Indeed, co-expression in insect cells showed that the subcomplexes 
TRF2-TIN2-TPP1-POT1, TIN2-TPP1-POT1 and TRF2-TIN2-TPP1 are able to form in solution. Additional 
expression of RAP1 revealed a stoichiometry of RAP12:TRF22:TIN21:TPP11:POT11, where RAP1 binding 
does not impact the TRF2:TIN2 ratio of 2:1 (Lim et al., 2017). However, TIN2 prefers binding to TRF1 
over TRF2 unless TPP1 is part of the complex indicating allosteric effects in vitro (Hu et al., 2017; Janovič 
et al., 2019). To gain insight into shelterin stoichiometry in vivo, a study was conducted that 
determined expression levels of the shelterin members by quantitative immunoblotting (Takai et al., 
2010). POT1 and TPP1 are 10-fold less abundant than the other members which indicates that not all 
members are constantly bound in the complex but could exist as subcomplexes or separate entities in 
vivo. TIN2, RAP1 and TRF2 showed highest expression levels in this study, suggesting that these 
proteins could form a shelterin-independent complex in vivo. Similarly, we observed stronger 
transcription of TRF2, RAP1, TIN2 and TPP1 in comparison to TRF1 and POT1 in our U2OS RNA-seq data 
set. As suitable antibodies for TPP1 and TIN2 were not at our disposal, we did not determine their 
abundance at telomeres in WT and ZNF524 KO conditions. Given the previously outlined in vitro data 
and their transcription levels, both TIN2 and TPP1 could putatively form subcomplexes of altering 
composition with the other shelterin members (Hu et al., 2017; Lim et al., 2017; Janovič et al., 2019). 
However, functional evidence linking TRF2/RAP1 to TIN2 or TPP1 in a shelterin independent manner is 
missing. Taken together, the parallels in expression patterns of TRF2 and RAP1 and their ZNF524-
mediated regulation indicate that the formation of this independent TRF2/RAP1 subcomplex in vivo 
seems likely. 
Binding patterns of shelterin members to telomeric sequences allow for differential regulation of 
subcomplexes 
ZNF524 specifically effects the binding of TRF2/RAP1 to telomeres. By Co-IP, we showed that a direct 
interaction between ZNF524 and TRF2 is unlikely, rendering a recruitment mechanism improbable. In 
support of this notion, ZNF524 was not among the ~6000 proteins identified in our proteome 
measurement while TRF2 and RAP1 abundance was clearly sufficient, indicating that ZNF524 is sub-
stoichiometric to TRF2/RAP1. Furthermore, the expression values of TRF2 and RAP1 do not decrease 
in ZNF524 KO clones, ruling out a TRF2/RAP1 specific transcription factor activity of ZNF524. These 
findings suggest an indirect ZNF524-mediated regulation of TRF2/RAP1 localization to telomeres and 
raise the question if TRF2-telomere binding properties allow for a differential regulation as compared 
to the other shelterin complex members. So how do telomere binding properties differ between the 
fully assembled shelterin complex and its separate member proteins? And how would these binding 
properties allow for a ZNF524-mediated differential regulation of TRF2/RAP1 as opposed to TRF1 or 
the fully assembled shelterin complex? In HeLa cells, early research on telomere binding properties 
showed that POT1 resides stably at telomeres while TRF1 interacts rather transiently. For TRF2, both 
behaviors were observed yet a potential influence of its interaction partners RAP1 or TIN2 was not 
55 
 
examined (Mattern et al., 2004). Both TRF1 and TRF2 form homodimers and bind telomeric sequences 
via their homeobox domains that have a high structural similarity. Despite homologous DNA binding 
domains, TRF1 and TRF2 apply different modes for 1D telomeric sequence search which has been 
linked to the basic N-terminus of TRF2 where TRF1 has an acidic N-terminus (Lin et al., 2014). This 
distinction in sequence specificity would allow for differential binding regulation of TRF1 and TRF2. 
Binary assays also demonstrated that TRF1 binds telomeric dsDNA with higher affinity (6 nM) than 
TRF2 (40 nM) despite structural similarity of the DNA binding domain. This difference in affinity might 
render TRF2 more susceptible to binding regulation than TRF1 further strengthening the aspect of 
differential regulation of the two dsDNA binders (Hanaoka, Nagadoi and Nishimura, 2009; Veverka, 
Janovič and Hofr, 2019). Additionally, it was shown that a 10-fold reduction of TRF2 levels did not affect 
TRF1 binding to telomeres (Takai et al., 2010). Taken together, the previously listed findings strongly 
suggest that the individual member proteins of the shelterin complex also have shelterin-independent 
binding properties and functions. The distinction between binding properties of TRF2/RAP1 and the 
fully assembled shelterin complex seems more nuanced. A comprehensive study of telomere 
recognition properties of both the fully assembled shelterin complex and the TRF2/RAP1 subcomplex 
was conducted in 2017 by Erdel et al. Mouse shelterin and mouse TRF2/RAP1 were expressed in 
HEK293T, purified and subjected to biochemical assays (Erdel et al., 2017). Similar binding properties 
were found for the shelterin complex and the TRF2/RAP1 subcomplex with regard to double-stranded 
telomeric repeats: They can specifically recognize telomeric sequences over non-telomeric sequences 
and do so by either 1D scanning of the DNA or by 3D diffusion. The latter is possible as the complexes 
can form in solution and do not need to assemble on the DNA. Especially for shelterin, 3D diffusion is 
the dominant mode of action. However, it is still controversial to which extend shelterin assembles in 
solution vs. at telomeric DNA (Lin et al., 2014; Erdel et al., 2017; Lim et al., 2017). A DNA-driven 
assembly could facilitate a differential regulation of the TRF2/RAP1 subcomplex as it remains an 
independent entity during sequence and structure recognition. Noticeably, neither shelterin nor 
TRF2/RAP1 bind telomeres cooperatively but act as separate entities (Erdel et al., 2017). This, too, 
allows for differential regulation of TRF2/RAP1 binding to telomeres without influencing the shelterin 
complex. Taken together, the TRF2/RAP1 subcomplex is able to form and associate with telomeric 
sequences and its binding properties differ from both TRF1 and the shelterin complex. In turn, this 
allows for an altered regulation of TRF2/RAP1 as compared to other telomeric proteins. One could also 
envision a stabilization of TRF2/RAP1 on telomeres by ZNF524 indicating a DNA-mediated crosstalk 
between the proteins rather than a direct protein-protein interaction.  
ZNF524-depleted telomeres resemble intermediate-state telomeres 
After the discovery of spontaneous DDR signaling at telomeres of viable cell lines, a three-state model 
was proposed to describe the different levels of telomere deprotection: closed-state telomeres, 
intermediate-state telomeres and uncapped-state telomeres (Cesare et al., 2009; Cesare and 
Karlseder, 2012). The closed-state telomeres are fully protected and neither fuse nor signal DNA 
damage. In stark contrast, the uncapped-state telomeres completely lack TRF2 either due to eroded 
telomeres or disruption of protein expression. This loss of TRF2 is detrimental to the cell as telomeres 
become unprotected and undergo NHEJ leading to chromosome fusions, kataegis, chromothripsis and 
genomic rearrangements. The intermediate-state, however, is characterized by the onset of DNA 
damage signaling and telomere aberrations, indicating that the telomere protection is hampered. Yet, 
telomere fusions are repressed and cells do not enter crisis.  
The early definition of the intermediate-state telomere was closely linked to inadequacies but not a 
complete loss of TRF2: spontaneous DNA damage responses were observed at ALT dependent 
telomeres that were postulated to have a lower TRF2 occupancy in comparison to telomere length 
56 
 
(Cesare et al., 2009). Upon closer investigation, intermediate-state telomeres induced by TRF2 knock 
down exhibit a less pronounced DDR than fully deprotected telomeres: telomere fusions are extremely 
rare, ATM activation and the subsequent CHK2 phosphorylation are markedly reduced. Also, cells with 
intermediate deprotection of telomeres continue to cycle without gene duplications. However, 
recruitment of γH2AX and 53BP1 was also found at intermediate-state telomeres (Cesare et al., 2013). 
These findings overlap with ours, as we also observed an increase in 53BP1 signaling at ZNF524-
depleted telomeres, while the more drastic effects like telomere fusions or amplified polyploidy did 
not occur. Furthermore, we did not see a strong increase in overall pATM or pCHK2 levels in U2OS 
ZNF524 KO clones, which complies with the intermediate-state of telomeres and its lack of telomere 
fusions. In mortal primary cells, the intermediate-state of telomeres is often accompanied by mild 
growth defects or even a senescence phenotype, as shown by partial TRF2 knock down. In general, 
poor proliferation and early onset of senescent become predominantly evident in primary cells as they 
have proficient p53 pathways that are often impaired in cancer cell lines (Takai et al., 2010; Cesare and 
Karlseder, 2012; Cesare et al., 2013). For example, TRF2 knock down in HeLa and HTC75 cancer cells 
did not affect the proliferation rate (Takai et al., 2010). The lack of growth defects in our findings could 
therefore be explained by the choice of cell line and future investigations towards the proliferative 
potential of ZNF524-depleted primary cells will provide more answers.  
The previously mentioned studies relied on TRF2 reduction by RNAi to induce intermediate 
deprotection of telomeres. Yet, TRF2 insufficiencies leading to intermediate-state telomeres can derive 
from different causes. For example, a RNAi-independent TRF2 reduction has also been observed upon 
treatment with the DSB inducing antibiotic Zeocin (Porro et al., 2014). In this context, the partial 
removal of TRF2 from telomeres has been linked to a prolonged mitotic arrest. By ChIP, a reduction of 
~60% after 72h treatment was demonstrated which also coincided with an increase of γH2AX at 
telomeres (Porro et al., 2014). Both the reduction level of TRF2 at telomeres and the observed mild 
increase in DNA damage signaling overlap with our findings. Nevertheless, a prolonged mitotic arrest 
has also been shown to activate cell cycle checkpoints or cause aneuploidy (Hayashi et al., 2012). As 
we have observed neither of these phenotypes, it seems unlikely that ZNF524 causes TRF2 loss at 
telomeres purely through cell cycle regulation. It would however be interesting to investigate 
prolonged mitotic arrest and Zeocin treatment in a ZNF524 KO background in search for synergistic 
effects. In addition, oxidative stress was proposed to cause TRF2 reduction and an increase in TIFs, t-
SCEs and APBs but no telomere fusions (Opresko et al., 2005; Kamranvar and Masucci, 2011). 
Interestingly, impairment of the TRFH dimerization domain of TRF2 also leads to DDR signaling of 
telomeres without inducing telomere fusions (Okamoto et al., 2013; Di Maro et al., 2014). These 
findings show that the intermediate deprotection of telomeres by TRF2 inadequacies can have variable 
causes and while the consequences might diverge (effects on APBs, telomere length, t-SCEs), they are 
always characterized by DDR signaling in the absence of fusions, a hallmark that we have also observed 
in the absence of ZNF524. A striking difference between our data and previous reports are the 
unaltered expression levels of TRF2 and RAP1 upon ZNF524 KO. While it is mostly assumed that the 
reduction in overall TRF2 protein levels would lead to a reduced TRF2 occupancy of telomeres, our 
data rather links the induction of the intermediate-state telomeres to the reduced binding capabilities 
of TRF2/RAP1 to the telomeres. To further strengthen this hypothesis, experimental proof needs to 
demonstrate the rescue of TIFs and t-SCEs by enhancing TRF2 binding to telomeres in the absence of 
ZNF524. At this point, we have not solved the mechanism by which ZNF524 influences TRF2 abundance 
at telomeres and cannot conclusively determine whether the intermediate-state is truly mediated by 
TRF2/RAP1 reduction or whether it is a direct effect of ZNF524 removal. 
When studying the function of TRF2 and RAP1 at telomeres in vivo, it is a challenge to keep these 
functions apart. As RAP1 relies on TRF2 for localization to telomeres, the removal of TRF2 will 
57 
 
automatically affect the abundance of RAP1 at telomeres thereby making a differentiation difficult. 
The removal of RAP1 on the other hand, does not have major effects in unchallenged human cell lines 
(Kabir, Hockemeyer and de Lange, 2014). However, TRF2 function relies on RAP1 whenever TRF2 is 
impaired. The TRF2 mutant “top-less” cannot support t-loop formation anymore but binding of RAP1 
can still prevent telomere fusions (Benarroch-Popivker et al., 2016). Similarly, critically short telomeres 
that might have lost their t-loop already but still harbor TRF2 are only susceptible to fusions when RAP1 
is removed. This was shown for critically short telomeres due to senescence in primary cells or 
telomerase inhibition in HeLa cells (Lototska et al., 2020). In mouse embryonic fibroblasts, RAP1 is a 
crucial repressor of HDR in a Ku negative background and removal of RAP1 from the telomeres results 
in t-SCEs (Sfeir et al., 2010; Rai et al., 2016). This is in line with our findings of increased recombination 
events in ZNF524 KO cells with reduced RAP1 localization to telomeres. While the increase in t-SCEs 
upon ZNF524 deletion is not as prominent as reported by Sfeir et al. in RAP-deficient mouse cells, this 
could be accounted for by the ~50% of RAP1 that remain bound to the telomeres in ZNF524 KO cells.  
Figure 25. ZNF524’s mode of action at telomeres 
Telomeres harboring ZNF524 remain intact. The loss of ZNF524 results in a reduction of TRF2/RAP1 at telomeres 
and compromises telomere integrity as seen by an increase in TIFs and unscheduled t-SCEs. 
The observed phenotypes in ZNF524 KO cells strongly suggest an intermediate deprotection state of 
telomeres. This telomeric state has previously been linked mainly to TRF2/RAP1 dysfunctions and it is 
plausible that this is also the case for ZNF524 depleted cells. Yet, experimental evidence directly linking 
the increase in TIFs and t-SCEs to the reduction of TRF2/RAP1 at ZNF524 depleted telomeres is still 
missing. Therefore, we need to consider the possibility that the compromised telomere integrity is 
rooted directly in the removal of ZNF524 rather than mediated by TRF2/RAP1 impairment and that in 
fact TRF2 reduction is a by-product of hampered ZNF524 activity.   
Potential involvement of ZNF524 in telomeric chromatin organization 
While ZNF524 clearly influences TRF2/RAP1 localization to telomeres and plays a role in preserving 
telomere integrity, the mechanism remains elusive. When taking the candidates identified in the 
synthetic lethality screen into account, some tempting speculations come to mind. 
ZNF524 as an epigenetic factor at telomeres 
The chromatin state of telomeres and its influence on telomere related processes remains 
controversial. Especially with regard to the ALT mechanism, it is not entirely clear if a heterochromatic 
state is beneficial for ALT or impedes it. Interestingly, several candidates identified in our SL screen are 
involved in histone modifications. CNOT2, CNOT4 and CNOT6 are members of the CCR4-NOT complex 
which promotes H3K4me3. Additionally, we identified DOT1L (Disruptor of telomeric silencing 1 like), 
58 
a methyltransferase targeting H3K79, and its cofactor AF10 (MLLT10) (Feng et al., 2002; Jones et al., 
2008; Chen et al., 2015). DOT1L dependent H3K79 dimethylation has in turn been proposed to 
promote H4K20me3 (Jones et al., 2008). The very same modification can be introduced by the 
methyltransferase SUV4-20H1 (or KMT5B) which has also been identified in our SL screen and was also 
suggested to perform this function at telomeres (Schoeftner and Blasco, 2010). Intriguingly, KO of 
SUV4-20H1 in MEFs leads to increased numbers of global sister chromatid exchanges as well as t-SCEs, 
reminiscent of the increase in t-SCEs upon ZNF524 depletion (Benetti et al., 2007). Similarly suggestive 
for ALT activity, the depletion of DOT1L in mouse cells increased the amount of PML bodies, aneuploidy 
and telomere length (Jones et al., 2008).  
But not only histone methylation factors have synergies with ZNF524: in gene ontology analysis of the 
synthetic lethality screen, the STAGA complex was identified which displays histone acetyltransferase 
activity. Interestingly, we also established a synthetic lethality between ZNF524 and the nuclear 
receptors NR2C2 and NR2F2. Both receptors have been implicated in the recruitment of the NuRD 
deacetylation complex to telomeres via ZNF827. If NuRD is not recruited to telomeres due to knock 
down of ZNF827 or NR2C2/NR2F2, hyperacetylation was observed but also an increase in H4K20me3. 
Furthermore, lack of NuRD at telomeres decreases the amount of t-SCEs and APBs, both hallmarks of 
ALT. Interestingly, a double knock-out of NR2C2 and NR2F2 also effects TRF2 and RAP1 localization to 
telomeres (Conomos, Reddel and Pickett, 2014). While NR2C2/NR2F2 knock-outs lead to an increase 
of TRF2/RAP1 at telomeres as opposed to the decrease observed upon ZNF524 removal, it is a potent 
example of shelterin (sub-)complexes being influenced by other telomere binders, potentially involving 
epigenetic marks. 
Furthermore, the presence of nucleosomal proteins itself already influences the binding behavior of 
TRF2 in vitro. Indeed, TRF2 displayed hampered binding to telomeric sequences associated with or in 
close proximity to nucleosomes, while TRF1 binding was unchanged. Again, the diverging binding 
behavior of TRF2 and TRF1 was linked to the N-terminal domains (Galati et al., 2015). Vice versa, TRF2 
actively influences nucleosome distribution in a cell cycle dependent manner (Galati et al., 2012). 
Interestingly, the crosstalk between TRF2 and nucleosomes is not limited to structural changes of the 
DNA but also involves direct interaction. For example, the N-terminal basic domain of TRF2 interacts 
with core histones and even displayed a preference for H3K9me3 over H3K27me3 (Galati et al., 2015; 
Konishi, Izumi and Shimizu, 2016). This allows for another layer of TRF2 regulation by epigenetic marks. 
A recent study showed that telomeric chromatin is packed in a specific columnar structure (mediated 
by histone tails) that exposes the DNA helix, putatively facilitating the binding of telomeric factors, 
especially TRF1 and TRF2. Alternatively, it can also adapt an open conformation, in turn exposing the 
nucleosome and strategically positioned histone tails for H3K56, H4K12, H4K16 acetylation and H3K9, 
H3K79, H4K20 methylation (Soman et al., 2022). So even though telomeres do not necessarily exhibit 
the same heterochromatic marks as other regions of the genome and do not rely on decompaction for 
DNA damage response, conformational and epigenetic changes take place at telomeres (Timashev et 
al., 2017). While there is still much to learn, it is tempting to speculate that ZNF524 takes part in the 
epigenetic pathways that determine telomeric chromatin structure, thereby influencing the binding of 
other telomeric proteins like TRF2/RAP1 and preserving telomere integrity. 
ZNF524 as a transcription factor at telomeres 
Telomeres are transcribed by RNA polymerase II into TERRA, starting from the subtelomeric region and 
involving CTCF.TRF1 and TRF2 also localize to the telomere proximal region of subtelomeres but this 
localization is reduced when CTCF is knocked down (Deng et al., 2012). As CTCF is a chromatin 
organizing factor, the loss of TRF2 binding might be a result of changes in chromatin topology.  
Additionally, the depletion of CTCF leads to impaired TERRA transcription at telomeres (Deng et al., 
59 
 
2012). TRF2 has also been implicated with the formation of TERRA R-loops at telomeres, both by 
interaction and mediation of R-loop formation via the basic domain. In contrast to the study by Deng 
et al., where both TRF1 and TRF2 are effected similarly by CTCF removal, TRF1 counteracts R-loops 
formation by TRF2 thereby keeping their occurrence in check (Lee et al., 2018). This is an interesting 
example of TRF2 regulation by TRF1 and indicates the need for controlled TRF2 activity. With regard 
to TERRA, a recent study demonstrated that TRF2 binds to TERRA G4 via its basic domain and that this 
interaction supports telomere integrity (Mei et al., 2021). Taken together, TERRA has the ability to 
introduce structural variation in telomeres by formation of telomeric R-loops and RNA G4 and these 
structures are mediated and recognized by TRF2. Here, one could envision the following ZNF524-
dependent mode of action: The “positive regulation of RNA polymerase II transcription preinitiation 
complex assembly/ positive regulation of transcription” gene onthology term came up in our synthetic 
lethality screen arguing that ZNF524, similar to CTCF, might promote TERRA transcription and the 
resulting R-loops serve as binding motifs for TRF2. As previously mentioned, TERRA needs to be tightly 
regulated and some of the mechanisms involved are not fully understood yet. Noteworthy, we also 
found RNaseH1, which specifically degrades R-loops, in our synthetic lethality screen. This could be an 
indicator of ZNF524 being involved both in TERRA transcription and the regulation of R-loops, for 
example via a feedback loop. Interestingly, TERRA promotes telomere histone methylation through 
the PCR2 complex, including H3K9me3, which can be recognized and bound by TRF2 (Konishi, Izumi 
and Shimizu, 2016; Montero et al., 2018). These findings provide a link to ZNF524’s potential 
involvement in epigenetic regulation of the telomeric chromatin landscape and its subsequent effects 
on TRF2. 
While there is some indication for ZNF524 being involved in TERRA transcription, a genome-wide 
transcription factor activity was not confirmed so far. Both proteome and transcriptome comparison 
of WT and ZNF524 KO clones resulted in few or no up- or downregulated genes. Furthermore, ChIP-
seq analysis did not reveal other binding sites but the telomeric repeats. This could either indicate that 
ZNF524 exclusively acts at telomeres and subtelomeres or that it is redundant in its global transcription 
factor activity. In fact, we identified TBP, TADA3, as well as other members of the STAGA and the CCR4-
NOT complex that have implications in transcription. While it is unlikely that ZNF524 acts at specific 
promoters or enhancer regions, the option remains that it positively influences transcription as part of 
the general transcription machinery. This sets ZNF524 apart from other zinc finger proteins with clear 
transcription factor activities, like ZBTB48, which acts on very few specific genes, or like CTCF, which is 
irreplaceable in its 3D genome organization function.  
ZNF524 as a telomere protection and maintenance factor 
Despite the reduction of TRF2 at ZNF524-depleted telomeres, we do not observe an activation of ATM, 
CHK2 or NHEJ with subsequent telomere fusions. We do, however, detect an increase in TIFs and t-
SCEs, indicating a compromised telomere integrity that is reminiscent of intermediate-state telomeres. 
Especially the effect of ZNF524 removal on t-SCE therefore hints at an involvement of ZNF524 in HR-
related pathways. At telomeres, a very prominent HR-dependent pathway is the alternative 
lengthening of telomeres, ALT. While ZNF524 itself is not essential for telomere length homeostasis in 
ALT positive cells, our data suggests involvement in this process with a potentially redundant function 
in actual length regulation. In addition to the slight deregulation of t-SCEs, we observed the 
colocalization of ZNF524-GFP with extraordinarily large telomeric foci in the ALT positive cell lines WI-
38 VA13, GM847 and Saos2. Telomeric foci this large often indicate APBs, hubs for telomere 
recombination events that contain multiple telomeres as well as a plethora of recombination factors. 
The fact that ZNF524 was primarily detected in these regions hints at an enrichment of ZNF524 at sites 
of telomere recombination where it might perform regulatory functions. Strikingly, in our synthetic 
lethality screen, we identified the nuclear orphan receptors (NOR) NR2C2 and NR2F2 and the synthetic 
60 
 
lethality/sickness with ZNF524 was confirmed for both proteins in the competitive proliferation assay. 
NR2C2 and NR2F2 predominantly bind to TCAGGG repeats inducing telomere cluster formation and 
establishing proximity to other NR2C/F2 binding sites which allows for recombination events to 
happen and can lead to interstitial telomeric sites (ITS)  (Aeby and Lingner, 2015; Marzec et al., 2015). 
Furthermore, they promote the formation of APBs, C-circles and t-SCEs (Conomos et al., 2012; Xu et 
al., 2019; Alhendi and Royle, 2020). These findings clearly link NR2C/F2 to HR at telomeres and 
postulate the nuclear orphan receptors as potential drivers of ALT. The synthetic lethality/sickness with 
ZNF524 therefore suggests an involvement of ZNF524 in ALT regulation. Additionally, NSMCE1, also 
known as NSE1, and NSMCE2, also known as NSE2 or MMS21, were identified as genetic interactors of 
ZNF524. Both proteins are members of the ‘structural maintenance of chromosomes’ SMC5/6 
complex. While the exact function of the SMC5/6 complex is still emerging, it has been shown to 
partake in eukaryotic DNA replication and repair and that these functions rely on NSE1 and NSE2 (Potts, 
Porteus and Yu, 2006; Chavez et al., 2010; Stephan, Kliszczak and Morrison, 2011; Gallego-Paez et al., 
2014; Kolesar et al., 2022). Noteworthy, SMC5/6 localizes to telomeres to support homologous 
recombination during ALT. Here, the sumoylation activity of NSE2 acts on TRF2 and TRF1 to promote 
APB formation (Potts and Yu, 2007). Again, ZNF524 is linked to HR at telomeres and ALT, potentially by 
a function involved in structural organization of chromatin, similarly to the SMC5/6 complex. 
So far, few studies have included altered chromatin structures like D-loops, HJs or G4 when 
characterizing telomere recognition (Lim and Cech, 2021). Especially in the case of TRF2 and RAP1 the 
influence of chromatin structure on telomere-protein interaction should not be neglected. TRF2 is 
known to not only bind telomeric dsDNA but also junctions. In vitro experiments showed that TRF2 
indeed preferred ds-/ss- junctions over dsDNA and that this preference is not influenced by the 
complex formation with RAP1 (Janoušková et al., 2015). Furthermore, TRF2 recognizes 3- and 4-way 
junctions and this interaction can even occur sequence-independent. TRF2 aids in the formation of 
these structures while also stabilizing them via the N-terminal basic domain (Fouché et al., 2006; Poulet 
et al., 2009). In vitro studies often regard 3-way junctions as substitutes for replication forks while 4-
way junctions represent Holliday junctions. With ZNF524’s genetic interaction with the NORs and 
NSMCE1/2 that are implicated in structural changes of chromosomes, which promote HR at telomeres 
and therefore ALT, it is tempting to speculate that ZNF524 might perform a telomere specific function 
that supports changes in chromatin structure, thereby promoting stronger TRF2/RAP1 binding to 
telomeric chromatin. This might include telomere positioning effects but could also be the stabilization 
of recombination intermediates or stalled replication forks allowing for HR and ALT activities as well as 
TRF2 binding. Destabilizing these structures by removal of ZNF524 would in turn compromise telomere 
integrity leading to an increase in TIFs. While this function might be prevalent in ALT positive cells, it 
can also effect HR and replication stress at telomerase positive cells, explaining the increase in TIFs 
seen in ZNF524 depleted HeLa cells. 
61 
Conclusion 
In this study, I characterize the previously undescribed zinc finger protein ZNF524 in a telomere 
context. ZNF524 directly binds to the canonical TTAGGG telomeric repeats and also shows reduced 
affinity for the TCAGGG, TGAGGG and TTGGG variant repeats. This interaction depends on zinc finger 2 
but maximum affinity binding involves all four of ZNF524’s zinc fingers.  Furthermore, we validated the 
localization of ZNF524 to telomeres within the cell.  
Interestingly, when ZNF524 is removed by knock-out, we observe a reduction of TRF2 and RAP1 at 
telomeres that does not derive from a reduction of TRF2 and RAP1 protein levels. Furthermore, ZNF524 
KO results in slightly increased levels of DNA damage signaling at telomeres at determined by 53BP1 
recruitment and the recombination frequency at ALT telomeres is elevated. These phenotypes 
resemble the previously described intermediate-state of deprotection at telomeres, yet the exact 
mode of action remains to be determined. An initial genome-wide synthetic lethality screen in ZNF524 
KO clones identified a plethora of genetic interactors that, upon closer validation, will provide deeper 
insight. 
Our data presents a diverse telomeric landscape that needs to be regulated in a coordinated manner 
including the shelterin complex, its subcomplexes, other telomere specific proteins like telomerase but 
also factors involved in replication, transcription and DDR. Our research places ZNF524 among these 
factors and considering this complex network will be beneficial to a better understanding of the unique 
processes at telomeres.   
  
62 
 
Appendix 
Table 1.  Differentially regulated genes in HeLa WT and ZNF524 KO clones identified in RNA-seq 
Gene Log2 fold Adjusted Gene Log2 fold Adjusted 
Gene ID name change p-values Gene ID name change p-values
ENSG00000090339 ICAM1 0.798711691 1.79E-12 ENSG00000127418 FGFRL1 0.570306012 0.001355867 
ENSG00000102048 ASB9 -1.017112722 1.37E-11 ENSG00000198517 MAFK 0.664143286 0.001406788 
ENSG00000140015 KCNH5 23.89969534 1.37E-11 ENSG00000089327 FXYD5 0.572499361 0.001410346 
ENSG00000128573 FOXP2 -0.925613071 1.38E-09 ENSG00000095383 TBC1D2 1.302273302 0.001410346 
ENSG00000124466 LYPD3 1.30102354 3.37E-08 ENSG00000178719 GRINA 0.674814876 0.001660215 
ENSG00000117984 CTSD 1.250904258 3.07E-07 ENSG00000185033 SEMA4B 1.224326108 0.001797896 
ENSG00000069399 BCL3 1.082029706 7.65E-07 ENSG00000167550 RHEBL1 1.626538242 0.001856139 
ENSG00000175592 FOSL1 1.200755224 2.18E-06 ENSG00000166025 AMOTL1 -0.698326536 0.001896965 
ENSG00000077238 IL4R 1.554686657 4.97E-06 ENSG00000198113 TOR4A 0.668265868 0.001896965 
ENSG00000172379 ARNT2 4.442764889 4.97E-06 ENSG00000081041 CXCL2 1.974076486 0.001896965 
ENSG00000186908 ZDHHC17 -0.544390061 2.11E-05 ENSG00000131370 SH3BP5 -0.515363267 0.002002281 
ENSG00000137710 RDX -0.449077937 2.34E-05 ENSG00000079337 RAPGEF3 1.926735833 0.002084705 
ENSG00000167470 MIDN 0.558339921 2.58E-05 ENSG00000118985 ELL2 0.832696031 0.002303587 
ENSG00000166741 NNMT 1.238524268 2.58E-05 ENSG00000254416 LINC02732 -1.57448604 0.002350713 
AC069277.
ENSG00000172216 CEBPB 0.99965827 7.64E-05 ENSG00000189229 1 -0.753924791 0.002350713 
ENSG00000170522 ELOVL6 -0.612632684 9.00E-05 ENSG00000107201 DDX58 1.879130538 0.002547283 
ENSG00000110719 TCIRG1 0.700021387 9.00E-05 ENSG00000149541 B3GAT3 0.73605149 0.002796442 
ENSG00000227467 LINC01537 3.44783297 9.00E-05 ENSG00000162729 IGSF8 1.054285541 0.002796442 
ENSG00000064932 SBNO2 0.755665859 0.0001312 ENSG00000126368 NR1D1 0.87862082 0.002852897 
ENSG00000182585 EPGN 2.159812176 0.0001312 ENSG00000134779 TPGS2 -0.324568219 0.003115186 
ENSG00000166979 EVA1C 0.925414834 0.000183947 ENSG00000063660 GPC1 0.429217818 0.003115186 
ENSG00000170412 GPRC5C 0.67296452 0.000301867 ENSG00000196411 EPHB4 0.446683181 0.003115186 
ENSG00000102265 TIMP1 0.943088186 0.000307807 ENSG00000109321 AREG 1.780265477 0.003115186 
AC156455.
ENSG00000145623 OSMR 1.624988315 0.000307807 
ENSG00000256546 1 -0.801697625 0.003179169 
ENSG00000113763 UNC5A 3.236874674 0.000307807 
ENSG00000152944 MED21 -0.375754692 0.003473673 
ENSG00000120889 TNFRSF10B 0.371932584 0.000341444 
ENSG00000171843 MLLT3 -0.312094445 0.003514993 
ENSG00000136379 ABHD17C 1.224357821 0.00041657 
ENSG00000070404 FSTL3 0.831267081 0.003514993 
ENSG00000164171 ITGA2 1.628455161 0.00041657 
ENSG00000108106 UBE2S 0.554746715 0.003565255 
ENSG00000143369 ECM1 1.168735892 0.000466299 
ENSG00000136048 DRAM1 1.233863781 0.003765597 
ENSG00000134107 BHLHE40 1.472475931 0.000466299 
ENSG00000168477 TNXB 2.425187147 0.003817033 
ENSG00000037042 TUBG2 0.888585439 0.000488089 
ENSG00000131408 NR1H2 0.446423571 0.003839838 
ENSG00000116016 EPAS1 1.175534534 0.000488089 
ENSG00000182704 TSKU 0.752039826 0.003839838 
ENSG00000151458 ANKRD50 0.457499675 0.000718463 
ENSG00000136244 IL6 4.851620391 0.003839838 
ENSG00000130589 HELZ2 0.783672276 0.000750372 
ENSG00000118418 HMGN3 -0.617627528 0.004000937 
ENSG00000221963 APOL6 0.519601264 0.000887609 
ENSG00000101972 STAG2 -0.366361536 0.004000937 
ENSG00000182010 RTKN2 -1.057174354 0.001165698 
ENSG00000214063 TSPAN4 0.652704836 0.004000937 
ENSG00000181045 SLC26A11 0.536281558 0.001165698 
ENSG00000173846 PLK3 0.836780383 0.004000937 
ENSG00000159216 RUNX1 1.036796082 0.001165698 
ENSG00000130066 SAT1 1.934920907 0.004000937 
ENSG00000237523 LINC00857 -0.883111805 0.001209443 
ENSG00000103888 CEMIP 6.076958648 0.004000937 
ENSG00000128487 SPECC1 -0.314861448 0.00125044 
ENSG00000129566 TEP1 0.478515927 0.004163929 
ENSG00000100342 APOL1 3.076081153 0.00125044 
ENSG00000175832 ETV4 1.251126744 0.004163929 
63 
Gene Log2 fold Adjusted Gene Log2 fold Adjusted 
Gene ID name change p-values Gene ID name change p-values 
ENSG00000142627 EPHA2 0.854141449 0.004252402 ENSG00000027847 B4GALT7 0.533391714 0.006168341 
ENSG00000102312 PORCN 0.970850109 0.004304087 ENSG00000110195 FOLR1 1.614083747 0.006168341 
ENSG00000184486 POU3F2 -1.797070924 0.004386658 ENSG00000170581 STAT2 0.731116837 0.006203167 
ENSG00000146242 TPBG 1.170798627 0.004386658 ENSG00000100284 TOM1 0.624270364 0.006527051 
ENSG00000161048 NAPEPLD -0.724084842 0.004453764 ENSG00000197136 PCNX3 0.31122744 0.006557185 
ENSG00000172985 SH3RF3 1.57293862 0.004571081 ENSG00000074527 NTN4 0.953780024 0.006557185 
AC079062.
ENSG00000263711 1 -0.555903267 0.004627664 ENSG00000172354 GNB2 0.374298035 0.006613666 
ENSG00000221955 SLC12A8 2.600547691 0.004627664 ENSG00000106829 TLE4 -0.565440719 0.006779074 
ENSG00000178028 DMAP1 -0.848350343 0.0046695 ENSG00000100644 HIF1A 0.85275346 0.006882203 
ENSG00000177951 BET1L 0.394859229 0.0046695 ENSG00000106366 SERPINE1 1.55237178 0.007006386 
ENSG00000161013 MGAT4B 0.410847991 0.0046695 ENSG00000184792 OSBP2 1.246719242 0.007047424 
ENSG00000115756 HPCAL1 0.699224564 0.004831816 ENSG00000106397 PLOD3 0.431701045 0.007082421 
ENSG00000161638 ITGA5 1.045295321 0.004831816 ENSG00000119917 IFIT3 1.800727914 0.007082421 
ENSG00000110057 UNC93B1 0.645233442 0.004848622 ENSG00000076351 SLC46A1 0.511431541 0.007211865 
ENSG00000003436 TFPI 0.731420472 0.004848622 ENSG00000177674 AGTRAP 0.49953364 0.00776359 
ENSG00000124762 CDKN1A 1.65381365 0.004848622 ENSG00000100983 GSS 0.45606808 0.00778349 
ENSG00000227191 TRGC2 -3.169393477 0.004950054 ENSG00000139289 PHLDA1 1.541621929 0.007851824 
ENSG00000125844 RRBP1 0.451895146 0.004950054 ENSG00000134070 IRAK2 1.93834459 0.007851824 
ENSG00000158863 FAM160B2 0.666121513 0.004950054 ENSG00000117226 GBP3 2.12084022 0.007879825 
ENSG00000156711 MAPK13 0.784933145 0.005312469 ENSG00000221926 TRIM16 -0.811298712 0.008226983 
ENSG00000148426 PROSER2 0.824076548 0.005312469 ENSG00000122299 ZC3H7A -0.531646303 0.008226983 
TNFRSF10
ENSG00000186866 POFUT2 0.655491993 0.008226983 
ENSG00000173530 D 0.898609397 0.005312469 
ENSG00000124216 SNAI1 1.317794093 0.008226983 
ENSG00000221869 CEBPD 0.96516737 0.005451621 
ENSG00000023608 SNAPC1 0.594750469 0.008493459 
ENSG00000100241 SBF1 0.470570752 0.005663963 
ENSG00000165915 SLC39A13 0.831390183 0.008493459 
ENSG00000136002 ARHGEF4 0.480276921 0.005665965 
ENSG00000163491 NEK10 -1.236026159 0.008600192 
ENSG00000120875 DUSP4 1.715136138 0.005665965 
ENSG00000116001 TIA1 -0.321435407 0.008639174 
ENSG00000189067 LITAF -0.655741192 0.005746281 
ENSG00000013364 MVP 1.035662868 0.008642115 
ENSG00000021645 NRXN3 -1.270428242 0.005746528 
ENSG00000005884 ITGA3 0.881619909 0.008648064 
ENSG00000130270 ATP8B3 0.552122808 0.005841886 
ENSG00000047597 XK -0.561923454 0.00866374 
ENSG00000165434 PGM2L1 1.055153363 0.005841886 
ENSG00000251322 SHANK3 0.888934806 0.008830831 
ENSG00000130558 OLFM1 5.10324969 0.005857186 
ENSG00000196639 HRH1 1.22257591 0.009075382 
ENSG00000164951 PDP1 0.936119705 0.005930941 
ENSG00000180900 SCRIB 0.246559068 0.009238619 
ENSG00000185000 DGAT1 0.624258647 0.005966144 
ENSG00000011422 PLAUR 0.89692215 0.0093117 
ENSG00000185022 MAFF 1.218912341 0.005966144 
ENSG00000169733 RFNG 0.610870541 0.009468957 
ENSG00000112511 PHF1 0.610396638 0.00609405 
AL390719. ENSG00000101224 CDC25B 0.642429234 0.009591995 
ENSG00000217801 1 1.461172135 0.00609405 
MAP3K2- ENSG00000077097 TOP2B -0.243430287 0.009930258 
ENSG00000236682 DT -0.787397457 0.006168341 
  
64 
 
 
Table 2. Proteins identified by proteome comparison of U2OS WT and ZNF524 KO clones 
Log2(Fold -Log10(p-
Protein names Full names 
change) value) 
ACD Adrenocortical dysplasia protein homolog 0.140 0.171 
TINF2 TERF1-interacting nuclear factor 2 0.072 0.084 
TERF1 Telomeric repeat-binding factor 1 0.479 0.973 
TERF2 Telomeric repeat-binding factor 2 0.303 1.137 
TERF2IP Telomeric repeat-binding factor 2-interacting protein 1 0.315 1.175 
RNF13 E3 ubiquitin-protein ligase RNF13 1.095 3.422 
RHOC Rho-related GTP-binding protein RhoC 0.746 2.987 
SNRPE Small nuclear ribonucleoprotein E 1.102 2.871 
HMGN1 Non-histone chromosomal protein HMG-14 0.860 2.713 
LAMTOR5 Ragulator complex protein LAMTOR5 0.834 2.705 
PET117 Protein PET117 homolog, mitochondrial 2.835 2.422 
PNPLA4 Patatin-like phospholipase domain-containing protein 4 0.860 2.336 
HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 0.673 2.320 
SURF2 Surfeit locus protein 2 1.936 2.290 
LSM14A Protein LSM14 homolog A 1.419 2.227 
Serine/threonine-protein phosphatase 6 catalytic 
PPP6C subunit;Serine/threonine-protein phosphatase 6 catalytic 0.672 2.057 
subunit, N-terminally processed 
Tubulin beta-8 chain;Tubulin beta-8 chain-like protein 
TUBB8 0.719 1.999 
LOC260334 
Histone H2B type 1-D;Histone H2B;Histone H2B type 1-
HIST1H2BD;HIST1H2BN;HIST1H2BM;HIST1H2BH;HIST2H2
M;Histone H2B type 1-N;Histone H2B type 1-H;Histone 0.775 1.964 
BF 
H2B type 2-F 
UCK1 Uridine-cytidine kinase 1 1.226 1.870 
PTPN14 Tyrosine-protein phosphatase non-receptor type 14 1.522 1.850 
Guanine nucleotide-binding protein G(I)/G(S)/G(O) 
GNG4 1.622 1.809 
subunit gamma-4 
CCDC85C Coiled-coil domain-containing protein 85C 0.921 1.773 
CROCC Rootletin 4.199 1.734 
POLR3H DNA-directed RNA polymerase III subunit RPC8 0.752 1.704 
NAPA Alpha-soluble NSF attachment protein 0.731 1.703 
FDX1L Adrenodoxin-like protein, mitochondrial 0.931 1.699 
65 
 
Log2(Fold -Log10(p-
Protein names Full names 
change) value) 
Myosin light chain 1/3, skeletal muscle isoform;Myosin 
MYL1;MYL3 0.760 1.681 
light chain 3 
LUC7L2 Putative RNA-binding protein Luc7-like 2 0.806 1.591 
PYURF Protein preY, mitochondrial 2.420 1.526 
PTBP2 Polypyrimidine tract-binding protein 2 1.819 1.489 
CD9 Tetraspanin;CD9 antigen 1.029 1.456 
SETX Probable helicase senataxin 1.077 1.445 
S100A10 Protein S100-A10 0.954 1.443 
DYNLL1 Dynein light chain 1, cytoplasmic 1.016 1.428 
ZMYM1 Zinc finger MYM-type protein 1 1.161 1.415 
FOSL1 Fos-related antigen 1 2.012 1.367 
MAN1A2 Mannosyl-oligosaccharide 1,2-alpha-mannosidase IB -1.785 4.861 
HAUS5 HAUS augmin-like complex subunit 5 -1.358 3.792 
CDK8;CDK19 Cyclin-dependent kinase 8;Cyclin-dependent kinase 19 -1.281 3.539 
MCEE Methylmalonyl-CoA epimerase, mitochondrial -0.806 2.998 
SFSWAP Splicing factor, suppressor of white-apricot homolog -0.852 2.942 
EML3 Echinoderm microtubule-associated protein-like 3 -0.694 2.767 
GOLGA5 Golgin subfamily A member 5 -1.256 2.682 
BRIP1 Fanconi anemia group J protein -0.652 2.519 
SYNE1 Nesprin-1 -2.149 2.316 
MANBAL Protein MANBAL -3.888 2.306 
GTPBP6 Putative GTP-binding protein 6 -1.238 2.258 
USE1 Vesicle transport protein USE1 -0.644 2.209 
Low molecular weight phosphotyrosine protein 
ACP1 -0.798 2.192 
phosphatase 
HEATR3 HEAT repeat-containing protein 3 -0.809 1.997 
C14orf1 Probable ergosterol biosynthetic protein 28 -1.341 1.925 
MED24 Mediator of RNA polymerase II transcription subunit 24 -0.882 1.906 
POLG2 DNA polymerase subunit gamma-2, mitochondrial -1.091 1.904 
Beta-1,4-galactosyltransferase 1;Lactose synthase A 
protein;N-acetyllactosamine synthase;Beta-N-
acetylglucosaminylglycopeptide beta-1,4-
B4GALT1 -0.752 1.879 
galactosyltransferase;Beta-N-acetylglucosaminyl-
glycolipid beta-1,4-galactosyltransferase;Processed beta-
1,4-galactosyltransferase 1 
ALDH1L2 Mitochondrial 10-formyltetrahydrofolate dehydrogenase -1.472 1.866 
66 
 
Log2(Fold -Log10(p-
Protein names Full names 
change) value) 
PCTP Phosphatidylcholine transfer protein -0.818 1.862 
SLC20A1 Sodium-dependent phosphate transporter 1 -0.739 1.841 
ATG7 Ubiquitin-like modifier-activating enzyme ATG7 -1.741 1.801 
Alpha-aminoadipic semialdehyde synthase, 
AASS mitochondrial;Lysine ketoglutarate -1.835 1.800 
reductase;Saccharopine dehydrogenase 
CYTL1 Cytokine-like protein 1 -1.675 1.768 
STXBP4 Syntaxin-binding protein 4 -1.146 1.764 
Glutamyl-tRNA(Gln) amidotransferase subunit B, 
PET112;GATB -1.170 1.719 
mitochondrial 
NBAS Neuroblastoma-amplified sequence -0.775 1.719 
UNC93B1 Protein unc-93 homolog B1 -1.227 1.689 
TOM1L2 TOM1-like protein 2 -0.930 1.679 
ZKSCAN1 Zinc finger protein with KRAB and SCAN domains 1 -1.357 1.677 
ADCK3 Atypical kinase ADCK3, mitochondrial -0.985 1.638 
PDE5A cGMP-specific 3,5-cyclic phosphodiesterase -1.838 1.630 
CDK5RAP2 CDK5 regulatory subunit-associated protein 2 -1.939 1.612 
CENPK Centromere protein K -1.280 1.602 
TTI1 TELO2-interacting protein 1 homolog -0.814 1.595 
GMEB1 Glucocorticoid modulatory element-binding protein 1 -1.153 1.571 
SPDL1 Protein Spindly -1.352 1.536 
TAF8 Transcription initiation factor TFIID subunit 8 -0.824 1.525 
PALD1 Paladin -1.021 1.504 
LPIN1 Phosphatidate phosphatase LPIN1 -1.448 1.479 
THNSL1 Threonine synthase-like 1 -0.963 1.466 
Calcium-transporting ATPase;Calcium-transporting 
ATP2C1 -1.106 1.465 
ATPase type 2C member 1 
MMGT1 Membrane magnesium transporter 1 -1.053 1.458 
PIK3R1 Phosphatidylinositol 3-kinase regulatory subunit alpha -1.074 1.443 
HSP90AB4P Putative heat shock protein HSP 90-beta 4 -2.506 1.395 
 
  
67 
 
Table 3  Genetic interactors of ZNF524 identified by SL screen 
gene Log2(Fold P value FDR gene Log2(Fold P value FDR 
change) change) 
AP2S1 -1.258054772 8.401E-06 0.00967428 ASNA1 -0.686606069 0.000815333 0.080766173 
SOCS3 -1.186374433 6.35932E-06 0.00901311 MRPS12 -0.685657371 0.000159366 0.051492689 
TRAF3 -1.101756023 3.34254E-06 0.007583272 RTCB -0.683245096 0.000362294 0.061810333 
SCAF8 -1.059779062 8.48061E-07 0.002604253 UTP15 -0.675156451 0.00012616 0.049457337 
TRAF2 -1.052225242 4.06172E-07 0.001870929 NSMCE1 -0.673346617 6.42671E-05 0.032892256 
DNM2 -0.922041498 0.000729891 0.077735526 TRAPPC5 -0.670425439 0.000973447 0.087897407 
CCNC -0.894122018 1.2138E-07 0.00074991 ANAPC2 -0.662786778 0.000739051 0.078258722 
MED12 -0.884332578 1.22102E-07 0.00074991 INTS8 -0.655597732 0.000545187 0.070226794 
SDHA -0.860124401 3.70418E-06 0.007583272 UBE2I -0.655540803 0.00039512 0.063058935 
PSMA3 -0.855467521 3.84024E-05 0.024037376 CLP1 -0.649403439 0.000802967 0.080100456 
TRIM49D1 -0.849758336 0.000671735 0.075208227 ARF6 -0.649325848 0.000533003 0.070147044 
EDF1 -0.844780526 0.000263232 0.057738578 TAF5L -0.643693219 0.000475374 0.068427919 
DOT1L -0.835804284 3.12307E-05 0.023367993 ARF4 -0.639692444 0.000759907 0.078552772 
CCDC101 -0.817388442 0.00015206 0.051492689 NAPG -0.634720168 0.000218466 0.054574138 
INTS7 -0.771969172 5.77667E-05 0.031304445 SCO2 -0.62784373 4.73849E-06 0.007943278 
FLCN -0.771906876 6.85579E-06 0.009022709 SMC2 -0.620077608 0.000138403 0.051267843 
SMARCB1 -0.77171567 1.3749E-06 0.003618939 SARS -0.619037293 7.86953E-05 0.037162609 
DKC1 -0.7716644 1.25671E-05 0.011577414 MVD -0.615657146 0.000689786 0.075985922 
UXT -0.750375249 0.000556072 0.070226794 CHD8 -0.61475912 0.000201834 0.054574138 
SPOP -0.749156931 1.06933E-05 0.010814809 SUPT20H -0.614300125 0.000507594 0.07014495 
VPS37A -0.742990448 0.000362308 0.061810333 ARHGAP2 -0.613728761 0.000213908 0.054574138 
1 
ADSL -0.74046162 3.0088E-05 0.023367993 
UBL5 -0.610103028 0.001020455 0.087897407 
STK11 -0.731540159 0.000246032 0.056297939 
RAB35 -0.608370944 7.72795E-05 0.037162609 
AP2M1 -0.725158627 0.000141908 0.051267843 
CNOT2 -0.606605337 2.03623E-05 0.017865517 
TADA1 -0.714554135 9.76774E-06 0.010586503 
MRPS6 -0.602085015 0.000931593 0.085822959 
TUFM -0.71217327 0.000125829 0.049457337 
SEC63 -0.601840126 0.000434699 0.065650242 
TRAPPC3 -0.706710387 0.00055869 0.070226794 
EIF2S3 -0.600097144 0.000288347 0.059500761 
PSMD14 -0.702331782 4.04428E-05 0.024037376 
LSM7 -0.599632139 0.00033112 0.060055437 
PNISR -0.700589793 0.000848151 0.081207178 
CDC26 -0.596159216 0.000602796 0.071654894 
TADA3 -0.69694247 7.45665E-06 0.009159248 
68 
 
gene Log2(Fold P value FDR gene Log2(Fold P value FDR 
change) change) 
RPS12 -0.595681251 0.000913824 0.084609085 SPTAN1 -0.514118866 0.000488579 0.069783524 
MRPL3 -0.587968393 1.11523E-05 0.010814809 NUFIP1 -0.513036444 0.00056029 0.070226794 
GGPS1 -0.586494697 6.06429E-06 0.00901311 POT1 -0.512327577 0.000475282 0.068427919 
C3orf17 -0.580009693 0.000278337 0.058508266 OLA1 -0.510124195 0.000407273 0.063058935 
TXN -0.579645069 3.29752E-05 0.023367993 SUV420H1 -0.50944105 0.001168608 0.090468884 
GRK6 -0.572826876 0.001004638 0.087897407 ZEB1 -0.508883652 0.00032432 0.060055437 
PHF5A -0.571577479 0.00076553 0.078552772 FCF1 -0.508088921 0.001206027 0.092203484 
SCFD1 -0.570898584 0.000167299 0.05202493 TLK2 -0.507803883 0.000879006 0.08263102 
YARS -0.569502848 0.00052918 0.07014495 MVK -0.50724779 0.000593339 0.071597909 
CAPN1 -0.569063401 0.001047924 0.087964594 DOHH -0.506054506 0.000800064 0.080100456 
FNTA -0.565662711 0.000631965 0.07277471 BTBD9 -0.504591561 0.001135814 0.089052648 
RPL7L1 -0.564221185 0.000156571 0.051492689 TBP -0.504448236 0.000321621 0.060055437 
WDR61 -0.563307311 0.001317646 0.096331914 HSPA14 -0.501868535 0.000299376 0.059745534 
BRF2 -0.554867819 0.000342243 0.060055437 IARS2 -0.501513381 0.00040144 0.063058935 
TTF1 -0.553795226 0.000453303 0.066549427 CEP97 -0.500621058 0.000212195 0.054574138 
RABGGTB -0.552626396 0.000339907 0.060055437 MYH9 -0.495464657 0.00059715 0.071597909 
COX7B -0.549219715 4.74226E-06 0.007943278 NSMCE2 -0.494779933 0.001353235 0.096718485 
ARIH1 -0.548149644 0.000833305 0.081149272 TMEM242 -0.493450176 0.000100565 0.042696498 
TBCE -0.537596814 0.000771672 0.078552772 ATP5E -0.489269007 0.001114838 0.088538321 
CDK8 -0.53570849 7.16638E-07 0.002604253 N4BP2L2 -0.489210007 0.000455101 0.066549427 
SLCO2B1 -0.534384107 0.000869616 0.082591116 NSRP1 -0.484626874 0.001092129 0.088392454 
MRPS14 -0.533662391 0.00030805 0.059745534 MRPL34 -0.480475905 0.000429984 0.065507953 
KARS -0.531451665 0.000516737 0.07014495 PPP6C -0.480062906 0.000430201 0.065507953 
PMPCB -0.530817823 0.000304326 0.059745534 FMR1 -0.479145241 0.000443788 0.066477979 
SDHAF2 -0.530376569 0.001086203 0.088392454 ANAPC5 -0.476590793 0.000331958 0.060055437 
POLG -0.529487659 0.000611493 0.071762828 AMDHD2 -0.475877099 0.001370069 0.096718485 
PICALM -0.520862104 0.000323366 0.060055437 DARS2 -0.475267674 4.40933E-05 0.024618761 
CNOT4 -0.520339705 3.85254E-05 0.024037376 RPLP0 -0.470485374 0.000566466 0.070521171 
ECD -0.518029857 0.000387974 0.062767475 NUF2 -0.470390656 0.00020625 0.054574138 
GID8 -0.517692026 0.001293135 0.095912241 MRPS21 -0.469912544 0.001074952 0.088392454 
CTPS1 -0.515272584 0.001005717 0.087897407 SMG6 -0.466143879 0.000526031 0.07014495 
69 
 
gene Log2(Fold P value FDR gene Log2(Fold P value FDR 
change) change) 
CAND1 -0.464534752 0.000682307 0.07573198 RNASEH1 -0.41913066 0.000524871 0.07014495 
PSAP -0.464315988 0.001093812 0.088392454 DNAJB1 -0.416037309 0.00027806 0.058508266 
COA5 -0.463711987 0.000190692 0.054574138 MLLT10 -0.415604487 2.63273E-05 0.022049073 
CMIP -0.46291754 0.000381124 0.062767475 FOXD4L1 -0.409983813 0.000895693 0.083349197 
OGFOD1 -0.459758168 3.94964E-05 0.024037376 TP53 -0.408718692 0.000762113 0.078552772 
DNAJC3 -0.457453112 0.000133011 0.051056869 MAEA -0.408041266 0.00110821 0.088392907 
NR2F2 -0.455700939 0.001316764 0.096331914 SOD1 -0.407212231 0.001014557 0.087897407 
MED18 -0.453751658 0.000383481 0.062767475 WDR25 -0.407091488 0.000212984 0.054574138 
VPS11 -0.451563854 6.61344E-05 0.032933122 BBS9 -0.403853834 0.000241869 0.056297939 
ZRSR2 -0.447757863 0.001042716 0.087964594 BCAS3 -0.401549814 0.000767482 0.078552772 
TARS2 -0.446790905 0.001365491 0.096718485 KCNK13 -0.400921511 0.00010172 0.042696498 
PEAK1 -0.446413047 6.36462E-05 0.032892256 USP15 -0.399341516 0.000239557 0.056297939 
MAT2A -0.445977587 0.000765809 0.078552772 SNX33 -0.399261697 0.001359995 0.096718485 
ITPK1 -0.443972673 0.000384864 0.062767475 DLX2 -0.395566113 0.00049281 0.069846283 
ANKRD36B -0.442436753 0.000253843 0.057037386 C11orf83 -0.394515285 3.17466E-05 0.023367993 
GATC -0.44082869 9.88688E-05 0.042696498 RBX1 -0.390713042 0.001296182 0.095912241 
AP2B1 -0.439484861 0.000211785 0.054574138 RASSF8 -0.389033515 0.000549197 0.070226794 
RARS2 -0.438094193 0.000398222 0.063058935 CDR1 -0.385696097 0.000503793 0.07014495 
MRPS9 -0.437397843 0.001187038 0.091511212 PAICS -0.385331941 0.001030439 0.087897407 
CINP -0.436989839 0.000822275 0.081018315 SLC26A10 -0.385149298 0.0002195 0.054574138 
TFG -0.435789467 0.001333439 0.096637498 MRPL37 -0.385106974 0.000337184 0.060055437 
FAM120C -0.433471675 0.001295587 0.095912241 KCNK7 -0.384718261 0.000673507 0.075208227 
KAT8 -0.433040655 0.00095908 0.087897407 EMCN -0.38433566 0.000342135 0.060055437 
ACTL7A -0.432525904 0.00064411 0.073712532 SWT1 -0.383175117 0.001028528 0.087897407 
PPIP5K2 -0.431347655 0.000720081 0.077136561 ABL1 -0.381526625 0.000169416 0.05202493 
CDCP2 -0.431344882 4.2755E-05 0.024617524 MARS2 -0.379076769 0.000304996 0.059745534 
CYB561A3 -0.428122443 0.001342697 0.096637498 ZBTB6 -0.375941171 4.00497E-05 0.024037376 
METAP1 -0.427586218 0.000546694 0.070226794 GOLT1B -0.375278272 0.001403919 0.098686396 
AP2A1 -0.426817377 0.000502636 0.07014495 UPRT -0.37410139 0.001228257 0.093130217 
ACBD5 -0.42269116 0.000219329 0.054574138 TDRD7 -0.37166703 0.000512934 0.07014495 
MRPS33 -0.421877694 0.000149798 0.051492689 PTGES2 -0.367761719 0.00013952 0.051267843 
70 
 
gene Log2(Fold P value FDR gene Log2(Fold P value FDR 
change) change) 
ZDHHC16 -0.366508986 0.000104279 0.042696498 RC3H2 -0.298314168 8.06787E-05 0.037162609 
ERVW-1 -0.366298634 0.00071708 0.077136561 CSTA -0.298105221 0.000206168 0.054574138 
ATP6V1H -0.363864178 0.000673278 0.075208227 GPR27 -0.298060908 0.001023003 0.087897407 
TXNDC5 -0.362299801 0.000211551 0.054574138 DNAJC19 -0.291194909 0.000990385 0.087897407 
GALNT8 -0.362109041 0.000174953 0.052844293 FLG -0.29044564 0.00098154 0.087897407 
PLA2G10 -0.360797913 0.00114074 0.089059857 CNOT6 -0.289880807 0.000279442 0.058508266 
SRGAP2D -0.358439742 0.000230382 0.055852412 C7orf71 -0.289517244 0.000147981 0.051492689 
MST4 -0.353591148 0.001368237 0.096718485 SARS2 -0.286044947 0.000833644 0.081149272 
CCAR2 -0.351996049 0.001338367 0.096637498 PTPN4 -0.284812318 0.001107659 0.088392907 
KIAA1147 -0.350988794 0.001409595 0.098686396 DNASE2B -0.282131994 0.000337498 0.060055437 
INPP5E -0.349760227 0.001081424 0.088392454 C3 -0.281131697 0.001099078 0.088392907 
CCDC176 -0.349239378 0.0001607 0.051492689 GPATCH8 -0.280504537 0.001279523 0.095834185 
TBX3 -0.347809068 0.001061378 0.088392454 ZNF675 -0.277481991 0.000659173 0.074970768 
MRPL49 -0.34487162 0.000996208 0.087897407 RPL5 -0.276059338 0.001050324 0.087964594 
LGI2 -0.342299401 0.00059843 0.071597909 LRRC63 -0.275682377 0.000801497 0.080100456 
PINX1 -0.34187055 0.001013387 0.087897407 MYLK4 -0.267832847 0.000290641 0.059500761 
MORN5 -0.341356481 0.000298348 0.059745534 C1orf158 -0.26508326 0.000385017 0.062767475 
PDSS1 -0.339525084 0.000836817 0.081149272 FBXO18 -0.26341914 0.000247497 0.056297939 
CACNG5 -0.337270554 0.00058071 0.071330558 UQCRC2 -0.262263415 0.000999899 0.087897407 
TMCO3 -0.33647062 0.000262134 0.057738578 UBP1 -0.257675945 0.001067901 0.088392454 
IRF1 -0.3344767 0.000556476 0.070226794 CEP76 -0.257240278 0.000222147 0.054574138 
RNF208 -0.322198786 0.000102052 0.042696498 BLOC1S6 -0.252231359 0.001130648 0.089026478 
TEX261 -0.317918466 0.000162094 0.051492689 MRRF -0.250832951 0.000626019 0.072543362 
SCAI -0.314913979 0.001322767 0.096331914 SHB -0.244911318 0.000610703 0.071762828 
DDX59 -0.313952943 0.000844244 0.081207178 PCDHB14 -0.239667933 0.000850637 0.081207178 
STAU1 -0.308951263 0.000388358 0.062767475 WWP2 -0.239080649 0.000277899 0.058508266 
RAB31 -0.307622018 0.000242023 0.056297939 SAT1 -0.232645842 0.000984303 0.087897407 
COL20A1 -0.305110496 0.000804265 0.080100456 PATE1 -0.230792035 0.001193884 0.091655483 
PHF19 -0.30332779 0.001049248 0.087964594 SEMA3C -0.226917022 0.001158435 0.090059803 
AP4E1 -0.301076278 0.00044742 0.066481622 FSTL5 -0.225359065 0.001414014 0.098686396 
CHIC1 -0.299875858 0.000523798 0.07014495 SPINK13 -0.220007747 0.000183613 0.054565726 
71 
 
gene Log2(Fold P value FDR gene Log2(Fold P value FDR 
change) change) 
COX17 -0.20855584 0.00131097 0.096331914 PITPNA -0.156922837 0.00071398 0.077136561 
FPGS -0.200317586 0.001029418 0.087897407 ZNF782 -0.15640992 0.000616254 0.071863774 
HMG20B -0.200251251 0.001089066 0.088392454 SVOPL -0.149641016 0.000586713 0.071590589 
CDK16 -0.199953953 0.000692843 0.075985922 PSMA8 -0.146635358 0.000875939 0.08263102 
ARFGAP1 -0.19091257 0.000575652 0.071183848 SECISBP2L -0.144088207 0.000405156 0.063058935 
NR2C2 -0.190682829 0.000702335 0.076571149 HEXB -0.137791157 0.001227345 0.093130217 
CKM -0.176491011 0.000746704 0.078552772 TTC3 -0.131160504 0.00089017 0.083255771 
CRBN -0.175119205 0.000359147 0.061810333 PBDC1 -0.119215683 0.001123182 0.088818113 
HORMAD2 -0.172272554 0.001247518 0.094202933 
  
72 
 
Materials and Methods 
Materials 
Cell lines created during this study 
Name of produced cell line parental cell antibiotic plasmid 
line 
HT1080ST ZNF524-GFP HT1080ST puromycin pLIX_403 ZNF524-GFP 
HT1080ST ZNF524-GFP ZF2 mut HT1080ST puromycin pLIX_403 ZNF524-GFP ZF2 mut 
HeLa ZNF524-GFP HeLa puromycin pLIX_403 ZNF524-GFP 
HeLa ZNF524-GFP ZF2 mut HeLa puromycin pLIX_403 ZNF524-GFP ZF2 mut 
U2OS ZNF524-GFP U2OS puromycin pLIX_403 ZNF524-GFP 
U2OS ZNF524-GFP ZF2 mut U2OS puromycin pLIX_403 ZNF524-GFP ZF2 mut 
HeLa1.3 ZNF524-GFP HeLa1.3 puromycin pLIX_403 ZNF524-GFP 
HeLa1.3 ZNF524-GFP ZF2 mut HeLa1.3 puromycin pLIX_403 ZNF524-GFP ZF2 mut 
WI-38 VA13 ZNF524-GFP WI-38 VA13 puromycin pLIX_403 ZNF524-GFP 
WI-38 VA13 ZNF524-GFP ZF2 mut WI-38 VA13 puromycin pLIX_403 ZNF524-GFP ZF2 mut 
GM847 ZNF524-GFP GM847 puromycin pLIX_403 ZNF524-GFP 
GM847 ZNF524-GFP ZF2 mut GM847 puromycin pLIX_403 ZNF524-GFP ZF2 mut 
Saos2 ZNF524-GFP Saos2 puromycin pLIX_403 ZNF524-GFP 
Saos2 ZNF524-GFP ZF2 mut Saos2 puromycin pLIX_403 ZNF524-GFP ZF2 mut 
U2OS ZNF524 KO clone 1 + U2OS G418 pInducer20 ZNF524 
 ZNF524-HA 
U2OS ZNF524 KO clone 2 +  U2OS G418 pInducer20 ZNF524 
ZNF524-HA 
U2OS ZNF524 KO clone 3 +  U2OS G418 pInducer20 ZNF524 
ZNF524-HA 
U2OS ZNF524 KO clone 4 +  U2OS G418 pInducer20 ZNF524 
ZNF524-HA 
U2OS ZNF524 KO clone 5 +  U2OS G418 pInducer20 ZNF524 
ZNF524-HA 
U2OS ZNF524 KO clone 1 +  U2OS G418 pInducer20 ZNF524 ZF2 mut 
ZNF524-HA ZF2 mut 
U2OS ZNF524 KO clone 2 +  U2OS G418 pInducer20 ZNF524 ZF2 mut 
ZNF524-HA ZF2 mut 
U2OS ZNF524 KO clone 3 + U2OS G418 pInducer20 ZNF524 ZF2 mut 
 ZNF524-HA ZF2 mut 
U2OS ZNF524 KO clone 4 +  U2OS G418 pInducer20 ZNF524 ZF2 mut 
ZNF524-HA ZF2 mut 
U2OS ZNF524 KO clone 5 +  U2OS G418 pInducer20 ZNF524 ZF2 mut 
ZNF524-HA ZF2 mut 
Plasmids 
internal insert backbone description 
number 
P695 H. sapiens ZNF524 pCoofy1 bacterial protein expression, N-
terminal His 
73 
 
P1113 H. sapiens ZNF524 ZF2 mut pCoofy1 bacterial protein expression, N-
terminal His 
P831 H. sapiens ZNF524 minimal pCoofy1 bacterial protein expression, N-
domain terminal His 
P760 H. sapiens ZNF524 pDest-pcDNA3.1 protein expression, N-terminal 
FLAG 
P813 H. sapiens ZNF524 ZF 1 mut pDest-pcDNA3.1 protein expression, N-terminal 
FLAG 
P814 H. sapiens ZNF524 ZF 2 mut pDest-pcDNA3.1 protein expression, N-terminal 
FLAG 
P815 H. sapiens ZNF524 ZF 3 mut pDest-pcDNA3.1 protein expression, N-terminal 
FLAG 
P816 H. sapiens ZNF524 ZF 4 mut pDest-pcDNA3.1 protein expression, N-terminal 
FLAG 
P851 H. sapiens ZNF524 ZF 1, 3 pDest-pcDNA3.1 protein expression, N-terminal 
mut FLAG 
P852 H. sapiens ZNF524 ZF 1, 4 pDest-pcDNA3.1 protein expression, N-terminal 
mut FLAG 
P853 H. sapiens ZNF524 ZF 3, 4 pDest-pcDNA3.1 protein expression, N-terminal 
mut FLAG 
P854 H. sapiens ZNF524 ZF 1, 3, 4 pDest-pcDNA3.1 protein expression, N-terminal 
mut FLAG 
P832 H. sapiens ZNF524 minimal pCoofy4 bacterial protein expression, N-
domain terminal His-MBP 
P872 H. sapiens ZNF524 CRISPR 1 pX459 V2 CRISPR gene editing 
new 
P873 H. sapiens ZNF524 CRISPR 2 pX459 V2 CRISPR gene editing 
new 
P874 H. sapiens ZNF524 CRISPR 3 pX459 V2 CRISPR gene editing 
n ew 
P1103 pMDLg/pRRE lentiviral packaging plasmid 
 
P1104 pRSV-Rev lentiviral packaging plasmid 
 
P1105 pMD2.G lentiviral packaging plasmid 
P1125 H. sapiens ZNF524 pLIX-403 with C- lentiviral, protein expression, C-
terminal GFP terminal GFP 
P1127 H. sapiens ZNF524 ZF 2 mut pLIX-403 with C- lentiviral, protein expression, C-
terminal GFP terminal GFP 
Kappei lab H. sapiens ZNF524 pTRIPZ lentiviral, N-terminal MYC-BirA* 
for BioID 
Kappei lab H. sapiens ZNF524 ZF 2 mut pTRIPZ lentiviral, N-terminal MYC-BirA* 
for BioID 
P1294 H. sapiens ZNF524 CRISPR pInducer20 lentiviral, protein expression, C-
resistant terminal HA 
P1295 H. sapiens ZNF524 ZF2 mut pInducer20 lentiviral, protein expression, C-
CRISPR resistant terminal HA 
P1383 H. sapiens NR2C2 sgRNA 1 plentiCRISPRv2_ lentiviral, CRISPR gene editing 
neo 
P1384 H. sapiens NR2C2 sgRNA 2 plentiCRISPRv2_ lentiviral, CRISPR gene editing 
neo 
74 
 
P1385 H. sapiens NR2F2 sgRNA 1 plentiCRISPRv2_ lentiviral, CRISPR gene editing 
neo 
P1386 H. sapiens NR2F2 sgRNA 2 plentiCRISPRv2_ lentiviral, CRISPR gene editing 
neo 
P1396 sgGal4 plentiCRISPRv2_ lentiviral, CRISPR gene editing 
neo 
P861 H. sapiens TRF2 pDest-pcDNA3.1 protein expression, N-terminal 
FLAG 
P1400 pLenti Lifeact-EGFP BlastR pLenti Lifeact fluorescent marker 
BlastR 
P1401 pLenti Lifeact-iRFP670 BlastR pLenti Lifeact fluorescent marker 
BlastR 
 
Oligonucleotides 
ZNF524 cloning  Primer sequence 5' - 3' 
Znf524_for ATGGACACCCCCAGCCCAGACCCGT 
Znf524_rev_noStop GGCCGGCTCCCCTTTCCCCTCTGTC 
ZNF524_SLIC_for AAGTTCTGTTCCAGGGGCCCATGGACAC 
CCCCAGCCCA GACCCGTTGC 
ZNF524_pCoofy_rev CCCCAGAACATCAGGTTAATGGCGTTAGGCCGGCTCC
CCTTTCCCCTCTGT 
ZNF524 ZNF1 mut for CCCACACTTCGCCCCGGTGTGCCTGC 
ZNF524 ZNF1 mut rev GAAGTGTGGGGCCTTCCTGG 
ZNF524 ZNF2 mut for GAAGCCGCACCAGGCCAAGGTTTGC 
ZNF524 ZNF2 mut rev CTGGTGCGGCTTCAGCTCTGAGTGC 
ZNF524 ZNF3 mut for CGGCCCTTCCGCGCCCCGCTGTGC 
ZNF524 ZNF3 mut rev GCGGAAGGGCCGCAGGCCGGCATG 
ZNF524 ZNF4 mut for GCGCCCGTACCAGGCCCCCATCTGC 
ZNF524 ZNF4 mut rev CTGGTACGGGCGCTCCCCCGAGTGC 
ZNF524 gRNA1 resistance for CAAATCGGACACTCAAGGCCTC 
ZNF524 gRNA1 resistance rev GTGTCCGATTTGAAGAGGTGGCTC 
ZNF524 gRNA2 resistance for GAGCGACCTCCTCTTGATCGATG 
ZNF524 gRNA2 resistance rev GAGGTCGCTCCCGCCAC 
ZNF524 gRNA3.1 resistance for GTGTGCCCTACACGGTCTCTG 
ZNF524 gRNA3.1 resistance rev TAGGGCACACCCTGATCATCG 
ZNF524 gRNA3.2 resistance for CTACACGGTCAGTGAAGGTTCAGC 
ZNF524 gRNA3.2 resistance rev GACCGTGTAGGGCACACC 
  
Knockout generation Primer sequence 5' - 3' 
ZNF524 ko 1 for new CACCGGGCCTTGAGTGTCCGATTTG 
ZNF524 ko 1 rev new AAACCAAATCGGACACTCAAGGCCC 
ZNF524 ko 2 for new CACCGGCACACCCTGATCATCGATC 
ZNF524 ko 2 rev new AAACGATCGATGATCAGGGTGTGCC 
75 
 
ZNF524 ko 3 for new CACCGGAACCTTCAGAGACCGTATA 
ZNF524 ko 3 rev new AAACTATACGGTCTCTGAAGGTTCC 
ZNF524 sequencing CRISPR CATGGATGTTGCAGTGCCG 
regions 
NR2C2 ko 1 for CACCGTCAGCCGGCAAAACTGACAG 
NR2C2 ko 1 rev AAACCTGTCAGTTTTGCCGGCTGAC 
NR2C2 ko 2 for CACCGAACTGACAGCCCCATAGTGA 
NR2C2 ko 2 rev AAACTCACTATGGGGCTGTCAGTTC 
NR2F2 ko 1 for CACCGGGCGCTGAAGAGCATCCTCG 
NR2F2 ko 1 rev AAACCGAGGATGCTCTTCAGCGCCC 
NR2F2 ko 2 for CACCGGGGCTCCGCGCGCAACAGCA 
NR2F2 ko 2 rev AAACTGCTGTTGCGCGCGGAGCCCC 
ZNF524 T7 endo for GGAGAGAGGGATGGGCGAGGTG 
ZNF524 T7 endo rev TGGCCCAGGGAGCGATGG 
T7E1 hNR2F2 for CGGTGCAGAGGGGCAGGATGC 
T7E1 hNR2F2 rev GAGGTGAACAGGACTATGGCCTTGAGGC 
ZNF524-P5 CTACACGACGCTCTTCCGATCTNNNNGCCTTATCTCCT
CCTGTTCCC 
ZNF524-P7 GACGTGTGCTCTTCCGATCTNNNNAGAAGTGTGGGGC
CTTCCTG 
Gal4 sgRNA control for CACCGAACGACTAGTTAGGCGTGTA 
Gal4 sgRNA control rev AAACTACACGCCTAACTAGTCGTTC 
  
DNA pull-down Oligonucleotide sequence 5' - 3' 
TTCGGG_for TTCGGGTTCGGGTTCGGGTTCGGGTTCGGGTTCGGGT
TCGGGTTCGGGTTCGGGTTCGGG 
TTCGGG_rev  AACCCGAACCCGAACCCGAACCCGAACCCGAACCCGA
ACCCGAACCCGAACCCGAACCCG 
TCAGGG_for GTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGG
GTCAGGGTCAGGGTCAGGGTCAGG 
TCAGGG_rev ACCCTGACCCTGACCCTGACCCTGACCCTGACCCTGA
CCCTGACCCTGACCCTGACCCTG 
TGAGGG_for GTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGTGAGG
GTGAGGGTGAGGGTGAGGGTGAGG 
TGAGGG_rev ACCCTCACCCTCACCCTCACCCTCACCCTCACCCTCAC
CCTCACCCTCACCCTCACCCTC 
TTGGGG_for TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGT
TGGGGTTGGGGTTGGGGTTGGGG 
TTGGGG_rev AACCCCAACCCCAACCCCAACCCCAACCCCAACCCCA
ACCCCAACCCCAACCCCAACCCC 
TTAGGG_for TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTT
AGGGTTAGGGTTAGGGTTAGGG 
TTAGGG_rev AACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA
CCCTAACCCTAACCCTAACCCT 
GTGAGT_for GTGAGTGTGAGTGTGAGTGTGAGTGTGAGTGTGAGTG
TGAGTGTGAGTGTGAGTGTGAGT 
GTGAGT_rev ACACTCACACTCACACTCACACTCACACTCACACTCAC
  ACTCACACTCACACTCACACTC 
76 
 
DIG labelled oligo for slot blot Oligonucleotide sequence 5' - 3' 
Alu_Dig TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCG
 A  
primers for restriction sites Oligonucleotide sequence 5' - 3' 
ZNF524 with 5' XhoI restriction CCGCTCGAGATGGACACCCCCAGCCCAGACCCGTTG 
site (forward) 
ZNF524 with 3' MluI restriction CGACGCGTTCAGGCCGGCTCCCCTTTCCCCTCTG 
 site (reverse)  
linearization primers Oligonucleotide sequence 5' - 3' 
Linearization of pCoofy vectors GGGCCCCTGGAACAGAACTTCCAG 
for  
Linearization of pCoofy vectors CGCCATTAACCTGATGTTCTGGGG 
rev 
  
TRAP assay primers Oligonucleotide sequence 5' - 3' 
TS primer  AATCCGTCGAGCAGAGTT 
ACX primer GCGCGGCTTACCCTTACCCTTACCCTAACC 
 
Media 
Medium Composition 
LB Luria 1% (w/v) Tryptone 
 
1% (w/v) NaCl 
 
0.5% (w/v) Yeast extract 
 
pH 7.0 w NaOH 
LB Agar plates 1% (w/v) Tryptone 
 
1% (w/v) NaCl 
 
0.5% (w/v) Yeast extract 
 
pH 7.0 with NaOH 
 
1.5% (w/v) Agar 
 
supplements dependent on plasmids 
 
100 μg/mL Ampicillin 
 
50 μg/mL Kanamycin 
 
50 μg/mL Spectinomycin 
YG medium 2% (w/v) Yeast extract 
 
0.5% (w/v) NaCl 
 
3.5% (v/v) Glycerol 
Autoinduction medium 2% (w/v) Peptone 
 
3% (w/v) Yeast extract 
 
25 mM Potassium phoshate buffer (from 1 M stock) 
77 
 
 
0.05% (w/v) Glucose 
 
2.2% (w/v) Lactose 
 
0.5% (v/v) Glycerin 
 
50 mM NH4Cl 
 
5 mM Na2SO4 
 
2 mM MgSO4 
 
1x TMS 
 
Solutions and buffers 
Buffer/Solution Composition 
RIPA 1% (v/v) Igepal 
 
0.1% (v/v) Sodium Deoxycholate 
 
150 mM NaCl 
 
50 mM Tris HCl pH 7.5 
 
1x cOmplete protease inhibitors (Roche) 
10x PBS 100 mM Na2HPO4 
 
20 mM KH2PO4 
 
1.37 M NaCl 
 
27 mM KCl 
PBS-T  1x PBS 
 
0.1% Tween-20 
10x TBS 0.5 M Tris HCl pH 7.6 
 
1.5 M NaCl 
TBS-T 1x TBS 
 
0.1% (v/v) Tween-20 
 
0.5% (v/v) Triton X-100 
EDTA pH8 500 mM disodium EDTA x2 H2O 
 
pH adjusted with NaOH 
Annealing buffer 200 mM Tris HCl pH 8.0 
 
100 mM MgCl2 
 
1 M KCl 
PBB Buffer 50 mM Tris HCl pH 7.5 
 
150 mM NaCl 
 
0.5% (v/v) Igepal CA-630 
 
5 mM MgCl2 
 
before use add: 
 
1 mM DTT 
78 
 
1000 x Trace Metal solution (TMS) 50 mM FeCl3 
 
20 mM CaCl2 
 
10 mM Mn(II)Cl2 
 
10 mM ZnCl2 
 
2 mM CoCl2 
 
2 mM Cu(II)Cl2 
 
2 mM NiCl2 
 
2 mM NaMoO4 
 
2 mM Na2SeO3 
Tris Buffer for E. coli harvest 50 mM Tris/HCl pH 7.5 
 
100 mM NaCl 
 
10 mM MgCl2 
 
1x cOmplete protease inhibitors (Roche) 
Western blot Transfer buffer 25 mM Tris 
 
192 mM Glycine 
 
20% (v/v) Methanol 
Permeabilization buffer (FISH)  20 mM Tris HCl pH 7.5 
 
50 mM NaCl 
 
3 mM MgCl2 
 
300 mM Sucrose 
 
0.5% Triton X-100 
Hybridization solution (FISH) 3x SSC 
 
50% (v/v) Formamide 
 
10% (v/v) Dextran sulfate 
 
50 μg/ml Heparin 
 
100 μg/ml Yeast tRNA 
 
100 μg/ml sheared Salmon sperm DNA 
Wash buffer 1 (FISH) 2x SSC 
 
50% (v/v) Formamide 
Wash buffer 2 (FISH) 50 mM Tris HCl pH 7.5 
 
150 mM NaCl 
 
0.05%  (v/v) Tween-20 
Wash buffer A (FISH) 10 mM Tris HCl pH 7.2 
 
70% (v/v) Formamide 
Hypotonic shock buffer 10 mM sodium citrate 
 
25 mM KCl 
Wash buffer (CO-FISH) 70% (v/v) Formamide 
79 
 
 
10 mM Tris HCl pH 7.4 
Lysis buffer 1 (ChIP) 140 mM NaCl 
 
50 mM Tris HCl pH 8 
 
250 mM Sucrose 
 
1 mM EDTA 
 
10% (v/v) Gycerol 
 
0.5% (v/v) Igepal CA-630 
 
0.25% (v/v) TritonX-100 
 
0.25% (v/v) Tween 20 
 
1x cOmplete protease inhibitors (Roche) 
Lysis buffer 2 (ChIP) 200 mM NaCl 
 
10 mM Tris HCl pH 8 
 
1 mM EDTA 
 
0.5 mM EGTA 
 
1x cOmplete protease inhibitors (Roche) 
Sonication buffer (ChIP) 50 mM Tris HCl pH 8 
 
10 mM EDTA 
 
1% (w/v) SDS 
 
1x cOmplete protease inhibitors (Roche) 
modified PBB buffer (ChIP) 180 mM NaCl 
 
50 mM Tris HCl pH 8 
 
0.25% (v/v) IGEPAL CA-630 
 
1 mM DTT 
 
5 mM MgCl2 
 
1x cOmplete protease inhibitors (Roche) 
Elution buffer (ChIP) 0.1 M NaHCO3 
 
1% (w/v) SDS 
Lysis buffer (TRAP) 50 mM Tris HCl pH 8.0 
 150 mM NaCl 
 1% NP40 
 1x cOmplete protease inhibitors (Roche) 
TE buffer 10 mM Tris HCl pH8 
 
1 mM EDTA pH8 
Buffer A (nuclear extracts) 10 mM Hepes KOH ph 7.9 
 
1.5 mM MgCl2 
 
10 mM KCl 
Buffer A+ (nuclear extracts) Buffer A 
80 
 
 
0.2% (v/v) Igepal CA-630 
 
1x cOmplete protease inhibitors (Roche) 
Buffer C+ (nuclear extracts) 420 mM NaCl 
 
20 mM Hepes KOH pH 7.9 
 
2 mM MgCl2 
 
0.2 mM EDTA pH 8 
 
20% (v/v) Glycerol 
 
0.2% (v/v) Igepal CA-630 
 
0.5 mM DTT 
 
1x cOmplete protease inhibitors (Roche) 
MS Destaining buffer 50% (v/v) 50 mM ABC 
 
50% (v/v) Ethanol 99.9% p.a 
MS Reduction buffer 50 mM ABC 
 
10 mM DTT 
MS Alkylation buffer 50 mM ABC 
 
50 mM IAA 
MS Digestion buffer 50 mM ABC 
MS Trypsin solution 50 mM ABC 
 
1 μg Trypsin (per sample) 
MS Extraction buffer 30% Acetonitrile 
MS Buffer A 0.1% (v/v) Formic Acid in HPLC grade H2O 
MS Buffer B 80% (v/v) Acetonitrile 
 
0.1% (v/v) Formic Acid 
Lysis buffer (protein expression) 50 mM Tris HCl pH 7.5 
 
150 mM NaCl 
 
5% (v/v) Glycerol 
 
2 mM 2-mercaptoethanol 
 
20 mM Imidazole 
 
40 μL smDNAse 
 
2 mM MgCl2 
 
0.1 mM ZnCl2 
 
1x cOmplete protease inhibitors (Roche) 
Elution buffer (protein expression) 50 mM Tris HCl pH 7.5 
 
150 mM NaCl 
 
5% (v/v) Glycerol 
 
2 mM 2-mercaptoethanol 
 
300 mM Imidazole 
81 
 
Buffer E (protein expression) 50 mM Tris HCl pH 7.5 
 
2 mM DTT 
 
5% (v/v) Glycerol 
Transfer buffer (Southern Blot) 0.6 M NaCl 
 
0.4 M NaOH 
Denaturing solution (for Southern Blot) 500 mM NaOH 
 
1.5 M NaCl 
Neutralizing solution (for Southern Blot) 0.5 M Tris HCl pH 7.5 
 
3 M NaCl 
Wash buffer 1 (Southern Blot) 2x SSC 
 
0.1% (w/v) SDS 
Wash buffer 2 (Southern Blot) 0.2x SSC 
 
0.1% (w/v) SDS 
GFP IP wash buffer 10 mM Tris HCl pH 7.5 
 
150 mM NaCl 
 
1x cOmplete protease inhibitors (Roche) 
Antibodies 
Target Host Catalogue nr. Company Dilution Linked to 
 
GFP Mouse 11814460001 Roche 1000  
 
FLAG Rabbit F7425 Sigma 800 
 
TRF2 Mouse NB100-56506  Novus 1000 and 250 
 
TRF2 Rabbit NB110-57130 Novus 1000 and 250 
 
RAP1 Mouse ab14404 Abcam 250 
 
53BP1 Rabbit NB100-304 Novus 250-500 
 
TRF1 Mouse PCRP-TERF1-1E5 DSHB 50 
 
POT1 Rabbit NB500-176 Novus 100 
 
Tubulin Mouse E7-s DSHB 200 
 
Actin Rabbit A2066 Sigma Aldrich 500 
 
GAPDH Mouse 2G7 DSHB 200 
  
ZNF524 Rabbit selfmade 300 
 
pATM Rabbit ab81292 Abcam 5000 
 
pCHK2 Rabbit 2661T Cell signaling 2000 
technology  
NR2C2 mouse sc-365895 Santa Cruz 500 
Biotechnology 
      
Rabbit Donkey NA934V GE Healthcare 3000 Horse-radish 
IgG peroxidase 
Mouse Sheep NA931V GE Healthcare 3000 Horse-radish 
IgG peroxidase 
Rabbit Goat 926-32211 LI-COR 15000 IRDye® 800CW 
IgG 
Mouse Goat 926-68070 LI-COR 15000 IRDye® 680RD 
IgG 
82 
 
Mouse Donkey A31571 Invitrogen  800 AlexaFluor647 
IgG 
Rabbit Donkey A31573 Invitrogen  800 AlexaFluor647 
IgG 
Mouse Goat A11017 Invitrogen  5000 AlexaFluor488 (F(ab)2) 
IgG 
Rabbit Goat A21246 Life technologies 5000 AlexaFluor647 (F(ab)2) 
IgG 
Mouse Goat A-11032 Invitrogen  500 AlexaFluor594 
IgG 
Rabbit Goat A-21206 Life technologies 300 AlexaFluor488 
IgG 
Mouse Donkey A21202 Life technologies 1000 AlexaFluor488 
IgG 
Rabbit Donkey A21207 Life technologies 1000 AlexaFluor594 
IgG 
 
Reagents 
Reagent Supplier Cat. No 
1 Kb extended DNA marker New England BioLabs #N3239 
2-mercaptoethanol Roth #4227.3 
4x NuPAGE LDS sample buffer Thermo Fisher Scientific #NP0008 
5x Protein Assay Dye Reagent concentrate BioRad #500-0006 
Acetic acid Sigma-Aldrich #33209 
Acetone Roth #9372.6 
Acetonitrile VWR #20048.320 
Adenosin-triphosphate (ATP) Sigma-Aldrich #A2383 
Agar Sigma-Aldrich #A5306 
Agarose Sigma-Aldrich #A9539 
alamarBlue™ Cell Viability Reagent Thermo Fisher Scientific #DAL1100 
Amicon Ultra 10kDa centrifugal filter unit Merck #UFC5010 
Ammonia solution Sigma-Aldrich #30501 
Ammonium chloride (NH4Cl) Roth #K298.1 
Ammoniumbicarbonate (ABC, NH4HCO3) Sigma-Aldrich #A6141-500G 
 
Ampicillin IMB Media lab 
BIBR1532 Absource Diagnostic  #S1186 
Biodyne B membrane (telomere southern blot) Pall #60207 
Biotin-7-dATP Jena Bioscience #NU-835-BIO 
Biotinylated 5x telomeric/control repeat Metabion - 
oligonucleotides 
BrdC Thermo Fisher Scientific  #J65456.03 
BrdU Sigma-Aldrich #B5002 
BSA Sigma-Aldrich #A3294 
83 
 
C18 MS column New Objective #FS360-75-8-N-5-
C30 
Calcium chloride (CaCl2) Roth #5239.1 
Chloroform Roth #3313.4 
Cobalt(II) chloride (CoCl2) Sigma-Aldrich #232696 
cOmplete Mini EDTA-free protease inhibitor Roche/Sigma-Aldrich #4693159001 
tablets 
Cover slips Langenbrinck #01-2222/5/get. 
Cy3-labeled G-rich telomere probe Eurogentec #PN-TG050-005 
Cytiva HiTrap™ Heparin HP-Säulen Thermo Fisher Scientific #10288944 
Dextran Sulfate Sigma-Aldrich #S4030 
di-Sodium hydrogen phosphate (Na2HPO4) Roth #4984.1 
Dithiothreitol (DTT) Sigma-Aldrich #D0632 
Dulbecco’s modified eagle medium (DMEM)  Gibco #21969035 
DMSO Sigma-Aldrich #D2650 
DnaseI New England BioLabs #M0303 
dNTPs (4x 100 mM) Jena Bioscience #NU-1005S 
Dulbecco's phosphate buffered saline (DPBS) Gibco #14190094 
DpnI New England BioLabs #R0176 
Dynabeads MyOne Streptavidin C1 Thermo Fisher Scientific #65001 
Dynabeads ProteinG Thermo Fisher Scientific #10004D 
 
EDTA IMB Media lab 
EGTA Sigma-Aldrich #E3889 
Empore C18 3M #15334911 
Ethanol 99.9% p.a Roth #9065.3 
Exonuclease III Promega #M1815 
fetal bovine serum (FBS) Gibco #10270106 
Fish Skin Gelatin Sigma-Aldrich #G7041 
FITC-labelled C-rich telomere probe Eurogentec #PN-TC011-005 
Formaldehyde solution Sigma-Aldrich #F8775 
Formamide Roth #6749.1 
Formic acid Merck #1.00264.1000 
G418 (suitable for cell culture)  Sigma-Aldrich #A1720 
Gel filtration size standard BioRad #1511901 
GeneRuler 1 Kb Thermo Fisher Scientific #SM0312 
GFPtrap MA, magnetic agarose GFP beads Chromotek #gtma-20 
Glucose Sigma-Aldrich #G7021 
Glycerol Honeywell #15523-1L-R-D 
Glycine Roth #3790.2 
84 
 
GoTaq® qPCR Master Mix Promega #A6001 
Heparin Sigma-Aldrich #H3393 
Hepes Roth #HN78.2 
HinfI New England BioLabs #R0155 
HisTrap™ High Performance GE Healthcare #GE17-5248-01 
Hoechst 33342 Solution Thermo Fisher Scientific #62249 
Hydrochloric acid (HCl) Roth #4625.1 
Igepal CA-630 Sigma-Aldrich #I8896 
illustra MicroSpin G-50 Columns GE Healthcare #27-5330-02 
Imidazole Sigma-Aldrich #56750 
Iodoacetamide (IAA) Sigma-Aldrich #I6125 
IPTG Roth #CN08.3 
Iron(III) chloride (FeCl3) Sigma-Aldrich #157740 
Ispopropanol Roth #9866.6 
 
Kanamycin IMB Media lab 
Klenow fragment -exo Thermo Fisher Scientific #EP0422 
Lactose Sigma-Aldrich #61341 
Leupeptin Serva #51867.03 
Lithium acetate (LiAc) Sigma-Aldrich #L4158 
LR Clonase II enzyme mix Thermo Fisher Scientific #11791020 
Magnesium chloride (MgCl2) Sigma-Aldrich #M2670 
Magnesium sulfate (MgSO4) Sigma-Aldrich #M7506 
Manganese(II) chloride tetrahydrate (Mn(II)Cl2) Roth #0276.2 
Methanol (MS grade) VWR #20864320 
MluI Thermo Fisher Scientific #ER0561 
Microspin sephadex G-50 columns GE Healthcare #GE27-5330-01 
Nickel(II) chloride (NiCl2) Sigma-Aldrich #339350 
Nitrocellulose Western Blot membrane Fisher Scientific GmbH #15259794 
Nocodazole Sigma-Aldrich #M1404 
NuPAGE 10% Bis-Tris gel, 10 well Thermo Fisher Scientific #NP0301 
NuPAGE 20x MES Running Buffer Thermo Fisher Scientific #11509166 
NuPAGE 20x MOPS Running Buffer Thermo Fisher Scientific #NP0001 
NuPAGE 4-12% Bis-Tris gel Thermo Fisher Scientific #NP0321 
OneTaq DNA polymerase New England BioLabs #M0480 
Opti-MEM Life Technologies #11058-021 
Paraformaldehyde, 16% w/v, methanol free Thermo Fisher Scientific #AA433689M 
PEG6000 part of Kit Thermo Fisher - 
Scientific 
85 
 
Penicillin-Streptomycin (suitable for cell culture) Sigma-Aldrich Chemie GmbH #P0781 
Pepstatin A Serva #52682.02 
Peptone Sigma-Aldrich #70173 
Pfu Ultra II polymerase Agilent #600672 
Phenol:Chloroform:isoamyl alcohol (25:24:1) Invitrogen/Thermo Fisher #15593049 
Scientific 
Phenylmethylsulfonyl fluoride (PMSF) Serva #32395.03 
Polybrene Santa Cruz Biotechnology #sc-134220 
Ponceau S solution Applichem #A2935 
Potassium chloride (KCl) Roth #6781.1 
Potassium di-hydrogen phosphate, monobasic Roth #P018.2 
(KH2PO4) 
Potassium hydroxide (KOH) Roth #7986.1 
ProLong® Gold Antifade Reagent with DAPI Thermo Fisher Scientific #P36941 
Propidium iodide Sigma-Aldrich #P4170 
Proteinase K Sigma-Aldrich #P2308 
Protran Nitrocellulose membrane (Western Blot) Amersham/VWR #10600002 
Puromycin (hydrochloride) Cayman #13884 
Recombinase A New England BioLabs #M0249 
RNase A Sigma-Aldrich #R5503 
RsaI New England BioLabs #R0167 
Salmon sperm Ambion/Thermo Fisher #AM9680 
Scientific 
SDS Roth #4360.1 
Skim Milk powder Sigma-Aldrich #70166 
Sm nuclease IMB Protein Production CF - 
Sodium azide Sigma-Aldrich #S2002 
Sodium chloride (NaCl) Thermo Fisher Scientific #15626770 
Sodium citrate Sigma-Aldrich #25114 
Sodium cyanoborohydride (NaBH3CN) Sigma-Aldrich #156159 
Sodium hydroxide (NaOH) Roth #6771.1 
Sodium hypochloride solution Roth #9062.3 
Sodium molybdate (NaMoO4) Sigma-Aldrich #243655 
Sodium selenite (Na2SeO3) Sigma-Aldrich #214485 
Sodium sulfate (Na2SO4) Sigma-Aldrich #S9627 
Sorbitol Sigma-Aldrich #85529 
Spectinomycin Sigma-Aldrich #S4014 
SSC 20x IMB Media lab 
Sucrose Sigma-Aldrich #S7903 
86 
SuperSignal West Pico plus Chemiluminescent Thermo Fisher Scientific #15626144 
Substrate 
Sybr Safe DNA stain Thermo Fisher Scientific #S33102 
T4 DNA Ligase Thermo Fisher Scientific #EL0011 
T4 Polynucleotid Kinase New England BioLabs #M0201 
T7 Endonuclease I New England BioLabs #M0302S 
TAMRA-labeled C-rich telomere probe Eurogentec #507207 
Taq polymerase homemade IMB Protein Production CF - 
TRAPeze 1x Chaps lysis buffer Sigma-Aldrich #S7705 
Trichloroacteic acid (TCA) Sigma-Aldrich #T6399 
Triethylammonium bicarbonate buffer (TEAB) Sigma-Aldrich #18597 
TritonX-100 Sigma-Aldrich #X100 
Trizma Base Sigma-Aldrich #T1503 
Trypan Blue solution Sigma-Aldrich #T8154 
Trypsin (proteomics grade) Sigma-Aldrich #T6567 
Trypsin (0.25%, sterile-filtered, suitable for cell Sigma-Aldrich #T4049-100ML 
culture) 
Tween-20 Sigma-Aldrich #P7949 
Vectashield mounting medium with DAPI Vector Laboratories #H-1200-10 
Whatman paper GE Healthcare #WHA10426892 
XhoI Thermo Fisher Scientific #ER0691 
Yeast Extract Sigma-Aldrich #70161 
yeast tRNA Thermo Fisher Scientific #11518736 
Zinc chloride (ZnCl2) Sigma-Aldrich #96468 
Zirkonia beads (0.1 mm) Roth #N033.1 
Instruments 
Instrument Supplier 
AF7000 widefield Leica 
Aktaprime Plus System GE Healthcare 
LSRFortessa SORP Becton Dickinson 
BioRuptor Plus Diagenode 
Branson sonifier 450 Branson Ultrasonics Corp. 
CHEF-DR III BioRad 
Chemidoc XRS+ BioRad 
EASY-nLC 1000 system Thermo Scientific 
Electrospray Ion source (Nanospray flex) Thermo Scientific 
Precellys 24 tissue homogenizer Bertin Instruments 
QExactive Plus mass spectrometer Thermo Scientific 
SP5 confocal microscope Leica 
87 
SPE STED confocal microscope Leica 
Tecan Reader Infinite 200 PRO Tecan 
Thermocycler BioRad/Biometra 
Ultracentrifuge Beckmann Coulter 
ViiA7 real-time PCR system Thermo Scientific 
Softwares 
Name Distributor 
Adobe Illustrator 2020 Adobe 
Image Lab 5.2 BioRad 
Mendeley Desktop Elsevier 
FlowJo V10.5.3. FlowJo 
Fiji Image J 
LAS X Leica 
Image Studio 3.1 LI-COR 
MaxQuant (V. 1.5.2.8) MaxQuant 
Excel 2016 Microsoft 
Powerpoint 2016 Microsoft 
Word 2016 Microsoft 
R-studio R Studio Inc 
R The R foundation 
SnapGene Viewer V5 SnapGene (R) 
Commercial assays 
Assay Supplier Cat. No 
Amaxa Cell Line Nucleofector Kit V  Lonza #VCA-1003 
First strand cDNA Synthesis Kit Thermo Fisher Scientific #K1612 
Gateway LR Clonase II Enzyme Mix Fisher Scientific  #10134992 
GenElute™ HP Plasmid Miniprep Kit Sigma-Aldrich Chemie GmbH #NA0160-1KT 
MinElute PCR Purification Kit Qiagen #28004 
Monarch DNA Gel Extraction Kit New England BioLabs #T1020 
pCR8 GW/TOPO Fisher Scientific #10532893 
Penta-His HRP Conjugate Kit Qiagen #34460 
QIAamp DNA Blood Mini Kit Qiagen #51104 
QIAamp DNA Blood Maxi Kit Qiagen #51194 
Qiagen Plasmid Midi Kit Qiagen #12143 
QIAprep Spin Miniprep Kit Qiagen #27106 
Qubit™ dsDNA HS- und BR-Assay-Kits Thermo Fisher Scientific #Q32854 
RNeasy MinElute Cleanup kit Qiagen #74104 
88 
 
SulfoLink™ Immobilization Kit for Peptides Thermo Scientific #44999 
TeloTAGGG Telomere Length Assay Roche #12209136001 
 
Methods 
Cell culture 
HeLa Kyoto (epitheloid carcinoma, telomerase positive), HeLa 1.3 (telomerase positive), HT1080ST 
(telomerase positive), U2OS (osteosarcoma, ALT positive), GM847 (ALT), Saos2 (ALT), and WI-38 VA-
13 (ALT) cells were cultivated in 4.5 g/L Dulbecco’s modified eagle medium (DMEM) supplemented 
with 10% fetal bovine serum (Gibco), 2 mM glutamine (Thermo Scientific), 100 U/mL penicillin and 
100 μg/mL streptomycin (Gibco). They were kept at 37 °C and 5% CO2 in a humidified incubator. For 
propagation of the cells, they were grown until ~90% confluence, washed once with 1x DPBS (Gibco) 
and detached by 5 min treatment with 0.25% trypsin (Sigma Aldrich) at 37 °C. Depending on the cell 
line and the experimental requirements the cells were seeded at 10-20% confluence. To monitor the 
proliferation of WT and ZNF524 KO clones, cells were cultured to a maximum confluency of 80%, 
counted every three days and the cumulative population doublings calculated. To examine the effect 
of telomerase inhibition, the medium was supplemented with BIBR1532 (Absource Diagnostics).  
Cloning and plasmids 
ZNF524 was obtained from the Orfeome collection (Q96C55; ENSG00000171443). Zinc finger point 
mutations were introduced by site-directed mutagenesis using specific primers and the sequences 
confirmed by Sanger sequencing (GATC). The constructs were LR recombined into pDest-pcDNA3.1 
with N-terminal FLAG tag or into pLIX_403 (Plasmid #41395, Addgene) with C-terminal GFP tag. pTRIPZ 
(Chojnowski et al., 2015)(gift by Oliver Dreesen, Cell Aging Laboratory, A*STAR Skin Research Labs) 
modified for 3rd generation lentivirus production was digested with XhoI (Thermo Fisher Scientific) and 
MluI (Thermo Fisher Scientific). The insert was PCR amplified to introduce the respective overhangs 
and ligated into the vector backbone using T4 DNA ligase (Thermo Fisher Scientific) according to 
manufacturer’s instructions. Cloning of pX459 V2 for CRISPR/Cas9 genome editing was done based on 
previous descriptions (Ran et al., 2013). pLIX_403 was a gift from David Root (Addgene plasmid #41395; 
http://n2t.net/addgene:41395; RRID:Addgene_41395). pInducer20 was a gift from Stephen Elledge 
(Addgene plasmid # 44012 ; http://n2t.net/addgene:44012 ; RRID:Addgene_44012). pSpCas9(BB)-2A-
Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988 ; 
http://n2t.net/addgene:62988 ; RRID:Addgene_62988)(Ran et al., 2013). 
TOPO cloning 
For subsequent LR recombination and holding purposes, constructs were introduced into pCR8 
GW/TOPO vector (Fisher Scientific) according to manufacturer’s instructions. In short, the insert was 
amplified from cDNA by PCR using Pfu Ultra II polymerase (Agilent) and subsequently incubated with 
OneTaq polymerase (New England BioLabs) to add A-overhangs. For the reaction, 0.6 μL PCR product 
were mixed with 0.3 μL salt solution and 0.1 μL pCR8 GW/TOPO vector, incubated at RT for 15 min and 
directly used for transformation into DH5α competent cells (New England BioLabs). The correct ORF 
sequence and orientation were confirmed by Sanger sequencing (GATC).  
LR recombination 
Following TOPO cloning, the constructs were transferred to destination vectors for protein expression. 
LR recombination was done using the Gateway LR Clonase II Enzyme Mix (Fisher Scientific) Kit 
according to manufacturer’s instructions. In short, 150 ng pCR8 GW/TOPO vector with respective 
89 
 
insert were mixed with 150 ng destination vector, 1 μL LR clonase II enzyme mix and TE buffer at a final 
volume of 5 μL and incubated at 25 °C for 1 h. Subsequently, 1 μL of ProteinaseK solution was added 
to the reaction and incubated at 37 °C for 10 min. For transformation, 1 μL of the reaction was added 
to 15 μL DH5α competent cells. 
SLIC cloning 
For bacterial expression of proteins, the construct was cloned into pCoofy1 or pCoofy4 using SLIC 
cloning as previously described (Scholz et al., 2013). First, the CDS was amplified from cDNA using 
sequence specific primers with SLIC overhangs and Pfu Ultra II polymerase (Agilent) according to 
manufacturer’s instructions. The PCR product was purified by ethanol precipitation. The vectors 
(pCoofy and pCoofy 4) were linearized by PCR reaction with backbone specific primers. Subsequently, 
120 ng linearized vector were incubated with 1200 ng PCR product and 1 μL RecombinaseA enzyme 
solution (1:1000 dilution in H2O, New England BioLabs) in RecA buffer and a total volume of 10 μL. 
After 30 min at 37 °C, 2.5 μL of the reaction were used for transformation of 25 μL DH5α competent 
cells. 
RNA preparation 
Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) according to manufacturer’s 
instructions including on-column DNA digestion. 
cDNA transcription 
cDNA was reverse transcribed from total RNA using the First Strand cDNA Synthesis Kit (Thermo Fisher 
Scientific) according the manufacturer’s instructions. The oligo (dT)18 primers were used in the 
reaction. 
Transformation of chemically competent E. coli and preparation of plasmids 
For transformation, chemically competent bacteria were thawed on ice. After adding the cloning 
product to the cells, the mixture was incubated on ice for another 30 min. The bacteria were then heat 
shocked at 42 °C for 45 sec, put on ice for 2 min and carefully suspended in 25 μL room temperature 
SOC Medium (New England BioLabs). After 1 h incubation at 37 °C, the bacteria were plated on LB Agar 
plates supplemented with the respective antibiotic and grown at 37 °C overnight. For propagation of 
ccdB containing LR recombination destination vectors, One Shot ccdB Survival 2 T1R Competent Cells 
(Thermo Fisher Scientific) were used. For bacterial expression of ZNF524 constructs, BL21(DE3)-T1R 
Competent Cells (Sigma-Aldrich) were used. DH5α E. coli (New England BioLabs) were used for cloning 
and holding of vectors while NEB® Stable Competent E. coli (High Efficiency) (New England BioLabs) 
were used for cloning and holding of lentiviral vectors. 
For plasmid preparation and sequencing, single colonies were picked and grown in 5 mL Luria broth 
Medium supplemented with the respective antibiotic at 37 °C overnight. Low copy plasmids were 
isolated using QiaPrep Spin miniprep Kit (Qiagen) and high copy plasmids were isolated using 
GenElute™ Plasmid Miniprep-Kit (Sigma Aldrich) according to manufacturer’s instruction. Lentiviral 
plasmids for transfection of HEK293T cells were purified using Qiagen Plasmid Midi Kit/ Qiagen Plasmid 
Maxi Kit (Qiagen) according to manufacturer’s instructions. Concentrations were measured on 
NanoDrop 2000 (Thermo Fisher Scientific) and sequences were confirmed by Sanger sequencing 
(GATC). 
Transfection 
Plasmids were transfected in HeLa Kyoto and HEK293 cells using linear polyethylenimine (PEI, MW 
25000; Polysciences). One day prior to transfection, cells were seeded in a 10 cm cell culture dish with 
90 
 
450000 cells/mL. For transfection, 48 μL PEI and 12 μg plasmid were diluted in DMEM and added to 
the cells. The medium was exchanged after 6-8 h. Plasmid transfection in U2OS cells was done using 
Amaxa Cell Line Nucleofector Kit V (Lonza) according to manufacturer’s instructions. 
Lentiviral transduction 
HEK293T cells were seeded to a confluence of 70% on the next day in a 10 cm cell culture dish. In 
preparation for the transfection, DMEM with 10% FBS but without any antibiotics was used. For 
transfection, 540 μL Opti-MEM (Gibco) were mixed with the packaging plasmids pMDLg/pRRE, pRSV-
Rev and pMD2.G (5 μg of each plasmid) and 10 μg of the expression vector (pLIX403_GFP for ZNF524-
GFP constructs, pInducer20 for ZNF524-HA constructs and pTRIPZ for BirA*-ZNF524 constructs). A 
separate mix of 540 μL Opti-MEM with 60 μg PEI was prepared and incubated for 5 min at RT. Both 
mixtures were combined and incubated for another 20 min at RT. Finally, the mixture was carefully 
added to the attached HEK293T cells. After 24 h, the medium was exchanged for DMEM with 10% FBS 
and Penicillin-Streptomycin. After another 48 h, the supernatant containing the virus was collected, 
filtered at a 0.45 μm cut-off and supplemented with 8 μg/mL polybrene (Santa Cruz Biotechnology) 
and 10 mM HEPES buffer pH 7.5. In parallel, receiving cells were seeded to 50% confluence at time 
point of treatment. The medium of the receiving cells was replaced with virus-containing supernatant, 
incubated for 24 h and then exchanged for fresh medium. 48 h after transduction, cells were treated 
with 2 μg/mL puromycin (Cayman, three days or until selection was completed) or with 400 μg/mL 
G418 (Sigma Aldrich, seven days or until the selection was completed).To test for protein expression, 
the cells were treated with increasing amounts of doxycycline for 48 h and protein levels determined 
by Western Blot or Immunofluorescence.  
Bacterial expression of recombinant proteins for DNA pull downs 
Autoinduction: BL21(DE3)-T1R E. coli carrying expression constructs in pCoofy vectors were grown in 
5 mL YG Medium with Kanamycin at 37 °C overnight. The overnight culture was diluted 1:50 in 2 mL 
YG Medium without antibiotics on the next day and grown to an optical density (OD600) of 0.7. 
Subsequently, 500 μL of the preculture were used to inoculate 100 mL Autoinduction Medium, which 
was incubated at 25 °C overnight. Cells were harvested by centrifugation. 
Lysis: The cell pellet was resuspended in 2 mL Tris buffer and divide into two flat lid micro tubes 
containing 500 μl of 0.1 mm zirconia beads (Roth). Bacteria were lysed at 5,600 rpm for 30 sec using 
Precellys 24 tissue homogenizer (Bertin Instruments). The lysate was centrifuged at 15,000 g for 2 min 
and incubated on ice for 5 min before repeating the lysis cycle. Finally, cell debris and beads were 
separated from lysate by centrifugation at 20,000 g for 5 min. The protein concentration of the 
supernatant was measured by Bradford assay.  
Lysis of human cell lines 
RIPA buffer: After detaching and washing the cells with DPBS, Radioimmunoprecipitation assay (RIPA) 
buffer supplemented with Protease inhibitor was added to the cell pellet and incubated on ice for 
30 min with occasional vigorous mixing. Cell debris was separated from the lysate by centrifugation 
(10 min at 10,000 g). Protein concentration of the supernatant was measured by Bradford assay. 
Lysates were subsequently used for Co-IP, Western Blot or DNA pull downs. 
LDS buffer: After detaching and washing the cells with DPBS, 4x NuPAGE LDS sample buffer (Thermo 
Fisher Scientific) diluted to 1x with H2O and 100 mM DTT (Sigma Aldrich) were added to the cell pellet 
and the mixture was boiled at for 10 min 95 °C. To shear the chromatin, the sample was sonicated in 
the water bath for 15 min. The lysate was subsequently used for quantitative Western Blots. 
91 
 
Bradford assay 
Protein concentrations of extracts were determined using the Bradford assay. In preparation of the 
standard curve, 0, 0.25, 0.5, 0.75 and 1 mg/mL BSA were diluted in H2O. The extracts were diluted in 
H2O if necessary. Of each sample, 20 μL were transferred to a cuvette, mixed with 980 μL 1xBradford 
reagent (diluted from 5x Protein Assay Dye Reagent Concentrate, Biorad) and incubated for 5 min. The 
samples were measured on a spectrophotometer at 595 nm in triplicates. The BSA dilution series was 
used to determine a standard curve from which the protein concentration of the samples were 
calculated. 
Western Blots 
Protein samples were size-separated by polyacrylamide gel electrophoresis in a 4-12% Bis-/Tris gel 
(NuPAGE, Thermo Scientific) in 1x MES/MOPS Running Buffer (NuPAGE, Thermo Fisher Scientific), run 
at 180 V for 45 min (70 min for pATM). Denatured proteins were then transferred to a Nitrocellulose 
membrane (Amersham Protran, VWR) by applying 300 mA for at least 60 min in a wet transfer 
chamber with ice cold Blotting buffer. The membrane was then blocked with 5% (w/v) skim milk (5% 
BSA (w/v) in TBS-T for pATM and pCHK2) at RT for 1 h and incubated with the respective antibody: 
αGFP (Roche, 1:1000 in 5% skim milk powder), αFLAG (Sigma Aldrich, 1:800 in 1% BSA (w/v) in PBS), 
αZNF524 (1:300 in 5% BSA (w/v) in PBS-T), αTRF2 (Novusbio, 1:500 in 5% (w/v) skim milk), αRAP1 
(abcam, 1:500 in 5% (w/v) skim milk), αtubulin (tubulin beta E7, DSHB, 1:500 in 5% (w/v) skim milk 
powder), αGAPDH (2G7 DHSB, 1:200 in 5% (w/v) skim milk powder), αactin (Sigma Aldrich, in 5% (w/v) 
skim milk powder). The corresponding secondary antibody was added in a 1:3000 dilution in 5% (w/v) 
skim milk (5% BSA (w/v) in TBS-T for pATM and pCHK2) for 1 h at RT followed by PBS-T washes (TBS-T 
for pATM and pCHK2). Bands were detected on a ChemiDoc Imaging Systems (BioRad) using Super 
Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to manufacturer’s 
instructions.  
The Penta-His HRP Conjugate Kit was used for detection of His-tagged proteins according to 
manufacturer’s instructions. 
qWB 
For quantitative Western blots, the membrane was blocked with the respective blocking buffer as 
previously described for Western Blots. After washing, the corresponding fluorescently labelled 
antibody was added in a 1:15000 dilution in respective blocking buffer and incubated for 1 h at RT in 
the dark. The membrane was washed in the dark and the bands detected using the LI-COR Odyssey (LI-
COR). The signal intensities were quantified using Image Studio 3.1 (LI-COR). 
DNA bait pull downs 
Biotinylated telomeric and control DNA for the DNA pulldown for detection of telomeric interactors 
was prepared as previously published (Kappei et al., 2013, 2017; Casas-Vila et al., 2015). To prepare 
the biotinylated bait DNA, 25 μL of the 10mer telomeric repeat primer or the scrambled control 
sequence primer were mixed with 25 μL of their reversed counterparts. After addition of 10 μL 
annealing buffer the reaction was brought to a final volume of 100 μL with ultra-pure water and heated 
at 80 °C for 5 min. Subsequently, the reaction remained in the switched off Eppendorf thermomixer 
until it cooled down to RT, thereby allowing for the annealing of the primers. For phosphorylation 55 μL 
ultra-pure water, 20 μL 10x T4 DNA ligase buffer (Thermo Scientific), 10 μL PEG 6000, 10 μL 100 mM 
ATP, 2 μL 1 M DTT and 27.5 μL of T4 Polynucleotide Kinase (Thermo Scientific) were added to the 
annealed oligos and the reaction was incubated at 37 °C for 2 h. For overnight ligation at RT, the 
reaction was treated with 4 μL T4 DNA ligase (Fermentas). Successful oligomerisation was confirmed 
by agarose gel electrophoresis. For Phenol/Chloroform extraction, one volume of ultra-pure water and 
92 
 
200 μL Phenol/Chloroform/IAA (25:24:1) pH 8.0 were added to the polymers followed by mixing and 
2 min centrifugation at 16000 g. The aqueous phase was transferred to a new Eppendorf tube and 
mixed with 1 mL 100% EtOH. Precipitation at -20 °C for 30 min was followed by 45 min centrifugation 
at 16000 g and 4 °C. The pellet was taken up in 74 μL ultra-pure water and biotinylated at 37 °C 
overnight through the addition of 10 µL 10x Polymerase buffer (Reaction buffer for Klenow fragment), 
10 µL 0.4 mM Biotin-7-dATP (Jena Bioscience) and 6 µL DNA polymerase 30 Units (klenow fragment 
exo-  5 U/µL). Finally, the biotinylated bait oligos were purified using Microspin Sephadex G-50 columns 
(GE Healthcare). 
The pull down with biotinylated bait DNA was performed as follows: Per sample 20 μL biotinylated 
oligo bait were diluted in 200 μL PBB buffer and mixed with 50 μL MyOne Streptavidin C1 Dynabeads 
(Thermo Fisher Scientific) that had been equilibrated in PBB buffer. After 15 min incubation at RT and 
continuous agitation, the DNA coupled beads were separated on a magnetic rack and washed three 
times with PBB buffer. Subsequently, the beads were taken up in 150 μL PBB buffer supplemented 
with 15 μg salmon sperm DNA to reduce unspecific binding. 400 μg bacterial or cell lysate were added 
to the DNA bait coupled beads and incubated on the rotating wheel at 4 °C for 90 min followed by 
three washes with 500 μL PBB buffer. The interacting proteins were eluted from the beads by adding 
25 μL 1x NuPAGE LDS sample buffer (NuPAGE, Thermo Scientific) supplemented with 100 mM DTT and 
heating at 70 °C for 10 min. The beads were again separated using a magnetic rack and the entire 
volume was then loaded onto a precast 10-well 4-12% Bis-/Tris NuPAGE gel (NuPAGE, Thermo 
Scientific) and denatured proteins were separated for 45 min and 180 V in 1x MES buffer (NuPAGE, 
Thermo Scientific).  
Nuclear extract preparation from adherent cells 
The amount of cells required depends on the cell line and the experimental procedure. The cells were 
harvested, pelleted for 15 min at 450 g, and washed once in PBS. The following steps were performed 
on ice or at 4 °C. Five volumes of cold buffer A were added to the pellet. A 10 min incubation step on 
ice allowed for the swelling of the cells before they were pelleted for 5 min at 450 g. The cells were 
brought into suspension in two pellet volumes of cold buffer A+ and the cell membrane was disrupted 
using a dounce homogenizer. The lysis was monitored in light field microscopy with 40x magnification. 
Subsequently, the lysate was centrifuged for 15 min at 3900 rpm and the supernatant collected as 
cytoplasmic fraction. The remaining pellet was washed with ten pellet volumes of PBS and then 
brought into suspension in two volumes of cold buffer C+. The cell suspension was incubated for 1 h 
at 4 °C under constant rotation. Finally, the chromatin was separated from the soluble nuclear fraction 
by 1 h centrifugation at 14800 rpm at 4 °C. The protein concentration of each fraction was determined 
by Bradford assay before snap freezing in liquid nitrogen and storing at -80 °C. 
BioID 
U2OS cell lines carrying BirA*-ZNF524 or BirA*-ZNF524 ZF2 mut were induced with 300 ng/mL 
doxycycline 48 h prior to harvest. After 42 h, the cells were additionally treated with 50 uM Biotin for 
6 h to allow for the biotinylation of proteins proximal to the target protein. Subsequently, nuclear 
extract was prepared as described above. Biotinylated proteins were isolated from the extract using 
MyOne Streptavidin C1 Dynabeads (Thermo Scientific). Therefore, 150 μl Dynabeads were washed 
with PBB buffer and mixed with 200 μL of 3 μg/μL nuclear extract and 500 μL PBB buffer. After 
2 incubation at 4 °C on a rotating wheel, the beads were pelleted on a magnetic rack and washed thrice 
with ice cold PBB buffer. Finally, the isolated proteins were eluted in 25 μL 2x Laemmli buffer (Sigma 
Aldrich) by boiling for 5 min at 95 °C and the supernatant prepared for mass spectrometry 
measurement. 
93 
 
MS sample preparation 
In-gel digestion was performed as previously described (Shevchenko et al., 2007). Essentially, 
denatured proteins were separated on a 4-12% Bis-/Tris gel (NuPAGE, Thermo Scientific) for 10 min 
(30 min for proteome) at 180 V in 1x MOPS buffer (NuPAGE, Thermo Scientific). Proteins were stained 
with Coomassie solution and lanes cut individually with a clean scalpel into 1 mm x 1 mm pieces. The 
gel pieces incubated with destaining buffer in several rounds to remove the Coomassie. Next, the gel 
pieces were twice dehydrated in 100% Acetonitrile and its remnants removed using a Concentrator 
Plus (Eppendorf, settings V-AQ). The gel pieces were incubated with reduction buffer for 60 min at 
56 °C followed by incubation with alkylation buffer for 45 min at RT in the dark. Again, two dehydration 
steps were performed and the acetonitrile completely removed. Subsequently, gel pieces were soaked 
in trypsin solution overnight at 37 °C. On the next day, the supernatant was collected and the digested 
peptides extracted by two rounds of incubation with extraction buffer and one round of 100% 
acetonitrile for 15 min at RT each. In each round, the supernatant was recovered and combined with 
the previous one. Using the Concentrator Plus, the acetonitrile was evaporated and the volume 
reduced to 100 μL.  
Stage tip purification of the samples was performed as previously described (Rappsilber, Mann and 
Ishihama, 2007). Therefore, two layers of Empore C18 material (3M) were stacked in a 200 μL pipet tip 
and the material activated by applying 50 μL methanol followed by 500 g centrifugation until the entire 
volume passed through the tip. In the same fashion, the material was equilibrated with Buffer B and 
then washed with Buffer A. After applying the sample, the tip was again washed with Buffer A and the 
sample eluted in 30 μL Buffer B. The excess acetonitrile was evaporated in the Concentrator Plus and 
the total volume finally adjusted to 14 μL with Buffer A. 
MS measurement and data analysis 
5 µL of sample were injected.  
For BioID: The desalted and eluted peptides were loaded on an in-house packed C18 column (New 
Objective, 25 cm long, 75 µm inner diameter) for reverse-phase chromatography. The EASY-nLC 1200 
system (Thermo Scientific) was mounted to a Q Exactive HF mass spectrometer (Thermo Scientific) and 
peptides were eluted from the column in an optimized 2 h gradient from 2-40% MS grade 
acetonitrile/0.5% formic acid solution at a flow rate of 225 nL min-1. The mass spectrometer was used 
in a data-dependent acquisition mode with one MS full scan and up to 20 MS/MS scans using HCD 
fragmentation. MS scans were conducted with 60,000 resolution at a maximum injection time of 20 
ms and MS/MS scans with 15,000 resolution at a maximum injection time of 75 ms.  
For proteome: The desalted and eluted peptides were loaded on an in-house packed C18 column (New 
Objective, 50 cm long, 75 µm inner diameter) for reverse-phase chromatography. The EASY-nLC 1200 
system (Thermo Scientific) was mounted to an Orbitrap Exploris 480 mass spectrometer (Thermo 
Scientific) and peptides were eluted from the column in an optimized 90-min gradient of 2.4-32% 
acetonitrile/0.1% formic acid solution at a flow rate of 250 nl min-1. The mass spectrometer was used 
in a data-dependent acquisition mode with one MS full scan followed by up to 20 MS/MS scans using 
HCD fragmentation. MS scans were conducted with 60,000 resolution at a maximum injection time of 
28 ms and MS/MS scans with 15,000 resolution at a maximum injection time of 28 ms. 
All raw files were processed with MaxQuant (for BioID: version 1.5.2.8; for proteome: version 1.6.5.0) 
and searched against the human Uniprot database (95,934 entries). Carbamidomethylation (Cys) was 
set as fixed modification, while oxidation (Met) and protein N-acetylation were considered as variable 
modifications. For enzyme specificity, trypsin was selected with a maximum of two miscleavages. 
94 
 
Search results were filtered with a false discovery rate of 0.01 and for known contaminants, proteins 
groups only identified by site, and reverse hits of the MaxQuant results. LFQ quantification (without 
fast LFQ) using at least 2 LFQ ratio counts and the match between run option were activated in the 
MaxQuant software. 
Immunofluorescence (IF) staining  
Cells were seeded on coverslips to a maximum confluency of 70%. After overnight incubation, the cells 
were washed with DPBS (Gibco) and fixed to the coverslips by 10 min incubation with 4% formaldehyde 
(Thermo Fisher Scientific) at RT. After washing with PBS (supplemented with 30 mM glycine for 
αTRF2/αGFP double staining), the cells were permeabilized with 0.5% Triton X-100, washed again and 
then blocked with 3% BSA and 0.3% Triton X-100 (or 0.2% fish skin gelatin for αTRF2/αGFP double 
staining) in PBS (blocking buffer) for 1 h at RT. The respective primary antibody was diluted in blocking 
buffer and added to cells for 1 h incubation at RT or overnight at 4 °C. Subsequently, cells were washed 
three times before addition of secondary antibody diluted in blocking buffer and 1 h incubation at RT. 
Following three washes with blocking buffer and one wash with PBS, the coverslip with the specimen 
was mounted onto the microscope slide using DAPI ProLong Diamond Antifade Reagent (Thermo 
Scientific) or Vectashield containing DAPI (Vector Laboratories). The slides were stored in the dark for 
24 h at RT, sealed and stored long-term at 4 °C.  
For quantification of shelterin complex members in interphase WT and ZNF524 KO clones, pictures 
were taken with a Leica TCS SP5 confocal microscope (pinhole 60.05 µm, 2x zoom). Z-stacks were taken 
with a distance of 0.13 µm between focal planes. The laser and gain settings were adjusted to the 
sample with the lowest signal intensity. Fiji (ImageJ) was used for quantification of signal intensities 
and areas of the telomere foci. Therefore, the channels split into the DAPI and red channel. A mask of 
the image was created to infer the volume of the imaged object. The threshold function of the software 
was used with activated plugins for identification of round objects (Otsu). After setting the threshold 
for the image in the histogram settings, the z-stack was converted to a binary mask and using the 3D 
OC Options menu the integrated density was calculated. Additionally, the 3D Object counter menu was 
used and the filters set to a minimum of 4. An additional filter to remove the lowest 10% was applied 
for stringency. 
For quantification of co-localization events in U2OS, we used a Zeiss LSM 880 with 100x/1.4 oil 
objective. Z-stacks were taken with 0.5 µm between focal planes. For quantification of co-localization 
events in other cells lines, we used Leica TCS SP5 confocal microscope (pinhole 60.05 µm, 2x zoom). 
The images were analyzed with Fiji (ImageJ). After maximum intensity projection, the channels were 
split and telomeric foci counted. Subsequently, the number of GFP foci overlapping with telomeric foci 
was visually determined.    
Fluorescence in situ hybridization (FISH) 
Cells were seeded 24 h prior to staining. After washing the slides with PBS, cells were fixed with 4% 
formaldehyde (Thermo Fisher Scientific) for 10 min and washed with PBS. U2OS cells were incubated 
with permeabilization buffer at 37 °C for 1 h, all other cell lines were treated with 0.5% Triton-X100 in 
PBS at RT for 7 min. The sample was dehydrated by successive immersion in 70%, 85% and 100% 
Ethanol for 3 min each. For U2OS cells, the TAMRA-labeled C-rich telomere probe (Eurogentec) was 
diluted in hybridization buffer. For all other cell lines, the probe was diluted 1:100 in 1x blocking 
reagent by Roche and added to the slides which were subsequently heated to 85 °C for 3 min and 
incubated either for 4 h at RT or overnight at 37 °C in a humidity chamber. In case of U2OS, the cells 
were washed with wash buffer 1 at 37 °C for 20 min followed by wash buffer 2 at RT for 15 min. All 
other cell lines were washed twice with wash buffer A (70% formamide, 10 mM Tris-HCl, pH 7.2), three 
95 
 
times with wash buffer 2 and twice with PBS. For TIF and Colocalization event scoring, cells were 
blocked (for U2OS: 10% FBS, 0.1% Triton X-100 in PBS; for other cell lines: 3% BSA and 0.3% TritonX-
100 in PBS) for 1 h at RT and incubated with 1:500 dilution of 53BP1 antibody (Novus)/ 1:1000 dilution 
of GFP antibody (Roche) for 2 h at RT or overnight at 4 °C followed by PBS washes and incubation with 
1:300 diluted secondary antibody (goat anti-rabbit coupled to Alexa488, Thermo Scientific). After a 
final PBS wash, DAPI ProLong Diamond Antifade Reagent (Thermo Scientific) or Vectashield containing 
DAPI (Vector Laboratories) was added to the cells. 
We analyzed the count data of TIF events using a generalized linear mixed model for negative 
binomially distributed data. For this purpose, we used the R package lme4. The factor genotype was 
implemented as fixed effect. The factor clone was implemented as random effect and played the role 
of a random perturbation of the fixed effect. The p-value for the influence of the factor genotype on 
the expected count was calculated using a Likelihood Ratio Test. The confidence intervals were 
calculated by endpoint transformation from Wald-type confidence intervals for linear combinations of 
the (fixed) model parameters. They can be interpreted as confidence intervals for the expected 
number of TIFs in the WT/KO group, with the random effect of the factor clone removed (Bates et al., 
2015). 
Chromosome orientation FISH 
U2OS WT and KO clones were seeded to 40% confluency in DMEM (Gibco) without antibiotics. After 
8 h, BrdC (Alfa Aesar, Fisher Scientific) and BrdU (Sigma Aldrich) were added in a 1:1000 dilution and 
incubated at 37 °C for 10 h followed by a treatment with 200 mM nocodazole (Sigma Aldrich) for 8 h. 
Medium was removed from the cells and collected to keep already detached mitotic cells. The 
remaining cells were detached by trypsin, collected with the supernatant and centrifuged at 200 g for 
5 min. The supernatant was discarded save 200 μL that were used to gently suspend the cells. A total 
of 10 mL hypotonic shock solution were added in a dropwise manner to the cell suspension during mild 
shaking. Subsequently, the suspension was incubated at 37 °C for 30 min. For fixation, 1 mL cold 
methanol/acetic acid (3:1 v/v) was added, gently mixed and centrifuged at 200 g for 5 min. The 
supernatant was discarded save 200 μL that were used to suspend the cell pellet. Another 7 mL of cold 
methanol/acetic acid were added in a dropwise manner during mild shaking. Cells were immediately 
spun at 200 g for 5 min and the previous step repeated twice. Finally, the cells were suspended in 
200 μL cold methanol/acetic acid, spread on microscope slides and dried for 1 h in the dark. Before 
staining, the metaphases were rehydrated in PBS for 5 min at RT, treated first with 0.5 mg/mL RNase A 
(in PBS, DNase free) at 37 °C for 10 min and then with 0.5 μg/mL Hoechst 33258 (Sigma) in 2xSSC at RT 
for 15 min. To degrade the newly synthesized strand, the slides were exposed to 365 nm UV light. The 
damaged BrdU/BrdC-substituted DNA strands were subsequently digested by 800 U Exonuclease III 
(Promega) in the dedicated buffer at 37°C for 30 min. The metaphases were washed in PBS, dehydrated 
in a series of 70%, 85% and 100% Ethanol and air-dried. Finally, the metaphases were hybridized first 
with Cy3-labeled G-rich telomere probe (1:100 dilution 5 nmol, Eurogentec) and then with FITC-
labelled C-rich telomere probe (1:100 dilution of 5 nmol, Eurogentec) at RT in the dark for 1.5 h. The 
slides were washed (70% formamide, 10 mM Tris-HCl pH 7.4). Again, dehydration was performed in 
the previously mentioned Ethanol series followed by drying. The metaphases were mounted with 
ProLong Gold Antifade Reagent with DAPI (Thermo Scientific). 
As previously described for the count data of TIF events, we analyzed the count data of t-SCE events in 
metaphase cells using a generalized linear mixed model for negative binomially distributed data. 
96 
 
Chromatin Immunoprecipitation 
U2OS stable cell lines carrying ZNF524-GFP, ZNF524-GFP  ZF2 mut or NLS-GFP were seeded in medium 
supplemented with 300 ng/mL doxycycline 48 h prior to the experiment to induce expression of the 
constructs. For crosslinking, the attached cells were washed with ice cold PBS twice and then incubated 
with 1% (v/v) formaldehyde without methanol in DMEM for exactly 20 min at RT. The reaction was 
quenched with 2.5 M Glycine in PBS for 5 min at RT. Subsequently, the entire medium was removed, 
the cells washed twice with ice cold PBS and then scraped from the cell culture dishes in 1 mL PBS. The 
cells were washed once with lysis buffer 1 for 15 min at 4 °C and centrifuged at 1000 g at 4 °C for 5 
min. The supernatant was removed, the cell pellet suspended in lysis buffer 2 and again washed for 15 
min at 4 °C. Cells were taken up in sonication buffer at a ratio of 10 Mio cells in 150 μL. During 
sonication, cells were kept on ice. To obtain chromatin fragments of about 200-500 bp, the following 
settings on the EpiShear probe sonicator (Active Motif) were used: Amplitude of 30%, 15 sec ON and 
30 sec OFF, 25 cycles. After sonication, the suspension was centrifuged at 20,000 g and 4 °C for 10 min 
and the supernatant kept as sonicate. To verify the successful sonication, 10 μL of sonicate were mixed 
with 200 mM NaCl and 1 mg/mL RNase A to a final volume of 100 μL and incubated at 37 °C for 1 h. 
The mixture was supplemented with 0.4 μg/mL Proteinase K and then incubated at 62 °C for 2 h. After 
purification using the Qiagen PCR purification Kit according to manufacturer’s instructions, the 
chromatin fragment size was determined on a 1.5% agarose gel. For immunoprecipitation, 36.5 μg 
chromatin were mixed with modified PBB buffer. Per replicate and construct 8 μL (35 μL for ChIP-seq) 
of GFP-Trap magnetic agarose beads (Chromotek) were equilibrated in PBB buffer, blocked in PBB 
buffer supplemented with 10 μg/mL BSA and sheared salmon sperm DNA (Thermo Scientific) and 
finally taken up in PBB buffer. The blocked and equilibrated beads were added to the chromatin and 
incubated at 4 °C on a rotating wheel overnight. After at least 16 h incubation, the 
Immunoprecipitation was washed with PBB buffer (150 mM NaCl) five times. Subsequently, the beads 
were washed once with TE buffer, suspended in filtered elution buffer and incubated at 60 °C for 30 
min. The supernatant containing the chromatin was kept and the elution step repeated once. To 
reverse the crosslink, the eluate was supplemented with NaCl to a final concentration of 200 mM and 
incubated at 65 °C overnight. On the following day, RNase A was added and the mixture incubated at 
37 °C for 30 min. Finally, 0.01 mM EDTA, 20 mM Tris-HCl pH 6.5 and separately 2.5 μg/mL Proteinase 
K were added and incubated at 45 °C for 2 h. The chromatin was then purified using the QIAquick PCR 
purification Kit (Qiagen) according to manufacturer’s instructions and the DNA eluted in TE buffer. 
Slot blot 
For detection of C-circles, 6 µL of the reaction were diluted to 100 µL in 2x SSC (3 M NaCl, 0.3 M sodium 
citrate) and slot-blotted on a Hybond XL nylon membrane (GE Healthcare). For detection of ChIP 
samples, the eluted chromatin was denatures at 95 °C. Subsequently, 10 µL of denatured chromatin 
were slot-blotted and hybridized with either telomeric ((CCCTAA)4) or Alu 
(TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGA) DIG-labeled probes. The TeloTAGGG Telomere 
Length Assay kit (Roche, Sigma-Aldrich) was used according to manufacturer’s instructions. After 
blotting, the membrane was UV cross-linked at 120 mJ using a Stratalinker UV Crosslinker (Stratagene), 
rinsed with H2O and twice with 2x SSC prior to incubation in pre-warmed DIG Easy Hyb Granules for 60 
min at 42 °C with gentle agitation for pre-hybridization. The DIG‐labelled probe (telomere or Alu), 
diluted 1:5000 in Hyb Granules, was added for hybridization and incubated for 3 h or overnight at 42 
°C with gentle agitation. The membrane was washed twice with stringent wash buffer 1 for 5 min at 
RT followed by two washes with pre-warmed stringent wash buffer 2 for 15 min at 50 °C and a wash 
in 1x washing buffer for 5 min at RT. Next, the membrane was incubated in 1x blocking solution for 30 
min followed by incubation with anti-DIG-AP antibody (1:10,000) diluted in 1x blocking solution for 
either 30 min at RT or overnight at 4 °C. Following two washes with 1x washing buffer for 15 min each, 
the membrane was incubated with 1x detection buffer for 5 min. For detection of the samples, CDP-
97 
 
star substrate solution was added to the membrane before visualization using either X-ray films or 
ChemiDoc Touch Imaging System (BioRad). Quantification of the signal intensity was done using Fiji 
(ImageJ). The background was subtracted and an equal area was measured for each slot. The 
integrated density was subjected to Student’s t-test analysis. 
Next-generation chromatin immunoprecipitation sequencing (ChIP-seq) 
ChIP samples were prepared by Alexia Hillairet (Kappei lab, Cancer Science Institute of Singapore). ChIP 
reactions were prepared as described above using 100 µg chromatin as starting material. The purified 
DNA fragments were submitted to NovogeneAIT for ChIP-seq sample preparation and sequencing. In 
brief, the DNA fragments were repaired, A-tailed and then ligated with Illumina adapters. After size 
selection and PCR amplification, the sequencing library was checked for size distribution using the 2100 
Bioanalyzer System (Agilent), quantified using real-time PCR and the Qubit dsDNA HS Assay kit on a 
Qubit 2.0 fluorometer (Thermo Scientific). The quantified libraries were pooled in equimolar ratio and 
sequenced on a NovaSeq 6000 (Illumina). 
Analysis was done by Vartika Khanchandani (Kappei lab, Cancer Science Institute of Singapore). For 
each sample 39 to 52 million reads were obtained as 150bp paired-end reads. The reads were mapped 
to the human reference genome version GRCh38 using Bowtie 2 version 2.3.5.1 with default settings 
and processed using samtools vesion 1.12 (Langmead and Salzberg, 2012; Danecek et al., 2021). 
Unique alignments were obtained by filtering alignments having a MAPQ score of 40 or more using 
samtools version 1.12. Bigwig tracks normalized to counts per million mapped reads were produced 
using deeptools 3.5.0 and peaks were called using MACS version 2.2.7.1 in paired-end mode with the 
default q-value cut-off of 0.05 (Zhang et al., 2008; Ramírez et al., 2016). 
Generation of knock-out cells 
For the generation of ZNF524 KO clones, three guide RNAs targeting different regions in the exon 
region of the ZNF524 gene were designed (see table). DNA oligonucleotides of these regions were 
cloned into the pX459 V2 vector containing the gRNA scaffolding as well as the Cas9 expression 
cassette. Successful cloning was confirmed by Sanger sequencing (GATC). U2OS and HeLa Kyoto cells 
were transfected and 48h later the cells were selected with 3 μg/mL puromycin for three days. After 
expansion, the selected cells were single-cell sorted on BD FACSAria III SORP. Sorting was performed 
by IMB Flow cytometry Core Facility. To confirm gene editing, T7 endonulcease 1 (T7E1) assay was 
performed on the unsorted cell pool. Therfeore, gDNA was isolated using the QIAamp DNA Mini Blood 
Kit (Qiagen) according to manufacturer’s instructions. The regions potentially carrying genomic 
modifications were PCR amplified from the gDNA using specific primers. Following denaturation of the 
PCR products for 10 min at 95 °C, a ramped reannealing (95 °C for 5 min, 95-85 °C at -2 °C/sec, 85-25 °C 
at -0.1 °C/sec) allowed for mismatches at modified sites. Treatment with 10 units T7E1 (New England 
Biolabs) for 30 min at 37 °C revealed successful genomic modifications. The resulting fragments were 
visualized on a 2% agarose gel. The single cells were expanded and checked for ZNF524 expression 
using our self-produced αZNF524 antibody. The clonal lines that were negative for ZNF524 expression 
were subjected to next generation sequencing for determination of the genomic modification. To this 
end, the region around the Cas9 cutting site was amplified from gDNA, followed by a second PCR 
reaction introducing P5 and P7 overhangs. The amplicons were purified using AMPure XP beads 
(Beckman Coulter) and the DNA concentration determined by Qubit dsDNA HS Assay Kit (Thermo 
Scientific) according to manufacturer’s instructions. In a third PCR, the P5 and P7 adaptors as well as 
sample specific indexes were added and the products again purified. Amplicons of all clones were 
pooled in equimolar ratios and sequenced on a MiSeq Nano Flowcell, paired-end for 2x 159 cycles plus 
7 cycles for the index read. DNA-Seq measurements of U2OS WT and ZNF524 KO samples yielded on 
average 57 K reads of 159 nt length per sample. We assessed the quality of the sequenced reads with 
98 
 
fastqc (Babraham Bioinformatics). Adapter sequences were removed from both ends of both reads 
using cutadapt version 1.14 (Martin, 2011). Paired reads were merged using pandaseq version 2.11 
(Masella et al., 2012) with the following parameters: -d BFSrk -A pear. Merged reads were mapped to 
chromosome 19 of the homo sapiens GRCh38 reference genome using gmap version 2017-02-15 (Wu 
and Watanabe, 2005) with the following parameters: --min-intron length=200 -f samse --nofails. For 
localization and visualization of the mutations we summarized mapped sequences using R version 3.4.3 
(R Core Team, 2017) and CrispRVariants bioconductor package version 1.6.0 (Lindsay et al., 2016). 
Variants within the region of interest were localized and mutation rates of all alleles were calculated 
for each sample. 
Protein and antibody purification 
His-MBP-ZNF524 was expressed from pCoofy4 (53) for immunization while His-ZNF524 was expressed 
from pCoofy1 for antibody purification. The E. coli BL21 pRARE strain carrying the expression vector 
were grown in 5 mL LB Medium at 37 °C overnight. The pre-culture was used for inoculation of 8x 1 L 
LB Medium. Bacteria was grown at 37 °C to an OD600 of 0.6-0.7. To induce expression of the 
recombinant protein, the bacteria was supplemented with 0.5 M IPTG (Roth), cultured for 3 days at 
18 °C and finally harvested at 5000 g for 30 min. To disrupt the bacterial membrane, pelleted cells were 
suspended in 250 mL lysis and exposed to sonication (Branson sonifier, Duty cycle: 40; output control: 
6; 2x 3 min). The lysate was kept on ice at all times. To remove any cell debris, the lysate was 
centrifuged at 19000 rpm for 30 min. The supernatant was carefully separated and His-MBP-ZNF524 
further purified on a HisTrapTM HP (GE Healthcare) via the Akta Prime Plus System. After washing with 
10% and 15% elution buffer the His-tagged target protein was eluted with 100% elution buffer at 
1 mL/min flow rate and the collected fractions checked for recombinant ZNF524 expression on 4-12% 
NuPAGE Novex Bis-Tris precast gels (Thermo Scientific). His-MBP-ZNF524 of about 75% purity was send 
to Pineda Antikoerper- Services, Berlin, for immunization of rabbits. 
For antibody purification from rabbit serum, the elution fractions containing His-ZNF524 were further 
purified on a HiTrap Heparin HPTM (GE Healthcare). Therefore, the eluted protein fractions were pooled 
and diluted in 10 volumes of Buffer E and applied to the Heparin column at a 1 mL/min flow rate. A 
gradient of 200 mM NaCl up to 1 M NaCl was used for elution and the collected fractions again 
examined by SDS-PAGE. Fractions containing His-ZNF524 were dialysed to coupling buffer (50 mM Tris 
HCl pH 8.5, 5 mM EDTA) for storage at 4 °C. Antibodies against ZNF524 were purified and enriched 
from the serum against this recombinantly expressed His-ZNF524 using the SulfoLink® Immobilization 
Kit for Peptides (Thermo Scientific) according to manufacturer’s instructions. 
Cell cycle analysis by FACS 
Per condition, 2 Mio cells were harvested and washed twice with PBS. The cell pellet was taken up in 
100 μL PBS and ice cold 70% ethanol was added in a dropwise manner under simultaneous agitation. 
The cells in ethanol were incubated at 4 °C for 1 h, subsequently washed with PBS and taken up in 
450 μL PBS. Propidium iodide (Sigma Aldrich) and RNase A were added to a final concentration of 
80 μg/mL and 40 μg/mL, respectively. After 30 min incubation at 4 °C in the dark, the DNA content of 
the cells was measured on LSRFortessa SORP (Becton Dickinson). For analysis, the cell cycle phases 
were assigned according to the DNA content and the percentage of cells per cell cycle phase was 
determined using FlowJo.   
 
99 
 
Southern blot analysis of telomere restriction fragment (TRF) lengths 
Genomic DNA was isolated using the QIAamp DNA blood Mini Kit (Qiagen) following the 
manufacturer’s instructions. TRF length analysis was performed using the TeloTAGGG telomere length 
assay kit (Roche, Sigma-Aldrich) with slight modifications to the manufacturer’s instructions (Kimura 
et al., 2010). 8 µg and 12 µg of DNA were digested for HeLa and U2OS cell line respectively, using 20 U 
of HinfI and RsaI each at 37°C for 4 h or overnight. Digested HeLa DNA was then resolved on an 0.8% 
agarose gel at 120 V for 4 h in 1x TAE buffer and the gel was visualized using RedSafe nucleic acid stain 
(iNtRON). For U2OS cells, we used pulsed field gel electrophoresis. The digested DNA was resolved on 
1% low-melt megabase agarose for 15 h with initial switch time 0.2 sec and final switch time 13 sec at 
6 V/cm using the CHEF-DRIII (BioRad). The DNA was visualized using SYBRSafe and the gel was 
incubated in 0.25 M HCl for 20 min for depurination, rinsed twice with distilled water followed by 
incubation in denaturation solution twice for 20 min. Subsequently, the gel was rinsed with distilled 
water twice before two washes with neutralizing solution for 20 min each. The digested DNA was then 
transferred to a positively charged nylon membrane (Hybond, N+, Amersham, UK) overnight by 
capillary osmosis in presence of 20x SSC  and fixed by UV-crosslinking at 120 mJ using a Stratalinker® 
UV Crosslinker (Stratagene). The membrane was rinsed twice with 2x SSC and incubated with pre-
warmed DIG Easy Hyb Granules for 1 h at 42°C before hybridization with DIG-labelled telomere probe 
(1 µl /5 ml of Hyb Granules) for 3 h at 42°C. Subsequently, the membrane was washed twice with 
stringent buffer 1 (2x SSC, 0.1% SDS) at RT for 5 min each, twice with pre-warmed stringent buffer 2 
(0.2x SSC, 0.1% SDS) at 50°C for 20 min each and rinsed with 1x wash buffer provided in the kit for 5 
min. The membrane was blocked with 1x blocking solution for 30 min at RT, followed by incubation 
with anti-DIG-AP antibody (1:10,000) diluted in blocking solution for 30 min at RT and subsequently 
washed twice with 1x washing buffer, 15 min each at RT. Followed by incubation in 1x detection 
solution for 5 min at RT, the TRF smear was detected using the digoxigenin luminescent detection (CDP 
star) system and developed on X-ray films. Average telomere length was calculated by comparison to 
the 1kb plus DNA ladder provided in the kit using telotool (Göhring et al., 2014). 
C-circle assay 
C-circle assays were done by Grishma Rane (Kappei Lab, Cancer Science Institute of Singapore). 
Genomic DNA was isolated from U2OS WT and ZNF524 KO clones using QIAamp DNA Blood Mini Kit 
(Qiagen) with RNase treatment. Following quantification with the Qubit dsDNA HS Assay Kit (Thermo 
Scientific) according to manufacturer’s instructions, 300 ng of DNA were digested using 10 U each of 
Hinf I and Rsa I at 37°C for 2 h. 7.5 ng and 15 ng of digested DNA were amplified with 7.5 U φ29 
polymerase (NEB) in 1X φ29 buffer (NEB) supplemented with 2 mM of dATP, dGTP and dTTP (Thermo 
Scientific) each and 0.1 mg mL-1 BSA for 6 h at 30 °C, followed by heat inactivated at 70 °C for 20 min. 
Reactions lacking either φ29 polymerase or gDNA template served as negative controls.  
Telomere Repeat Amplification Protocol (TRAP) 
HeLa WT and ZNF524 KO cells were lysed in TRAP lysis buffer for 30 min on ice and cell debris separated 
from the lysate by centrifugation for 30 min at full 20,000 g. The protein concentration was measured 
by Bradford and equal amounts of each clones were diluted in TRAPeze Chaps buffer (Millipore). 
Subsequently, 200 nM of the TS and the ACX primers were added, as well as the GoTaq qPCR Master 
Mix (Promega) and H2O to a final volume of 20 uL. The TRAP assay reaction was run and measured on 
the ViiA7 real-time PCR system (Thermo Scientific) with 20 min at 25 °C followed by 10 min at 95 °C 
and 40 cycles of 30 sec at 95 °C, 30 sec at 60 °C and one minute at 72 °C. The amplification products 
were separated in a 20% TBE gel. 
 
100 
 
Alamar blue viability assay 
The assay was performed in a 96-well format. 2000 cells were seeded per well and incubated for one 
day. The indicated molarity of BIBR1532 was added to a final volume of 180 μL. After three days, 20 μL 
alamarBlue™ Cell Viability Reagent (Thermo Fisher Scientific) were added per well and the cells 
incubated for 3 h at 37 °C in the dark. Subsequently, the fluorescence intensity was measured on a 
Tecan Reader Infinite 200 PRO (Tecan). 
RNA sequencing (RNA-seq) 
Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) according to manufacturer’s 
instructions including on-column DNA digestion. Sample preparation and sequencing were performed 
by IMB Genomics Core Facility. NGS library prep was performed with Illumina's TruSeq stranded mRNA 
LT Sample Prep Kit following Illumina’s standard protocol (Part # 15031047 Rev. E). Libraries were 
prepared by using only ¼ of the reagents with a starting amount of 250ng and they were amplified in 
11 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent 
technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life 
technologies). Libraries were pooled in equimolar ratio and sequenced on 1 NextSeq 500 Highoutput 
Flowcell, SR for 1x75 cycles plus 2x 8 cycles for dual index read. 
The analysis of the sequencing results was performed by Albert Fradera-Sola. 
mRNA read processing and mapping: Library quality was assessed with FastQC version 0.11.8 before 
being aligned against the H. sapiens genome assembly Homo_sapiens.GRCh38.98 and its associated 
.GTF and .BED files annotations. Such alignment was performed with STAR 81 version 2.7.3a (options:-
-runMode alignReads --outStd SAM --outSAMattributes Standard --outSJfilterReads Unique --
outSAMunmapped Within --outReadsUnmapped None --outFilterMismatchNoverLmax 0.04 --
outFilterMismatchNmax 999 --sjdbOverhang 75) (Dobin et al., 2013). Reads mapping to annotated 
features in the .GTF file were counted with featureCounts version 1.6.2 (options: --donotsort -t 
exon)(Liao, Smyth and Shi, 2014). Coverage tracks were generated with deepTools version 3.1 
(bamCoverage --binSize 1 --skipNonCoveredRegions --normalizeUsing CPM) and plotted using Gviz on 
an R framework (R Development Core Team, 2014; Hahne and Ivanek, 2016; Ramírez et al., 2016). 
Finally, overall quality of the reads and the alignment was assessed with MultiQC version 1.7 (Ewels et 
al., 2016).  
Differential expression analysis: Further filtering and exploratory analysis were performed in an R 
framework including ggplot2 (Wickham, 2016). Pairwise differential expression comparisons were 
performed with DESeq2 (Love, Huber and Anders, 2014). Gene expression in RPKM was used to filter 
out individuals with a replicate average lower than 0 thus considering them as non-expressed. 
Differentially expressed genes (DEGs) were selected with an adjusted p-value (FDR) of less than 0.01 
and a threshold of at least 1 log2 fold-change difference between conditions was applied. Overlapping 
genes between conditions were assessed for significance with a hypergeometric distribution test (p-
value < 0.01) as implemented in R base stats. 
Co-Immunoprecipitation 
Expression of ZNF524-GFP WT and NLS-GFP in U2OS was induced by 48 h treatment with 500 ng/mL 
doxycycline. After 24 h, the cells were transfected for FLAG-TRF2 expression as previously described. 
Cells were harvested and lysed in Radioimmunoprecipitation assay (RIPA) buffer supplemented with 
cOmplete protease inhibitor by Roche. The GFP-tagged proteins were targeted using 10 µL GFP-Trap 
magnetic beads (Chromotek) per IP equilibrated in GFP IP wash buffer, while FLAG-TRF2 was targeted 
by αFLAG in PBB buffer. Per IP, 400 µg lysate were diluted in the respective IP buffer and incubated on 
101 
 
the rotating wheel at 4 °C overnight. Samples incubated with FLAG were subsequently supplemented 
with 12.5 μL Dynabeads Protein G (Invitrogen) per IP and incubated for another 2 h. Using a magnetic 
rack, each samples was washed three times with the respective IP buffer and finally eluted in LDS 
buffer followed by 10 min at 70 °C. 
Synthetic lethality screen 
For stable integration of Cas9 in U2OS WT clones 2, 3, 4 and ZNF524 KO clones 1, 3, 4 , the cells were 
lentivirally transduced with lentiCas9-Blast (Addgene, #52962) as previously described and selected 
with 10 μg/mL blasticidin. 
To identify genetic interactors of ZNF524 genome-wide, we chose a pooled sgRNA library targeting 
18,543 genes with a total of 187,536 gRNAs (Addgene, #1000000095)(Park et al., 2017). The library 
came split into three parts, with library 1 and 2 of equal sizes. Viral particles of the library were 
produced by transfection of HEK293T in a 15 cm dish format, essentially as previously described. Here, 
3.3 μg of each packaging plasmid and of library 1 and library 2 were used as well as 0.18 μg of library 
3. After harvesting the virus-containing supernatant from HEK293T cells, the supernatant was 
centrifuged at 4,000 g and 4 °C overnight to pellet the viral particles and concentrate the titer 10-fold. 
To determine the titer, untreated U2OS cells were seeded and treated with a dilution series of viral 
particles from 1:100 to 1:106. Start selection with 3 μg/mL puromycin two days after transduction. 
After 10 days, the cells were fixed with methanol:acetic acid (3:1 v/v) and stained with trypan blue 
(Sigma Aldrich). The colonies were counted and the titer calculated by multiplying the number of 
colonies with the dilution factor and dividing by the volume of virus solution used. U2OS WT and 
ZNF524 KO clones with stable expression of Cas9 were seeded to 8x107 cells per clone and transduced 
as previously described with a multiplicity of infection below one. The cells were selected with 
7.5 μg/mL puromycin for one week and then maintained in medium supplemented with 2.5 μg/mL 
puromycin. Cells were collected three days after selection start as initial timepoint and after one month 
as final timepoint. From these cells, gDNA was isolated using the QIAamp DNA Blood Maxi Kit (Qiagen) 
according to manufacturer’s instructions. In a first PCR, Herculase II Fusion DNA polymerase (Agilent) 
was used according to manufacturer’s instructions to amplify the integrated sgRNA region from a total 
of 300 μg gDNA input per sample. Per sample, 100 PCR reactions were run with general primers. The 
PCR products were purified and the residual primers removed using AMPure XP beads (Beckman 
Coulter). In a second PCR reaction, adapter sequences as well as sample specific indeces were 
introduced using a general forward primer and a designated reverse primer. Again, the PCR products 
were purified and the residual primers removed using AMPure XP beads (Beckman Coulter). After 
measuring the DNA concentration by Qubit, the samples were mixed in equimolar ratios to 4 nM per 
sample. IMB Genomics Core Facility performed sequencing of the pool using the Illumina NextSeq 500 
platform for high output with custom Sequence read 1 primer and Index read primer.  
The analysis of the sequencing results was done by Michal Levin: Raw RNA-sequencing reads were cut 
to retrieve only the 20 first nucleotides of the read using cutadapt (version 1.15). The resulting 
sequences were mapped to the sgRNA library sequences using bowtie2 (version 2.3.4.3) and read per 
sgRNA entity was counted and summarized. The raw counts were normalized using the cpm (counts 
per million) approach. Then the CRISPRBetaBinomial (CB2) algorithm available with the cb2 
bioconductor R package was used to detect genes with significant sgRNA changes between WT and KO 
after 1 month of sgRNA introduction. The statistical test is based on the beta-binomial distribution, 
which is optimally suited to sgRNA data. The details of the algorithm are summarized in (Jeong et al., 
2019). For GO enrichment analyses the bioconductor R package topGO was used. For the enrichment 
analysis Fisher’s Test was used. Further the weighting algorithm was applied which prevents GO terms 
102 
 
on very high levels of the ontology (i.e. more general terms) from appearing in and overloading the list 
of significant terms. Only terms with p-values below 0.01 are shown. 
Competitive proliferation assay in NR2C2 or NR2F2 depleted cells 
U2OS WT clones were virally transduced with pLenti Lifeact-EGFP BlastR (Addgene, #84383) and 
ZNF524 KO clones were virally transduced with pLenti Lifeact-iRFP670 BlastR (Addgene, #84385) as 
previously described. After selection with 10 μg/mL blasticidin, a WT and a KO clone were mixed in 1:1 
ratios and virally transduced with a mix of either plentiCRISPRv2_neo NR2C2 sgRNA 1 and 
plentiCRISPRv2_neo NR2C2 sgRNA 2 or plentiCRISPRv2_neo NR2F2 sgRNA 1 and plentiCRISPRv2_neo 
NR2F2 sgRNA 2. As negative control, the WT:KO mix was virally transduced with plentiCRISPRv2_neo 
sgGal4. After selection with 400 ug/mL G418, the remaining NR2C2 protein amounts in the NR2C2 KO 
mixes were determined by WB as previously described. To confirm genome editing in cells treated with 
NR2F2 sgRNA, the T7E1 assay was performed as previously described with NR2F2 locus specific 
primers. The ratios of WT:KO clones were determined every three to four days over the course of 
30 days. Therefore, the cells were detached by trypsinization, suspended in medium and directly 
measured by flow cytometry on the FACSCelesta Cell Analyzer (Becton Dickinson). A minimum of 
50.000 cells per condition and time point were measured. The percentage of EGFP positive cells/WT 
and iRFP positive cells/ZNF524 KO was calculated using FlowJo. The ratios of NR2C2 KO cells or NR2F2 
KO cells were normalized to Gal4 controls. 
 
  
103 
 
References 
Abreu, E. et al. (2010) ‘TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo’, 
Molecular and Cellular Biology. American Society for Microbiology, 30(12), pp. 2971–2982. doi: 
10.1128/mcb.00240-10. 
Aeby, E. and Lingner, J. (2015) ‘ALT telomeres get together with nuclear receptors’, Cell. Cell Press, pp. 
811–813. doi: 10.1016/j.cell.2015.02.006. 
Aguado, J. et al. (2019) ‘Inhibition of DNA damage response at telomeres improves the detrimental 
phenotypes of Hutchinson–Gilford Progeria Syndrome’, Nature Communications. Nature Publishing 
Group, 10(1), pp. 1–11. doi: 10.1038/s41467-019-13018-3. 
Ahmed, M. S. et al. (2018) ‘Hutchinson–Gilford Progeria Syndrome: A Premature Aging Disease’, 
Molecular Neurobiology. Humana Press Inc., pp. 4417–4427. doi: 10.1007/s12035-017-0610-7. 
Alhendi, A. S. N. and Royle, N. J. (2020) ‘The absence of (TCAGGG)n repeats in some telomeres, 
combined with variable responses to NR2F2 depletion, suggest that this nuclear receptor plays an 
indirect role in the alternative lengthening of telomeres’, Scientific Reports. Nature Research, 10(1). 
doi: 10.1038/s41598-020-77606-w. 
Allshire, R. C., Dempster, M. and Hastie, N. D. (1989) ‘Human telomeres contain at least three types of 
G-rich repeat distributed non-randomly’, Nucleic Acids Research, 17(12), pp. 4611–4627. doi: 
10.1093/nar/17.12.4611. 
AlSabbagh, M. M. (2020) ‘Dyskeratosis congenita: a literature review’, JDDG: Journal der Deutschen 
Dermatologischen Gesellschaft. Wiley-VCH Verlag, 18(9), pp. 943–967. doi: 10.1111/ddg.14268. 
Alter, B. P. et al. (2012) ‘Telomere length is associated with disease severity and declines with age in 
dyskeratosis congenita’, Haematologica. Ferrata Storti Foundation, 97(3), pp. 353–359. doi: 
10.3324/haematol.2011.055269. 
Arat, N. Ö. and Griffith, J. D. (2012) ‘Human Rap1 interacts directly with telomeric DNA and regulates 
TRF2 localization at the telomere’, Journal of Biological Chemistry. American Society for Biochemistry 
and Molecular Biology, 287(50), pp. 41583–41594. doi: 10.1074/jbc.M112.415984. 
Armanios, M. (2012) ‘Telomerase and idiopathic pulmonary fibrosis’, Mutation Research - 
Fundamental and Molecular Mechanisms of Mutagenesis. Mutat Res, pp. 52–58. doi: 
10.1016/j.mrfmmm.2011.10.013. 
Arnoult, N., Van Beneden, A. and Decottignies, A. (2012) ‘Telomere length regulates TERRA levels 
through increased trimethylation of telomeric H3K9 and HP1α’, Nature Structural and Molecular 
Biology. Nat Struct Mol Biol, 19(9), pp. 948–956. doi: 10.1038/nsmb.2364. 
Arnoult, N. and Karlseder, J. (2015) ‘Complex interactions between the DNA-damage response and 
mammalian telomeres’, Nature Structural and Molecular Biology. Nature Publishing Group, pp. 859–
866. doi: 10.1038/nsmb.3092. 
Arora, R. et al. (2014) ‘RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in 
ALT tumour cells’, Nature Communications. Nature Publishing Group, 5. doi: 10.1038/ncomms6220. 
Arora, R. and Azzalin, C. M. (2015) ‘Telomere elongation chooses TERRA ALTernatives’, RNA Biology. 
Taylor and Francis Inc., 12(9), pp. 938–941. doi: 10.1080/15476286.2015.1065374. 
104 
 
Azzalin, C. M. et al. (2007) ‘Telomeric repeat-containing RNA and RNA surveillance factors at 
mammalian chromosome ends’, Science. Science, 318(5851), pp. 798–801. doi: 
10.1126/science.1147182. 
Azzalin, C. M. and Lingner, J. (2008) ‘Telomeres: The silence is broken’, Cell Cycle. Taylor and Francis 
Inc., pp. 1161–1165. doi: 10.4161/cc.7.9.5836. 
Babraham Bioinformatics - FastQC A Quality Control tool for High Throughput Sequence Data (no date). 
Available at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (Accessed: 10 March 
2021). 
Bae, N. S. and Baumann, P. (2007) ‘A RAP1/TRF2 Complex Inhibits Nonhomologous End-Joining at 
Human Telomeric DNA Ends’, Molecular Cell. Mol Cell, 26(3), pp. 323–334. doi: 
10.1016/j.molcel.2007.03.023. 
Baird, D. M., Jeffreys, A. J. and Royle, N. J. (1995) ‘Mechanisms underlying telomere repeat turnover, 
revealed by hypervariable variant repeat distribution patterns in the human Xp/Yp telomere.’, The 
EMBO Journal. Wiley, 14(21), pp. 5433–5443. doi: 10.1002/j.1460-2075.1995.tb00227.x. 
Ballew, B. J. and Savage, S. A. (2013) ‘Updates on the biology and management of dyskeratosis 
congenita and related telomere biology disorders’, Expert Review of Hematology. Expert Rev Hematol, 
pp. 327–337. doi: 10.1586/ehm.13.23. 
Barbaro, P. M., Ziegler, D. S. and Reddel, R. R. (2016) ‘The wide-ranging clinical implications of the short 
telomere syndromes’, Internal Medicine Journal. Blackwell Publishing, 46(4), pp. 393–403. doi: 
10.1111/imj.12868. 
Barefield, C. and Karlseder, J. (2012) ‘The BLM helicase contributes to telomere maintenance through 
processing of late-replicating intermediate structures’, Nucleic acids research. 2012/05/10. Oxford 
University Press, 40(15), pp. 7358–7367. doi: 10.1093/nar/gks407. 
Barthel, F. P. et al. (2017) ‘Systematic analysis of telomere length and somatic alterations in 31 cancer 
types’, Nature Genetics. Nature Publishing Group, 49(3), pp. 349–357. doi: 10.1038/ng.3781. 
Bates, D. et al. (2015) ‘Fitting linear mixed-effects models using lme4’, Journal of Statistical Software. 
American Statistical Association, 67(1), pp. 1–48. doi: 10.18637/jss.v067.i01. 
Beauséjour, C. M. et al. (2003) ‘Reversal of human cellular senescence: Roles of the p53 and p16 
pathways’, EMBO Journal. EMBO J, 22(16), pp. 4212–4222. doi: 10.1093/emboj/cdg417. 
Beishline, K. et al. (2017) ‘CTCF driven TERRA transcription facilitates completion of telomere DNA 
replication’, Nature Communications. Nature Publishing Group, 8(1). doi: 10.1038/s41467-017-02212-
w. 
Benarroch-Popivker, D. et al. (2016) ‘TRF2-Mediated Control of Telomere DNA Topology as a 
Mechanism for Chromosome-End Protection’, Molecular Cell. Cell Press, 61(2), pp. 274–286. doi: 
10.1016/j.molcel.2015.12.009. 
Benetti, R. et al. (2007) ‘Suv4-20h deficiency results in telomere elongation and derepression of 
telomere recombination’, Journal of Cell Biology. The Rockefeller University Press, 178(6), pp. 925–
936. doi: 10.1083/jcb.200703081. 
Benson, E. K., Lee, S. W. and Aaronson, S. A. (2010) ‘Role of progerin-induced telomere dysfunction in 
HGPS premature cellular senescence’, Journal of Cell Science. J Cell Sci, 123(15), pp. 2605–2612. doi: 
105 
 
10.1242/jcs.067306. 
Bernadotte, A., Mikhelson, V. M. and Spivak, I. M. (2016) ‘Markers of cellular senescence. Telomere 
shortening as a marker of cellular senescence’, Aging. Impact Journals LLC, 8(1), pp. 3–11. doi: 
10.18632/aging.100871. 
Bianchi, A. et al. (1997) ‘TRF1 is a dimer and bends telomeric DNA’, EMBO Journal. John Wiley & Sons, 
Ltd, 16(7), pp. 1785–1794. doi: 10.1093/emboj/16.7.1785. 
Biffi, G. et al. (2013) ‘Quantitative visualization of DNA G-quadruplex structures in human cells’, Nature 
Chemistry. Europe PMC Funders, 5(3), pp. 182–186. doi: 10.1038/nchem.1548. 
Bilaud, T. et al. (1996) ‘The telobox, a Myb-related telomeric DNA binding motif found in proteins from 
yeast, plants and human’, Nucleic Acids Research. Nucleic Acids Res, 24(7), pp. 1294–1303. doi: 
10.1093/nar/24.7.1294. 
Bilaud, T. et al. (1997) ‘Telomeric localization of TRF2, a novel human telobox protein’, Nature Genetics. 
Nat Genet, p. 239. doi: 10.1038/ng1097-236. 
Blackburn, E. H. and Gall, J. G. (1978) ‘A tandemly repeated sequence at the termini of the 
extrachromosomal ribosomal RNA genes in Tetrahymena’, Journal of Molecular Biology. J Mol Biol, 
120(1), pp. 33–53. doi: 10.1016/0022-2836(78)90294-2. 
Blackford, A. N. and Jackson, S. P. (2017) ‘ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA 
Damage Response’, Molecular Cell. Cell Press, pp. 801–817. doi: 10.1016/j.molcel.2017.05.015. 
Bluhm, A. et al. (2019) ‘ZBTB10 binds the telomeric variant repeat TTGGGG and interacts with TRF2’, 
Nucleic Acids Research. Oxford University Press, 47(4), pp. 1896–1907. doi: 10.1093/nar/gky1289. 
Bombarde, O. et al. (2010) ‘TRF2/RAP1 and DNA-PK mediate a double protection against joining at 
telomeric ends’, EMBO Journal. Nature Publishing Group, 29(9), pp. 1573–1584. doi: 
10.1038/emboj.2010.49. 
Boyle, J. M. et al. (2020) ‘Telomere length set point regulation in human pluripotent stem cells critically 
depends on the shelterin protein TPP1’, Molecular Biology of the Cell. American Society for Cell Biology, 
31(23), pp. 2583–2596. doi: 10.1091/MBC.E19-08-0447. 
Broccoli, D. et al. (1997) ‘Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2’, 
Nature Genetics. Nature Publishing Group, p. 235. doi: 10.1038/ng1097-231. 
Brown, J. P., Wei, W. and Sedivy, J. M. (1997) ‘Bypass of senescenoe after disruption of 
p21(CIP1)/(WAF1) gene in normal diploid human fibroblasts’, Science. Science, 277(5327), pp. 831–
834. doi: 10.1126/science.277.5327.831. 
Bryan, T. M. et al. (1995) ‘Telomere elongation in immortal human cells without detectable telomerase 
activity’, EMBO Journal. Wiley-VCH Verlag, 14(17), pp. 4240–4248. doi: 10.1002/j.1460-
2075.1995.tb00098.x. 
Canela, A. et al. (2007) ‘High-throughput telomere length quantification by FISH and its application to 
human population studies’, Proceedings of the National Academy of Sciences of the United States of 
America. National Academy of Sciences, 104(13), pp. 5300–5305. doi: 10.1073/pnas.0609367104. 
Capper, R. et al. (2007) ‘The nature of telomere fusion and a definition of the critical telomere length 
in human cells’, Genes and Development. Cold Spring Harbor Laboratory Press, 21(19), pp. 2495–2508. 
106 
 
doi: 10.1101/gad.439107. 
Casas-Vila, N. et al. (2015) ‘Identification of TTAGGG-binding proteins in Neurospora crassa, a fungus 
with vertebrate-like telomere repeats’, BMC Genomics. BioMed Central Ltd., 16(1), p. 965. doi: 
10.1186/s12864-015-2158-0. 
Casteel, D. E. et al. (2009) ‘A DNA polymerase-α·primase cofactor with homology to replication protein 
A-32 regulates DNA replication in mammalian cells’, Journal of Biological Chemistry. American Society 
for Biochemistry and Molecular Biology, 284(9), pp. 5807–5818. doi: 10.1074/jbc.M807593200. 
Celli, G. B., Denchi, E. L. and de Lange, T. (2006) ‘Ku70 stimulates fusion of dysfunctional telomeres yet 
protects chromosome ends from homologous recombination’, Nature Cell Biology. Nature Publishing 
Group, 8(8), pp. 885–890. doi: 10.1038/ncb1444. 
Celli, G. B. and de Lange, T. (2005) ‘DNA processing is not required for ATM-mediated telomere damage 
response after TRF2 deletion’, Nature Cell Biology. Nature Publishing Group, 7(7), pp. 712–718. doi: 
10.1038/ncb1275. 
Cesare, A. J. et al. (2009) ‘Spontaneous occurrence of telomeric DNA damage response in the absence 
of chromosome fusions’, Nature Structural and Molecular Biology. Nature Publishing Group, 16(12), 
pp. 1244–1251. doi: 10.1038/nsmb.1725. 
Cesare, A. J. et al. (2013) ‘The Telomere deprotection response is functionally distinct from the 
Genomic DNA damage response’, Molecular Cell. Cell Press, 51(2), pp. 141–155. doi: 
10.1016/j.molcel.2013.06.006. 
Cesare, A. J. and Griffith, J. D. (2004) ‘Telomeric DNA in ALT Cells Is Characterized by Free Telomeric 
Circles and Heterogeneous t-Loops’, Molecular and Cellular Biology. American Society for 
Microbiology, 24(22), pp. 9948–9957. doi: 10.1128/mcb.24.22.9948-9957.2004. 
Cesare, A. J. and Karlseder, J. (2012) ‘A three-state model of telomere control over human proliferative 
boundaries’, Current Opinion in Cell Biology. NIH Public Access, pp. 731–738. doi: 
10.1016/j.ceb.2012.08.007. 
Cesare, A. J. and Reddel, R. R. (2010) ‘Alternative lengthening of telomeres: Models, mechanisms and 
implications’, Nature Reviews Genetics. Nat Rev Genet, pp. 319–330. doi: 10.1038/nrg2763. 
Chang, H. H. Y. et al. (2017) ‘Non-homologous DNA end joining and alternative pathways to double-
strand break repair’, Nature Reviews Molecular Cell Biology. Nature Publishing Group, pp. 495–506. 
doi: 10.1038/nrm.2017.48. 
Chang, M., Arneric, M. and Lingner, J. (2007) ‘Telomerase repeat addition processivity is increased at 
critically short telomeres in a Tel1-dependent manner in Saccharomyces cerevisiae’, Genes and 
Development. Genes Dev, 21(19), pp. 2485–2494. doi: 10.1101/gad.1588807. 
Chavez, A. et al. (2010) ‘Sumoylation and the structural maintenance of chromosomes (Smc) 5/6 
complex slow senescence through recombination intermediate resolution’, Journal of Biological 
Chemistry. J Biol Chem, 285(16), pp. 11922–11930. doi: 10.1074/jbc.M109.041277. 
Chen, J. L., Blasco, M. A. and Greider, C. W. (2000) ‘Secondary structure of vertebrate telomerase RNA’, 
Cell. Cell Press, 100(5), pp. 503–514. doi: 10.1016/S0092-8674(00)80687-X. 
Chen, L. Y., Redon, S. and Lingner, J. (2012) ‘The human CST complex is a terminator of telomerase 
activity’, Nature. Nature, 488(7412), pp. 540–544. doi: 10.1038/nature11269. 
107 
 
Chen, S. et al. (2015) ‘The PZP domain of AF10 senses unmodified H3K27 to regulate DOT1L-mediated 
methylation of H3K79’, Molecular cell. NIH Public Access, 60(2), p. 319. doi: 
10.1016/J.MOLCEL.2015.08.019. 
Chen, Y. et al. (2008) ‘A shared docking motif in TRF1 and TRF2 used for differential recruitment of 
telomeric proteins’, Science. American Association for the Advancement of Science, 319(5866), pp. 
1092–1096. doi: 10.1126/science.1151804. 
Chen, Y. et al. (2011) ‘A conserved motif within RAP1 has diversified roles in telomere protection and 
regulation in different organisms’, Nature Structural and Molecular Biology. NIH Public Access, 18(2), 
pp. 213–223. doi: 10.1038/nsmb.1974. 
Cheung, D. H.-C. et al. (2012) ‘PinX1 is involved in telomerase recruitment and regulates telomerase 
function by mediating its localization’, FEBS Letters. John Wiley & Sons, Ltd, 586(19), pp. 3166–3171. 
doi: 10.1016/j.febslet.2012.06.028. 
Cho, N. W. et al. (2014) ‘Interchromosomal homology searches drive directional ALT telomere 
movement and synapsis’, Cell. Cell Press, 159(1), pp. 108–121. doi: 10.1016/j.cell.2014.08.030. 
Chojnowski, A. et al. (2015) ‘Progerin reduces LAP2α-telomere association in hutchinson-gilford 
progeria’, eLife. eLife Sciences Publications Ltd, 4(AUGUST2015), pp. 1–21. doi: 10.7554/eLife.07759. 
Chong, L. et al. (1995) ‘A human telomeric protein’, Science. American Association for the 
Advancement of Science, 270(5242), pp. 1663–1667. doi: 10.1126/science.270.5242.1663. 
Chow, T. T. et al. (2012) ‘Early and late steps in telomere overhang processing in normal human cells: 
The position of the final RNA primer drives telomere shortening’, Genes and Development, 26(11), pp. 
1167–1178. doi: 10.1101/gad.187211.112. 
Chu, H. P. et al. (2017) ‘TERRA RNA Antagonizes ATRX and Protects Telomeres’, Cell. Cell Press, 170(1), 
pp. 86-101.e16. doi: 10.1016/j.cell.2017.06.017. 
Ciccia, A. and Elledge, S. J. (2010) ‘The DNA Damage Response: Making It Safe to Play with Knives’, 
Molecular Cell. NIH Public Access, pp. 179–204. doi: 10.1016/j.molcel.2010.09.019. 
Conomos, D. et al. (2012) ‘Variant repeats are interspersed throughout the telomeres and recruit 
nuclear receptors in ALT cells’, Journal of Cell Biology, 199(6), pp. 893–906. doi: 
10.1083/jcb.201207189. 
Conomos, D., Reddel, R. R. and Pickett, H. A. (2014) ‘NuRD-ZNF827 recruitment to telomeres creates a 
molecular scaffold for homologous recombination’, Nature Structural and Molecular Biology. Nature 
Publishing Group, 21(9), pp. 760–770. doi: 10.1038/nsmb.2877. 
Coppé, J. P. et al. (2008) ‘Senescence-associated secretory phenotypes reveal cell-nonautonomous 
functions of oncogenic RAS and the p53 tumor suppressor.’, PLoS biology. Public Library of Science, 
6(12). doi: 10.1371/journal.pbio.0060301. 
Corriveau, M. et al. (2013) ‘Coordinated interactions of multiple POT1-TPP1 proteins with telomere 
DNA’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 
288(23), pp. 16361–16370. doi: 10.1074/jbc.M113.471896. 
Court, R. et al. (2005) ‘How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: A 
view from high-resolution crystal structures’, EMBO Reports. EMBO Rep, 6(1), pp. 39–45. doi: 
10.1038/sj.embor.7400314. 
108 
 
Crabbe, L. et al. (2004) ‘Defective telomere lagging strand synthesis in cells lacking WRN helicase 
activity’, Science. Science, 306(5703), pp. 1951–1953. doi: 10.1126/science.1103619. 
Cusanelli, E. and Chartrand, P. (2015) ‘Telomeric repeat-containing RNA TERRA: A noncoding RNA 
connecting telomere biology to genome integrity’, Frontiers in Genetics. Frontiers Media S.A., 6(MAR), 
p. 143. doi: 10.3389/fgene.2015.00143. 
D’Adda Di Fagagna, F. et al. (2003) ‘A DNA damage checkpoint response in telomere-initiated 
senescence’, Nature. Nature Publishing Group, 426(6963), pp. 194–198. doi: 10.1038/nature02118. 
Danecek, P. et al. (2021) ‘Twelve years of SAMtools and BCFtools’, GigaScience. NLM (Medline), 10(2), 
pp. 1–4. doi: 10.1093/gigascience/giab008. 
Davoli, T. and de Lange, T. (2012) ‘Telomere-Driven Tetraploidization Occurs in Human Cells 
Undergoing Crisis and Promotes Transformation of Mouse Cells’, Cancer Cell. NIH Public Access, 21(6), 
pp. 765–776. doi: 10.1016/j.ccr.2012.03.044. 
Decker, M. L. et al. (2009) ‘Telomere length in Hutchinson-Gilford Progeria Syndrome’, Mechanisms of 
Ageing and Development. Elsevier, 130(6), pp. 377–383. doi: 10.1016/j.mad.2009.03.001. 
Dehé, P.-M. and Cooper, J. P. (2010) ‘Fission yeast telomeres forecast the end of the crisis’, FEBS 
Letters. John Wiley & Sons, Ltd, 584(17), pp. 3725–3733. doi: 10.1016/j.febslet.2010.07.045. 
Déjardin, J. and Kingston, R. E. (2009) ‘Purification of Proteins Associated with Specific Genomic Loci’, 
Cell. Cell, 136(1), pp. 175–186. doi: 10.1016/j.cell.2008.11.045. 
Demaria, M. et al. (2014) ‘An essential role for senescent cells in optimal wound healing through 
secretion of PDGF-AA’, Developmental Cell. Cell Press, 31(6), pp. 722–733. doi: 
10.1016/j.devcel.2014.11.012. 
Denchi, E. L. and De Lange, T. (2007) ‘Protection of telomeres through independent control of ATM 
and ATR by TRF2 and POT1’, Nature. Nature Publishing Group, 448(7157), pp. 1068–1071. doi: 
10.1038/nature06065. 
Deng, Z. et al. (2009) ‘TERRA RNA Binding to TRF2 Facilitates Heterochromatin Formation and ORC 
Recruitment at Telomeres’, Molecular Cell. NIH Public Access, 35(4), pp. 403–413. doi: 
10.1016/j.molcel.2009.06.025. 
Deng, Z. et al. (2012) ‘A role for CTCF and cohesin in subtelomere chromatin organization, TERRA 
transcription, and telomere end protection’, EMBO Journal. European Molecular Biology Organization, 
31(21), pp. 4165–4178. doi: 10.1038/emboj.2012.266. 
Deng, Z. et al. (2013) ‘Inherited mutations in the helicase RTEL1 cause telomere dysfunction and 
Hoyeraal-Hreidarsson syndrome’, Proceedings of the National Academy of Sciences of the United 
States of America. Proc Natl Acad Sci U S A, 110(36). doi: 10.1073/pnas.1300600110. 
Van Deursen, J. M. (2014) ‘The role of senescent cells in ageing’, Nature. Nature Publishing Group, pp. 
439–446. doi: 10.1038/nature13193. 
Dilley, R. L. et al. (2016) ‘Break-induced telomere synthesis underlies alternative telomere 
maintenance’, Nature. Nature Publishing Group, 539(7627), pp. 54–58. doi: 10.1038/nature20099. 
Diman, A. et al. (2016) ‘Nuclear respiratory factor 1 and endurance exercise promote human telomere 
transcription’, Science Advances. American Association for the Advancement of Science, 2(7). doi: 
109 
 
10.1126/sciadv.1600031. 
Diman, A. and Decottignies, A. (2018) ‘Genomic origin and nuclear localization of TERRA telomeric 
repeat‐containing RNA: from Darkness to Dawn’, The FEBS Journal. Blackwell Publishing Ltd, 285(8), 
pp. 1389–1398. doi: 10.1111/febs.14363. 
Ding, H. et al. (2004) ‘Regulation of murine telomere length by Rtel: An essential gene encoding a 
helicase-like protein’, Cell, 117(7), pp. 873–886. doi: 10.1016/j.cell.2004.05.026. 
Diotti, R. et al. (2015) ‘DNA-directed polymerase subunits play a vital role in human telomeric overhang 
processing’, Molecular Cancer Research. American Association for Cancer Research Inc., 13(3), pp. 
402–410. doi: 10.1158/1541-7786.MCR-14-0381. 
Dobin, A. et al. (2013) ‘STAR: ultrafast universal RNA-seq aligner’, Bioinformatics. Oxford Academic, 
29(1), pp. 15–21. doi: 10.1093/bioinformatics/bts635. 
Doench, J. G. et al. (2014) ‘Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene 
inactivation’, Nature Biotechnology. Nature Publishing Group, 32(12), pp. 1262–1267. doi: 
10.1038/nbt.3026. 
Doksani, Y. et al. (2013) ‘XSuper-resolution fluorescence imaging of telomeres reveals TRF2-dependent 
T-loop formation’, Cell. Cell Press, 155(2), p. 345. doi: 10.1016/j.cell.2013.09.048. 
Doksani, Y. (2019) ‘The response to dna damage at telomeric repeats and its consequences for 
telomere function’, Genes. MDPI AG. doi: 10.3390/genes10040318. 
Draskovic, I. et al. (2009) ‘Probing PML body function in ALT cells reveals spatiotemporal requirements 
for telomere recombination’, Proceedings of the National Academy of Sciences of the United States of 
America. National Academy of Sciences, 106(37), pp. 15726–15731. doi: 10.1073/pnas.0907689106. 
Drosopoulos, W. C., Kosiyatrakul, S. T. and Schildkraut, C. L. (2015) ‘BLM helicase facilitates telomere 
replication during leading strand synthesis of telomeres’, Journal of Cell Biology. Rockefeller University 
Press, 210(2), pp. 191–208. doi: 10.1083/jcb.201410061. 
Dunham, M. A. et al. (2000) ‘Telomere maintenance by recombination in human cells’, Nature 
Genetics. Nature Publishing Group, 26(4), pp. 447–450. doi: 10.1038/82586. 
Erdel, F. et al. (2017) ‘Telomere Recognition and Assembly Mechanism of Mammalian Shelterin’, Cell 
Reports. Elsevier B.V., 18(1), pp. 41–53. doi: 10.1016/j.celrep.2016.12.005. 
Ernst, P. and Vakoc, C. R. (2012) ‘WRAD: Enabler of the SET1-family of H3K4 methyltransferases’, 
Briefings in Functional Genomics. Brief Funct Genomics, 11(3), pp. 217–226. doi: 10.1093/bfgp/els017. 
Ewels, P. et al. (2016) ‘MultiQC: summarize analysis results for multiple tools and samples in a single 
report’, Bioinformatics. Oxford University Press, 32(19), pp. 3047–3048. doi: 
10.1093/bioinformatics/btw354. 
Fairall, L. et al. (2001) ‘Structure of the TRFH dimerization domain of the human telomeric proteins 
TRF1 and TRF2’, Molecular Cell. Cell Press, 8(2), pp. 351–361. doi: 10.1016/S1097-2765(01)00321-5. 
Fasching, C. L. et al. (2007) ‘DNA damage induces alternative lengthening of telomeres (ALT)-associated 
promyelocytic leukemia bodies that preferentially associate with linear telomeric DNA’, Cancer 
Research. American Association for Cancer Research, 67(15), pp. 7072–7077. doi: 10.1158/0008-
5472.CAN-07-1556. 
110 
 
Feng, Q. et al. (2002) ‘Methylation of H3-lysine 79 is mediated by a new family of HMTases without a 
SET domain’, Current Biology. Cell Press, 12(12), pp. 1052–1058. doi: 10.1016/S0960-9822(02)00901-
6. 
Feuerhahn, S. et al. (2010) ‘TERRA biogenesis, turnover and implications for function’, FEBS Letters. 
John Wiley & Sons, Ltd, 584(17), pp. 3812–3818. doi: 10.1016/j.febslet.2010.07.032. 
Flynn, R. L. et al. (2011) ‘TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-
stranded DNA’, Nature. NIH Public Access, 471(7339), pp. 532–538. doi: 10.1038/nature09772. 
Flynn, R. L. et al. (2015) ‘Alternative lengthening of telomeres renders cancer cells hypersensitive to 
ATR inhibitors’, Science. American Association for the Advancement of Science, 347(6219), pp. 273–
277. doi: 10.1126/science.1257216. 
Fouché, N. et al. (2006) ‘The basic domain of TRF2 directs binding to DNA junctions irrespective of the 
presence of TTAGGG repeats’, Journal of Biological Chemistry. Elsevier, 281(49), pp. 37486–37495. doi: 
10.1074/jbc.M608778200. 
Fumagalli, M. et al. (2012) ‘Telomeric DNA damage is irreparable and causes persistent DNA-damage-
response activation’, Nature Cell Biology. NIH Public Access, 14(4), pp. 355–365. doi: 10.1038/ncb2466. 
Galati, A. et al. (2012) ‘TRF2 controls telomeric nucleosome organization in a cell cycle phase-
dependent manner’, PLoS ONE. Public Library of Science, 7(4), p. 34386. doi: 
10.1371/journal.pone.0034386. 
Galati, A. et al. (2015) ‘TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization’, 
Nucleic Acids Research. Oxford University Press, 43(12), pp. 5824–5837. doi: 10.1093/nar/gkv507. 
Gallego-Paez, L. M. et al. (2014) ‘Smc5/6-mediated regulation of replication progression contributes 
to chromosome assembly during mitosis in human cells’, Molecular Biology of the Cell. Mol Biol Cell, 
25(2), pp. 302–317. doi: 10.1091/mbc.E13-01-0020. 
García‐Beccaria, M. et al. (2015) ‘ Therapeutic inhibition of TRF 1 impairs the growth of p53 ‐deficient 
K‐Ras  G12V  ‐ induced lung cancer by induction of telomeric DNA damage ’, EMBO Molecular Medicine. 
EMBO, 7(7), pp. 930–949. doi: 10.15252/emmm.201404497. 
Giannone, R. J. et al. (2010) ‘The Protein Network Surrounding the Human Telomere Repeat Binding 
Factors TRF1, TRF2, and POT1’, PLoS ONE. Edited by B. A. Sullivan. Public Library of Science, 5(8), p. 
e12407. doi: 10.1371/journal.pone.0012407. 
Glousker, G. et al. (2015) ‘Unraveling the pathogenesis of Hoyeraal-Hreidarsson syndrome, a complex 
telomere biology disorder’, British Journal of Haematology. Blackwell Publishing Ltd, pp. 457–471. doi: 
10.1111/bjh.13442. 
Glousker, G. et al. (2020) ‘Human shelterin protein <scp>POT</scp> 1 prevents severe telomere 
instability induced by homology‐directed <scp>DNA</scp> repair’, The EMBO Journal. EMBO, 39(23), 
p. e104500. doi: 10.15252/embj.2020104500. 
Göhring, J. et al. (2014) ‘TeloTool: A new tool for telomere length measurement from terminal 
restriction fragment analysis with improved probe intensity correction’, Nucleic Acids Research. 
Nucleic Acids Res, 42(3). doi: 10.1093/nar/gkt1315. 
Greider, C. W. and Blackburn, E. H. (1985) ‘Identification of a specific telomere terminal transferase 
activity in tetrahymena extracts’, Cell. Cell Press, 43(2 PART 1), pp. 405–413. doi: 10.1016/0092-
111 
 
8674(85)90170-9. 
Greider, C. W. and Blackburn, E. H. (1987) ‘The telomere terminal transferase of tetrahymena is a 
ribonucleoprotein enzyme with two kinds of primer specificity’, Cell. Cell Press, 51(6), pp. 887–898. 
doi: 10.1016/0092-8674(87)90576-9. 
Griffith, J. D. et al. (1999) ‘Mammalian telomeres end in a large duplex loop’, Cell. Cell Press, 97(4), pp. 
503–514. doi: 10.1016/S0092-8674(00)80760-6. 
Grill, S. et al. (2019) ‘Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase 
Regulation in Somatic and Germ Cells’, Cell Reports. Elsevier B.V., 27(12), pp. 3511-3521.e7. doi: 
10.1016/j.celrep.2019.05.073. 
Grolimund, L. et al. (2013) ‘A quantitative telomeric chromatin isolation protocol identifies different 
telomeric states’, Nature Communications. Nature Publishing Group, 4. doi: 10.1038/ncomms3848. 
Guo, X. et al. (2007) ‘Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response 
to suppress tumorigenesis’, EMBO Journal. European Molecular Biology Organization, 26(22), pp. 
4709–4719. doi: 10.1038/sj.emboj.7601893. 
Hahne, F. and Ivanek, R. (2016) ‘Visualizing genomic data using Gviz and bioconductor’, in Methods in 
Molecular Biology. Humana Press Inc., pp. 335–351. doi: 10.1007/978-1-4939-3578-9_16. 
Hanaoka, S. et al. (2001) ‘NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle 
lacking the positive surface charge typical of Myb DNA-binding domains’, Journal of Molecular Biology. 
Academic Press, 312(1), pp. 167–175. doi: 10.1006/jmbi.2001.4924. 
Hanaoka, S., Nagadoi, A. and Nishimura, Y. (2009) ‘Comparison between TRF2 and TRF1 of their 
telomeric DNA-bound structures and DNA-binding activities’, Protein Science. Wiley, 14(1), pp. 119–
130. doi: 10.1110/ps.04983705. 
Hayashi, M. T. et al. (2012) ‘A telomere-dependent DNA damage checkpoint induced by prolonged 
mitotic arrest’, Nature Structural and Molecular Biology. Nature Publishing Group, 19(4), pp. 387–394. 
doi: 10.1038/nsmb.2245. 
Hayashi, M. T. et al. (2015) ‘Cell death during crisis is mediated by mitotic telomere deprotection’, 
Nature. Nature Publishing Group, 522(7557), pp. 492–496. doi: 10.1038/nature14513. 
Hayflick, L. (1965) ‘The limited in vitro lifetime of human diploid cell strains’, Experimental Cell 
Research. doi: 10.1016/0014-4827(65)90211-9. 
Hayflick, L. and Moorhead, P. S. (1961) ‘The serial cultivation of human diploid cell strains’, 
Experimental Cell Research. doi: 10.1016/0014-4827(61)90192-6. 
Heaphy, C. M. et al. (2011) ‘Altered telomeres in tumors with ATRX and DAXX mutations’, Science. NIH 
Public Access, p. 425. doi: 10.1126/science.1207313. 
Heiss, N. S. et al. (1998) ‘X-linked dyskeratosis congenita is caused by mutations in a highly conserved 
gene with putative nucleolar functions’, Nature Genetics. Nat Genet, 19(1), pp. 32–38. doi: 
10.1038/ng0598-32. 
Hemann, M. T. et al. (2001) ‘The shortest telomere, not average telomere length, is critical for cell 
viability and chromosome stability’, Cell. Elsevier B.V., 107(1), pp. 67–77. doi: 10.1016/S0092-
8674(01)00504-9. 
112 
 
Hemann, M. T. and Greider, C. W. (2000) ‘Wild-derived inbred mouse strains have short telomeres’, 
Nucleic Acids Research. Oxford University Press, 28(22), pp. 4474–4478. doi: 10.1093/nar/28.22.4474. 
Henson, J. D. et al. (2009) ‘DNA C-circles are specific and quantifiable markers of alternative- 
lengthening-of-telomeres activity’, Nature Biotechnology. Nat Biotechnol, 27(12), pp. 1181–1185. doi: 
10.1038/nbt.1587. 
Henson, J. D. and Reddel, R. R. (2010) ‘Assaying and investigating Alternative Lengthening of Telomeres 
activity in human cells and cancers’, FEBS Letters. John Wiley & Sons, Ltd, 584(17), pp. 3800–3811. doi: 
10.1016/j.febslet.2010.06.009. 
Hewitt, G. et al. (2012) ‘Telomeres are favoured targets of a persistent DNA damage response in ageing 
and stress-induced senescence’, Nature Communications. Nature Publishing Group, 3, p. 708. doi: 
10.1038/ncomms1708. 
Hiyama, E. and Hiyama, K. (2007) ‘Telomere and telomerase in stem cells’, British Journal of Cancer. 
Nature Publishing Group, pp. 1020–1024. doi: 10.1038/sj.bjc.6603671. 
Ho, S. T. et al. (2019) ‘The PinX1/NPM interaction associates with hTERT in early-S phase and facilitates 
telomerase activation’, Cell and Bioscience. BioMed Central Ltd., 9(1), p. 47. doi: 10.1186/s13578-019-
0306-y. 
Hockemeyer, D. et al. (2006) ‘Recent Expansion of the Telomeric Complex in Rodents: Two Distinct 
POT1 Proteins Protect Mouse Telomeres’, Cell. Elsevier B.V., 126(1), pp. 63–77. doi: 
10.1016/j.cell.2006.04.044. 
Hockemeyer, D. et al. (2008) ‘Engineered telomere degradation models dyskeratosis congenita’, Genes 
and Development. Genes Dev, 22(13), pp. 1773–1785. doi: 10.1101/gad.1679208. 
Holohan, B., Wright, W. E. and Shay, J. W. (2014) ‘Telomeropathies: An emerging spectrum disorder’, 
Journal of Cell Biology. Rockefeller University Press, pp. 289–299. doi: 10.1083/jcb.201401012. 
Høyeraal, H. M., Lamvik, J. and Moe, P. J. (1970) ‘CONGENITAL HYPOPLASTIC THROMBOCYTOPENIA 
AND CEREBRAL MALFORMATIONS IN TWO BROTHERS’, Acta Pædiatrica. Acta Paediatr Scand, 59(2), 
pp. 185–191. doi: 10.1111/j.1651-2227.1970.tb08986.x. 
Hreidarsson, S. et al. (1988) ‘A syndrome of progressive pancytopenia with microcephaly, cerebellar 
hypoplasia and growth failure’, Acta Paediatrica Scandinavica. Acta Paediatr Scand, 77(5), pp. 773–
775. doi: 10.1111/j.1651-2227.1988.tb10751.x. 
Hu, C. et al. (2017) ‘Structural and functional analyses of the mammalian TIN2-TPP1-TRF2 telomeric 
complex’, Cell Research. Nature Publishing Group, 27(12), pp. 1485–1502. doi: 10.1038/cr.2017.144. 
Huang, C., Dai, X. and Chai, W. (2012) ‘Human Stn1 protects telomere integrity by promoting efficient 
lagging-strand synthesis at telomeres and mediating C-strand fill-in’, Cell Research. Cell Res, 22(12), pp. 
1681–1695. doi: 10.1038/cr.2012.132. 
Huber, M. D. (2002) ‘G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific 
inhibition’, Nucleic Acids Research. Oxford University Press (OUP), 30(18), pp. 3954–3961. doi: 
10.1093/nar/gkf530. 
Huffman, K. E. et al. (2000) ‘Telomere shortening is proportional to the size of the G-rich telomeric 3’-
overhang’, Journal of Biological Chemistry. JBC Papers in Press, 275(26), pp. 19719–19722. doi: 
10.1074/jbc.M002843200. 
113 
 
Hwang, H., Opresko, P. and Myong, S. (2014) ‘Single-molecule real-time detection of telomerase 
extension activity’, Scientific Reports. Nature Publishing Group, 4. doi: 10.1038/srep06391. 
Iwano, T. et al. (2004) ‘Importance of TRF1 for Functional Telomere Structure’, Journal of Biological 
Chemistry. J Biol Chem, 279(2), pp. 1442–1448. doi: 10.1074/jbc.M309138200. 
Jacobs, J. J. L. and De Lange, T. (2004) ‘Significant role for p16INK4a in p53-independent telomere-
directed senescence’, Current Biology. Cell Press, 14(24), pp. 2302–2308. doi: 
10.1016/j.cub.2004.12.025. 
Jády, B. E., Bertrand, E. and Kiss, T. (2004) ‘Human telomerase RNA and box H/ACA scaRNAs share a 
common Cajal body-specific localization signal’, Journal of Cell Biology. J Cell Biol, 164(5), pp. 647–652. 
doi: 10.1083/jcb.200310138. 
Jahn, A. et al. (2017) ‘ ZBTB 48 is both a vertebrate telomere‐binding protein and a transcriptional 
activator ’, EMBO reports. EMBO, 18(6), pp. 929–946. doi: 10.15252/embr.201744095. 
Janoušková, E. et al. (2015) ‘Human Rap1 modulates TRF2 attraction to telomeric DNA’, Nucleic Acids 
Research. Oxford University Press, 43(5), pp. 2691–2700. doi: 10.1093/nar/gkv097. 
Janovič, T. et al. (2019) ‘Human Telomere Repeat Binding Factor TRF1 Replaces TRF2 Bound to 
Shelterin Core Hub TIN2 when TPP1 Is Absent’, Journal of Molecular Biology. Academic Press, 431(17), 
pp. 3289–3301. doi: 10.1016/j.jmb.2019.05.038. 
Jansson, L. I. et al. (2019) ‘Telomere DNA G-quadruplex folding within actively extending human 
telomerase’, Proceedings of the National Academy of Sciences of the United States of America. National 
Academy of Sciences, 116(19), pp. 9350–9359. doi: 10.1073/pnas.1814777116. 
Jeong, H. H. et al. (2019) ‘Beta-binomial modeling of CRISPR pooled screen data identifies target genes 
with greater sensitivity and fewer false negatives’, Genome Research. Cold Spring Harbor Laboratory 
Press, 29(6), pp. 999–1008. doi: 10.1101/gr.245571.118. 
Jones, B. et al. (2008) ‘The Histone H3K79 Methyltransferase Dot1L Is Essential for Mammalian 
Development and Heterochromatin Structure’, PLoS Genetics. Edited by W. A. Bickmore. Public Library 
of Science, 4(9), p. e1000190. doi: 10.1371/journal.pgen.1000190. 
Kabir, S., Hockemeyer, D. and de Lange, T. (2014) ‘TALEN gene knockouts reveal no requirement for 
the conserved human shelterin protein Rap1 in telomere protection and length regulation’, Cell 
Reports. Elsevier, 9(4), pp. 1273–1280. doi: 10.1016/j.celrep.2014.10.014. 
Kamranvar, S. A. and Masucci, M. G. (2011) ‘The Epstein-Barr virus nuclear antigen-1 promotes 
telomere dysfunction via induction of oxidative stress’, Leukemia. Nature Publishing Group, 25(6), pp. 
1017–1025. doi: 10.1038/leu.2011.35. 
Kappei, D. et al. (2013) ‘HOT1 is a mammalian direct telomere repeat-binding protein contributing to 
telomerase recruitment’, The EMBO Journal. John Wiley & Sons, Ltd, 32(12), pp. 1681–1701. doi: 
10.1038/emboj.2013.105. 
Kappei, D. (2013) Novel telomere binding proteins. Technische Universität Dresden. 
Kappei, D. et al. (2017) ‘Phylointeractomics reconstructs functional evolution of protein binding’, 
Nature Communications. Nature Publishing Group, 8(1), pp. 1–9. doi: 10.1038/ncomms14334. 
Karlseder, J. et al. (2003) ‘Targeted Deletion Reveals an Essential Function for the Telomere Length 
114 
 
Regulator Trf1’, Molecular and Cellular Biology. American Society for Microbiology, 23(18), pp. 6533–
6541. doi: 10.1128/mcb.23.18.6533-6541.2003. 
Karlseder, J., Smogorzewska, A. and De Lange, T. (2002) ‘Senescence induced by altered telomere state, 
not telomere loss’, Science. American Association for the Advancement of Science, 295(5564), pp. 
2446–2449. doi: 10.1126/science.1069523. 
Karremann, M. et al. (2020) ‘Revesz syndrome revisited’, Orphanet Journal of Rare Diseases. BioMed 
Central Ltd. doi: 10.1186/s13023-020-01553-y. 
Kaul, Z. et al. (2012) ‘Five dysfunctional telomeres predict onset of senescence in human cells’, EMBO 
Reports. EMBO Rep, 13(1), pp. 52–59. doi: 10.1038/embor.2011.227. 
Kim, N. W. et al. (1994) ‘Specific association of human telomerase activity with immortal cells and 
cancer’, Science. American Association for the Advancement of Science, 266(5193), pp. 2011–2015. 
doi: 10.1126/science.7605428. 
Kimura, M. et al. (2010) ‘Measurement of telomere length by the southern blot analysis of terminal 
restriction fragment lengths’, Nature Protocols. Nature Publishing Group, 5(9), pp. 1596–1607. doi: 
10.1038/nprot.2010.124. 
Kipling, D. and Cooke, H. J. (1990) ‘Hypervariable ultra-long telomeres in mice’, Nature. Nature 
Publishing Group, 347(6291), pp. 400–402. doi: 10.1038/347400a0. 
Kirwan, M. et al. (2011) ‘Dyskeratosis congenita and the DNA damage response’, British Journal of 
Haematology. John Wiley & Sons, Ltd, 153(5), pp. 634–643. doi: 10.1111/j.1365-2141.2011.08679.x. 
Kolesar, P. et al. (2022) ‘Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase’, Cells. MDPI, 
11(1). doi: 10.3390/cells11010165. 
König, P., Fairall, L. and Rhodes, D. (1998) ‘Sequence-specific DNA recognition by the Myb-like domain 
of the human telomere binding protein TRF1: A model for the protein - DNA complex’, Nucleic Acids 
Research. Nucleic Acids Res, 26(7), pp. 1731–1740. doi: 10.1093/nar/26.7.1731. 
Konishi, A., Izumi, T. and Shimizu, S. (2016) ‘TRF2 protein interacts with core histones to stabilize 
chromosome ends’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular 
Biology Inc., 291(39), pp. 20798–20810. doi: 10.1074/jbc.M116.719021. 
Krizhanovsky, V. et al. (2008) ‘Senescence of Activated Stellate Cells Limits Liver Fibrosis’, Cell. Elsevier 
B.V., 134(4), pp. 657–667. doi: 10.1016/j.cell.2008.06.049. 
Kudlow, B. A. et al. (2008) ‘Suppression of proliferative defects associated with processing-defective 
lamin A mutants by hTERT or inactivation of p53’, Molecular Biology of the Cell. American Society for 
Cell Biology, 19(12), pp. 5238–5248. doi: 10.1091/mbc.E08-05-0492. 
Lam, E. Y. N. et al. (2013) ‘G-quadruplex structures are stable and detectable in human genomic DNA’, 
Nature Communications. Europe PMC Funders, 4, p. 1796. doi: 10.1038/ncomms2792. 
Lam, Y. C. et al. (2010) ‘SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated 
repair’, EMBO Journal. EMBO J, 29(13), pp. 2230–2241. doi: 10.1038/emboj.2010.58. 
de Lange, T. (2018) ‘Shelterin-Mediated Telomere Protection’, Annual Review of Genetics. Annual 
Reviews, 52(1), pp. 223–247. doi: 10.1146/annurev-genet-032918-021921. 
115 
 
Langmead, B. and Salzberg, S. L. (2012) ‘Fast gapped-read alignment with Bowtie 2’, Nature Methods. 
Nature Publishing Group, 9(4), pp. 357–359. doi: 10.1038/nmeth.1923. 
Lansdorp, P. and van Wietmarschen, N. (2019) ‘Helicases FANCJ, RTEL1 and BLM act on guanine 
quadruplex DNA in vivo’, Genes. MDPI AG. doi: 10.3390/genes10110870. 
Lapasset, L. et al. (2011) ‘Rejuvenating senescent and centenarian human cells by reprogramming 
through the pluripotent state’, Genes and Development. Cold Spring Harbor Laboratory Press, 25(21), 
pp. 2248–2253. doi: 10.1101/gad.173922.111. 
Laprade, H. et al. (2020) ‘Single-Molecule Imaging of Telomerase RNA Reveals a Recruitment-Retention 
Model for Telomere Elongation’, Molecular Cell. Cell Press, 79(1), pp. 115-126.e6. doi: 
10.1016/j.molcel.2020.05.005. 
Latrick, C. M. and Cech, T. R. (2010) ‘POT1-TPP1 enhances telomerase processivity by slowing primer 
dissociation and aiding translocation’, EMBO Journal. European Molecular Biology Organization, 29(5), 
pp. 924–933. doi: 10.1038/emboj.2009.409. 
Lee, O. H. et al. (2011) ‘Genome-wide YFP fluorescence complementation screen identifies new 
regulators for telomere signaling in human cells’, Molecular and Cellular Proteomics. American Society 
for Biochemistry and Molecular Biology, 10(2). doi: 10.1074/mcp.M110.001628. 
Lee, Y. W. et al. (2018) ‘TRF1 participates in chromosome end protection by averting TRF2-dependent 
telomeric R loops’, Nature Structural and Molecular Biology. Nature Publishing Group, 25(2), pp. 147–
153. doi: 10.1038/s41594-017-0021-5. 
LeGuen, T. et al. (2013) ‘Human RTEL1 deficiency causes hoyeraal-hreidarsson syndrome with short 
telomeres and genome instability’, Human Molecular Genetics. Hum Mol Genet, 22(16), pp. 3239–
3249. doi: 10.1093/hmg/ddt178. 
Lei, M., Podell, E. R. and Cech, T. R. (2004) ‘Structure of human POT1 bound to telomeric single-
stranded DNA provides a model for chromosome end-protection’, Nature Structural and Molecular 
Biology. Nature Publishing Group, 11(12), pp. 1223–1229. doi: 10.1038/nsmb867. 
Lenain, C. et al. (2006) ‘The Apollo 5’ exonuclease functions together with TRF2 to protect telomeres 
from DNA repair.’, Current biology : CB. Cell Press, 16(13), pp. 1303–10. doi: 
10.1016/j.cub.2006.05.021. 
Li, B. and De Lange, T. (2003) ‘Rap1 Affects the Length and Heterogeneity of Human Telomeres’, 
Molecular Biology of the Cell. American Society for Cell Biology, 14(12), pp. 5060–5068. doi: 
10.1091/mbc.E03-06-0403. 
Li, B. and Lustig, A. J. (1996) ‘A novel mechanism for telomere size control in Saccharomyces cerevisiae’, 
Genes and Development. Cold Spring Harbor Laboratory Press, 10(11), pp. 1310–1326. doi: 
10.1101/gad.10.11.1310. 
Li, B., Oestreich, S. and De Lange, T. (2000) ‘Identification of human Rap1: Implications for telomere 
evolution’, Cell. Cell, 101(5), pp. 471–483. doi: 10.1016/S0092-8674(00)80858-2. 
Li, J. S. Z. et al. (2017) ‘TZAP: A telomere-associated protein involved in telomere length control’, 
Science. American Association for the Advancement of Science, 355(6325), pp. 638–641. doi: 
10.1126/science.aah6752. 
Liao, Y., Smyth, G. K. and Shi, W. (2014) ‘FeatureCounts: An efficient general purpose program for 
116 
 
assigning sequence reads to genomic features’, Bioinformatics, 30(7), pp. 923–930. doi: 
10.1093/bioinformatics/btt656. 
Lillard-Wetherell, K. et al. (2004) ‘Association and regulation of the BLM helicase by the telomere 
proteins TRF1 and TRF2’, Human Molecular Genetics, 13(17), pp. 1919–1932. doi: 
10.1093/hmg/ddh193. 
Lim, C. J. et al. (2017) ‘Reconstitution of human shelterin complexes reveals unexpected stoichiometry 
and dual pathways to enhance telomerase processivity’, Nature Communications. Nature Publishing 
Group, 8(1). doi: 10.1038/s41467-017-01313-w. 
Lim, C. J. and Cech, T. R. (2021) ‘Shaping human telomeres: from shelterin and CST complexes to 
telomeric chromatin organization’, Nature Reviews Molecular Cell Biology. Springer Science and 
Business Media LLC, pp. 1–16. doi: 10.1038/s41580-021-00328-y. 
Lin, J. et al. (2014) ‘TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel 
mechanism to search for protein partners at telomeres’, Nucleic Acids Research. Oxford Academic, 
42(4), pp. 2493–2504. doi: 10.1093/NAR/GKT1132. 
Lindsay, H. et al. (2016) ‘CrispRVariants charts the mutation spectrum of genome engineering 
experiments’, Nature Biotechnology. Nature Publishing Group, pp. 701–702. doi: 10.1038/nbt.3628. 
London, T. B. C. et al. (2008) ‘FANCJ is a structure-specific DNA helicase associated with the 
maintenance of genomic G/C tracts’, Journal of Biological Chemistry. J Biol Chem, 283(52), pp. 36132–
36139. doi: 10.1074/jbc.M808152200. 
Londoño-Vallejo, J. A. et al. (2004) ‘Alternative Lengthening of Telomeres Is Characterized by High 
Rates of Telomeric Exchange’, Cancer Research. American Association for Cancer Research, 64(7), pp. 
2324–2327. doi: 10.1158/0008-5472.CAN-03-4035. 
Lototska, L. et al. (2020) ‘ Human RAP 1 specifically protects telomeres of senescent cells from DNA 
damage ’, EMBO reports. EMBO, 21(4). doi: 10.15252/embr.201949076. 
Love, M. I., Huber, W. and Anders, S. (2014) ‘Moderated estimation of fold change and dispersion for 
RNA-seq data with DESeq2’, Genome Biology. BioMed Central Ltd., 15(12), p. 550. doi: 
10.1186/s13059-014-0550-8. 
Van Ly, D. et al. (2018) ‘Telomere Loop Dynamics in Chromosome End Protection’, Molecular Cell. Cell 
Press, 71(4), pp. 510-525.e6. doi: 10.1016/j.molcel.2018.06.025. 
Maciejowski, J. et al. (2015) ‘Chromothripsis and Kataegis Induced by Telomere Crisis’, Cell. Cell Press, 
163(7), pp. 1641–1654. doi: 10.1016/j.cell.2015.11.054. 
Maciejowski, J. and De Lange, T. (2017) ‘Telomeres in cancer: Tumour suppression and genome 
instability’, Nature Reviews Molecular Cell Biology. Nature Publishing Group, pp. 175–186. doi: 
10.1038/nrm.2016.171. 
Maestroni, L., Matmati, S. and Coulon, S. (2017) ‘Solving the telomere replication problem’, Genes. 
MDPI AG. doi: 10.3390/genes8020055. 
Makarov, V. L., Hirose, Y. and Langmore, J. P. (1997) ‘Long G tails at both ends of human chromosomes 
suggest a C strand degradation mechanism for telomere shortening’, Cell. Elsevier B.V., 88(5), pp. 657–
666. doi: 10.1016/S0092-8674(00)81908-X. 
117 
 
Di Maro, S. et al. (2014) ‘Shading the TRF2 recruiting function: A new horizon in drug development’, 
Journal of the American Chemical Society. American Chemical Society, 136(48), pp. 16708–16711. doi: 
10.1021/ja5080773. 
Martin, M. (2011) ‘Cutadapt removes adapter sequences from high-throughput sequencing reads’, 
EMBnet.journal. EMBnet Stichting, 17(1), p. 10. doi: 10.14806/ej.17.1.200. 
Martinez, P. et al. (2010) ‘Mammalian Rap1 controls telomere function and gene expression through 
binding to telomeric and extratelomeric sites’, Nature Cell Biology. Europe PMC Funders, 12(8), pp. 
768–780. doi: 10.1038/ncb2081. 
Martínez, P. et al. (2009) ‘Increased telomere fragility and fusions resulting from TRF1 deficiency lead 
to degenerative pathologies and increased cancer in mice’, Genes and Development. Genes Dev, 
23(17), pp. 2060–2075. doi: 10.1101/gad.543509. 
Martínez, P. et al. (2016) ‘A genetic interaction between RAP1 and telomerase reveals an unanticipated 
role for RAP1 in telomere maintenance’, Aging Cell. Blackwell Publishing Ltd, 15(6), pp. 1113–1125. 
doi: 10.1111/acel.12517. 
Marzec, P. et al. (2015) ‘Nuclear-Receptor-Mediated Telomere Insertion Leads to Genome Instability 
in ALT Cancers’, Cell. Cell Press, 160(5), pp. 913–927. doi: 10.1016/j.cell.2015.01.044. 
Masella, A. P. et al. (2012) ‘PANDAseq: Paired-end assembler for illumina sequences’, BMC 
Bioinformatics. BMC Bioinformatics, 13(1). doi: 10.1186/1471-2105-13-31. 
Mattern, K. A. et al. (2004) ‘Dynamics of Protein Binding to Telomeres in Living Cells: Implications for 
Telomere Structure and Function’, Molecular and Cellular Biology. American Society for Microbiology, 
24(12), pp. 5587–5594. doi: 10.1128/mcb.24.12.5587-5594.2004. 
McClintock, B. (1941) ‘The Stability of Broken Ends of Chromosomes in Zea Mays.’, Genetics, 26(2), pp. 
234–82. doi: 10.1093/genetics/26.2.234. 
McElligott, R. and Wellinger, R. J. (1997) ‘The terminal DNA structure of mammalian chromosomes’, 
EMBO Journal. John Wiley & Sons, Ltd, 16(12), pp. 3705–3714. doi: 10.1093/emboj/16.12.3705. 
McHugh, D. and Gil, J. (2018) ‘Senescence and aging: Causes, consequences, and therapeutic avenues’, 
Journal of Cell Biology. Rockefeller University Press, pp. 65–77. doi: 10.1083/jcb.201708092. 
Mei, Y. et al. (2021) ‘TERRA G-quadruplex RNA interaction with TRF2 GAR domain is required for 
telomere integrity’, Scientific Reports. Springer Science and Business Media LLC, 11(1). doi: 
10.1038/s41598-021-82406-x. 
Mendez-Bermudez, A. et al. (2018) ‘Genome-wide Control of Heterochromatin Replication by the 
Telomere Capping Protein TRF2’, Molecular Cell. Cell Press, 70(3), pp. 449-461.e5. doi: 
10.1016/j.molcel.2018.03.036. 
Min, J., Wright, W. E. and Shay, J. W. (2017) ‘Alternative Lengthening of Telomeres Mediated by Mitotic 
DNA Synthesis Engages Break-Induced Replication Processes’, Molecular and Cellular Biology. 
American Society for Microbiology, 37(20). doi: 10.1128/mcb.00226-17. 
Montero, J. J. et al. (2016) ‘Telomeric RNAs are essential to maintain telomeres’, Nature 
Communications. Nature Publishing Group, 7. doi: 10.1038/ncomms12534. 
Montero, J. J. et al. (2018) ‘TERRA recruitment of polycomb to telomeres is essential for histone 
118 
 
trymethylation marks at telomeric heterochromatin’, Nature Communications. Nature Publishing 
Group, 9(1). doi: 10.1038/s41467-018-03916-3. 
Morin, G. B. (1989) ‘The human telomere terminal transferase enzyme is a ribonucleoprotein that 
synthesizes TTAGGG repeats’, Cell. Cell, 59(3), pp. 521–529. doi: 10.1016/0092-8674(89)90035-4. 
Muller, J. H. (1938) ‘The remaking of chromosomes’, The collectiong Net, XIII(116). 
Nabetani, A. and Ishikawa, F. (2009) ‘Unusual Telomeric DNAs in Human Telomerase-Negative 
Immortalized Cells’, Molecular and Cellular Biology. American Society for Microbiology, 29(3), pp. 703–
713. doi: 10.1128/mcb.00603-08. 
Nandakumar, J. et al. (2012) ‘The TEL patch of telomere protein TPP1 mediates telomerase recruitment 
and processivity’, Nature. NIH Public Access, 492(7428), pp. 285–289. doi: 10.1038/nature11648. 
Nassour, J. et al. (2019) ‘Autophagic cell death restricts chromosomal instability during replicative 
crisis’, Nature. Nature Publishing Group, 565(7741), pp. 659–663. doi: 10.1038/s41586-019-0885-0. 
Nečasová, I. et al. (2017) ‘Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas 
Rap1 restores it’, Nucleic Acids Research. Oxford University Press, 45(21), pp. 12170–12180. doi: 
10.1093/nar/gkx812. 
Nergadze, S. G. et al. (2009) ‘CpG-island promoters drive transcription of human telomeres’, RNA. RNA, 
15(12), pp. 2186–2194. doi: 10.1261/rna.1748309. 
Nguyen, T. H. D. et al. (2018) ‘Cryo-EM structure of substrate-bound human telomerase holoenzyme’, 
Nature. Nature Publishing Group, 557(7704), pp. 190–195. doi: 10.1038/s41586-018-0062-x. 
Nishikawa, T. et al. (2001) ‘Solution structure of a telomeric DNA complex of human TRF1’, Structure. 
Cell Press, 9(12), pp. 1237–1251. doi: 10.1016/S0969-2126(01)00688-8. 
Nittis, T. et al. (2010) ‘Revealing novel telomere proteins using in vivo cross-linking, tandem affinity 
purification, and label-free quantitative LC-FTICR-MS’, Molecular and Cellular Proteomics. American 
Society for Biochemistry and Molecular Biology, 9(6), pp. 1144–1156. doi: 10.1074/mcp.M900490-
MCP200. 
van Nuland, R. et al. (2013) ‘Quantitative Dissection and Stoichiometry Determination of the Human 
SET1/MLL Histone Methyltransferase Complexes’, Molecular and Cellular Biology. American Society 
for Microbiology, 33(10), pp. 2067–2077. doi: 10.1128/mcb.01742-12. 
O’Sullivan, R. J. et al. (2014) ‘Rapid induction of alternative lengthening of telomeres by depletion of 
the histone chaperone ASF1’, Nature Structural and Molecular Biology. NIH Public Access, 21(2), pp. 
167–174. doi: 10.1038/nsmb.2754. 
Okamoto, K. et al. (2013) ‘A two-step mechanism for TRF2-mediated chromosome-end protection’, 
Nature. Nature Publishing Group, 494(7438), pp. 502–505. doi: 10.1038/nature11873. 
Oliva-Rico, D. and Herrera, L. A. (2017) ‘Regulated expression of the lncRNA TERRA and its impact on 
telomere biology’, Mechanisms of Ageing and Development. Elsevier Ireland Ltd, pp. 16–23. doi: 
10.1016/j.mad.2017.09.001. 
Olovnikov, A. M. (1971) ‘[Principle of marginotomy in template synthesis of polynucleotides].’, Doklady 
Akademii nauk SSSR, 201(6), pp. 1496–9. Available at: 
http://www.ncbi.nlm.nih.gov/pubmed/5158754. 
119 
 
Olovnikov, A. M. (1996) ‘Telomeres, telomerase, and aging: Origin of the theory’, Experimental 
Gerontology. Elsevier Inc., 31(4), pp. 443–448. doi: 10.1016/0531-5565(96)00005-8. 
Opresko, P. L. et al. (2002) ‘Telomere-binding protein TRF2 binds to and stimulates the Werner and 
Bloom syndrome helicases’, Journal of Biological Chemistry. J Biol Chem, 277(43), pp. 41110–41119. 
doi: 10.1074/jbc.M205396200. 
Opresko, P. L. et al. (2005) ‘Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2’, 
Nucleic Acids Research. Oxford University Press, 33(4), pp. 1230–1239. doi: 10.1093/nar/gki273. 
Ouellette, M. M. et al. (2000) ‘Subsenescent telomere lengths in fibroblasts immortalized by limiting 
amounts of telomerase’, Journal of Biological Chemistry. Elsevier, 275(14), pp. 10072–10076. doi: 
10.1074/jbc.275.14.10072. 
van Overbeek, M. and de Lange, T. (2006) ‘Apollo, an Artemis-Related Nuclease, Interacts with TRF2 
and Protects Human Telomeres in S Phase’, Current Biology, 16(13), pp. 1295–1302. doi: 
10.1016/j.cub.2006.05.022. 
Palm, W. and De Lange, T. (2008) ‘How shelterin protects mammalian telomeres’, Annual Review of 
Genetics. Annu Rev Genet, pp. 301–334. doi: 10.1146/annurev.genet.41.110306.130350. 
Park, R. J. et al. (2017) ‘A genome-wide CRISPR screen identifies a restricted set of HIV host dependency 
factors’, Nature Genetics. Nature Publishing Group, 49(2), pp. 193–203. doi: 10.1038/ng.3741. 
Pickett, H. A. et al. (2009) ‘Control of telomere length by a trimming mechanism that involves 
generation of t-circles’, EMBO Journal. European Molecular Biology Organization, 28(7), pp. 799–809. 
doi: 10.1038/emboj.2009.42. 
Pickett, H. A. et al. (2011) ‘Normal mammalian cells negatively regulate telomere length by telomere 
trimming’, Human Molecular Genetics. Oxford Academic, 20(23), pp. 4684–4692. doi: 
10.1093/hmg/ddr402. 
Pike, A. M. et al. (2019) ‘TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity’, 
Molecular and Cellular Biology. American Society for Microbiology, 39(21). doi: 10.1128/mcb.00593-
18. 
Pinzaru, A. M. et al. (2016) ‘Telomere Replication Stress Induced by POT1 Inactivation Accelerates 
Tumorigenesis’, Cell Reports. Elsevier B.V., 15(10), pp. 2170–2184. doi: 10.1016/j.celrep.2016.05.008. 
Pinzaru, A. M. et al. (2020) ‘Replication stress conferred by POT1 dysfunction promotes telomere 
relocalization to the nuclear pore’, Genes and Development. Cold Spring Harbor Laboratory Press, 
34(23–24), pp. 1619–1636. doi: 10.1101/gad.337287.120. 
Popuri, V. et al. (2014) ‘Human RECQL1 participates in telomere maintenance’, Nucleic Acids Research. 
Oxford University Press, 42(9), pp. 5671–5688. doi: 10.1093/nar/gku200. 
Porro, A. et al. (2010) ‘Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils 
the Presence of Distinct and Multiple Regulatory Pathways’, Molecular and Cellular Biology. American 
Society for Microbiology, 30(20), pp. 4808–4817. doi: 10.1128/mcb.00460-10. 
Porro, A. et al. (2014) ‘Functional characterization of the TERRA transcriptome at damaged telomeres’, 
Nature Communications. Nature Publishing Group, 5, p. 5379. doi: 10.1038/ncomms6379. 
Porro, A., Feuerhahn, S. and Lingner, J. (2014) ‘TERRA-Reinforced Association of LSD1 with MRE11 
120 
 
Promotes Processing of Uncapped Telomeres’, Cell Reports. Elsevier, 6(4), pp. 765–776. doi: 
10.1016/j.celrep.2014.01.022. 
Potts, P. R., Porteus, M. H. and Yu, H. (2006) ‘Human SMC5/6 complex promotes sister chromatid 
homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks’, 
EMBO Journal. EMBO J, 25(14), pp. 3377–3388. doi: 10.1038/sj.emboj.7601218. 
Potts, P. R. and Yu, H. (2007) ‘The SMC5/6 complex maintains telomere length in ALT cancer cells 
through SUMOylation of telomere-binding proteins’, Nature Structural and Molecular Biology. Nature 
Publishing Group, 14(7), pp. 581–590. doi: 10.1038/nsmb1259. 
Poulet, A. et al. (2009) ‘TRF2 promotes, remodels and protects telomeric Holliday junctions’, The EMBO 
Journal. Nature Publishing Group, 28(6), pp. 641–651. doi: 10.1038/emboj.2009.11. 
R Core Team (2017) R: A Language and Environment for Statistical Computing. (no date). Available at: 
http://www.r-project.org/. 
R Development Core Team (2014) ‘R: A language and evironment for statistical computing’. R 
Foundation for Statistical Computing, Vienna, Austria. 
Rai, R. et al. (2016) ‘TRF2-RAP1 is required to protect telomeres from engaging in homologous 
recombination-mediated deletions and fusions’, Nature Communications. Nature Publishing Group, 7. 
doi: 10.1038/ncomms10881. 
Rajavel, M., Mullins, M. R. and Taylor, D. J. (2014) ‘Multiple facets of TPP1 in telomere maintenance’, 
Biochimica et Biophysica Acta - Proteins and Proteomics. Elsevier, pp. 1550–1559. doi: 
10.1016/j.bbapap.2014.04.014. 
Ramírez, F. et al. (2016) ‘deepTools2: a next generation web server for deep-sequencing data analysis’, 
Nucleic acids research. Nucleic Acids Res, 44(W1), pp. W160–W165. doi: 10.1093/nar/gkw257. 
Ran, F. A. et al. (2013) ‘Genome engineering using the CRISPR-Cas9 system’, Nature Protocols. Nat 
Protoc, 8(11), pp. 2281–2308. doi: 10.1038/nprot.2013.143. 
Rappsilber, J., Mann, M. and Ishihama, Y. (2007) ‘Protocol for micro-purification, enrichment, pre-
fractionation and storage of peptides for proteomics using StageTips’, Nature Protocols. Nature 
Publishing Group, 2(8), pp. 1896–1906. doi: 10.1038/nprot.2007.261. 
Ray, S. et al. (2014) ‘G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA 
binding’, Proceedings of the National Academy of Sciences of the United States of America. National 
Academy of Sciences, 111(8), pp. 2990–2995. doi: 10.1073/pnas.1321436111. 
Richards, E. J. and Ausubel, F. M. (1988) ‘Isolation of a higher eukaryotic telomere from Arabidopsis 
thaliana’, Cell. Cell Press, 53(1), pp. 127–136. doi: 10.1016/0092-8674(88)90494-1. 
Riethman, H., Ambrosini, A. and Paul, S. (2005) ‘Human subtelomere structure and variation’, 
Chromosome Research. Chromosome Res, pp. 505–515. doi: 10.1007/s10577-005-0998-1. 
Ritschka, B. et al. (2017) ‘The senescence-associated secretory phenotype induces cellular plasticity 
and tissue regeneration’, Genes and Development. Cold Spring Harbor Laboratory Press, 31(2), pp. 
172–183. doi: 10.1101/gad.290635.116. 
Rivera, T. et al. (2017) ‘A balance between elongation and trimming regulates telomere stability in 
stem cells’, Nature Structural and Molecular Biology. Nature Publishing Group, 24(1), pp. 30–39. doi: 
121 
 
10.1038/nsmb.3335. 
Rossiello, F. et al. (2017) ‘DNA damage response inhibition at dysfunctional telomeres by modulation 
of telomeric DNA damage response RNAs’, Nature Communications. Nature Publishing Group, 8. doi: 
10.1038/ncomms13980. 
Roumelioti, F. et al. (2016) ‘ Alternative lengthening of human telomeres is a conservative DNA 
replication process with features of break‐induced replication ’, EMBO reports. EMBO, 17(12), pp. 
1731–1737. doi: 10.15252/embr.201643169. 
Sarek, G. et al. (2015) ‘TRF2 recruits RTEL1 to telomeres in s phase to promote t-loop unwinding’, 
Molecular Cell. Cell Press, 57(4), pp. 622–635. doi: 10.1016/j.molcel.2014.12.024. 
Sarek, G. et al. (2019) ‘CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle’, 
Nature. Nature Research, 575(7783), pp. 523–527. doi: 10.1038/s41586-019-1744-8. 
Sarthy, J. et al. (2009) ‘Human RAP1 inhibits non-homologous end joining at telomeres’, EMBO Journal. 
European Molecular Biology Organization, 28(21), pp. 3390–3399. doi: 10.1038/emboj.2009.275. 
Schmutz, I. et al. (2017) ‘TRF2 binds branched DNA to safeguard telomere integrity’. doi: 
10.1038/nsmb.3451. 
Schoeftner, S. and Blasco, M. A. (2008) ‘Developmentally regulated transcription of mammalian 
telomeres by DNA-dependent RNA polymerase II’, Nature Cell Biology. Nat Cell Biol, 10(2), pp. 228–
236. doi: 10.1038/ncb1685. 
Schoeftner, S. and Blasco, M. A. (2010) ‘Chromatin regulation and non-coding RNAs at mammalian 
telomeres’, Seminars in Cell and Developmental Biology. Elsevier Ltd, pp. 186–193. doi: 
10.1016/j.semcdb.2009.09.015. 
Scholz, J. et al. (2013) ‘A new method to customize protein expression vectors for fast, efficient and 
background free parallel cloning’, BMC Biotechnology. BMC Biotechnol, 13. doi: 10.1186/1472-6750-
13-12. 
Sekne, Z. et al. (2022) ‘Structural basis of human telomerase recruitment by TPP1-POT1’, Science. 
American Association for the Advancement of Science, 375(6585), pp. 1173–1176. doi: 
10.1126/science.abn6840. 
Sfeir, A. et al. (2009) ‘Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient 
Replication’, Cell. Cell, 138(1), pp. 90–103. doi: 10.1016/j.cell.2009.06.021. 
Sfeir, A. et al. (2010) ‘Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA 
damage signal’, Science. Science, 327(5973), pp. 1657–1661. doi: 10.1126/science.1185100. 
Sfeir, A. J. et al. (2005) ‘Telomere-end processing: The terminal nucleotidesof human chromosomes’, 
Molecular Cell. Cell Press, 18(1), pp. 131–138. doi: 10.1016/j.molcel.2005.02.035. 
Sfeir, A. and De Lange, T. (2012) ‘Removal of shelterin reveals the telomere end-protection problem’, 
Science. American Association for the Advancement of Science, 336(6081), pp. 593–597. doi: 
10.1126/science.1218498. 
Shay, J. W. and Wright, W. E. (2011) ‘Role of telomeres and telomerase in cancer’, Seminars in Cancer 
Biology. NIH Public Access, pp. 349–353. doi: 10.1016/j.semcancer.2011.10.001. 
122 
 
Shevchenko, A. et al. (2007) ‘In-gel digestion for mass spectrometric characterization of proteins and 
proteomes’, Nature Protocols. Nat Protoc, 1(6), pp. 2856–2860. doi: 10.1038/nprot.2006.468. 
De Silanes, I. L. et al. (2014) ‘Identification of TERRA locus unveils a telomere protection role through 
association to nearly all chromosomes’, Nature Communications. Nature Publishing Group, 5. doi: 
10.1038/ncomms5723. 
De Silanes, I. L., D’Alcontres, M. S. and Blasco, M. A. (2010) ‘TERRA transcripts are bound by a complex 
array of RNA-binding proteins’, Nature Communications. Nature Publishing Group, 1(3). doi: 
10.1038/ncomms1032. 
Simboeck, E. et al. (2013) ‘DPY30 regulates pathways in cellular senescence through ID protein 
expression’, EMBO Journal. European Molecular Biology Organization, 32(16), pp. 2217–2230. doi: 
10.1038/emboj.2013.159. 
Smogorzewska, A. et al. (2000) ‘Control of Human Telomere Length by TRF1 and TRF2’, Molecular and 
Cellular Biology. American Society for Microbiology, 20(5), pp. 1659–1668. doi: 
10.1128/mcb.20.5.1659-1668.2000. 
Soman, A. et al. (2022) ‘Columnar structure of human telomeric chromatin’, Nature. Nature Publishing 
Group, pp. 1–8. doi: 10.1038/s41586-022-05236-5. 
Stansel, R. M., De Lange, T. and Griffith, J. D. (2001) ‘T-loop assembly in vitro involves binding of TRF2 
near the 3′ telomeric overhang’, EMBO Journal. John Wiley & Sons, Ltd, 20(19), pp. 5532–5540. doi: 
10.1093/emboj/20.19.5532. 
Van Steensel, B. and De Lange, T. (1997) ‘Control of telomere length by the human telomeric protein 
TRF1’, Nature. Nature, 385(6618), pp. 740–743. doi: 10.1038/385740a0. 
Van Steensel, B., Smogorzewska, A. and De Lange, T. (1998) ‘TRF2 protects human telomeres from end-
to-end fusions’, Cell. Cell Press, 92(3), pp. 401–413. doi: 10.1016/S0092-8674(00)80932-0. 
Stephan, A. K., Kliszczak, M. and Morrison, C. G. (2011) ‘The Nse2/Mms21 SUMO ligase of the Smc5/6 
complex in the maintenance of genome stability’, FEBS Letters. FEBS Lett, pp. 2907–2913. doi: 
10.1016/j.febslet.2011.04.067. 
Summers, P. A. et al. (2021) ‘Visualising G-quadruplex DNA dynamics in live cells by fluorescence 
lifetime imaging microscopy’, Nature Communications. Nature Research, 12(1). doi: 10.1038/s41467-
020-20414-7. 
Sun, H. et al. (1998) ‘The Bloom’s syndrome helicase unwinds G4 DNA’, Journal of Biological Chemistry. 
J Biol Chem, 273(42), pp. 27587–27592. doi: 10.1074/jbc.273.42.27587. 
Sundquist, W. I. and Klug, A. (1989) ‘Telomeric DNA dimerizes by formation of guanine tetrads between 
hairpin loops’, Nature. Nature Publishing Group, 342(6251), pp. 825–829. doi: 10.1038/342825a0. 
Tacconi, E. M. C. and Tarsounas, M. (2015) ‘How homologous recombination maintains telomere 
integrity’, Chromosoma. Springer Science and Business Media Deutschland GmbH, pp. 119–130. doi: 
10.1007/s00412-014-0497-2. 
Takai, K. K. et al. (2010) ‘In vivo stoichiometry of shelterin components’, Journal of Biological Chemistry. 
American Society for Biochemistry and Molecular Biology, 285(2), pp. 1457–1467. doi: 
10.1074/jbc.M109.038026. 
123 
 
Takai, K. K. et al. (2011) ‘Telomere Protection by TPP1/POT1 Requires Tethering to TIN2’, Molecular 
Cell. Cell Press, 44(4), pp. 647–659. doi: 10.1016/j.molcel.2011.08.043. 
Tang, J. et al. (2008) ‘G-quadruplex preferentially forms at the very 3′ end of vertebrate telomeric DNA’, 
Nucleic Acids Research. Oxford University Press, 36(4), pp. 1200–1208. doi: 10.1093/nar/gkm1137. 
Taylor, D. J. et al. (2011) ‘Multiple POT1-TPP1 proteins coat and compact long telomeric single-
stranded DNA’, Journal of Molecular Biology. Academic Press, 410(1), pp. 10–17. doi: 
10.1016/j.jmb.2011.04.049. 
Tejera, A. M. et al. (2010) ‘TPP1 is required for TERT recruitment, telomere elongation during nuclear 
reprogramming, and normal skin development in mice’, Developmental Cell. Europe PMC Funders, 
18(5), pp. 775–789. doi: 10.1016/j.devcel.2010.03.011. 
Theimer, C. A. et al. (2007) ‘Structural and Functional Characterization of Human Telomerase RNA 
Processing and Cajal Body Localization Signals’, Molecular Cell. Mol Cell, 27(6), pp. 869–881. doi: 
10.1016/j.molcel.2007.07.017. 
Timashev, L. A. et al. (2017) ‘The DDR at telomeres lacking intact shelterin does not require substantial 
chromatin decompaction’, Genes & development. Genes Dev, 31(6), pp. 578–589. doi: 
10.1101/gad.294108.116. 
Touzot, F. et al. (2012) ‘Heterogeneous telomere defects in patients with severe forms of dyskeratosis 
congenita’, Journal of Allergy and Clinical Immunology. Mosby Inc., 129(2), pp. 473-482.e3. doi: 
10.1016/j.jaci.2011.09.043. 
Tremblay, V. et al. (2014) ‘Molecular basis for DPY-30 association to COMPASS-like and NURF 
complexes’, Structure. Cell Press, 22(12), pp. 1821–1830. doi: 10.1016/j.str.2014.10.002. 
Turner, K., Vasu, V. and Griffin, D. (2019) ‘Telomere Biology and Human Phenotype’, Cells. MDPI AG, 
8(1), p. 73. doi: 10.3390/cells8010073. 
Uringa, E.-J. et al. (2012) ‘RTEL1 contributes to DNA replication and repair and telomere maintenance’, 
Molecular Biology of the Cell. Edited by A. G. Matera.  The American Society for Cell Biology , 23(14), 
pp. 2782–2792. doi: 10.1091/mbc.e12-03-0179. 
Vannier, J.-B. et al. (2013) ‘RTEL1 is a replisome-associated helicase that promotes telomere and 
genome-wide replication.’, Science (New York, N.Y.). American Association for the Advancement of 
Science, 342(6155), pp. 239–42. doi: 10.1126/science.1241779. 
Vannier, J. B. et al. (2012) ‘RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain 
telomere integrity’, Cell. Cell, 149(4), pp. 795–806. doi: 10.1016/j.cell.2012.03.030. 
Venteicher, A. S. et al. (2009) ‘A human telomerase holoenzyme protein required for Cajal body 
localization and telomere synthesis’, Science. Science, 323(5914), pp. 644–648. doi: 
10.1126/science.1165357. 
Vera, E. et al. (2012) ‘The Rate of Increase of Short Telomeres Predicts Longevity in Mammals’, Cell 
Reports. Cell Press, 2(4), pp. 732–737. doi: 10.1016/j.celrep.2012.08.023. 
Veverka, Janovič and Hofr (2019) ‘Quantitative Biology of Human Shelterin and Telomerase: Searching 
for the Weakest Point’, International Journal of Molecular Sciences. MDPI AG, 20(13), p. 3186. doi: 
10.3390/ijms20133186. 
124 
 
Viceconte, N. et al. (2017) ‘Highly Aggressive Metastatic Melanoma Cells Unable to Maintain Telomere 
Length’, Cell Reports. Elsevier B.V., 19(12), pp. 2529–2543. doi: 10.1016/j.celrep.2017.05.046. 
Victorelli, S. and Passos, J. F. (2017) ‘Telomeres and Cell Senescence - Size Matters Not’, EBioMedicine. 
Elsevier B.V., pp. 14–20. doi: 10.1016/j.ebiom.2017.03.027. 
De Vitis, M., Berardinelli, F. and Sgura, A. (2018) ‘Telomere length maintenance in cancer: At the 
crossroad between telomerase and alternative lengthening of telomeres (ALT)’, International Journal 
of Molecular Sciences. MDPI AG. doi: 10.3390/ijms19020606. 
Voronina, N. et al. (2020) ‘The landscape of chromothripsis across adult cancer types’, Nature 
Communications. Nature Research, 11(1). doi: 10.1038/s41467-020-16134-7. 
Vulliamy, T. J. et al. (2006) ‘Mutations in dyskeratosis congenita: Their impact on telomere length and 
the diversity of clinical presentation’, Blood. Blood, 107(7), pp. 2680–2685. doi: 10.1182/blood-2005-
07-2622. 
Wan, M. et al. (2009) ‘OB fold-containing protein 1 (OBFC1), a human homolog of yeast Stn1, associates 
with TPP1 and is implicated in telomere length regulation’, Journal of Biological Chemistry. J Biol Chem, 
284(39), pp. 26725–26731. doi: 10.1074/jbc.M109.021105. 
Wang, F. et al. (2012) ‘Human CST Has Independent Functions during Telomere Duplex Replication and 
C-Strand Fill-In’, Cell Reports, 2(5), pp. 1096–1103. doi: 10.1016/j.celrep.2012.10.007. 
Watson, J. D. (1972) ‘Origin of concatemeric T7 DNA’, Nature New Biology. Nat New Biol, 239(94), pp. 
197–201. doi: 10.1038/newbio239197a0. 
Wellinger, R. J. and Zakian, V. A. (2012) ‘Everything you ever wanted to know about Saccharomyces 
cerevisiae telomeres: Beginning to end’, Genetics. Oxford University Press, pp. 1073–1105. doi: 
10.1534/genetics.111.137851. 
Whittemore, K. et al. (2019) ‘Telomere shortening rate predicts species life span’, Proceedings of the 
National Academy of Sciences of the United States of America. National Academy of Sciences, 116(30), 
pp. 15122–15127. doi: 10.1073/pnas.1902452116. 
Wickham, H. (2016) HadleyyWickham ggplot2 Elegant Graphics for Data Analysis Second Edition. 
Available at: http://www.springer.com/series/6991 (Accessed: 14 February 2022). 
Wicky, C. et al. (1996) ‘Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function 
in Caenorhabditis elegans’, Proceedings of the National Academy of Sciences of the United States of 
America. National Academy of Sciences, 93(17), pp. 8983–8988. doi: 10.1073/pnas.93.17.8983. 
Williamson, J. R., Raghuraman, M. K. and Cech, T. R. (1989) ‘Monovalent cation-induced structure of 
telomeric DNA: The G-quartet model’, Cell. Cell Press, 59(5), pp. 871–880. doi: 10.1016/0092-
8674(89)90610-7. 
Wood, A. M. et al. (2014) ‘TRF2 and lamin A/C interact to facilitate the functional organization of 
chromosome ends’, Nature Communications. Nature Publishing Group, 5(1), pp. 1–9. doi: 
10.1038/ncomms6467. 
Wright, W. E. et al. (1997) ‘Normal human chromosomes have long G-rich telomeric overhangs at one 
end’, Genes and Development. Cold Spring Harbor Laboratory Press, 11(21), pp. 2801–2809. doi: 
10.1101/gad.11.21.2801. 
125 
 
Wu, G. et al. (2014) ‘PinX1, a novel target gene of p53, is suppressed by HPV16 E6 in cervical cancer 
cells’, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms. Elsevier B.V., 1839(2), pp. 88–96. 
doi: 10.1016/j.bbagrm.2014.01.004. 
Wu, L. et al. (2006) ‘Pot1 Deficiency Initiates DNA Damage Checkpoint Activation and Aberrant 
Homologous Recombination at Telomeres’, Cell. Elsevier B.V., 126(1), pp. 49–62. doi: 
10.1016/j.cell.2006.05.037. 
Wu, P. et al. (2010) ‘Apollo Contributes to G Overhang Maintenance and Protects Leading-End 
Telomeres’, Molecular Cell. Mol Cell, 39(4), pp. 606–617. doi: 10.1016/j.molcel.2010.06.031. 
Wu, P., Takai, H. and De Lange, T. (2012) ‘Telomeric 3′ overhangs derive from resection by Exo1 and 
apollo and fill-in by POT1b-associated CST’, Cell. Cell, 150(1), pp. 39–52. doi: 
10.1016/j.cell.2012.05.026. 
Wu, T. D. and Watanabe, C. K. (2005) ‘GMAP: A genomic mapping and alignment program for mRNA 
and EST sequences’, Bioinformatics. Bioinformatics, 21(9), pp. 1859–1875. doi: 
10.1093/bioinformatics/bti310. 
Wu, Y., Shin-ya, K. and Brosh, R. M. (2008) ‘FANCJ Helicase Defective in Fanconia Anemia and Breast 
Cancer Unwinds G-Quadruplex DNA To Defend Genomic Stability’, Molecular and Cellular Biology. 
American Society for Microbiology, 28(12), pp. 4116–4128. doi: 10.1128/mcb.02210-07. 
Xi, L. and Cech, T. R. (2014) ‘Inventory of telomerase components in human cells reveals multiple 
subpopulations of hTR and hTERT’, Nucleic Acids Research. Oxford University Press, 42(13), pp. 8565–
8577. doi: 10.1093/nar/gku560. 
Xin, H. et al. (2007) ‘TPP1 is a homologue of ciliate TEBP-β and interacts with POT1 to recruit 
telomerase’, Nature. Nature Publishing Group, 445(7127), pp. 559–562. doi: 10.1038/nature05469. 
Xu, M. et al. (2019) ‘Nuclear receptors regulate alternative lengthening of telomeres through a novel 
noncanonical FANCD2 pathway’, Science Advances. American Association for the Advancement of 
Science, 5(10). doi: 10.1126/sciadv.aax6366. 
Ye, J. et al. (2010) ‘TRF2 and Apollo Cooperate with Topoisomerase 2α to Protect Human Telomeres 
from Replicative Damage’, Cell. Elsevier B.V., 142(2), pp. 230–242. doi: 10.1016/j.cell.2010.05.032. 
Ye, J. Z. S. et al. (2004) ‘TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on 
telomeres’, Journal of Biological Chemistry. Elsevier, 279(45), pp. 47264–47271. doi: 
10.1074/jbc.M409047200. 
Yeager, T. R. et al. (1999) ‘Telomerase-negative Immortalized Human Cells Contain a Novel Type of 
Promyelocytic Leukemia (PML) Body’, Cancer Research, 59(17), pp. 4175 LP – 4179. Available at: 
http://cancerres.aacrjournals.org/content/59/17/4175.abstract. 
Yoo, J. E., Oh, B. K. and Park, Y. N. (2009) ‘Human PinX1 Mediates TRF1 Accumulation in Nucleolus and 
Enhances TRF1 Binding to Telomeres’, Journal of Molecular Biology. Academic Press, 388(5), pp. 928–
940. doi: 10.1016/j.jmb.2009.02.051. 
Yoo, J. E., Park, Y. N. and Oh, B. K. (2014) ‘Pinx1, a telomere repeat-binding factor 1 (TRF1)-interacting 
protein, maintains telomere integrity by modulating TRF1 homeostasis, the process in which human 
telomerase reverse transcriptase (hTERT) plays dual roles’, Journal of Biological Chemistry. American 
Society for Biochemistry and Molecular Biology Inc., 289(10), pp. 6886–6898. doi: 
10.1074/jbc.M113.506006. 
126 
 
Zahler, A. M. et al. (1991) ‘Inhibition of telomerase by G-quartet DMA structures’, Nature. Nature, 
350(6320), pp. 718–720. doi: 10.1038/350718a0. 
Zaug, A. J. et al. (2010) ‘Functional interaction between telomere protein TPP1 and telomerase’, Genes 
and Development. Cold Spring Harbor Laboratory Press, 24(6), pp. 613–622. doi: 
10.1101/gad.1881810. 
Zhang, B. et al. (2009) ‘Silencing PinX1 compromises telomere length maintenance as well as 
tumorigenicity in telomerase-positive human cancer cells’, Cancer Research. American Association for 
Cancer Research, 69(1), pp. 75–83. doi: 10.1158/0008-5472.CAN-08-1393. 
Zhang, J. M. et al. (2019) ‘Alternative Lengthening of Telomeres through Two Distinct Break-Induced 
Replication Pathways’, Cell Reports. Elsevier B.V., 26(4), pp. 955-968.e3. doi: 
10.1016/j.celrep.2018.12.102. 
Zhang, Y. et al. (2008) ‘Model-based analysis of ChIP-Seq (MACS)’, Genome Biology. Genome Biol, 9(9). 
doi: 10.1186/gb-2008-9-9-r137. 
Zhao, Y. et al. (2009) ‘Telomere Extension Occurs at Most Chromosome Ends and Is Uncoupled from 
Fill-In in Human Cancer Cells’, Cell. Cell, 138(3), pp. 463–475. doi: 10.1016/j.cell.2009.05.026. 
Zhao, Y. et al. (2018) ‘The 11th C2H2 zinc finger and an adjacent C-terminal arm are responsible for 
TZAP recognition of telomeric DNA’, Cell Research. Nature Publishing Group, pp. 130–134. doi: 
10.1038/cr.2017.141. 
Zhong, F. L. et al. (2012) ‘TPP1 OB-fold domain controls telomere maintenance by recruiting 
telomerase to chromosome ends’, Cell. Elsevier B.V., 150(3), pp. 481–494. doi: 
10.1016/j.cell.2012.07.012. 
Zhong, Z. et al. (1992) ‘A mammalian factor that binds telomeric TTAGGG repeats in vitro’, Molecular 
and Cellular Biology. American Society for Microbiology, 12(11), pp. 4834–4843. doi: 
10.1128/mcb.12.11.4834-4843.1992. 
Zhou, X. Z. and Lu, K. P. (2001) ‘The Pin2/TRF1-interacting protein PinX1 is a potent telomerase 
inhibitor’, Cell. Elsevier B.V., 107(3), pp. 347–359. doi: 10.1016/S0092-8674(01)00538-4. 
Zimmermann, M. et al. (2014) ‘TRF1 negotiates TTAGGG repeatassociated replication problems by 
recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling’, Genes and Development. 
Cold Spring Harbor Laboratory Press, 28(22), pp. 2477–2491. doi: 10.1101/gad.251611.114. 
Zou, Y. et al. (2004) ‘Does a sentinel or a subset of short telomeres determine replicative senescence?’, 
Molecular Biology of the Cell. American Society for Cell Biology, 15(8), pp. 3709–3718. doi: 
10.1091/mbc.E04-03-0207. 
127 
iv 
v