5-formylcytosine is an activating epigenetic mark for Pol III during Xenopus zygotic reprogramming

Abstract

Active DNA demethylation proceeds by the sequential oxidation of 5mC to 5hmC, 5fC and 5caC by TET proteins, followed by TDG-mediated removal of 5fC and 5caC to restore unmodified cytosine. While 5mC is an established epigenetic mark with known gene repression function, whether its oxidative derivatives have an instructive, physiologically relevant role of their own, apart from being DNA demethylation intermediates, remains unclear. Previous work from this lab showed that 5fC forms transient chromocenters at the perinucleolar compartment of Xenopus embryos during zygotic genome activation (ZGA). Moreover, 5fC was highly enriched on Pol III target genes at ZGA, notably at oocyte-type tandem arrayed tRNA genes. In this thesis, I address the question of what is the role of 5fC during Xenopus ZGA. I used methylation-assisted bisulfite sequencing to detect 5fC on selected tRNA genes at ZGA with base resolution. I found that 5fC marks all CpGs in and around tRNA-iMet and tRNA-Gly at ZGA at comparable levels. To erase global 5fC from early Xenopus embryos, I overexpressed human Tdg mRNA, which led to delayed embryonic development and lethality during gastrulation. Pol III chromatin immunoprecipitation in control and Tdg overexpressing embryos revealed that 5fC is required for Pol III binding at ZGA. By manipulating Tet and Tdg enzymes, I showed that 5fC is required for tRNA transcription during ZGA. To complement and extend this finding, I established a synthetic tRNA-iMet transgene assay, which showed that 5fC specifically promotes tRNA transcription during ZGA in vivo. Finally, efforts to identify a 5fC reader did not lead to the discovery of the mechanism by which 5fC exerts its gene activating role, which remains unresolved. Together, these results establish 5fC as an activating epigenetic mark for Pol III during zygotic reprogramming.