Elucidation of the cell-specific regulation of the novel human tumor gene NOT
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
License
Abstract
The human NOT gene is the homologue of the l(2)not gene from Drosophil and suggested to be relevant for diverse cellular processes including tumorous growth and N-glycosylation. This PhD thesis provides the first insight into the cell-specific mechanisms controlling the expression of the NOT gene. The first part focuses on the identification and characterization of NOT splice variants. This analysis revealed the existence of 17 NOT transcripts which, depending on the alternative first exon, can be separated into two groups. The transcripts NOT-1 and NOT-4 are translated. Most of the other NOT isoforms harbor premature stop codons and are degraded by the nonsense-mediated mRNA decay (NMD) pathway. The discovery of two different transcription start sites suggests the existence of two different NOT promoters that regulate the transcription of two pre-mRNAs from which all other isoforms are generated via alternative splicing.
The second part describes the identification of transcription factor binding sites within a putative 1.15kb NOT promoter region. Bioinformatics tools predicted 7 potential binding sites for the transcription factor EGR-1, 22 for NFκB, 16 for CREB, and 10 for MYCN. From these putative sites one EGR-1-binding site (nts 330-312), two MYCN-binding sites (nts 184-173 and 80-69), one CREB3-binding site (nts 217-206), and six NFκB binding sites (nts 973-951, 789-776, 768-755, 638-624, 339-330, and 223-214) were confirmed in vitro using the yeast one-hybrid, EMSA, ELISA, and affinity chromatography techniques. In vivo-binding analysis using chromatin immunoprecipitation (ChIP) showed cell-specific occupancy of the EGR-1 and MYCN binding elements, whereas CREB3 and NFκB bind in all investigated cell lines. In the case of NFκB, the number of occupied sites as well as the composition and phosphorylation of the bound NFκB dimers varies between cells. The ChIP results also showed that all NFκB signals are accompanied by signals of its cofactor RPS3. In accordance with previous results obtained in our laboratory, the data presented here substantiates the DNA binding of RPS3. Furthermore, the paralogue histone acetyltransferases CBP and p300, which are described as transcriptional coactivators of NFκB and EGR-1, are bound in all investigated cell lines. The impact of the detected protein-DNA interactions on NOT expression was assessed using RNAi (for EGR-1 and NFκB), pharmacological activation or inhibition of the respective transcription factor (TNFα and Parthenolide for NFκB, BFA for CREB3), or overexpression (CREB3). Comparative studies in murine cell lines identified homologues of the human splice variants and showed that the promoter architecture is identical. Moreover, the murine NOT gene is also regulated by NFκB, CREB3, CBP, p300, and RPS3.