BioProject

DNA faces continuous threats from various endogenous and exogenous stresses. More...

DNA faces continuous threats from various endogenous and exogenous stresses. When damaged, DNA requires repair to enable proper cell functioning. Cells have evolved multiple repair pathways to maintain genome stability. Transcription mediated by RNA polymerase II (RNA Pol II), which is conventionally linked with gene expression, plays an integral role in DNA repair. Transcription occurring at sites of DNA damage facilitates repair by generating functional transcripts, recruiting DNA repair factors, or modulating chromatin architecture. Conversely, the shutdown of transcription in nearby genes is crucial to ensure effective DNA repair. UV-induced DNA lesions have been shown to lead to a genome-wide suppression of transcription through RNA Pol II ubiquitylation and subsequent degradation. However, the global transcriptional response to double-strand breaks remains unclear.In this study, we employed mNET-Seq analysis to examine nascent transcripts bound to the CTD Y1P, S2P and S5P of RNA Pol II. Our data reveal a global transcriptional shutdown, as evidenced by a greater number of downregulated protein coding genes in response to irradiation. Interestingly, we observed a significant increase in Y1P antisense transcripts near promoters, known as PROMPTs, which are associated with the downregulated protein coding genes upon irradiation. Furthermore, an increased promoter pausing index correlated with elevated expression of PROMPTs in response to double strand breaks induction. Motif enrichment analysis uncovered binding sites for the Polycomb repressive complex 2 (PRC2) in upregulated PROMPTs. Using proximity ligation assays, we demonstrated an increased proximity between Y1P RNA Pol II and PRC2 upon irradiation. Finally, we also report that inhibition of Y1P or depletion of PRC2 results in the loss of transcriptional repression of protein coding genes, associated with PROMPTs. Overall design: mNet-Seq sample preparation using Tyrosine 1 phosphorylated (Y1P) RNA Pol II, Serine 2 phosphorylated(S2P) RNA Pol II,Serine 5 phosphorylated(S5P) RNA Pol II antibodies. Samples were either irradiated or non-irradiated for each these antibody types. The extracted RNA Pol II was then processed using mNET-Seq protocol (Nojima 2016) to study the nascent RNA transcripts bound to them Less...