| Summary: | RNA Polymerase II (RNAPII) transcribes protein-coding and small structural genes, and its functions are critical for appropriate gene regulation in the cell. Post-translational modifications of RNAPII integrate transcription, chromatin modification and co-transcriptional RNA processing to allow correct and efficient gene expression. Lineage-specific genes have complex regulation in Embryonic Stem (ES) cells. They are repressed by Polycomb-repressive complexes (PRC) but primed with RNAPII complexes that have atypical modification, and occupy promoter and coding regions, in the absence Serine2 phosphorylation (Brookes et al., 2012) suggesting chromatin modification and RNA processing may be compromised. Low-level transcription has been detected at a panel of PRC-repressed genes (Stock et al., 2007) but little else is known about the transcriptional activity of RNAPII at these genes, to shed light on the mechanisms of PRC repression. I have investigated features of RNAPII transcription in ES cells focussing on understanding transcription at PRC-repressed genes. To capture nascent transcripts, I optimised RNA-ChIP using RNAPII antibodies to extract RNA associated with chromatin at active and PRC-repressed genes. Genome-wide analysis by Next Generation Sequencing (NGS) showed that PRC-repressed genes are transcribed throughout coding regions, and in a sense orientation, but at lower levels than expected from RNAPII abundance detected by DNA-ChIP. Promoter-associated antisense transcription is lacking, in contrast to active genes. Minimal transcript capping and lack of RNAPII transcription beyond transcription end sites (TES) at PRC-repressed genes suggest multiple regulatory mechanisms may accompany PRC repression, which are linked with compromised RNA processing. Exosome regulates a proportion of PRC-repressed genes at the level of transcription and capping although the extent of involvement is still under scrutiny. The present work and ongoing analysis add valuable information to help elucidate mechanisms of gene regulation associated with Polycomb repression to increase our knowledge of pluripotency and ES cell biology.
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