Interplay between polycomb repression and RNA polymerase II regulation genome-wide in murine ES cells

Abstract

Gene regulation is a complex process that involves recruitment of transcription machinery, chromatin modification, and co-transcriptional RNA processing. RNA polymerase II (RNAPII) transcribes protein-coding genes. It contains a carboxy- terminal domain (CTD) consisting of multiple repeats. Specific patterns of CTD modifications promote the recruitment of appropriate factors at different stages of the transcription cycle, integrating transcription with chromatin modification and co-transcriptional RNA processing. In pluripotent ES cells, Polycomb repressive complexes (PRCs) silence important developmental regulator genes. I have investigated RNAPII regulation at PRC-target genes genome-wide in murine ES cells by mining ChIP-Seq and mRNA-Seq datasets, using computational approaches. PRC targets exhibit different expression and RNAPII states: Silent genes lacking RNAPII, silent genes associated with RNAPII which is phosphorylated on the serine 5 residue of the CTD (S5P) but lacking serine 2 phosphorylated residues (S2P), and expressed genes with both S5P and S2P. At silent PRC-target genes, S5P and PRCs co-occupy the same promoters and the complexes extend into coding regions in the absence of S2P, suggesting disruption of co-transcriptional RNA processing. Sequential ChIP demonstrates simultaneous association of PRC and RNAPII to the same chromatin. Most expressed PRC-target genes display CTD marks of activity, including presence of S2P throughout the gene body; however S2P and PRCs do not co-immunoprecipitate, suggesting that active and PRC-repression are two separate chromatin states, that occur in different cells of the ES population, or within different alleles in the same cell. Finally, I have developed a software, SeqGI, for the analysis and interpretation of sequencing data. SeqGI provides a GUI framework for the simultaneous visualisation and statistical evaluation of read profiles over genomic features. This work provides valuable insights on the biology of ES cells, and elucidates combinations of different factors across the genome shedding light on different mechanisms of gene activation, poising and silencing.