Epigenetic regulation of gene transcription: Jhdm1b characterization and genome-wide localization of H2A ubiquitylation

Histone methylation and ubiquitylation play important roles in regulating gene expression and form part of the epigenetic memory system that regulates cellular fate. Two general classes of enzymes linked to covalent modification are the JmjC domain-containing proteins capable of histone demethylation and the E3 ubiquitin ligases. Here we report the evolutionary conservation of JmjC domain-containing proteins with the goal of predicting active demethylases. We demonstrate that one of these proteins, Jhdm1b, is an H3K36 demethylase both in vitro and in vivo. Knock-down of Jhdm1b in primary MEFs inhibits cell proliferation and induces cellular senescence in a pRb and p53 pathway-dependent manner that is mediated through direct de-repression of p15Ink4b. Jhdm1b binds to the p15Ink4b promoter and maintains low levels of local H3K36me2. Importantly, knock-down followed by rescue experiments demonstrate that this function is mediated through an active JmjC domain.;Transcriptional regulation by PcG proteins is achieved at least partly through the PRC1-mediated ubiquitylation of histone H2A (uH2A). Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome-wide binding patterns of some of the PRC1 components there have been no reports describing genome-wide localization of uH2A. Using the recently developed ChIP-Seq technology, here we report genome wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site (TSS) of well annotated genes that is enriched at promoters containing H3K27me3. Peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower level expression when compared to genes that do not contain promoter peaks of uH2A. We demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout and local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. Our work not only reveals Bmi1-dependent H2A ubiquitylation but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells.