Dynamic Loss of H2B Ubiquitylation without Corresponding Changes in H3K4 Trimethylation during Myogenic Differentiation

Abstract

c Ubiquitylation of H2B on lysine 120 (H2Bub) is associated with active transcriptional elongation. H2Bub has been implicated in histone cross talk and is generally regarded to be a prerequisite for trimethylation of histone 3 lysine 4 (H3K4me3) and H3K79 in both yeast and mammalian cells. We performed a genome-wide analysis of epigenetic marks during muscle differentiation, and strikingly, we observed a near-complete loss of H2Bub in the differentiated state. We examined the basis for global loss of this mark and found that the H2B ubiquitin E3 ligase, RNF20, was depleted from chromatin in differentiated myotubes, indicating that recruitment of this protein to genes substantially decreases upon differentiation. Remarkably, during the course of myogenic differentiation, we observed retention and acquisition of H3K4 trimethylation on a large number of genes in the absence of detectable H2Bub. The Set1 H3K4 trimethylase complex was efficiently recruited to a subset of genes in myotubes in the absence of detectable H2Bub, accounting in part for H3K4 trimethylation in myotubes. Our studies suggest that H3K4me3 deposition in the absence of detectable H2Bub in myotubes is mediated via Set1 and, perhaps, MLL complexes, whose recruitment does not require H2Bub. Thus, muscle cells represent a novel setting in which to explore mechanisms that regulate histone cross talk. Modifications of histones, including lysine methylation, acetylation, and ubiquitylation, are closely associated with the control and modulation of gene transcription. Chromatin modifications are often asymmetrically deposited with respect to transcription start sites (TSS) of genes. In mammalian cells, lysine 4 trimethylation of histone H3 (H3K4me3) is associated with the TSS and 5= ends of genes, whereas H2B monoubiquitylation at lysine 120 (H2Bub) and H3K36 trimethylation are associated with transcribed regions of genes (22). H2Bub is catalyzed by a heterodimeric E3 ubiquitin ligase complex comprised of RNF20 and RNF40 (Bre1a/b in yeast) (11,46) and the E2 ubiquitinconjugating enzyme, Rad6 (12,13,30). H2Bub has been associated with active transcription and, more specifically, with transcriptional elongation (43). Several groups have independently demonstrated that H2Bub is a prerequisite for H3K79 and H3K4 methylation in yeast through a transtail mechanism (6,8,24,37). While monomethylation of H3K4 and H3K79 was found to be H2Bub independent (7, 33), H2Bub has been shown to direct diand trimethylation of H3K4 and H3K79 through the recruitment of relevant enzymes, Set1 and Dot1, respectively, facilitating histone cross talk in both yeast and mammals (12,19,37).The PAF1 complex (Paf1C) has also been shown to play a role in the regulation of H2B ubiquitylation in both yeast and mammals via recruitment and activation of Rad6/RNF20 on transcribed regions of chromatin in a manner that is dependent on elongating RNA polymerase (24,43,46). In addition, the Bur1 kinase in yeast and CDK9 in humans were shown to promote deposition of H2Bub (15,29). Thus, H2B mono...

H2B monoubiquitylation is a 5′-enriched active transcription mark and correlates with exon–intron structure in human cells

“…Similar results were obtained in HeLa cells (Supplemental Fig. 4B,C) and mouse myoblast cells (Vethantham et al 2012; Supplemental Fig. 4D,E).…”

“…For mouse myoblast cells, the H2Bub1 and H3K36me3 ChIPseq data were downloaded from ArrayExpress with accession number E-GEOD-25308 (Asp et al 2011;Vethantham et al 2012), and the RNA-seq data were downloaded from the GEO database with accession number GSM521256 (Trapnell et al 2010). Gene definitions were given by their Refseq gene track (mm9) in the UCSC Genome Browser, and the other processes were the same as those used for the human genome data.…”

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription

“…We first analyzed the H2B DUB of the human SAGA complex. Former analyses in mammals revealed that H2Bub was extensively enriched in the transcribed region of expressed genes, in agreement with an association of the H2B ubiquitination enzymes with the elongating Pol II Shema et al 2008;Vethantham et al 2012). However, it remains to be determined whether SAGA can deubiquitinate all H2Bub-containing nucleosomes, which represent a large fraction of the genome, or whether its DUB activity would be restricted to the promoters of SAGA-regulated genes.…”

“…Former ChIP-on-chip experiments in S. cerevisiae revealed a colocalization of H2Bub with H3K79me3 in the coding region of only a subset of genes and suggested that Ubp8 would mainly remove ubiquitin from H2Bub in the 59 region of genes at H3K4me3 peaks (Schulze et al 2009. This would indicate different regulations for H2B ubiquitination between yeast and other eukaryotes, as indicated by differential H2Bub distribution (Kharchenko et al 2011;Roudier et al 2011;Vethantham et al 2012). To compare the activity of the evolutionarily well-conserved DUB module of the yeast and the human SAGA complexes, we aimed to compare H2Bub profiles in wild-type and ubp8D strains.…”

Codependency of H2B monoubiquitination and nucleosome reassembly on Chd1

“…These findings in yeast are highly relevant in metazoans, as a recent study reported low levels of H2B monoubiquitination, but normal H3K4 trimethylation levels, during myogenic differentiation (Vethantham et al 2012). Understanding the mechanism of Chd1's regulation of H2B monoubiquitination will require much further analyses in both yeast and mammalian systems.…”