Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells

Abstract

Histone modifications have emerged as important regulators of transcription. Histone H2B monoubiquitination has also been implicated in transcription; however, better understanding of the biological significance of this modification in mammalian cells has been hindered by the lack of suitable reagents, particularly antibodies capable of specifically recognizing ubiquitinated H2B (ubH2B). Here, we report the generation of anti-ubH2B monoclonal antibodies using a branched peptide as immunogen. These antibodies provide a powerful tool for exploring the biochemical functions of H2B monoubiquitination at both a genome-wide and gene-specific level. Application of these antibodies in high resolution chromatin immunoprecipitation (ChIP)-chip experiments in human cells, using tiling arrays, revealed preferential association of ubiquitinated H2B with the transcribed regions of highly expressed genes. Unlike dimethylated H3K4, ubH2B was not associated with distal promoter regions. Furthermore, experimental modulation of the transcriptional activity of the tumour suppressor p53 was accompanied by rapid changes in the H2B ubiquitination status of its p21 target gene, attesting to the dynamic nature of this process. It has recently been demonstrated that the apparent extent of gene expression often reflects elongation rather than initiation rates; thus, our findings suggest that H2B ubiquitination is intimately linked with global transcriptional elongation in mammalian cells.

RNF20–RNF40: A ubiquitin‐driven link between gene expression and the DNA damage response

“…Another technical breakthrough came with the generation of antibodies that specifically recognize H2Bub [57]. These antibodies enabled the determination of H2Bub genomic distribution by chromatin immunoprecipitation (ChIP), followed by genomic DNA microarray analysis (ChIP/chip) or DNA sequencing (ChIP/Seq).…”

“…A more feasible strategy to investigate the role of H2Bub in mammalian transcription regulation is to deplete the cells of the responsible E3 ligase, RNF20, via RNAi [61]. Surprisingly, expression microarray analysis of human HeLa cells depleted of RNF20 revealed that, despite the positive correlation between H2Bub and expression levels [57], the expression of most genes was essentially independent on H2Bub levels and was unaffected by RNF20 depletion [57]. Thus, although H2Bub is positively correlated with the transcription process in general, it is not rate-limiting for the transcription of most genes.…”

“…These two proteins form a tight heterodimer that acts as the major E3 ligase responsible for histone H2B monoubiquitylation on K120 in mammalian cells. Accordingly, depletion of either RNF20 or RNF40 leads to almost complete loss of H2Bub.Another technical breakthrough came with the generation of antibodies that specifically recognize H2Bub [57]. These antibodies enabled the determination of H2Bub genomic distribution by chromatin immunoprecipitation (ChIP), followed by genomic DNA microarray analysis (ChIP/chip) or DNA sequencing (ChIP/Seq).…”

Monoubiquitination of Histone H2B Blocks Eviction of Histone Variant H2A.Z from Inducible Enhancers

“…In line with this hypothesis, estrogen-dependent breast cancer cells are able to proliferate without estrogen when RNF40 is knocked down (Prenzel et al, 2011). Surprisingly, the inactivation of RNF20/40 only has a moderate effect on overall gene expression levels (Shema et al, 2008), raising the possibility that the H2Bub1 chromatin mark could be dedicated to the regulation of specific groups of genes rather than the regulation of the whole genome. Indeed, H2Bub1 has been associated with the regulation of inducible genes involved in cell differentiation (Zhu et al, 2005;Fuchs et al, 2012;Karpiuk et al, 2012;Materne et al, 2016) and inflammation (Tarcic et al, 2016), suggesting a role for H2Bub1 in the regulation of inducible rather than constitutive transcription.…”

“…Genomic analyses showed that H2Bub1 is enriched in actively transcribed regions of human genes (Minsky et al, 2008) and it has been proposed to be involved in the regulation of global transcriptional elongation (Pavri et al, 2006;Minsky et al, 2008).…”