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.

Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.[Keywords: RNF20; BRE1; H2B ubiquitylation; tumor suppressor; transcription] Supplemental material is available at http://www.genesdev.org. Received June 6, 2008; revised version accepted August 12, 2008. Eukaryotic DNA is packaged into a chromatin structure of repeating nucleosomes consisting of DNA wrapped around an octamer of core histone proteins (H2A, H2B, H3, and H4). The histone tails, which protrude from the nucleosome, are subjected to a multitude of covalent modifications believed to play a vital role in chromatin remodeling and transcriptional regulation (Jenuwein and Allis 2001;Berger 2007;. One such modification is histone H2B monoubiquitylation. In the yeast S. cerevisiae this process is mediated by the E3 ligase BRE1 (Hwang et al. 2003). In mammals, the hBRE1(RNF20)/RNF40 complex was shown to function as the relevant E3 ligase (Kim et al. 2005;Zhu et al. 2005). In yeast, transcription of several inducible genes is impaired in the absence of ubiquitylated H2B (H2Bub) (Kao et al. 2004). Increased levels of H2Bub occur on the GAL1 core promoter and throughout the transcribed region upon transcriptional activation, with both ubiquitylation and deubiquitylation being required for optimal transcription (Henry et al. 2003;Xiao et al. 2005). Moreover, H2B monoubiquitylation was shown to lead to H3 methylation on Lys 4 and Lys 79, considered marks of actively transcribed genes (Briggs et al. 2002;Sun and Allis 2002). Yet, a recent study suggests that H2B ubiquitylation in S. pombe controls transcriptional elongation by RNA polymerase II (Pol II) independently of H3 methylation (Tanny et al. 2007).Along with the studies linking H2Bub positively with active transcription, other reports suggest a link between

Post-translational histone modifications have essential roles in controlling nuclear processes; however, the specific mechanisms regulating these modifications and their combinatorial activities remain elusive. Cyclin-dependent kinase 9 (CDK9) regulates gene expression by phosphorylating transcriptional regulatory proteins, including the RNA polymerase II carboxy-terminal domain. Here, we show that CDK9 activity is essential for maintaining global and gene-associated levels of histone H2B monoubiquitination (H2Bub1). Furthermore, CDK9 activity and H2Bub1 help to maintain correct replication-dependent histone messenger RNA (mRNA) 3 0 -end processing. CDK9 knockdown consistently resulted in inefficient recognition of the correct mRNA 3 0 -end cleavage site and led to increased read-through of RNA polymerase II to an alternative downstream polyadenylation signal. Thus, CDK9 acts to integrate phosphorylation during transcription with chromatin modifications to control co-transcriptional histone mRNA processing.

Histone modifications play a pivotal role in regulating transcription and other chromatin-associated processes. In yeast, histone H2B monoubiquitylation affects gene silencing. However, mammalian histone ubiquitylation remains poorly understood. We report that the Mdm2 oncoprotein, a RING domain E3 ubiquitin ligase known to ubiquitylate the p53 tumor suppressor protein, can interact directly with histones and promote in vitro monoubiquitylation of histones H2A and H2B. Moreover, Mdm2 induces H2B monoubiquitylation in vivo. Endogenous Mdm2 is tethered in vivo, presumably via p53, to chromatin comprising the p53-responsive p21(waf1) promoter, and Mdm2 overexpression enhances protein ubiquitylation in the vicinity of a p53 binding site within that promoter. Moreover, when recruited to a promoter in the absence of p53, Mdm2 can repress transcription dependently on its RING domain, suggesting that its E3 activity contributes to repression. Histone ubiquitylation may thus constitute a novel mechanism of transcriptional repression by Mdm2, possibly underlying some of its oncogenic activities.