Of all the proteins in eukaryotic cells, histones, first recognized in the late 1800s, are the proteins most frequently bound to DNA. Histones are small, highly conserved basic proteins. The interaction between histones and DNA is fundamental for the packaging of DNA around nucleosomes to form chromatin. Nucleosomes consist of a histone octamer comprised of two copies of each of the core histone proteins H2A, H2B, H3, and H4, around which approximately 147 nucleotides of DNA is wrapped. Within the nucleosome, histones H2A and H2B consist as two H2A:H2B dimers and the H3:H4 relationship is a tetramer (Kornberg and Lorch 1999). Histone H1 distinguishes itself by falling outside of the core histone group, functioning to link strands of DNA on entry and exit of the nucleosome. NH2-terminal tails of core histones extend outside of the core structure, exposing residues on these tails to dynamic posttranslational modifications (PTMs) that are critical to controlling gene transcription and other cellular functions such as DNA repair (Dawson and Kouzarides 2012). Along with DNA methylation at CpG islands, histone PTMs constitute major aspects of epigenomic gene regulation in the mammalian cell.
Genes Encoding Histone H2B
Genes encoding core histone proteins are necessarily highly conserved but also numerous with the presence of histone gene or splice variants contributing, along with PTMs of histone tails, to the complexity required for epigenetic regulation (Singh et al. 2013). Replication-dependent histone variants are required rapidly just prior to S phase as the cell prepares to divide. The genes encoding replication-dependent histone variants are located in clusters and their mRNAs lack a polyadenylated, poly(A), tail. Replication-dependent histones are regulated by the cell cycle. All of these facts about these variants ensure that chromatin structure can be rapidly built as required in the nuclei of newly generated cells. Replication-independent histones also exist, generally dispersed as single genes throughout the genome and with poly(A) mRNAs; however, in mammals these are not reported to encode histone H2B (Marzluff et al. 2002). Between 17 and 22 human H2B genes, including pseudogenes, have been reported, the majority being histone H2A/H2B pairs, encoding multiple different isoforms (Albig et al. 1999; Marzluff et al. 2002; Singh et al. 2013). Examples of genes encoding histone H2B include HIST1H2AB in the large HIST1 cluster located on human chromosome 6 (6p21-p22) and HIST2H2AB in the smaller HIST2 cluster in humans on chromosome 1 (1q21) (Marzluff et al. 2002; Singh et al. 2013). Compared to other histones, histone H2B has fewer variants, with variants that do exist believed to have specific roles in gametogenesis and at specific developmental stages, regulating chromatin condensation and so playing important roles in accessibility of chromatin for gene transcription (Kamakaka and Biggins 2005).
Posttranslational Modifications of Histone H2B
The H2Bub1 chromatin mark is written by conjugation of the 8.5 kDa ubiquitin molecule to K120 of histone H2B by specific E3 ubiquitin ligases, the main one of which is accepted as the E3 ubiquitin ligase complex made up of the RING finger proteins RNF20 and RNF40. Upstream of these E3s that play a role in substrate recognition is an E1 (activating ATP-dependent ubiquitin enzyme) and E2 (ubiquitin conjugating enzyme (UBE2A and UBE2E1)) that function with the E3 to covalently attach the single ubiquitin to its lysine substrate. Other E3 ligases known to perform this function at this site include BRCA1/BARD1, MDM2, and BAF250B (reviewed in (Johnsen 2012; Fuchs and Oren 2014; Cole et al. 2015)). As are all histone PTMs, monoubiquitination of histone H2B is a dynamic process, being erased by as many as eleven deubiquitinases from the ubiquitin-specific protease USP sub-family, including USP7, USP36, and USP44 (reviewed in (Johnsen 2012; Fuchs and Oren 2014; Cole et al. 2015)). H2Bub1 is of particular interest amongst histone marks in that the size and positioning of this mark within the nucleosome structure physically separates chromatin strands, opening this structure for transcription (Fierz et al. 2011). Yet, its function is not only dictated by size and position, as H2Bub1 serves to act as a platform, uniquely recruiting members of complexes involved in histone cross talk, as well as DNA damage and transcription (Shema-Yaacoby et al. 2013).
When the cell experiences DNA damage, some of the RNF20 and RNF40 that normally functions in transcription becomes phosphorylated by the DNA damage sensing serine/threonine protein kinase Ataxia telangiectasia mutated (ATM) at sites of double-strand breaks. Here, RNF20 and RNF40 work as the E3 ligase complex to monoubiquitinate histone H2B at K120, creating open chromatin fibers that are accessible to DNA repair factors. In transcription, cyclin-dependent kinase 9, CDK9, phosphorylates the H2Bub1 E2 and serine 2 in the carboxy-terminus of RNA polymerase II, generating a binding domain for RNF20, RNF40, and associated proteins. RNF20 and RNF40 then function to monoubiquitinate K120 on histone H2B. H2Bub1 recruits the chromatin remodeling factor FACT that removes one of the H2A-H2B dimers from the nucleosome, opening up the chromatin and allowing RNA polymerase II to move along the DNA enabling transcriptional elongation and gene expression (reviewed in Cole et al. 2015).
Histone H2B in Malignancy
Cancer is frequently described as a disease of aberrant gene transcription, and H2Bub1 specifically has been described as a “master switch” of gene regulation (Kim et al. 2005; Espinosa 2008). Normal tissue and some benign tumors have been shown by immunohistochemistry to express H2Bub1 (Prenzel et al. 2011; Wang et al. 2013; Melling et al. 2016). There is a growing list, however, of aggressive malignancies reported to have lost global expression of H2Bub1, including breast cancer (Prenzel et al. 2011), parathyroid cancer (Hahn et al. 2012), colorectal cancer (Melling et al. 2016), gastric carcinoma (Wang et al. 2013), and lung cancer (Urasaki et al. 2012). In a number of these malignancies, loss of global H2Bub1 was reported to be associated with a worse prognosis (Prenzel et al. 2011; Melling et al. 2016). One study has linked aberrant glucose metabolism seen in tumors to H2Bub1 loss (Urasaki et al. 2012). The fundamental cause of global loss of H2Bub1, however, remains to be elucidated for the majority of tumors in which it is lost, although it has been associated with loss-of-function mutations of the tumor suppressor CDC73 in sporadic and familial parathyroid cancer (Hahn et al. 2012). CDC73 is a binding partner of the main E3 ligase complex, RNF20/RNF40, responsible for monoubiquitinating histone H2B. It is also part of the human RNA polymerase II-associated factor 1 (PAF1) transcriptional complex. It is proposed that disturbance of the RNF20/RNF40/CDC73 association abrogates the ability of RNF20/RNF40 to function as an E3 ligase for the histone H2B substrate (Hahn et al. 2012). To date, no other specific gene mutations in associated ubiquitin machinery, including E3 ligases, deubiquitinases (DUBs) or members of the complexes in which they function have been associated with loss of H2Bub1. It is likely, however, that genetic mutations or variants in genes encoding ubiquitination machinery and/or epigenetic regulation of these genes will have a role in loss of global H2Bub1 in different tumor types.
While a body of literature is building exploring global levels of H2Bub1 in malignancy, the fact that H2Bub1 works at the level of the individual gene influencing whether chromatin is open and transcriptionally active or closed and transcriptionally silent is key to understanding the importance of this histone mark. In fact, H2Bub1 has been referred to as a “master switch” of gene regulation. In this context, decrease in H2Bub1 as the result of treatment with proteasome inhibitors has been shown to lead to defects in transcriptional elongation of estrogen target genes in cancer models (Prenzel et al. 2011). Decreases in H2Bub1 via downregulation of its E3 ligase show decreased accumulation of H2Bub1 at the coding sequences of the cyclin-dependent kinase inhibitor 1A, CDKN1A (the gene encoding the cell cycle regulator p21), that correlates with lower expression of this gene (Minsky et al. 2008).
FDA-approved epigenomic therapies targeting histone modifications are already emerging, for example, the histone deacetylase inhibitors Vorinostat (Zolinza®) and Romidepsin (Istodax®). The association of aberrant H2Bub1 with malignancy identifies this histone mark, and the factors controlling it, as potential targets for molecular therapy as we move further inside the era of precision medicine. A substantial part of the appeal of H2Bub1 as a target lies in its fundamental roles in the control of gene transcription and DNA repair. A further reason for its appeal is its apparent loss across a broad range of different tumor types. It is likely that targeting of H2Bub1 for therapy might best accompany DNA damage-based chemotherapeutics given the key roles of H2Bub1 in DNA repair.
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