RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells

Abstract

SUMMARY H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (i) that hRAD6 serves as the cognate E2 conjugating enzyme, (ii) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120, (iii) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery, (iv) that H2B ubiquitylation per se does not affect the level of hPAF-, SII- and p300-dependent transcription and likely functions downstream and (v) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and tri-methylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

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

“…Nevertheless, several lines of evidence strongly suggest that this is indeed the case. For example, depletion of hRAD6A, the E2 enzyme that serves in RNF20-mediated H2B monoubiquitylation [52], leads to a strong drop in H2Bub levels and reproduces the transcriptional effects of RNF20 depletion on both gene groups [62]. A similar effect is seen upon knockdown of WAC1, a functional partner of RNF20-RNF40 and regulator of H2B ubiquitylation levels [63] (data not shown).…”

“…Notably, H2B monoubiquitylation was subsequently found to be required for di-and trimethylation of lysine 4 and lysine 79 of histone H3 at transcribed chromatin [42][43][44][45][46][47][48]. This pathway is conserved from yeast to mammals, and is dependent on a host of additional proteins that converge at the elongating RNA Pol II [41,[49][50][51][52][53][54]. Subsequently, the mammalian orthologs of the yeast Bre1, RNF20 and RNF40, were identified [55,56].…”

“…Thus, while carrying out its regular enzymatic activity in the DDR context, it was presumably physically and functionally detached from its regular context. In the transcription context, H2B monoubiquitylation in yeast and mammals is dependent on the early steps in transcription initiation and elongation, and the corresponding E3 ligase closely interacts with many proteins that take part in this process, some of which associate directly with RNA Pol II [41,[49][50][51][52][53][54]99,100]. It is not entirely clear how many of the proteins surrounding RNF20-RNF40 in the transcription context accompany this heterodimer to the DNA damage sites.…”

An acetylation-monoubiquitination switch on Lysine 120 of H2B

“…[5][6][7] In human cells, the mono-ubiquitination mechanism includes the function of hRAD6 and RNF20/40. 8 Indeed, the modification of this residue is essential for recruitment of histone methylating activities that act on K4 and K79 of histone H3. 9,10 However, K120 is also acetylated in bulk histones preparations.…”

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

“…In Saccharomyces cerevisiae, monoubiquitylation of lysine residue 123 in histone H2B was shown to play a positive role in H3K4 and H3K79 methylations, which are involved in transcription initiation and elongation, respectively (Briggs et al 2002;Sun and Allis 2002). In mammals, H2Bub1 coupled to RAD6-mediated transcription has been reported to directly stimulate H3K4 methylation ), and synthetically ubiquitylated H2B stimulates DOT1L-mediated H3K79 methylations in vitro (McGinty et al 2008;Kim et al 2009;Oh et al 2010). Despite many efforts to infer the roles of histone modifications, however, the crosstalk between H2Bub1 and the multiple methylations of H3K4 and H3K79 is not fully understood, especially in mammals.…”