Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

Abstract

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The mono-ubiquitylation of a conserved lysine in H2B (K123 inSaccharomyces cerevisiae; K120 in humans) occurs co-transcriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its Histone Modification Domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ubin vivoandin vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Usingin vitrocrosslinking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic and biochemical experiments, we identified separation-of-function mutations inS. cerevisiae RAD6that greatly impair H2BK123 ubiquitylation but not other Rad6 functions. Finally, by employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.Significance StatementTranscription by RNAPII is tightly coordinated with mechanisms that control chromatin structure. Disruption of this interplay leads to deleterious effects on gene expression and genome architecture. Proteins that associate with RNAPII during transcription elongation play an important role in coupling histone modifications to active transcription. Paf1C, a conserved member of the RNAPII active elongation complex, is required for the ubiquitylation of histone H2B, a modification with effects on nucleosome stability and the methylation and acetylation state of chromatin. Here, we provide new insights into how a conserved domain in Paf1C, which we previously showed to be necessary and sufficient for Paf1C-mediated stimulation of H2B ubiquitylation, interacts with the ubiquitin conjugase for H2B thereby guiding its specificity.

Multiple direct and indirect roles of Paf1C in elongation, splicing, and histone post-translational modifications

“…In rtf1∆ conditions, SRAT transcription increases, which is consistent with previous observations of antisense upregulation in fission and budding yeast mutants unable to ubiquitylate H2B. 16,61,62 The absence of notable defects in 4tU-seq completion score, intron accumulation, or antisense transcription upon Rtf1 depletion suggests that H2Bub may not intrinsically impact these phenotypes, although further work will be needed to rule out compensatory effects of removing other Rtf1 functions. In contrast to the rapid H2Bub turnover, H3K4me3, H3K79me2/3, and H3K36me3 persist over the depletion time course, and therefore, we cannot readily attribute the transcription defects observed during acute Paf1C depletion to the global loss of these modifications.…”

“…We found that the acute depletion of any Paf1C subunit does not lead to the upregulation of antisense transcripts (Figure 2E), suggesting that Paf1C is not directly or solely responsible for preventing their expression. H2Bub has also been implicated in the repression of antisense transcription, 16,61,62 but despite the striking loss of H2Bub upon depletion of Rtf1, we see no significant upregulation of SRATs. These results suggest that Paf1C and likely H2Bub do not intrinsically repress antisense transcription.…”

“…The strongest effect on H2Bub levels was caused by Rtf1 depletion, which was expected given the direct role of the Rtf1 histone modification domain in stimulating H2B ubiquitylation through its interaction with Rad6. 15,16 Some Paf1C∆ strains are deficient in H3 methylation marks. For example, paf1∆, ctr9∆, and rtf1∆ strains have severely reduced levels of the H2Bub-dependent modifications H3K4me2/3 and H3K79me2/3, and paf1∆, ctr9∆, and cdc73∆ strains are additionally defective for H3K36me3.…”

Rtf1 HMD domain facilitates global histone H2B monoubiquitination and regulates morphogenesis and virulence in the meningitis-causing pathogenCryptococcus neoformans

“…The posttranslational modifications of core histones, including acetylation, methylation, and ubiquitination, represent major mechanisms by which cells alter the chromatin structural properties and regulate gene transcription [1,2]. Among them, the monoubiquitination of a lysine (K) residue on the C-terminal of histone H2B (H2Bub1) is a conserved modification that occurs on H2B K120 residue in Homo sapiens and K123 residue in Saccharomyces cerevisiae [3,4]. H2Bub1 is enriched at regions of active transcription but plays roles in both gene activation and repression [5][6][7].…”

“…Interestingly, the histone modification domain (HMD) within Rtf1 is both necessary and sufficient for stimulating H2Bub1 in yeast [4, 18]. Expression of the Rtf1 HMD alone restores H2Bub1 levels in S. cerevisiae mutants deleted for the RTF1 gene or all five Paf1C subunits-encoding genes [3, 4, 18]. These studies show that Rtf1 is the only Paf1C subunit that is strictly required for deposition of H2Bub1 in vivo.…”