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 monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae ; K120 in humans) occurs cotranscriptionally 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 H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking 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, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6–HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. 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.

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 Paf1CΔ strains are deficient in H3 methylation marks. For example, paf1Δ , ctr9Δ , and rtf1Δ strains have severely reduced levels of the H2Bub-dependent modifications H3K4me2/3 and H3K79me2/3, and paf1Δ , ctr9Δ, and cdc73Δ strains are additionally defective for H3K36me3.…”

“…14 In stimulating H2Bub, Rtf1 interacts directly with Rad6, the ubiquitin conjugase for H2B. 15,16 Rtf1 also governs the occupancy of a chromatin remodeler, Chd1, on gene bodies in yeast and stimulates elongation rate after RNAPII pause release in metazoans. 8,15,[17][18][19][20] Leo1 is structurally intertwined with Paf1 to form a module capable of interacting with RNA and the N-terminal tail of histone H3.…”

Mechanisms and Functions of the RNA Polymerase II General Transcription Machinery during the Transcription Cycle

“…Studies have implicated Paf1C in chromatin modifications and epigenetic control through direct contacts with related factors, including FACT [226,227] and Chd1 [228,229]. Additionally, Paf1C has been associated with the maintenance of several histone marks for active chromatin, further emphasizing its role as an important regulator of Pol II productive elongation [229][230][231][232].…”