The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6

Abstract

Summary The five-subunit yeast Paf1 Complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

Emerging Insights into the Roles of the Paf1 Complex in Gene Regulation

“…At most genes in S. cerevisiae , Paf1C occupancy peaks downstream of the transcription start site near the +2 and +3 nucleosomes [37, 38]. This enrichment pattern is consistent with an ordered recruitment model in which Paf1C joins the elongation machinery after Spt5 has associated with Pol II.…”

“…In both yeast and human cells, Paf1C localizes to active genes at levels that correlate with transcriptional output [15, 16, 37, 38]. Several studies have explored the mechanisms that couple Paf1C and its various activities to Pol II (Figure 1, Key Figure).…”

“…H2Bub is catalyzed by the ubiquitin conjugase (E2) Rad6 and the dimeric ubiquitin ligase (E3) Bre1 (UBE2A/2B and RNF20/40 in humans, respectively). Interestingly, a domain within the Rtf1 subunit required for H2Bub [73, 74] interacts directly with Rad6 in budding yeast cells and stimulates Rad6 and Bre1-dependent H2Bub in a minimal, transcription-free in vitro system [37]. Transcription is thought to be a prerequisite for H2Bub in vivo [75], perhaps reflecting transcription-dependent localization of the ubiquitylation machinery.…”

The Ulp2 SUMO protease promotes transcription elongation through regulation of histone sumoylation

“…Consistent with the hypothesis that histone H2B was the critical ubiquitylation target responsible for this genetic interaction, the htb1-K123R mutation, which specifically eliminates H2B ubiquitylation, was also lethal in ulp2D cells (Fig 4C). Furthermore, disruption of RTF1, encoding a component of the RNAPII-associated chromatin remodeling PAF complex, which promotes H2B ubiquitylation (Van Oss et al, 2016), was also lethal in combination with ulp2D ( Fig 4E). Loss of Ulp2 catalytic activity was sufficient for the synthetic interaction with bre1D ( Fig 4D).…”

Shaping the cellular landscape with Set2/SETD2 methylation

“…Specifically, the Paf1 complex is required for H3K36me3 [40, 41] in addition to H2BK123 mono-ubiquitylation (H2BK123ub1) [57, 58] – a chromatin mark that is necessary for H3K4 and H3K79 methylation. In the case of H2BK123ub1, the histone modification domain of Rtf1 (a member of the Paf1 complex that links Paf1 to RNAPII; [59]) directly associates with Rad6/Bre1, thus providing a molecular basis for how Paf1 regulates H2Bub1 [60]. However, in the case of Set2 methylation, the role of Paf1 is likely indirect.…”