Identification of a Role for Histone H2B Ubiquitylation in Noncoding RNA 3′-End Formation Through Mutational Analysis of Rtf1 in Saccharomyces cerevisiae

Abstract

The conserved eukaryotic Paf1 complex regulates RNA synthesis by RNA polymerase II at multiple levels, including transcript elongation, transcript termination, and chromatin modifications. To better understand the contributions of the Paf1 complex to transcriptional regulation, we generated mutations that alter conserved residues within the Rtf1 subunit of the Saccharomyces cerevisiae Paf1 complex. Importantly, single amino acid substitutions within a region of Rtf1 that is conserved from yeast to humans, which we termed the histone modification domain, resulted in the loss of histone H2B ubiquitylation and impaired histone H3 methylation. Phenotypic analysis of these mutations revealed additional defects in telomeric silencing, transcription elongation, and prevention of cryptic initiation. We also demonstrated that amino acid substitutions within the Rtf1 histone modification domain disrupt 39-end formation of snoRNA transcripts and identify a previously uncharacterized regulatory role for the histone H2B K123 ubiquitylation mark in this process. Cumulatively, our results reveal functionally important residues in Rtf1, better define the roles of Rtf1 in transcription and histone modification, and provide strong genetic support for the participation of histone modification marks in the termination of noncoding RNAs.

Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast

“…The coprecipitation of H3 with FLAG-H2B indicated that the FLAG-H2B was incorporated into nucleosomes. Importantly, two mutant forms of the HMD, NLS-Myc-HMD E104K and NLS-Myc-HMD F123S-which abrogate the histone modification activity of full-length Rtf1 (32) and are expressed at levels similar to the wild-type HMD (Fig. S6B)-showed reduced association with FLAG-H2B (Fig.…”

“…Mutational studies indicated a primary role for the Rtf1 HMD in controlling H2B K123 ubiquitylation (32). We therefore asked whether the NLS-Myc-HMD could restore H2B K123 ubiquitylation in an rtf1Δ strain.…”

“…Therefore, although overexpression of the HMD is likely required for high levels of histone modifications, it does not bypass the canonical pathway for H2B K123 ubiquitylation and downstream H3 modifications. Null alleles of RTF1 or mutations within the HMD-coding region suppress the transcriptional defects caused by a Ty-δ element insertion mutation within the HIS4 promoter, his4-912δ, and thus confer a Suppressor of Ty or Spt − phenotype (32,44). We examined the Spt − phenotype of an rtf1Δ his4-912δ strain that was transformed with the NLS-Myc-HMD or Myc-Rtf1 2-micron expression plasmids.…”

“…Whether Paf1C has a more direct role in promoting histone modifications is not known. We previously reported that deletions and amino acid substitutions within a small region of the S. cerevisiae Rtf1 protein dramatically reduce global levels of H3 K4 and K79 dimethylation and trimethylation and H2B K123 ubiquitylation, leading us to define residues 62-152 as the histone modification domain (HMD) of Rtf1 (32,44). Here we show that the Rtf1 HMD interacts with chromatin and is sufficient to promote H2B K123 ubiquitylation and H3 K4 and K79 methylation.…”

“…Paf1C-which in budding yeast consists of the subunits Paf1, Ctr9, Cdc73, Rtf1, and Leo1-impacts RNA synthesis at multiple stages (21). Paf1C associates with RNA pol II from the 5′ end of a gene to the poly(A) site (22,23); interacts functionally and physically with the transcription elongation factors Spt4-Spt5/DSIF, Spt16-Pob3/FACT, and TFIIS (8,(24)(25)(26)(27); regulates the phosphorylation state of RNA pol II (28,29); and is required for proper 3′ end formation of certain transcripts (30)(31)(32). Important to this study, deletion of RTF1 from yeast cells causes dramatic reductions in H2B K123 ubiquitylation and H3 K4 and K79 methylation (33)(34)(35)(36), and this role in histone modification, like other Paf1C functions, is conserved in higher eukaryotes (37)(38)(39)(40).…”