The positioning of histone modifications at genes has been associated with numerous co-transcriptional processes ranging from initiation to elongation to 3' end processing. In this report, we investigated the genome-wide distribution of H2B monoubiquitylation in budding yeast. We found that H2BK123ub1, a mark of active transcription, is predominantly localized across gene coding regions, consistent with its postulated roles in transcription elongation [20, 22, 53]. The mark was also proportional to transcription rate, a likely consequence of the association of the H2B ubiquitylation machinery with elongating RNA polymerase II [32, 54, 55]. Both of these features are similar to the distribution of H2B monoubiquitylation in the human genome, supporting the view that both the regulation of this histone modification and its functional roles in transcription are evolutionarily conserved [34, 56]. We also found a correlation between the levels of H2BK123ub1 and gene length, with the mark enriched on long genes. Because long genes frequently contain cryptic transcription initiation sites in their coding regions, this correlation supports the view that the presence of H2BK123ub1 contributes to the restoration of chromatin structure during transcription elongation to suppress the utilization of these sites .
A novel finding of our studies was the discovery that H2BK123ub1 is present in both the introns and exons of the yeast ribosomal protein genes which accounts for the majority of splicing events in yeast cells. Bioinformatic analyses of genome-wide data sets from worms and humans revealed that exons are enriched with active histone modifications while introns are generally depleted of these same marks [5, 7, 9]. This bias has been attributed primarily to chromatin architecture, with nucleosome occupancy higher in exons compared to introns [5–7, 9, 10]. Using published data sets on nucleosome occupancy in yeast , we found that yeast introns also have reduced nucleosome levels in introns compared to exons. However, this difference does not correlate with a reduction in H2BK123ub1 levels in the introns of the RP genes, which account for ~90% of the splicing activity of intron-containing genes . The finding that H2BK123ub1 is a feature of yeast introns is similar to a recent finding, again from bioinformatic analysis of chromatin modification data sets, that H2B ubiquitylation is one of 10 marks of 5' introns in humans . Most yeast genes with introns contain a single intron that is located close to the 5' end of the coding region [57–59]. Thus, the presence of H2B ubiquitylation in the 5' introns of human genes may reflect a similarity in the chromatin architecture of promoter-proximal introns between yeast and human intron-containing genes. We also found that H2BK123ub1 peaked at the 3' intron-exon boundary, particularly in the RP genes. We speculate that the chromatin structure of 3' intron-exon boundaries in these genes could carry a signal that enhances the accessibility of the enzymatic machinery that mediates H2B ubiquitylation (Rad6-Bre1) [27, 28]. Alternatively, enzymes that target H2B for de-ubiquitylation (Ubp8 and Ubp10) [15, 29] could be preferentially prevented from associating with these regions. It is currently unclear what feature of chromatin architecture at these boundaries regulates either the deposition or removal of H2B ubiquitylation. Likewise, it is not known if this distinct chromatin structure plays a role in transcriptional regulation, including pre-mRNA splicing.
H2B ubiquitylation controls the methylation of three lysine residues in histone H3 in trans. A comparison of the genome-wide distributions of H2BK123ub1, H3K4me3, H3K79me2/me3, and H3K36me3 showed that the four modifications occupy distinct regions in genes. As previously reported, H3K123ub1 and H3K79me3 co-localize across coding regions, while H2BK123ub1 and H3K79me2 show an anti-correlation at intergenic regions . H3K4me3 is localized predominantly at 5' gene regions, while H3K36me3, like H2BK123ub1, spreads across coding regions, but with a more pronounced enrichment at the 3' end of genes. How H2BK123ub1 controls these particular distribution patterns remains an area of intense investigation [16, 18, 24, 60–63]. H3K4me3 and H3K79me3, like H2BK123ub1, were also present in the introns of five ribosomal protein genes that were analyzed, and all of these marks were separable from nucleosome occupancy. Together, the results are similar to the reported presence of H2B ubiquitylation and H3K79me3 in the 5' introns of humans .
Unlike the situation in humans and worms [6, 7, 12], H3K36me3 is present in both the introns and exons of yeast genes. The restricted presence of H3K36me3 in exons in higher eukaryotic genomes has been correlated with the regulation of alternative mRNA splicing [6, 7, 28]. These observations suggest two possible roles for H3K36me3 in pre-mRNA splicing, and specifically in the regulation of pre-RNA splicing. First, H3K36me3 could mark exons as a part of a gene structure and along with cis-splicing elements facilitate the decision of whether to include a specific exon [6, 12]. Alternatively, H3K36me3 could act as an anchor site for recruiting splicing factors that regulate alternative mRNA splicing [7, 28]. However, in the yeast genome, the majority of intron-containing genes contain a single intron, and the regulation of splicing efficiency is thus more important than alternative mRNA splicing. It has been suggested that splicing efficiency is a function of the rate of RNA polymerase II elongation. This scenario is supported by a recent finding that RNAP II pauses transiently around the 3'end of introns and that this pause coincides with splicing factor recruitment . Thus, the presence of histone marks in both introns and exons might promote splicing efficiency by controlling RNA polymerase II elongation. The finding that H2BK123ub1 levels are enhanced at 3' intron-exon boundaries could provide a mechanism to couple RNAPII elongation to the recruitment of splicing factors. Moreover, the dynamic relationship between the levels of H2BK123ub1 and H3K36me3 in introns and exons supports a redundant mechanism to ensure optimal RNAP II elongation, in turn promoting efficient pre-mRNA splicing.
Because the loss of H2B ubiquitylation, H3K4/K79 methylation, or H3K36 methylation does not compromise cell viability, the histone modifications cannot play an essential role in splicing. We suggest that the presence of these marks in introns, together with reduced nucleosome occupancy in these regions, are part of a chromatin architecture that facilitates the recognition of exons and introns by splicing regulators. Further support for this mechanism comes from the observation that a synthetic lethal phenotype resulted from combining an htb-K123R mutation with deletions of genes with roles in pre-mRNA splicing, specifically in U2 splicesome assembly. For example, the histone modifications might serve as binding sites for proteins that in turn interact with splicing factors. Such a scenario has been proposed for H3K4me3 and the Chd1 protein, which contains a chromodomain that recognizes the methyl mark and interacts with the U2 snRNP complex in both humans and yeast to promote efficient splicing [65–67].