H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

Abstract

Background: The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA.

Results: Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality.

Conclusion: These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

Figure 1

H2B ubiquitylation is enriched at gene coding regions and correlates with transcriptional activity and gene length. (A) H2BK123ub1 is enriched at gene coding regions. H2BK123ub1 ChDIP-chip was performed using a high-resolution oligonucleotide tiling array. The log2 ratio of ubiquitylated H2B in wild type yeast cells was subjected to the averaged gene analysis (detailed in Materials and Methods). (B) H2BK123ub1 levels at various genomic locations calculated as in (A). ORF: open read frame; intergenic region; telomere: the average of the last 20 Kb of each chromosome; rDNA: ribosomal DNA; HM: the silent HMRa and HMLa loci. (C) H2BK123ub1 levels are proportional to transcription rate. All yeast genes were divided into five subclasses according to transcription rate [35]. Composite H2BK123ub1 profiles for each subclass are shown on averaged genes based on the analysis from Pokholok et al. (2005). (D) H2BK123ub1 levels are correlated with gene length. All yeast genes were divided into 8 subclasses according to the length of their coding regions. Composite H2BK123ub1 profiles for each subclass are shown on averaged genes as in (C). (E) Genes with transcription rates below 16 mRNA/hour were selected and divided into subclasses based on gene length as in (D). The average enrichment of H2BK123ub1in each subclass was plotted as a function of the averaged genes. (F) Genes with transcription rates greater than or equal to 16 mRNA/hour were selected and divided into subclasses based on gene length as in (D) and then plotted as in (E).

Figure 2

H2B ubiquitylation marks introns and exons. (A-C) Localization of H2BK123ub1 across the coding region of all intron-containing, RP and non-RP genes. The DNA sequences of exon1, intron and exon2 of all intron-containing genes in yeast were divided into 10, 30 and 40 bins, respectively. The composite of the averaged H2BK123ub1 level was aligned in the order: exon1-intron-exon2. (D-F) Nucleosome occupancy across the coding region of all intron-containing RP and non-RP genes. The composite profiles were analyzed as in (A-C). The average occupancy of nucleosomes in introns and exons was derived from data in Jiang and Pugh (2009) [42] and calculated as described in Materials and Methods. (G) H2B and (H) H3 occupancies at several intron- containing ribosomal protein (RP) genes were assayed by ChIP using an anti-Flag antibody to detect FLAG-H2B and an antibody against the C-terminus of H3. The relative H2B and H3 occupancies were calculated by comparison to the occupancy of these histones at the INT-V region.

Figure 3

H2BK123ub1, H3K36me3, H3K4me3, and H3K79me3 levels in introns and exons of ribosomal protein genes. (A) ChDIP was performed to detect the level of HA-ubiquitin tagged Flag-H2B. Antibodies against (B) H3K36me3 (C) H3K4me3 and (D) H3K79me3 were used in ChIP to detect the levels of these three marks. Probe-based PCR primers were designed to amplify promoter (P), intron, exon, and 3' intergenic regions for the genes analyzed. H2BK123ub1 levels were normalized to H2B, and H3K36me3, H3K4me3 and H3K79me3 levels were normalized to H3. In (B) the levels of H3K36me3 were also measured in an htb-K123R mutant (K123R) that lacks H2BK123ub1. Error bars represent the standard deviation of at least three independent experiments.

Figure 4

Genetic interactions between the Bre1/H2B ubiquitylation and pre-RNA splicing pathways. (A) Synthetic genetic interactions of bre1Δ were derived from BioGRID, and its interactions with RNA processing mutants were selected and displayed using the Osprey network visualization system [47]. Colored lines connect bre1Δ to mutations in genes leading to synthetic interactions (either positive or negative). The red triangle indicates genes that function in pre-mRNA splicing. (B) Growth analysis of double mutant cells of lea1Δ htb-K123R and msl1Δ htb-K123R. Cells carrying a URA3 plasmid expressing wild type HTB1 were transformed with a HIS3 plasmid carrying HTB1 or htb-K123R. After selection, transformants were grown at 30°C in SC-histidine medium for 24 hours. Cells were then spotted in 10-fold serial dilution onto SC-histidine plates or SC-histidine plates containing 1 mg of 5-FOA/ml, and the plates were incubated at 30°C for 2-3 days. Cells that were unable to lose the wild type HTB1 gene failed to grow on 5-FOA.