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The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae.

Cucinotta CE, Young AN, Klucevsek KM, Arndt KM - PLoS Genet. (2015)

Bottom Line: In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3.Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination.Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.

No MeSH data available.


Related in: MedlinePlus

The H2A substitutions affect histone levels on genes but do not greatly affect transcription of genes that are sensitive to nucleosome occupancy.(A) Western analysis of H2B, H2A, and H3 levels in the H2A mutant strains. G6PDH levels served as a loading control. (B, C, D) Analysis of H2B (KY2674), H2A (KY2675), and H3 (KY943) occupancy at the 5’- and 3’ ends of PYK1 and at TELVI by ChIP. The error bars denote SEM of three independent experiments. (E) Northern analysis assessing the effects of the H2A substitutions and H2B-K123R (KY2044) on SER3, SRG1, FLO8 and FLO8 cryptic transcript levels. Upper band (*) corresponds to the full-length FLO8 transcript and the lower band (**) corresponds to the cryptic internally initiated transcript. The spt6-1004 (KY2678) and spt16-197 (KY2679) temperature-sensitive alleles serve as positive controls for cryptic initiation and SER3 derepression and are isogenic to the wild-type strain KY2677. SCR1 was used as a loading control.
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pgen.1005420.g003: The H2A substitutions affect histone levels on genes but do not greatly affect transcription of genes that are sensitive to nucleosome occupancy.(A) Western analysis of H2B, H2A, and H3 levels in the H2A mutant strains. G6PDH levels served as a loading control. (B, C, D) Analysis of H2B (KY2674), H2A (KY2675), and H3 (KY943) occupancy at the 5’- and 3’ ends of PYK1 and at TELVI by ChIP. The error bars denote SEM of three independent experiments. (E) Northern analysis assessing the effects of the H2A substitutions and H2B-K123R (KY2044) on SER3, SRG1, FLO8 and FLO8 cryptic transcript levels. Upper band (*) corresponds to the full-length FLO8 transcript and the lower band (**) corresponds to the cryptic internally initiated transcript. The spt6-1004 (KY2678) and spt16-197 (KY2679) temperature-sensitive alleles serve as positive controls for cryptic initiation and SER3 derepression and are isogenic to the wild-type strain KY2677. SCR1 was used as a loading control.

Mentions: Previous studies have shown that H2B K123ub is required for proper histone occupancy [32,33], that the docking domain of H2A is important for the association of H2A and H2B with H3 and H4 [33–35], and that the acidic patch lies at the interface of H2A and H2B [1,35]. Therefore, we examined global and local levels of histones by western analysis and ChIP, respectively (Fig 3). Global levels of H2B, H3, and H2A were unaffected in the mutants, with two exceptions (Fig 3A). The two exceptions, H2A-E93A and H2A-L94A, were detected at levels that were lower than wild-type H2A, indicating a potential defect in the expression, stability, or antibody recognition of these H2A mutant proteins. H2B, H2A, and H3 occupancy levels were assessed at both the highly transcribed gene PYK1 and a non-transcribed telomeric region using ChIP analysis (Fig 3B–3D). Four of the alanine substitutions in H2A resulted in lower occupancy levels of H2B at PYK1 (Fig 3B). H2A occupancy was not as drastically affected in the mutant strains; however, the signals for H2A-E57A and H2A-E93A enrichment were reduced at all loci tested (Fig 3C). For H2A-E93A, this could be due to reduced H2A protein levels or reduced immunoreactivity (Fig 3A). H3 occupancy levels at PYK1 were also slightly affected in some of the mutant strains, particularly at the 5’ end of the gene (Fig 3D). Importantly, the reduced histone occupancy levels do not account for the reduced H2B K123ub levels in the H2A mutant strains, as we have normalized the H2B K123ub levels to total histone levels in our assays (Fig 2).


The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae.

Cucinotta CE, Young AN, Klucevsek KM, Arndt KM - PLoS Genet. (2015)

The H2A substitutions affect histone levels on genes but do not greatly affect transcription of genes that are sensitive to nucleosome occupancy.(A) Western analysis of H2B, H2A, and H3 levels in the H2A mutant strains. G6PDH levels served as a loading control. (B, C, D) Analysis of H2B (KY2674), H2A (KY2675), and H3 (KY943) occupancy at the 5’- and 3’ ends of PYK1 and at TELVI by ChIP. The error bars denote SEM of three independent experiments. (E) Northern analysis assessing the effects of the H2A substitutions and H2B-K123R (KY2044) on SER3, SRG1, FLO8 and FLO8 cryptic transcript levels. Upper band (*) corresponds to the full-length FLO8 transcript and the lower band (**) corresponds to the cryptic internally initiated transcript. The spt6-1004 (KY2678) and spt16-197 (KY2679) temperature-sensitive alleles serve as positive controls for cryptic initiation and SER3 derepression and are isogenic to the wild-type strain KY2677. SCR1 was used as a loading control.
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Related In: Results  -  Collection

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pgen.1005420.g003: The H2A substitutions affect histone levels on genes but do not greatly affect transcription of genes that are sensitive to nucleosome occupancy.(A) Western analysis of H2B, H2A, and H3 levels in the H2A mutant strains. G6PDH levels served as a loading control. (B, C, D) Analysis of H2B (KY2674), H2A (KY2675), and H3 (KY943) occupancy at the 5’- and 3’ ends of PYK1 and at TELVI by ChIP. The error bars denote SEM of three independent experiments. (E) Northern analysis assessing the effects of the H2A substitutions and H2B-K123R (KY2044) on SER3, SRG1, FLO8 and FLO8 cryptic transcript levels. Upper band (*) corresponds to the full-length FLO8 transcript and the lower band (**) corresponds to the cryptic internally initiated transcript. The spt6-1004 (KY2678) and spt16-197 (KY2679) temperature-sensitive alleles serve as positive controls for cryptic initiation and SER3 derepression and are isogenic to the wild-type strain KY2677. SCR1 was used as a loading control.
Mentions: Previous studies have shown that H2B K123ub is required for proper histone occupancy [32,33], that the docking domain of H2A is important for the association of H2A and H2B with H3 and H4 [33–35], and that the acidic patch lies at the interface of H2A and H2B [1,35]. Therefore, we examined global and local levels of histones by western analysis and ChIP, respectively (Fig 3). Global levels of H2B, H3, and H2A were unaffected in the mutants, with two exceptions (Fig 3A). The two exceptions, H2A-E93A and H2A-L94A, were detected at levels that were lower than wild-type H2A, indicating a potential defect in the expression, stability, or antibody recognition of these H2A mutant proteins. H2B, H2A, and H3 occupancy levels were assessed at both the highly transcribed gene PYK1 and a non-transcribed telomeric region using ChIP analysis (Fig 3B–3D). Four of the alanine substitutions in H2A resulted in lower occupancy levels of H2B at PYK1 (Fig 3B). H2A occupancy was not as drastically affected in the mutant strains; however, the signals for H2A-E57A and H2A-E93A enrichment were reduced at all loci tested (Fig 3C). For H2A-E93A, this could be due to reduced H2A protein levels or reduced immunoreactivity (Fig 3A). H3 occupancy levels at PYK1 were also slightly affected in some of the mutant strains, particularly at the 5’ end of the gene (Fig 3D). Importantly, the reduced histone occupancy levels do not account for the reduced H2B K123ub levels in the H2A mutant strains, as we have normalized the H2B K123ub levels to total histone levels in our assays (Fig 2).

Bottom Line: In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3.Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination.Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.

No MeSH data available.


Related in: MedlinePlus