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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.


Recruitment of histone modification and elongation machinery is impaired in the H2A mutants.ChIP analyses of HSV-Bre1 (KY2674) (A), Rtf1 (KY2674) (B), Spt5 (KY943) (C), Spt16 (KY2675) (D), and HSV-Set1 (KY2719) (E) at the 5’- and 3’-ends of transcribed loci (PYK1 and PMA1) and at TELVI. The error bars represent SEM of three independent experiments.
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pgen.1005420.g006: Recruitment of histone modification and elongation machinery is impaired in the H2A mutants.ChIP analyses of HSV-Bre1 (KY2674) (A), Rtf1 (KY2674) (B), Spt5 (KY943) (C), Spt16 (KY2675) (D), and HSV-Set1 (KY2719) (E) at the 5’- and 3’-ends of transcribed loci (PYK1 and PMA1) and at TELVI. The error bars represent SEM of three independent experiments.

Mentions: In addition to decreased stability of the ubiquitylation mark conferred by Ubp8, the reduction in histone modification levels in the H2A mutants could be due to impaired recruitment of the modification enzymes required for the H2B K123ub cascade, such as the ubiquitin-protein ligase Bre1. To analyze the effects of the H2A substitutions on recruitment of Bre1 to actively transcribed genes, we performed ChIP analysis of HSV-tagged Bre1 (Fig 6A). All five of the H2A mutants tested showed reduced recruitment of HSV-Bre1 to PYK1 and PMA1, particularly at their 5’ ends (Fig 6A). With the exception of the H2A-E57A mutant, Bre1 occupancy was also reduced at ADH1 (S2 Fig). As expected, HSV-Bre1 levels at the non-transcribed TELVI region were similar to those of the untagged control strain. Also in agreement with previous observations [7], Bre1 levels at the 5’ ends of PMA1 and PYK1 were higher than those at the 3’ ends of the genes. To determine whether reduced levels of HSV-Bre1 could account for the reduced HSV-Bre1 occupancy in the H2A mutant strains, we performed western analysis. Our results show that total HSV-Bre1 levels in the H2A mutants are similar to those in a wild-type strain (S4 Fig). These results indicate that residues in the H2A acidic patch are required for proper Bre1 recruitment to active genes.


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)

Recruitment of histone modification and elongation machinery is impaired in the H2A mutants.ChIP analyses of HSV-Bre1 (KY2674) (A), Rtf1 (KY2674) (B), Spt5 (KY943) (C), Spt16 (KY2675) (D), and HSV-Set1 (KY2719) (E) at the 5’- and 3’-ends of transcribed loci (PYK1 and PMA1) and at TELVI. The error bars represent SEM of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524731&req=5

pgen.1005420.g006: Recruitment of histone modification and elongation machinery is impaired in the H2A mutants.ChIP analyses of HSV-Bre1 (KY2674) (A), Rtf1 (KY2674) (B), Spt5 (KY943) (C), Spt16 (KY2675) (D), and HSV-Set1 (KY2719) (E) at the 5’- and 3’-ends of transcribed loci (PYK1 and PMA1) and at TELVI. The error bars represent SEM of three independent experiments.
Mentions: In addition to decreased stability of the ubiquitylation mark conferred by Ubp8, the reduction in histone modification levels in the H2A mutants could be due to impaired recruitment of the modification enzymes required for the H2B K123ub cascade, such as the ubiquitin-protein ligase Bre1. To analyze the effects of the H2A substitutions on recruitment of Bre1 to actively transcribed genes, we performed ChIP analysis of HSV-tagged Bre1 (Fig 6A). All five of the H2A mutants tested showed reduced recruitment of HSV-Bre1 to PYK1 and PMA1, particularly at their 5’ ends (Fig 6A). With the exception of the H2A-E57A mutant, Bre1 occupancy was also reduced at ADH1 (S2 Fig). As expected, HSV-Bre1 levels at the non-transcribed TELVI region were similar to those of the untagged control strain. Also in agreement with previous observations [7], Bre1 levels at the 5’ ends of PMA1 and PYK1 were higher than those at the 3’ ends of the genes. To determine whether reduced levels of HSV-Bre1 could account for the reduced HSV-Bre1 occupancy in the H2A mutant strains, we performed western analysis. Our results show that total HSV-Bre1 levels in the H2A mutants are similar to those in a wild-type strain (S4 Fig). These results indicate that residues in the H2A acidic patch are required for proper Bre1 recruitment to active genes.

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.