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Identification of lysine 37 of histone H2B as a novel site of methylation.

Gardner KE, Zhou L, Parra MA, Chen X, Strahl BD - PLoS ONE (2011)

Bottom Line: Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks.While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it.Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, School of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.

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Methylation of lysine 37 of histone H2B is conserved.(A) Multiple sequence alignment of histone H2B from different species reveals that budding yeast histone H2B lysine 37 is conserved from yeast to humans. Sequence alignment was completed using ClustalX [103]. NCBI accession numbers are as follows: Saccharomyces cerevisiae: NP_010510.1; Schizosaccharomyces pombe: NP_588181.1; Drosophila melanogaster: NP_724342.1; Caenorhabditis elegans: NP_507031.1; Xenopus laevis: NP_001086753.1; Mus musculus: NP_783594.1; Gallus gallus: CAA40537.1; Bos taurus: DAA31692.1; Homo sapiens: NP_733759.1. Asterisk (*) denotes position of conserved lysine residue. (B) Increasing amounts of oligonucleosomes purified from chicken erythrocyte nuclei and mononucleosomes isolated from HeLa cell nuclei were run against histones extracted from yeast strains harboring wild-type Flag-H2B (YKG001), Flag-H2B K37A (YKG007), and wild-type H2B (untagged) (BY4742), as shown by Coomassie brilliant blue (CBB) staining of histone proteins electrophoresed on 15% SDS-polyacrylamide gels (lower panel). An equivalent loading of histone substrate was used for Western blot analysis using purified α-H2BK37me2 antibody (upper panel). Similar signals are detected for chicken- and human-derived histone substrates to that observed for yeast harboring wild-type H2B (either tagged or untagged), but not yeast H2B with an K37A mutation, thus demonstrating that dimethylation of histone H2B lysine 37 is conserved among species.
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pone-0016244-g005: Methylation of lysine 37 of histone H2B is conserved.(A) Multiple sequence alignment of histone H2B from different species reveals that budding yeast histone H2B lysine 37 is conserved from yeast to humans. Sequence alignment was completed using ClustalX [103]. NCBI accession numbers are as follows: Saccharomyces cerevisiae: NP_010510.1; Schizosaccharomyces pombe: NP_588181.1; Drosophila melanogaster: NP_724342.1; Caenorhabditis elegans: NP_507031.1; Xenopus laevis: NP_001086753.1; Mus musculus: NP_783594.1; Gallus gallus: CAA40537.1; Bos taurus: DAA31692.1; Homo sapiens: NP_733759.1. Asterisk (*) denotes position of conserved lysine residue. (B) Increasing amounts of oligonucleosomes purified from chicken erythrocyte nuclei and mononucleosomes isolated from HeLa cell nuclei were run against histones extracted from yeast strains harboring wild-type Flag-H2B (YKG001), Flag-H2B K37A (YKG007), and wild-type H2B (untagged) (BY4742), as shown by Coomassie brilliant blue (CBB) staining of histone proteins electrophoresed on 15% SDS-polyacrylamide gels (lower panel). An equivalent loading of histone substrate was used for Western blot analysis using purified α-H2BK37me2 antibody (upper panel). Similar signals are detected for chicken- and human-derived histone substrates to that observed for yeast harboring wild-type H2B (either tagged or untagged), but not yeast H2B with an K37A mutation, thus demonstrating that dimethylation of histone H2B lysine 37 is conserved among species.

Mentions: Sequence alignment of histone H2B from Saccharomyces cerevisiae against multiple species reveals that lysine 37 is conserved along evolution, despite lower sequence similarity of surrounding amino acid residues (Figure 5A). To determine if we could detect the presence of methylated lysine 37 in higher eukaryotes, we performed Western blot analysis comparing oligonucleosomes isolated from chicken erythrocyte nuclei and core histones from HeLa cell nuclei to yeast histones. Western blot analysis using the α-H2BK37me2 antibody revealed that this mark is indeed conserved in higher eukaryotes (Figure 5B), as a comparable species is observed in both the chicken and human histone samples as to histones extracted from yeast harboring wild-type, but K37A mutant, H2B. The presence of a discernable signal in samples derived from higher eukaryotic species suggests that, despite the lack of an obvious cellular phenotype in yeast to date, this mark is likely to be biologically important since it was retained during evolution.


Identification of lysine 37 of histone H2B as a novel site of methylation.

Gardner KE, Zhou L, Parra MA, Chen X, Strahl BD - PLoS ONE (2011)

Methylation of lysine 37 of histone H2B is conserved.(A) Multiple sequence alignment of histone H2B from different species reveals that budding yeast histone H2B lysine 37 is conserved from yeast to humans. Sequence alignment was completed using ClustalX [103]. NCBI accession numbers are as follows: Saccharomyces cerevisiae: NP_010510.1; Schizosaccharomyces pombe: NP_588181.1; Drosophila melanogaster: NP_724342.1; Caenorhabditis elegans: NP_507031.1; Xenopus laevis: NP_001086753.1; Mus musculus: NP_783594.1; Gallus gallus: CAA40537.1; Bos taurus: DAA31692.1; Homo sapiens: NP_733759.1. Asterisk (*) denotes position of conserved lysine residue. (B) Increasing amounts of oligonucleosomes purified from chicken erythrocyte nuclei and mononucleosomes isolated from HeLa cell nuclei were run against histones extracted from yeast strains harboring wild-type Flag-H2B (YKG001), Flag-H2B K37A (YKG007), and wild-type H2B (untagged) (BY4742), as shown by Coomassie brilliant blue (CBB) staining of histone proteins electrophoresed on 15% SDS-polyacrylamide gels (lower panel). An equivalent loading of histone substrate was used for Western blot analysis using purified α-H2BK37me2 antibody (upper panel). Similar signals are detected for chicken- and human-derived histone substrates to that observed for yeast harboring wild-type H2B (either tagged or untagged), but not yeast H2B with an K37A mutation, thus demonstrating that dimethylation of histone H2B lysine 37 is conserved among species.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020972&req=5

pone-0016244-g005: Methylation of lysine 37 of histone H2B is conserved.(A) Multiple sequence alignment of histone H2B from different species reveals that budding yeast histone H2B lysine 37 is conserved from yeast to humans. Sequence alignment was completed using ClustalX [103]. NCBI accession numbers are as follows: Saccharomyces cerevisiae: NP_010510.1; Schizosaccharomyces pombe: NP_588181.1; Drosophila melanogaster: NP_724342.1; Caenorhabditis elegans: NP_507031.1; Xenopus laevis: NP_001086753.1; Mus musculus: NP_783594.1; Gallus gallus: CAA40537.1; Bos taurus: DAA31692.1; Homo sapiens: NP_733759.1. Asterisk (*) denotes position of conserved lysine residue. (B) Increasing amounts of oligonucleosomes purified from chicken erythrocyte nuclei and mononucleosomes isolated from HeLa cell nuclei were run against histones extracted from yeast strains harboring wild-type Flag-H2B (YKG001), Flag-H2B K37A (YKG007), and wild-type H2B (untagged) (BY4742), as shown by Coomassie brilliant blue (CBB) staining of histone proteins electrophoresed on 15% SDS-polyacrylamide gels (lower panel). An equivalent loading of histone substrate was used for Western blot analysis using purified α-H2BK37me2 antibody (upper panel). Similar signals are detected for chicken- and human-derived histone substrates to that observed for yeast harboring wild-type H2B (either tagged or untagged), but not yeast H2B with an K37A mutation, thus demonstrating that dimethylation of histone H2B lysine 37 is conserved among species.
Mentions: Sequence alignment of histone H2B from Saccharomyces cerevisiae against multiple species reveals that lysine 37 is conserved along evolution, despite lower sequence similarity of surrounding amino acid residues (Figure 5A). To determine if we could detect the presence of methylated lysine 37 in higher eukaryotes, we performed Western blot analysis comparing oligonucleosomes isolated from chicken erythrocyte nuclei and core histones from HeLa cell nuclei to yeast histones. Western blot analysis using the α-H2BK37me2 antibody revealed that this mark is indeed conserved in higher eukaryotes (Figure 5B), as a comparable species is observed in both the chicken and human histone samples as to histones extracted from yeast harboring wild-type, but K37A mutant, H2B. The presence of a discernable signal in samples derived from higher eukaryotic species suggests that, despite the lack of an obvious cellular phenotype in yeast to date, this mark is likely to be biologically important since it was retained during evolution.

Bottom Line: Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks.While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it.Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, School of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.

Show MeSH
Related in: MedlinePlus