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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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Related in: MedlinePlus

Phenotypic analysis of strains harboring H2B K37R/A mutations.(A) Phenotypic spotting assays indicate that cells harboring mutations at lysine 37 in histone H2B to arginine (YKG006) or alanine (YKG007) do not show sensitivity to YPD media containing 100 mM hydroxyurea (HU; a DNA damaging agent that leads to replication fork collapse), as is observed in an H2B K123R mutant strain (YKG002) [101], but rather grow similarly to yeast containing wild-type H2B (YKG001). (B) Telomeric silencing assay demonstrates that reporter strains harboring H2B K37R and H2B K37A mutations (YKG028 and YKG029, respectively) exhibit normal silencing like that observed for reporter strains expressing wild-type H2B (YKG027), but not strains that express an H2B K123R mutation (YZS274) or are deleted for SIR2 (YZS275), which have known defects in telomeric silencing [48]. Growth on SC-HIS serves as a plating control, as all strains express H2B-containing plasmids carrying a HIS3 auxotrophic marker. (C) Introduction of H2B K37R or K37A mutations (YKG033 and YKG034, respectively) into strains containing a temperature-sensitive allele of SPT16 (spt16–197) does not affect cellular growth at the semi- and non-permissive temperatures (32°C and 34°C, respectively), as cells grow at a similar rate to those harboring wild-type H2B (YKG031). Introduction of an H2B K123R mutation (YKG032) exacerbates growth in the spt16–197 background at the semi-permissive temperature, in agreement with previously published results [102]. The isogenic parental strain Y131 expressing wild-type SPT16 grows phenotypically normal at the non-permissive temperature for the spt16–197 strain.
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pone-0016244-g004: Phenotypic analysis of strains harboring H2B K37R/A mutations.(A) Phenotypic spotting assays indicate that cells harboring mutations at lysine 37 in histone H2B to arginine (YKG006) or alanine (YKG007) do not show sensitivity to YPD media containing 100 mM hydroxyurea (HU; a DNA damaging agent that leads to replication fork collapse), as is observed in an H2B K123R mutant strain (YKG002) [101], but rather grow similarly to yeast containing wild-type H2B (YKG001). (B) Telomeric silencing assay demonstrates that reporter strains harboring H2B K37R and H2B K37A mutations (YKG028 and YKG029, respectively) exhibit normal silencing like that observed for reporter strains expressing wild-type H2B (YKG027), but not strains that express an H2B K123R mutation (YZS274) or are deleted for SIR2 (YZS275), which have known defects in telomeric silencing [48]. Growth on SC-HIS serves as a plating control, as all strains express H2B-containing plasmids carrying a HIS3 auxotrophic marker. (C) Introduction of H2B K37R or K37A mutations (YKG033 and YKG034, respectively) into strains containing a temperature-sensitive allele of SPT16 (spt16–197) does not affect cellular growth at the semi- and non-permissive temperatures (32°C and 34°C, respectively), as cells grow at a similar rate to those harboring wild-type H2B (YKG031). Introduction of an H2B K123R mutation (YKG032) exacerbates growth in the spt16–197 background at the semi-permissive temperature, in agreement with previously published results [102]. The isogenic parental strain Y131 expressing wild-type SPT16 grows phenotypically normal at the non-permissive temperature for the spt16–197 strain.

Mentions: We also performed assays to screen for phenotypes related to DNA replication and repair. To that end, wild-type H2B and the H2B K37 mutant strains were spotted on media containing the agents hydroxyurea (HU, an agent which blocks replication leading to replication fork collapse) or methyl methanesulfonate (MMS, an alkylating agent that causes DNA lesions and ultimately DNA strand breaks). However lysine 37 mutations in histone H2B did not alter cellular growth compared to an isogenic wild-type parent on media containing 0.05% MMS (data not shown) or 100 mM HU (Figure 4A), where cells bearing a H2B K123R mutation were sensitive to both. Moreover, to assess the ability of lysine 37 mutant strains to carry out replication, plasmid maintenance assays were completed, where the ability of a cell to replicate a reporter plasmid containing a single origin of replication and a selectable marker is measured [84]. Mutation of lysine 37 on histone H2B to either arginine or alanine did not affect the ability of yeast strains to faithfully replicate the reporter plasmid as compared to isogenic wild-type cells (data not shown). Taken together, the results from these screening assays suggest that histone H2B lysine 37 does not have a significant role in DNA replication or repair.


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)

Phenotypic analysis of strains harboring H2B K37R/A mutations.(A) Phenotypic spotting assays indicate that cells harboring mutations at lysine 37 in histone H2B to arginine (YKG006) or alanine (YKG007) do not show sensitivity to YPD media containing 100 mM hydroxyurea (HU; a DNA damaging agent that leads to replication fork collapse), as is observed in an H2B K123R mutant strain (YKG002) [101], but rather grow similarly to yeast containing wild-type H2B (YKG001). (B) Telomeric silencing assay demonstrates that reporter strains harboring H2B K37R and H2B K37A mutations (YKG028 and YKG029, respectively) exhibit normal silencing like that observed for reporter strains expressing wild-type H2B (YKG027), but not strains that express an H2B K123R mutation (YZS274) or are deleted for SIR2 (YZS275), which have known defects in telomeric silencing [48]. Growth on SC-HIS serves as a plating control, as all strains express H2B-containing plasmids carrying a HIS3 auxotrophic marker. (C) Introduction of H2B K37R or K37A mutations (YKG033 and YKG034, respectively) into strains containing a temperature-sensitive allele of SPT16 (spt16–197) does not affect cellular growth at the semi- and non-permissive temperatures (32°C and 34°C, respectively), as cells grow at a similar rate to those harboring wild-type H2B (YKG031). Introduction of an H2B K123R mutation (YKG032) exacerbates growth in the spt16–197 background at the semi-permissive temperature, in agreement with previously published results [102]. The isogenic parental strain Y131 expressing wild-type SPT16 grows phenotypically normal at the non-permissive temperature for the spt16–197 strain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020972&req=5

pone-0016244-g004: Phenotypic analysis of strains harboring H2B K37R/A mutations.(A) Phenotypic spotting assays indicate that cells harboring mutations at lysine 37 in histone H2B to arginine (YKG006) or alanine (YKG007) do not show sensitivity to YPD media containing 100 mM hydroxyurea (HU; a DNA damaging agent that leads to replication fork collapse), as is observed in an H2B K123R mutant strain (YKG002) [101], but rather grow similarly to yeast containing wild-type H2B (YKG001). (B) Telomeric silencing assay demonstrates that reporter strains harboring H2B K37R and H2B K37A mutations (YKG028 and YKG029, respectively) exhibit normal silencing like that observed for reporter strains expressing wild-type H2B (YKG027), but not strains that express an H2B K123R mutation (YZS274) or are deleted for SIR2 (YZS275), which have known defects in telomeric silencing [48]. Growth on SC-HIS serves as a plating control, as all strains express H2B-containing plasmids carrying a HIS3 auxotrophic marker. (C) Introduction of H2B K37R or K37A mutations (YKG033 and YKG034, respectively) into strains containing a temperature-sensitive allele of SPT16 (spt16–197) does not affect cellular growth at the semi- and non-permissive temperatures (32°C and 34°C, respectively), as cells grow at a similar rate to those harboring wild-type H2B (YKG031). Introduction of an H2B K123R mutation (YKG032) exacerbates growth in the spt16–197 background at the semi-permissive temperature, in agreement with previously published results [102]. The isogenic parental strain Y131 expressing wild-type SPT16 grows phenotypically normal at the non-permissive temperature for the spt16–197 strain.
Mentions: We also performed assays to screen for phenotypes related to DNA replication and repair. To that end, wild-type H2B and the H2B K37 mutant strains were spotted on media containing the agents hydroxyurea (HU, an agent which blocks replication leading to replication fork collapse) or methyl methanesulfonate (MMS, an alkylating agent that causes DNA lesions and ultimately DNA strand breaks). However lysine 37 mutations in histone H2B did not alter cellular growth compared to an isogenic wild-type parent on media containing 0.05% MMS (data not shown) or 100 mM HU (Figure 4A), where cells bearing a H2B K123R mutation were sensitive to both. Moreover, to assess the ability of lysine 37 mutant strains to carry out replication, plasmid maintenance assays were completed, where the ability of a cell to replicate a reporter plasmid containing a single origin of replication and a selectable marker is measured [84]. Mutation of lysine 37 on histone H2B to either arginine or alanine did not affect the ability of yeast strains to faithfully replicate the reporter plasmid as compared to isogenic wild-type cells (data not shown). Taken together, the results from these screening assays suggest that histone H2B lysine 37 does not have a significant role in DNA replication or repair.

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