Identification of Lysine 37 of Histone H2B as a Novel Site of Methylation

Texas A&M University, United States of America
PLoS ONE (Impact Factor: 3.23). 01/2011; 6(1):e16244. DOI: 10.1371/journal.pone.0016244
Source: PubMed

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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Available from: Brian D Strahl, Sep 27, 2015
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    • "The novel types of modifications include tyrosine hydroxylation [30], serine and threonine acetylation [32], lysine crotonylation (Kcr) [30], lysine N-formylation [33], lysine succinylation [34], lysine malonylation [34], lysine propionylation [35], lysine butyrylation [35], O-GlcNAcylation (beta-N-acetylglucosamine) [36-38], lysine 5-hydroxylation [39] and cysteine glutathionylation [40]. Novel sites include Ub [41], phosphorylation [42,43], ADP-ribosylation [44], lysine acetylation and mono-, di- and tri- lysine methylations [30,45]. The degree of characterization varies for each PTM identified; however, there are some interesting findings and themes that emerge. "
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    ABSTRACT: Histone post-translational modifications (PTMs) have been linked to a variety of biological processes and disease states, thus making their characterization a critical field of study. In the last 5 years, a number of novel sites and types of modifications have been discovered, greatly expanding the histone code. Mass spectrometric methods are essential for finding and validating histone PTMs. Additionally, novel proteomic, genomic and chemical biology tools have been developed to probe PTM function. In this snapshot review, proteomic tools for PTM identification and characterization will be discussed and an overview of PTMs found in the last 5 years will be provided.
    Epigenetics & Chromatin 08/2013; 6(1):24. DOI:10.1186/1756-8935-6-24 · 5.33 Impact Factor
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    • "Briefly, 63 deletion strains (Table 3) were mated to the temperaturesensitive cdc6-1 strain (JCY332), and haploids carrying both mutations were isolated by growth on selective media. All of the deletion strains originated from the Yeast Knock Out library (Open Biosystems) except strains lacking SET1 or DOT1; the set1D strains were created de novo while the dot1D strain was previously published (Gardner et al. 2011). Additionally, SET1 and BRE1 deletions were recreated de novo in the cdc6-1 mutant in the BY4741 background (yLF058). "
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    ABSTRACT: DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.
    Genetics 07/2012; 192(2):371-84. DOI:10.1534/genetics.112.142349 · 5.96 Impact Factor
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    • "One would imagine that the simplest solution to analyze all the modified sites on histone H3 would be to sequence the intact non-digested protein, and indeed, such an approach has already been performed and is termed top down MS analysis. Although not as widely implemented as bottom up or middle down mass spectrometry, top down mass spectrometry has successfully been used recently to discover H2BK37me2 as a novel modified residue in S. cerevisiae and various phosphorylation sites on histone H1 variants, such as H1.4S187ph, enriched on promoters for rDNA and glucocorticoid response elements in HeLa cells [56, 57]. However, the same reasons why middle down MS analysis are less sensitive compared to bottom up analysis are exacerbated for top down analysis, where charge state and combinatorial PTM-dilution effects are compounded when considering the full length protein. "
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    ABSTRACT: Histone post-translational modifications (PTMs) positively and negatively regulate gene expression, and are consequently a vital influence on the genomic profile of all eukaryotic species. The study of histone PTMs using classical methods in molecular biology, such as immunofluorescence and Western blotting, is challenging given the technical issues of the approaches, and chemical diversity and combinatorial patterns of the modifications. In light of these many technical limitations, mass spectrometry (MS) is emerging as the most unbiased and rigorous experimental platform to identify and quantify histone PTMs in a high-throughput manner. This review covers the latest developments in mass spectrometry for the analysis of histone PTMs, with the hope of inspiring the continued integration of proteomic, genomic and epigenetic research.
    Current Chemical Genomics 08/2011; 5(Suppl 1):106-14. DOI:10.2174/1875397301005010106
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