Global turnover of histone post-translational modifications and variants in human cells

Department of Molecular Biology, Princeton University, Princeton NJ, 08544, USA. .
Epigenetics & Chromatin (Impact Factor: 5.33). 12/2010; 3(1):22. DOI: 10.1186/1756-8935-3-22
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Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement.
In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC) pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants.
Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.

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Available from: Rebecca Levin, Oct 19, 2015
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    • "Reestablishing H3K27me3 on the duplicated chromatin depends on the methyltransferase E(z), a member of the PRC2 complex [43]. Several studies exploiting mass spectrometry approaches show that H3K27me3 appears stable following replication, supporting the view that parental histones and marks are recycled [60] [61]. However, studies in Drosophila have challenged this view. "
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    ABSTRACT: In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin is critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process. This article is part of a Special Issue entitled: Methylation Multifaceted Modification - looking at transcription and beyond, edited by Dr. Johnathan Whetstine.
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 12/2014; DOI:10.1016/j.bbagrm.2014.03.009 · 6.33 Impact Factor
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    • "We thus explored its role in heterochromatin assembly during RAF-induced senescence of human fibroblasts. Histone acetylation has a rapid turnover due to the highly dynamic equilibrium between histone acetyl transferase (HAT) and histone deacetylase (HDAC) activities [36]. Previous work suggested that MOF (MYST1/KAT8) is the key HAT responsible for the bulk global acetylation of H4-K16 in mammals [11,12]. "
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    ABSTRACT: Background Cellular senescence is a stress response of mammalian cells leading to a durable arrest of cell proliferation that has been implicated in tumor suppression, wound healing, and aging. The proliferative arrest is mediated by transcriptional repression of genes essential for cell division by the retinoblastoma protein family. This repression is accompanied by varying degrees of heterochromatin assembly, but little is known regarding the molecular mechanisms involved. Results We found that both deacetylation of H4-K16Ac and expression of HMGA1/2 can contribute to DNA compaction during senescence. SIRT2, an NAD-dependent class III histone deacetylase, contributes to H4-K16Ac deacetylation and DNA compaction in human fibroblast cell lines that assemble striking senescence-associated heterochromatin foci (SAHFs). Decreased H4-K16Ac was observed in both replicative and oncogene-induced senescence of these cells. In contrast, this mechanism was inoperative in a fibroblast cell line that did not assemble extensive heterochromatin during senescence. Treatment of senescent cells with trichostatin A, a class I/II histone deacetylase inhibitor, also induced rapid and reversible decondensation of SAHFs. Inhibition of DNA compaction did not significantly affect the stability of the senescent state. Conclusions Variable DNA compaction observed during senescence is explained in part by cell-type specific regulation of H4 deacetylation and HMGA1/2 expression. Deacetylation of H4-K16Ac during senescence may explain reported decreases in this mark during mammalian aging and in cancer cells.
    Epigenetics & Chromatin 08/2012; 5(1):15. DOI:10.1186/1756-8935-5-15 · 5.33 Impact Factor
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    • "A full list of antibodies used in this study is available in Supplemental Table 2. HeLa cells were cultured in suspension between 5−10 × 105 cells/ml in minimum essential Joklik modified media supplemented with 10% new-born calf serum, 2 mM L-glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin34. mESCs were cultured in DMEM high glucose medium supplemented with 15% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 100 units/mL penicillin, 0.1 mg/mL streptomycin/mL, and 1000 units/mL LIF/ESGRO35. MEFs were derived from 14.5 d mouse embryos and cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin35. "
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    ABSTRACT: Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.
    Scientific Reports 07/2012; 2:489. DOI:10.1038/srep00489 · 5.58 Impact Factor
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