Abstract
BackgroundPost-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.ResultsIn 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.ConclusionsQuantitative 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.
Highlights
Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events
It is known that the majority of histone synthesis is synchronized with Sphase, and that H3.1 and H3.3 are deposited in a replication-dependent and-independent manner, respectively [8,9]
Because we studied endogenous histones, there are no tags to interfere with higher-order chromatin structure and our measurements accurately capture global in vivo global histone turnover
Summary
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. 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. Acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, 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. 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. We observed little difference in the turnover between most modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me has a faster turnover than this mark on the other H3 variants
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