In Vivo Residue-specific Histone Methylation Dynamics

Abstract

Methylation of specific histone residues is capable of both gene activation and silencing. Despite vast work on the function of methylation, most studies either present a static snapshot of methylation or fail to assign kinetic information to specific residues. Using liquid chromatography-tandem mass spectrometry on a high-resolution mass spectrometer and heavy methyl-SILAC labeling, we studied site-specific histone lysine and arginine methylation dynamics. The detection of labeled intermediates within a methylation state revealed that mono-, di-, and trimethylated residues generally have progressively slower rates of formation. Furthermore, methylations associated with active genes have faster rates than methylations associated with silent genes. Finally, the presence of both an active and silencing mark on the same peptide results in a slower rate of methylation than the presence of either mark alone. Here we show that quantitative proteomic approaches such as this can determine the dynamics of multiple methylated residues, an understudied portion of histone biology.Histones are decorated extensively with numerous posttranslational modifications (PTMs) 2 on several different residues (1). Located mostly in the unstructured N-terminal tails, these PTMs influence the expression of genes bound to the histones by the recruitment or displacement of non-histone transcriptional or regulatory proteins (2). Lysine methylation is notable among histone PTMs for its diversity of forms and for its binary-like influence over gene expression. Methyltransferases and demethylases catalyze specific conversions between unmodified (me0), mono-(me1), di-(me2), and trimethylation (me3) states. For instance, while G9a and Suv39h1 direct histone H3 lysine 9 (H3K9) me1/me2 and me3 production, respectively (3), JHDM2A (4), and JHDM3A (5) promote demethylation of H3K9me2 and H3K9me3, respectively. The binary influence on gene expression can be illustrated with chromatin immunoprecipitation (ChIP) studies that revealed H3K4 to be enriched in euchromatic regions and H3K9 in heterochromatic regions (6). Another residue that can be mono-and dimethylated on histones is arginine. Furthermore, whereas H4R3 methylation (7) and H3R2me1 (8) are associated with gene activation, H3R2me2 is associated with gene silencing (8).These features suggest that histone lysine and arginine methylations are dynamically regulated processes. Nevertheless, most studies present histone methylation as a static condition at a particular time, with little regard to turnover or dynamics of the PTM. Indeed, the inability to resolve between transient and more long-lived methylations likely contributed to the traditional conception of histone methylation as irreversible (9). Past studies have used radiolabeling to track methylation turnover on a non-residue specific basis (10, 11) and other studies monitored the correlation between histone methylation and cell cycle progression (12-14), including a recent report (15) that tracked the modification profile of newly synthes...

Epigenetic inheritance: Uncontested?

“…Current results indicated that histone lysine methylations in general have a much longer half-life than acetylation or phosphorylation [51,60,61], and that certain methylation states might be established on newly deposited histones in a chromatin assembly-coupled way. But clearly, modification states like H4K20me2 are established by a mechanism independent of DNA replication [51,52].…”

“…Surprisingly, old H3 histones continue to be methylated at K79 at a rate comparable to that of newly deposited H3 histones, a clear indication that H3K79 methylation cannot be reestablished in a replicationcoupled manner. Given that H3K79me2 shares a similar half-life with the histone modifications most likely to carry epigenetic information (H3K27me3 and H3K9me3) [60,61], it was proposed that there might be a certain degree of positional "scrambling" of K79 methylation through the cell cycle [59].…”

H3K36 Methylation Antagonizes PRC2-mediated H3K27 Methylation

“…For quantification, the membranes were stained with Coomassie Blue G-250 followed by liquid scintillation counting for each stained histone band. Are Mostly Methylated at Lys-27, Except for Newly Synthesized Histones-We initially intended to assess the establishment of histone lysine methylation on newly synthesized histones during the cell cycle using stable isotope labeling-based quantitative MS (25,(32)(33)(34). In brief, HeLa cells were arrested by double thymidine block and then released into lysine 8 ([ 13 C 6 , 15 N 2 ] heavy isotope-labeled L-lysine)-supplemented medium, which labels newly synthesized proteins.…”

Chemical and biochemical approaches in the study of histone methylation and demethylation

“…168,169 Various studies have applied the SILAC method for the analysis of histone modifications. 170133,171,172 For example, Bonenfant and colleagues analyzed the core histone modifications occurring through the cell cycle. 173 A complex pattern of cycledependent methylation was observed: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased.…”