Global Regulation of Post-translational Modifications on Core Histones

Abstract

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose-and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.

Chromosomal Protein HMGN1 Modulates the Phosphorylation of Serine 1 in Histone H2A

“…Second, H3 and H2A histone tails reach outside of the nucleosomal particle at opposite sites (25). Third, H3 histone is much more intensively modified in vivo than histone H2A (21). Despite the numerous differences between histones H2A and H3, the modifications of both histones are modulated by nucleosomal-bound HMGN1.…”

Liquid chromatography mass spectrometry profiling of histones

“…In principle, the characterization of these two amino acids (considering acetylation, and 1−3 methylations at K9 with/without S10 phosphorylation) would require 10 antibodies of which only 4 are currently available commercially. Histone characterization is further complicated due to the existence of sequence variants [8][9][10][11][12]. These variants often differ by only a few amino acids, which may not be in the antigenic epitope.…”

“…Thus, detection of specific variants using antibodies is challenging. More direct approaches have been employed including micro-sequencing and sequencing by mass spectrometry [9][10][11][12][13]. However, sequencing based methodologies are usually tedious and time-consuming.…”

Application of mass spectrometry to the identification and quantification of histone post‐translational modifications

“…As expected, phosphorylated isoforms of histones H2A, H3, and H4 increased following OA treatment. However, a dramatic decrease in an isoform of histone H4 that represents a combination of mono-acetylation and di-methylation preceded the appearance of phosphorylated H4 isoforms suggesting that OA can also effect histone acetylation [Galasinski et al, 2002].…”