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.
Highlights
Histones H2A, H2B, H3, and H4 form the nucleosome core and are important targets for post-translational modification in eukaryotic cells
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
Identification of Histones: Mass Determination and Peptide Mapping—Histones were enriched by acid extraction and analyzed by reversed-phase chromatography in-line with ion spray mass spectrometry (LC/MS)
Summary
Cell Culture and Treatment—K562 human erythroleukemia cells were obtained from the American Type Culture Collection. Histone Isolation—Histones were isolated from 2.5 ϫ 107 cells by acid extraction. Lysates were prepared by 20 strokes in a tight fitting glass/glass Dounce homogenizer, followed by three 20-s pulses of sonication on ice. Insoluble material was collected by centrifugation (17,000 ϫ g for 10 min), and the pellets were resuspended in buffer B (8 M urea, 100 mM dithiothreitol, 1% Triton X-100) and incubated 10 min at room temperature. Histones and chromatin-associated proteins were precipitated from the supernatant by the addition of 10 volumes of cold acetone and incubation overnight at Ϫ20 °C. The proteins were collected by centrifugation (17,000 ϫ g for 10 min), and the pellets were lyophilized and resuspended in 100 l of 10 mM HEPES, pH 8.0, plus protease inhibitors. In some experiments, isolated histones were treated with 20 units of calf alkaline intestinal phosphatase (Promega, with no additional buffers). The proteins were eluted from the column with a gradient of
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