Global regulation of post-translational modifications on core histones.
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.
Galasinski
Scott C
SC
Howard Hughes Medical Institute, the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Louie
Donna F
DF
Gloor
Kristen K
KK
Resing
Katheryn A
KA
Ahn
Natalie G
NG
D000107:Acetylation; D002843:Chromatin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D006657:Histones / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007477:Ions; D020014:K562 Cells; D013058:Mass Spectrometry; D019319:Okadaic Acid / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013997:Time Factors
D002843:Chromatin; D004791:Enzyme Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D007477:Ions; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D019319:Okadaic Acid; C012589:trichostatin A; D010744:Phosphoric Monoester Hydrolases