Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.
Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.
Talasz
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Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Innsbruck, Austria. Heribert.Talasz@uibk.ac.at
Helliger
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W
Sarg
B
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Debbage
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PL
Puschendorf
B
B
Lindner
H
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D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002193:Cantharidin / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D002352:Carrier Proteins; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; C411023:anti-Fas monoclonal antibody; C015482:protein kinase modulator; D002193:Cantharidin