Histone carbonylation in vivo and in vitro

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups i nto histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium bor ohydride). Histone H1 extracted from bovine thymus, liver and spleen was fo und to contain significantly elevated amounts of protein-bound carbonyl gro ups as compared with core histones. The carbonyl content of nuclear protein s of rat pheochromocytoma cells (PC12 cells) was not greatly increased foll owing oxidative stress induced by H2O2, but was significantly increased fol lowing alkylating stress induced by N-methyl-N'-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing s ugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei . Studies of the mechanism of histone H1 modification by ADP-ribose indicat e that carbonylation involves formation of a stable acyclic ketoamine, Our results demonstrate preferential histone H1 carbonylation in vivo, with pot entially important consequences for chromatin structure and function.