Poly(ADP-ribosylation) of histones in intact human keratinocytes

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Abstract
The poly(ADP-ribosylation)of chromosomal proteins is an epigenetic consequence of clastogenic DNA damaging agents which affects chromatin structure and function. We studied the poly(ADP-ribosylation) of the major classes of histones in response to DNA breakage induced by an extracellular burst of active oxygen (AO) or the alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG) in the immortalized human keratinocytes HaCaT using a combination of affinity chromatography on phenylboronate resin and immunoblotting with polyclonal antibodies against histones H1, H2B, H2A, H3, and H4. The following findings characterized the poly(ADPR) reaction: (1) pretreatment of nuclear extracts with snake venom phosphodiesterase which removes poly(ADPR) chains strongly reduced the material which was retained by phenylboronate; (2) the ADPR transferase inhibitor benzamide (100 .mu.M) suppressed AO-induced poly(ADP-ribosylation); (3) poly(ADP-ribosylation) reduced the electrophoretic mobility of the modified histones. Several histones were constitutively poly(ADP-ribosylated) in untreated controls: 0.03% of H2A, 0.04-0.06% of H2B, and 0.04% of H3.1 carried at least one poly(ADPR) chain of undetermined length. AO transiently increased the poly(ADPR) levels of all major histones with the exception of H1. The extent of substitution 30 min after exposure to AO generated by 50 .mu.g/mL xanthine and 5 .mu.g/mL xanthine oxidase was 0.8% for A24 > 0.3% for H4 > 0.1% for H3.1 = 0.1% for H3.2 = 0.1% for H2B.2 > 0.09% for H2A. Within 60 min, poly(ADPR) substitution had decreased to control levels for H3 and H4 and below control levels for H2A and H2B. The addition of benzamide (100 .mu.M) prevented AO-induced poly(ADP-ribosylation) as well as the drop in cellular NAD levels (at 30 min). MNNG was more effective than AO in inducing histone poly(ADP-ribosylation), and to judge from the significant decreases in electrophoretic mobility, the lengths of the ADPR chains were longer. In increasing order, the extents of substitution 20 min after treatment with 5 .mu.g/mL MNNG were 1.8% for H3 > 1.2% for H1 > 0.95% for H2B > 0.36% for H4. In addition, the variant form H2B.x and unidentified low-mobility H2B- and H4-related antigens were substantially poly(ADP-ribosylated). Our results indicate that AO and MNNG induce distinct patterns of histone poly(ADP-ribosylation). Consequently, different DNA strand breaking agents are expected to affect chromatin structure and function in a characteristic fashion.

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