In vitro poly(ADP-ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP-ribosyl)ated in vitro. The modifying p roduct, poly(ADP-ribose), was found covalently bound to each histone varian t at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other ti ssues, and the testis-specific H1t, which appears only at the pachytene spe rmatocyte stage of germ cell development. These H1s were modified with poly (ADP-ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP-ribos e)polymerase in the presence of [P-32] NAD. In parallel, poly(ADP-ribosyl)a ted H1s were also prepared following incubation of intact rat testis nuclei with [P-32] NAD. In both experiments, the poly(ADP-ribosyl)ated proteins w ere purified from the native forms by means of phenyl boronic agarose chrom atography. The results from both analyses were in agreement and showed qual itative differences with regard to the poly(ADP-ribose) covalently associat ed with H1a and H1t. Comparison of the bound polymers clearly indicated tha t the oligomers associated with H1a were within 10-12 units long, whereas l onger chains (less than or equal to 20 ADP-R units) were linked to H1t. Ind ividual poly(ADP-ribosyl)ated H1s were complexed with homologous H1-deplete d oligonucleosomes (0.5-2.5 kbp) in order to measure their ability to conde nsate chromatin, in comparison with the native ones. Circular dichroism sho wed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA-binding properties of the an alyzed H1s. In particular, the poly(ADP-ribosyl)ated H1a and H1t had opposi te effects on the condensation of H1-depleted oligonucleosomes. J. Cell. Bi ochem. 76:20-29, 1999. (C) 1999 Wiley-Liss, Inc.