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 product, poly(ADP-ribose), was found covalently bound to each histone variant 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 tissues, and the testis-specific H1t, which appears only at the pachytene spermatocyte 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-ribose)polymerase in the presence of [(32)P] NAD. In parallel, poly(ADP-ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [(32)P] NAD. In both experiments, the poly(ADP-ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP-ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10-12 units long, whereas longer chains (</=20 ADP-R units) were linked to H1t. Individual poly(ADP-ribosyl)ated H1s were complexed with homologous H1-depleted oligonucleosomes (0.5-2.5 kbp) in order to measure their ability to condensate chromatin, in comparison with the native ones. Circular dichroism showed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA-binding properties of the analyzed H1s. In particular, the poly(ADP-ribosyl)ated H1a and H1t had opposite effects on the condensation of H1-depleted oligonucleosomes.
Faraone-Mennella
M R
MR
Dipartimento di Chimica Organica e Biologica, Università Federico II, 16-80134 Napoli, Italy. faraone@cds.unina.it
De Lucia
F
F
Gentile
N
N
Quesada
P
P
Farina
B
B
D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D008297:Male; D009690:Nucleic Acid Conformation; D010761:Phosphorus Radioisotopes; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D051381:Rats; D013737:Testis / Q000378:metabolism*
D002843:Chromatin; D006657:Histones; D010761:Phosphorus Radioisotopes; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA