The DNA Binding and Catalytic Domains of Poly(ADP-Ribose) Polymerase 1 Cooperate in the Regulation of Chromatin Structure and Transcription

Abstract

We explored the mechanisms of chromatin compaction and transcriptional regulation by poly(ADP-ribose) polymerase 1 (PARP-1), a nucleosome-binding protein with an NAD⁺-dependent enzymatic activity. By using atomic force microscopy and a complementary set of biochemical assays with reconstituted chromatin, we showed that PARP-1 promotes the localized compaction of chromatin into supranucleosomal structures in a manner independent of the amino-terminal tails of core histones. In addition, we defined the domains of PARP-1 required for nucleosome binding, chromatin compaction, and transcriptional repression. Our results indicate that the DNA binding domain (DBD) of PARP-1 is necessary and sufficient for binding to nucleosomes, yet the DBD alone is unable to promote chromatin compaction and only partially represses RNA polymerase II-dependent transcription in an in vitro assay with chromatin templates (∼50% of the repression observed with wild-type PARP-1). Furthermore, our results show that the catalytic domain of PARP-1, which does not bind nucleosomes on its own, cooperates with the DBD to promote chromatin compaction and efficient transcriptional repression in a manner independent of its enzymatic activity. Collectively, our results have revealed a novel function for the catalytic domain in chromatin compaction. In addition, they show that the DBD and catalytic domain cooperate to regulate chromatin structure and chromatin-dependent transcription, providing mechanistic insights into how these domains contribute to the chromatin-dependent functions of PARP-1.

We acknowledge and thank Michael Hottiger for PARP-1 constructs; Jim Kadonaga and Takashi Ito for the recombinant ACF chromatin assembly system; Mi Young Kim for reagents and advice; Kit Umbach, Magnus Bergkvist, and the Cornell University Nanobiotechnology Center for assistance with AFM; Matthew Gamble for assistance with the statistical analyses; and members of the Kraus and Lis laboratories for critical reading of the manuscript.

This work was supported by a grant from the NIH/NIDDK (DK069710 to W.L.K.), postdoctoral fellowships from the Susan G. Komen Breast Cancer Foundation (to D.D.R.) and the New York State Health Research Science Board (to T.Z.), and Cornell University's Nanobiotechnology Center, an STC Program of the NSF (Agreement no. ECS-9876771).