A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks

Ismail Hassan Ismail; Darin McDonald; Hilmar Strickfaden; Zhizhong Xu; Michael J. Hendzel

doi:10.1074/jbc.M113.461699

A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks*

From the ^‡Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada and
the ^§Biophysics Department, Faculty of Science, Cairo University, Giza 12613, Egypt

↵2 Alberta Innovates Health Solutions Senior Scholar. To whom correspondence should be addressed: Dept. of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Cross Cancer Institute, 11560 University Ave., Edmonton, AB T6G 1Z2, Canada. Tel.: 780-432-8439; Fax: 780-432-8892; E-mail: mhendzel{at}ualberta.ca.

Background: DNA damage-induced ubiquitylation is important in regulating the DNA damage response.

Results: PRT4165 inhibits histone H2A ubiquitylation and the accumulation of ubiquitin at the DNA double-strand break (DSB) sites.

Conclusion: PRT4165 is a novel drug for studying DSB response.

Significance: PRT4165 may constitute a novel approach for studying DSB response and for development of new cancer therapy.

Abstract

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

Footnotes

↵1 Recipient of Alberta Cancer Foundation postdoctoral fellowships.
↵* This work was supported by grants from the Alberta Cancer Foundation and the Canadian Institute of Health Research.