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.
Highlights
DNA damage-induced ubiquitylation is important in regulating the DNA damage response
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 Polycomb-repressive complex 1 (PRC1) complexes, but not RNF8 nor RNF168
Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to double-strand break (DSB), our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation
Summary
DNA damage-induced ubiquitylation is important in regulating the DNA damage response. Mutation of histone H2AX lysines 119 and 120 to arginine and knockdown of RNF2 were both shown to inhibit histone H2AX phosphorylation [18, 19], whereas at the other extreme, defects in signaling were not observed, but homologous recombination was impaired [15] The differences between these studies may reflect compensation by paralogues [10], differences in knockdown efficiency, and the potential reprogramming of gene expression that could accompany the lengthy incubation periods required in knockdown experiments. We found that the drug was an effective inhibitor of H2AX ubiquitylation taking place in response to DNA double-strand breaks This allowed us to test whether or not transient inhibition of PRC1 could reproduce any of the results obtained using knockdown and knock-out approaches. We find that inhibition of PRC1 dramatically reduces the accumulation of ubiquitin at sites of DSBs and strongly inhibits the repair of DSBs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
