2-pyridine-3-yl-methylene-indan-1,3-dione (PRT-4165) 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 PRT-4165 inhibits both RNF2 and RING 1A. PRT-4165 is an inhibitor of Bmi1/Ring1A, subunits of the polycomb repressive complex 1 (PRC1); inhibits self-ubiquitination (IC50 = 3.9 uM) but does not increase cellular levels of either subunit. Prevents Bmi1/Ring1A-mediated ubiquitination and drug-induced degradation of topoisomerase 2α (Top2α). Also shown to inhibit the in vitro E3 ubiquitin ligase activity of RNF2 and a Bmi1/RNF2 complex, inhibiting H2A/H2AX ubiquitination. Blocks polycomb repressor complex (PRC) 1-mediated histone H2A ubiquitination in vitro and in vivo. PRT-4165 can be used torapidly inhibit ongoing histone H2A ubiquitylation in cells. This drug represents the first chemical inhibitor that is available to inhibit the PRC1 E3 ubiquitin ligase and should provide an important tool for gene expression studies.