The cellular response to highly genotoxic DNA double-strand breaks (DSBs) involves the exquisite coordination of multiple signaling and repair factors. Here, we conducted a functional RNAi screen and identified BAP1 as a deubiquitinase required for efficient assembly of the homologous recombination (HR) factors BRCA1 and RAD51 at ionizing radiation (IR) -induced foci. BAP1 is a chromatin-associated protein frequently inactivated in cancers of various tissues. To further investigate the role of BAP1 in DSB repair, we used a gene targeting approach to knockout (KO) this deubiquitinase in chicken DT40 cells. We show that BAP1-deficient cells are (i) sensitive to IR and other agents that induce DSBs, (ii) defective in HR-mediated immunoglobulin gene conversion, and (iii) exhibit an increased frequency of chromosomal breaks after IR treatment. We also show that BAP1 is recruited to chromatin in the proximity of a single site-specific I-SceI-induced DSB. Finally, we identified six IR-induced phosphorylation sites in BAP1 and showed that mutation of these residues inhibits BAP1 recruitment to DSB sites. We also found that both BAP1 catalytic activity and its phosphorylation are critical for promoting DNA repair and cellular recovery from DNA damage. Our data reveal an important role for BAP1 in DSB repair by HR, thereby providing a possible molecular basis for its tumor suppressor function.

No abstract

The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.Posttranslational modification of proteins with ubiquitin plays a central role in a wide variety of biological processes in eukaryotic cells (44,64). Depending on the nature of the modification (e.g., poly-versus monoubiquitination), modified substrates can be either degraded by the proteasome or regulated at the level of their activity and function (4, 45). Ubiquitination is reversible, and a significant repertoire of proteases, termed deubiquitinating enzymes (DUBs), are emerging as critical regulators of ubiquitin signaling (40,46). BAP1 (BRCA1-associated protein 1) was originally isolated as a nuclear DUB that interacts with, and enhances the growthsuppressive effect of, the tumor suppressor BRCA1 (19). BAP1 also acts in a BRCA1-independent manner; its overexpression in cells lacking BRCA1 has been shown to inhibit cell proliferation and tumor growth (60). Interestingly, recent studies indicate that RNA interference (RNAi)-mediated depletion of BAP1 can also exert an inhibitory effect on cell proliferation (31,36,41). Although the exact molecular mechanisms are largely unknown, these data suggest that BAP1 controls cell cycle progression. In further support of this notion, homozygous inactivating mutations in BAP1 have been found in subsets of lung carcinoma and breast cancer cell lines, suggesting that this DUB is a tumor suppressor (19,67).