Wendy W. Au

Nuclear–cytoplasmic shuttling of BARD1 contributes to its proapoptotic activity and is regulated by dimerization with BRCA1

The breast cancer-associated protein, BARD1, colocalizes with BRCA1 in nuclear foci in the S phase and after DNA damage, and the two proteins form a stable heterodimer implicated in DNA repair, protein ubiquitination, and control of mRNA processing. BARD1 has a BRCA1-independent proapoptotic activity; however, little is known about its regulation. Here, we show that BARD1 localization and apoptotic activity are regulated by nuclear–cytoplasmic shuttling. We identified a functional CRM1-dependent nuclear export sequence (NES) near the N-terminal RING domain of BARD1. The NES forms part of the BRCA1 dimerization domain, and coexpression of BRCA1 resulted in masking of the NES and nuclear retention of BARD1. In transient expression assays, BARD1 apoptotic activity was stimulated by nuclear export, and both apoptotic function and nuclear export were markedly reduced by BRCA1. Similar findings were obtained for endogenous BARD1. Silencing BRCA1 expression by siRNA, or disrupting the endogenous BARD1/BRCA1 interaction by peptide competition caused a reduction in BARD1 nuclear localization and foci formation, and increased the level of cytoplasmic BARD1 correlating with increased apoptosis. Our findings suggest that BRCA1/BARD1 heterodimer formation is important for optimal nuclear targeting of BARD1 and its role in DNA repair and cell survival.

The BRCA1 RING and BRCT domains cooperate in targeting BRCA1 to ionizing radiation-induced nuclear foci.

BRCA1 accumulates in nuclear foci during S-phase and reassembles into DNA repair-associated foci after DNA damage, reflecting its role in genome maintenance. BRCA1 comprises a RING domain at the N terminus and a BRCT domain at the C terminus, through which it associates with DNA repair proteins. The key sequences that target BRCA1 to DNA damage-induced foci have not been identified. Here, we mapped the BRCA1 foci-targeting domains of yellow fluorescence protein (YFP)-tagged BRCA1 in MCF-7 breast cancer cells exposed to ionizing radiation (IR). Cancer mutations specific to the BRCT domain, but not the RING domain, abolished BRCA1 recruitment to IR-induced foci. The YFP-BRCT domain itself, however, localized poorly at IR-induced foci, and the RING domain and other sequences were negative. We discovered that only when the RING and BRCT domains were combined was foci targeting restored to levels observed for wild-type BRCA1. The RING-BRCT fusion co-localized at foci with the MDC1 DNA damage response factor and inhibited entry of endogenous BRCA1 into nuclear foci. Our results explain why exon 11-deficient BRCA1 splice variants are targeted to IR-induced foci even though they are incapable of repairing DNA damage. We propose that both RING and BRCT domains together target BRCA1 to large focal assemblies at DNA double-stranded breaks.