Mutations in the breast cancer susceptibility gene, BRCA1, account for a significant proportion of hereditary breast and ovarian cancers. The BRCA1 C-terminal (BRCT) domain, which can activate transcription when fused to a heterologous DNA binding domain, is required for BRCA1 function in suppression of tumorigenesis. Here, we provide evidence for a new activation domain in BRCA1 that lies adjacent to the BRCT domain. We name the two domains AD1 and AD2, respectively. Like AD2, the newly

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... red AD1 can act independently as an activation domain in both yeast and human cells. However, unlike AD2, AD1 activity in mammalian cells is cell type context-dependent. Furthermore, combination of these two domains in mammalian cells can result in a robust synergy in transcriptional activation. A highly conserved coiled-coil motif in AD1 is required for the cooperative transcription activation. Interestingly, the functional cooperativity between AD1 and AD2 is absent in certain breast and ovarian cancer cell lines, although each domain can still activate transcription. Therefore, the differential and cooperative actions of the two activation modules may contribute to the heterogeneous risk of BRCA1 mutations in different tissues. Between 5 and 10% of all breast cancers are hereditary, and most of these are caused by germline mutations in two breast cancer susceptibility genes, BRCA1 and BRCA2 (1, 2). The remaining 90 -95% of breast cancers are classified as sporadic. The human BRCA1 gene encodes a 1863-amino acid protein with a highly conserved RING finger domain at the N terminus and two repeats of the BRCT 1 domain at the C terminus (see Fig. 1A ). Although most cancer-predisposing mutations of BRCA1 result in gross truncation of the protein, 5-10% of the disease-associated mutations lead to single amino acid substitutions (5). Many of the cancer-predisposing single-point mutations occur in the RING or BRCT domains, underscoring the importance of these two domains to BRCA1 function in tumor suppression. The exact biochemical function of the BRCA1 protein has been the focus of intense research. Several lines of evidence suggest that BRCA1 is involved in DNA repair (6 -9). Embryonic stem cells from BRCA1-deficient mice are hypersensitive to ionizing radiation, presumably because of defects in transcription-coupled repair of oxidative DNA damage as well as double-strand break-induced homologous recombination (6, 8, 10). The role of BRCA1 in DNA repair is further supported by the observation that it associates with several well known repair and recombination proteins such as RAD51 (11) , RAD50/ MRE11/NBS1 (12, 13), and MSH2/MSH6 (13). BRCA1 also interacts with and is phosphorylated by ATM and CHK2 (7, 14), two protein kinases that are key players in damage checkpoint control. It has also been suggested that BRCA1 is involved in regulation of transcription. When tethered to a transcriptional promoter via a heterologous DNA binding domain, the C-terminal 304-amino acid region (aa 1560 -1863) including the BRCT domain can act as a trans-activation domain (3, 4). The same C-terminal region of BRCA1 can remodel chromatin when tethered to chromosomal DNA (15). Consistent with its potential role in transcriptional regulation, the BRCA1 polypeptide is associated with the RNA polymerase II holoenzyme via RNA helicase A (16, 17). It has also been reported that, when overexpressed in mammalian cells, the full-length BRCA1 protein can potentiate transcription from several natural promoters in both a p53-dependent and -independent manner (18 -21). Finally, BRCA1 is associated with histone-modifying enzymes (p300 and histone deacetylase) (17, 21, 22) and an ATP-dependent chromatin remodeling machine (hSNF/SWI) (23). Thus, BRCA1 may utilize the BRCT domain to increase chromatin accessibility and facilitate multiple nuclear processes. During the course of further characterization of BRCA1mediated transcriptional activation, we discovered a novel trans-activation domain of BRCA1 that resides immediately upstream of the BRCT domain. We designated this new and previously known domain as AD1 and AD2, respectively. Although both domains can act as trans-activation domains, AD1 activity is restricted by cellular contexts to a greater extent than AD2 activity. Furthermore, the two activation domains can cooperatively activate transcription in many cell lines tested. A highly conserved coiled-coil region in BRCA1 is critical for the functional synergy between these two activation domains. Thus, our findings imply that other cellular and molecular modifiers could influence the biochemical property of BRCA1. EXPERIMENTAL PROCEDURES Plasmids and Cell Lines-The mammalian luciferase reporter plasmid was described previously (24). To construct the mammalian expression vectors for the GAL4 fusion proteins, the sequences encoding