Inherited mutations in the BRCA1 gene, presumably leading to loss of function, confer susceptibility to breast and ovarian neoplasms and are thought to be responsible for approximately 2.5-5% of all breast cancers. It has been suggested that alternative mechanisms, such as disruption of transcription, may also be involved in the suppression of BRCA1 gene expression/ function in breast cancers. Therefore, we initiated studies on the BRCA1 transcriptional promoter. Utilizing systematic promoter

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... letions and transient transfection assays, a 36-base pair region was determined to be important for the positive regulation of BRCA1 transcription. Deletion of this positive regulatory region resulted in a significant loss of promoter activity. Utilizing DNA binding assays, proteins with specific affinities for the positive regulatory region were detected. Disruption of the DNA-protein complexes could affect normal BRCA1 transcription and may contribute to breast cancer susceptibility. Breast cancer is the second leading cause of death in American women, accounting for more than 50,000 deaths each year. Current estimates place the average American woman's lifetime risk of developing breast cancer at approximately 11%. However, women with two or more first degree relatives with breast cancer have an estimated 13-fold increased risk over the general population (1). Breast cancer in such families has an inheritance pattern consistent with a highly penetrant autosomal dominant allele (2, 3). BRCA1, the first breast cancer susceptibility gene to be identified (4), was isolated in 1994 (5). An interesting fact regarding BRCA1 is that although the mutations in the gene in familial breast cancers are of high penetrance, very few mutations in the BRCA1 gene have been found in sporadic forms of the cancer. These findings are prompting researchers to study the possibility of disruption of BRCA1 function through epigenetic mechanisms. Consistent with this notion, it has been suggested that transcriptional dysregulation of BRCA1 may play a role in suppressing BRCA1 expression in breast cells, perhaps contributing to the development of a neoplastic phenotype. Two studies have demonstrated a decrease of BRCA1 expression in sporadic breast cancer (6, 7). Another set of studies describe CpG methylation of BRCA1 transcriptional promoter in a number of sporadic breast cancers, in contrast to the lack of methylation in normal breast tissues samples (8, 9) . In light of these observations, we initiated a study to characterize the BRCA1 transcriptional promoter. Previously, the structural features of the BRCA1 promoter were described (10, 11), and preliminary descriptions of modest and indirect effects of estrogen on the BRCA1 promoter activity were reported (12, 13). However, no information was available regarding regulatory sites and specific regulatory factors. In this report we provide evidence for a positive regulatory region (PRR) 1 in the BRCA1 promoter and show data that suggest that multiple proteins bind specifically to the site. EXPERIMENTAL PROCEDURES Isolation of the BRCA1 Promoter-The BRCA1 promoter was subcloned from a bacterial artificial chromosome clone, BAC 694 (kindly provided by Dr. Sean Tavtigian, Myriad Genetics) (14). Briefly, PstI linker (5Ј-GCTGCAGC-3Ј) was ligated into the blunted HindIII site in the pGL2 vector (Promega vector with the firefly luciferase reporter gene). BAC 694 was digested with PstI, and the resulting fragments were shotgun cloned into the pGL2 vector and transformed into competent DH5-␣ Escherichia coli cells. The transformed bacterial colonies were screened by colony hybridization with a radiolabeled BRCA1 cDNA probe (BRCA1 cDNA was kindly provided by Frank Calzone) (15) labeled by the random hexamer method (16), and clones with a 3.8kilobase insert containing the BRCA1 5Ј genomic fragment were selected. The cloned fragment was sequenced completely and is identical to the previously described genomic fragment encompassing the BRCA1 promoter (GenBank TM accession number U37574) (11). The nucleotide position of mutants are numbers from the P1 promoter initiation site at nucleotide 1582. Mutants of BRCA1 Promoter-Systematic promoter deletions were constructed by unidirectional exonuclease III digestion. 10 g of BRCA1 promoter-luciferase construct was digested with MluI restriction enzyme and blunted with ␣-phosphorothioate nucleotides using Klenow enzyme. This treatment rendered the ends of the linearized plasmid resistant to exonuclease III digestion. The linearized DNA was purified by phenol/chloroform/isoamyl alcohol extraction and digested with XhoI restriction enzyme, generating a 5Ј end susceptible to the exonuclease digestion. The fragment was purified and then subjected to exonuclease III digestion. Aliquots of the reaction were removed at regular intervals, and the reactions were terminated. Finally, the fragments were blunted using S1 nuclease, religated, and transformed into DH5-␣ competent cells. DNA preparations (using Qiagen columns) made from selected colonies were screened by analytical restriction enzyme digestions. Mutants Ϫ202 and ϩ20 were constructed by exploiting the restriction enzyme sites present on the BRCA1 promoter (EcoRI, Ϫ202; SacI, ϩ20) and also the sites present in the polylinker of the luciferase vector. The BRCA1-luciferase construct was digested with EcoRI/XhoI (XhoI site is present in the polylinker) and SacI (SacI site is also present in the polylinker), respectively, and blunted with Klenow enzyme, and the larger fragment gel was purified and religated. Additional mutants to generate progressive deletions were constructed by a polymerase chain reaction based strategy. 5Ј primers were designed at regular intervals along the sequence of the BRCA1 promoter: Ϫ245, 5Ј-CTC ACG CGT TAG AGG CTA GAG GGC AGG-3Ј; Ϫ198, 5Ј-CTC ACG CGT TCC TCT TCC GTC TCT TTC-3Ј; Ϫ177,