Positive Regulation of the BRCA1 Promoter*

Inherited mutations in the BRCA1gene, 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 deletions and transient transfection assays, a 36-base pair region was determined to be important for the positive regulation ofBRCA1 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.

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 deletions 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, 5Ј-CTC ACG CGT TTA CGT CAT CCG GGG GCA-3Ј; Ϫ162, 5Ј-CTC ACG CGT GCA GAC TGG GTG GCC AAT-3Ј; Ϫ152, 5Ј-CTC ACG CGT TGG CCA ATC CAG AGC CCC-3Ј; Ϫ118, 5Ј-CTC ACG CGT CTT TCT GTC CCT CCC ATC-3Ј; Ϫ86, 5Ј-CTC ACG CGT GAT TTC GTA TTC TGA GAG-3Ј; Ϫ43, 5Ј-CTC ACG CGT GGT TTC CGT GGC AAC GGA-3Ј; Ϫ34, 5Ј-CTC ACG CGT GGC AAC GGA AAA GCG CGG-3Ј; Ϫ26, 5Ј-CTC ACG CGT AAA GCG CGG GAA TTA CAG-3Ј; Ϫ17, 5Ј-CTC ACG CGT GAA TTA CAG ATA AAT TAA-3Ј; Ϫ7, 5Ј-CTC ACG CGT TAA ATT AAA ACT GCG ACT-3Ј. The 5Ј primers included three bases, CTC, followed by the MluI enzyme site, which is underlined. The single 3Ј primer used was: ϩ36, 5Ј-TAG CTC GAG GGA AGT CTC AGC GAG CTC-3Ј. This primer included TAG followed by a XhoI site, which is underlined, at the 5Ј end. The amplification conditions used were as follows (1 cycle for 2 min at 94°C and 35 cycles at 94°C for 15 s, 60°C for 15 s, 72°C for 30 s), and 1 ng of the cloned BRCA1 promoter plasmid was used as template DNA. The amplified products were digested with MluI and XhoI restriction enzymes and ligated into the restricted MluI-XhoI site of the pGL3 basic vectors (Promega). In addition, two synthetic primers (ϩ5, 5Ј-CGC GTT GCG ACT GCG CGG CGT GAG CTC GCT GAG ACT TCC TC-3Ј and ϩ36, 5Ј-TCG AGA GGA AGT CTC AGC GAG CTC ACG CCG CGC AGT CGC AA-3Ј) designed to include the BRCA1 promoter region from ϩ5 to ϩ36 were annealed. The annealed primers generated a MluI compatible site at the 5Ј end and a XhoI compatible site at the 3Ј end, which were utilized in ligation of the annealed primers directly into the MluI and XhoI site of the pGL3 vector. All the constructs were verified by sequencing.
Cell Culture, Transfections, and Reporter Gene Assays-TK-TS13 cells (hamster kidney cells kindly provided by Dr. Bruno Calabretta) were transfected by calcium phosphate precipitation. A total of 3 g of DNA was used in each transfection (1 g of BRCA promoter, 1 g of pUC19 plasmid, and 1 g of pRL-CMV Renilla luciferase vector purchased from Promega). The cells were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 100 g/liter gentamycin sulfate. MCF-7 cells (maintained in minimum essential medium, 10% fetal bovine serum, 100 g/ml gentamycin sulfate, 2 mM sodium pyruvate and glutamine) were transfected using Fugene-6 reagent manufactured by Boehringer Mannheim. A total of 2 g of DNA (0.5 g of promoter construct and 1.5 g of pRL-CMV) with 10 l of Fugene reagent was used in each transfection. All the transfections were optimized for six-well plates. The transfected cells were lysed after 48 h by addition of 250 l of passive lysis buffer (Promega). 20 l of the lysed cell extract was added to 100 l of luciferase substrate, and the light emissions were measured in a scintillation counter. Renilla luciferase readings (which were utilized to normalize the transfection efficiencies) were measured in the same tube using conditions recommended by the manufacturer.
In experiments involving UV radiation-induced cross-linking of DNA and protein, each electrophoretic mobility shift assay reaction was transferred into a 96-well plate at 4°C and exposed to UV radiation in a UV-Stratalinker 1800 (Stratagene) for 20 min. After the addition of sample buffer (1 M Tris, pH 6.8, 10% SDS, 50% glycerol, 10% ␤-mer-captoethanol, and 0.2% bromphenol blue) and boiling for 5 min, the reaction was resolved in a 9% SDS-polyacrylamide gel. The dried gel was exposed to film. Negative control experiments with a random probe were also performed. The random probe was synthesized (in a manner similar to the PRR probe) by the extension of a DNA oligonucleotide (5Ј-CCC GGG AGT AGA-3Ј) annealed to a longer complementary oligonucleotide (5Ј-CTA GTC AGA CAC GTA GAC TCT ACT CCC GGG-3Ј) in the presence of BrdUrd and dCTP containing ␣-32 P label.

BRCA1 Minimal
Promoter-In order to identify the minimal promoter containing the essential regulatory regions, systematically deleted mutants of the BRCA1 promoter region were constructed (Fig. 1). The transcriptional activities of these mutants were tested in TK-TS13 and MCF-7 cells by luciferase reporter gene assays (Fig. 2). The TK-TS13 cells were used in initial studies ( Fig. 2A), due to the ease with which they are transfected and because of their ability to support high levels of BRCA1 promoter activities. Subsequent detailed studies were performed in MCF-7 breast epithelial cell line (Figs. 2B  and 3A).
Results of transfections of the mutants in TK-TS13 cell indicated that on the deletion of 1380 bases from mutant Ϫ1582 (and generating Ϫ202), there is a 2.5-fold drop of luciferase activity, which is not significant considering the high sensitivity of the luciferase assays and the number of bases deleted ( Fig. 2A). Within the same tract of promoter DNA (Ϫ1582 to Ϫ202), the two most wide ranging luciferase values were 6127 (for Ϫ1244) and 308 (for Ϫ329) normalized light units, a difference of 20-fold. However, this difference was accompanied by a loss of 915 bases from Ϫ1244.
Overall, the data suggest that short deletions within the segment Ϫ1582 to Ϫ202 do not cause a significant change in promoter activity. However, gross deletions (Ϫ1582 to Ϫ202) do alter the configuration of the promoter sufficiently to affect the promoter activities significantly, because several weak regulatory sites (both enhancers and repressors) with additive effects may be deleted.
Transfections of selected BRCA1 promoter mutants in MCF-7 cells also indicated that essential transcriptional regulatory sites (which could have a strong effect on the transcription of BRCA1) were not present in the tract from Ϫ1582 to Ϫ202 (Fig. 2B). Interestingly, in MCF-7 cells mutant Ϫ202 was observed to possess a transcriptional activity that was comparable (and slightly higher) to that of construct Ϫ1582.
Finally, results from both cell lines indicated the presence of a sensitive region of 222 bases (Ϫ202 to ϩ20), which on deletion resulted in a 100% loss of BRCA1 promoter activities (Fig. 2). These experiments strongly suggested that the essential regulatory elements of the BRCA1 promoter reside within the deleted segment. Furthermore, the mapped segment encompassed the P1 promoter region, suggesting that the activity of the P1 promoter was predominant in both the transfected cell lines. Curiously, P2 did not demonstrate any functional activity in either of the cell lines tested (Fig. 2). It is possible that sequences within the P1 promoter region may regulate the transcriptional initiation from P2.
Identification of the BRCA1 PRR-Following the identification of the Ϫ202 to ϩ20 segment as essential for BRCA1 transcription, detailed characterization of the segment was undertaken. Additional unidirectional, polymerase chain reactionbased deletion mutants were constructed, and their activities were tested (Fig. 3A). With the aid of these promoter mutants, it was determined that deletion of a short 22-base pair region between Ϫ198 and Ϫ177 (Fig. 3B) resulted in a significant loss (14.5-fold) of luciferase activity. Further removal of 15 more nucleotides (Ϫ162) led to an additional 4-fold loss in activity.
Overall, the removal of 36 bases (from Ϫ198 to Ϫ162) results in a 56-fold loss in luciferase activity, indicating a PRR.
Interestingly, the PRR contains a short polypyrimidine-polypurine (Py-Pu) tract (the majority of nucleotides on the sense strand are pyrimidines and by extension the complementary strand is mostly purine) (Fig. 3B). The first 22 bases in the site are almost exclusively Py-Pu (21 of 22 or 95%). This Py-Pu-rich tract is followed by a putative CREB site. Overall the pyrimidines in the sense strand make up 75% (27 of 36) of PRR.
DNA Binding Assays-In order to characterize the proteins binding to the PRR site, electrophoretic mobility shift assays were performed using MCF-7 nuclear extracts. Retarded protein-DNA complexes were detected (Fig. 4A, lanes 2-8). Lane 2 lacked poly(dI⅐dC), and therefore the factors binding the probe largely represented nonspecific proteins. Addition of poly(dI⅐dC) (lanes 3-9), cleared nonspecific bands, and three protein-DNA complexes were detected (except in lane 9); a single intense, higher mobility band and two weaker bands with lower mobility, were observed. Addition of 50-fold excess of double-stranded nonlabeled oligonucleotides or sheared salmon sperm DNA (lanes 5-8) did not compete away the protein-DNA complexes. In sharp contrast, the proteins binding the labeled probe were efficiently competed out by a 50-fold excess of nonlabeled PRR probe (lane 9). This experiment indicates that the PRR-binding proteins bind in a sequencespecific manner.
In order to further characterize the components of the protein complex that assembled on the PRR, UV cross-linking experiments (involving DNA-protein linkage) were performed (Fig. 4B). This involved incubation of radiolabeled, BrdUrdcontaining PRR probe, with MCF-7 nuclear extracts in the presence of UV radiation. The radiation induced covalent linking of proteins to the BrdUrd residues present in the PRR probe. All the reactions contained 3 g of poly(dI⅐dC), and one of the reactions was supplemented with 0.5 g of doublestranded oligonucleotides to increase the stringency of binding to the probe (lane 3). Lane 2 exhibits three diffuse and indistinct bands. The intensity of these bands increased on the addition of 0.5 g of double-stranded oligonucleotides (lane 3), reinforcing the possibility that they represent specific protein-DNA interactions. The approximate molecular masses of the protein-DNA complexes observed were 55, 150, and 230 kDa.
Additional experiments were performed to exclude the possibility that the covalent protein-DNA complexes were formed due to nonspecific interactions. A random oligonucleotide labeled with ␣-32 P and BrdUrd residues was used in UV crosslinking experiments (Fig. 4C). Lane 2 of the figure demonstrates the characteristic three-band pattern representing proteins bound to the PRR. In contrast, no significant protein-DNA complexes were observed when the random probe was incubated with MCF-7 nuclear extract (lane 4). This experiment suggests that the proteins detected by cross-linking experiments recognized and bound the PRR specifically.
Finally, it is probable that each of the DNA-protein complexes observed do not represent multiple proteins linked to a single DNA probe but a single protein molecule bound to one molecule of the PRR probe. This conclusion is based on the fact that the UV-induced protein-DNA cross-linking is inefficient; therefore it is unlikely that more than one protein molecule will link to a single molecule of DNA probe. DISCUSSION Evidence for important roles of BRCA1 in normal functioning of cells is accumulating. Specifically, a strong role for BRCA1 in DNA repair mechanisms (18 -20) as well as in transcriptional regulation (21-23) has been suggested. This theme is reinforced in a recent report that cites the involvement of BRCA1 in transcription-coupled repair of DNA (24). Therefore, FIG. 1. BRCA1 promoter region and mutants. The upper panel depicts the BRCA1 promoter region, with two previously described initiation sites P1 (nucleotide 1582) and P2 (nucleotide 1858) (11). The lower panel depicts the generation of unidirectional deletions within the promoter region, which leave the upstream KpnI site in the polylinker intact (the adjacent PstI is within the BRCA1 promoter). Cleavage of the digested fragment with KpnI and PstI releases the promoter fragment. The numbers of the mutants correspond to the 5Ј deletions catalogued and characterized in Fig. 2A. it has been suggested that suppression of BRCA1 expression may cause defects in the DNA repair machinery, leading to chromosomal defects and tumorigenesis.
BRCA1 expression may be suppressed through transcriptional silencing in a subset of sporadic breast cancers, underscoring the importance of studies to elucidate the transcriptional mechanisms involved in the regulation of BRCA1 expression. The present studies strongly suggest that intact and functional PRR may be crucial for normal transcription of BRCA1. Hindrance of the PRR function may occur either by methylation of proximate sequences or by alterations in the properties of the regulatory factors, leading to suppression of BRCA1 expression. Furthermore, elucidation of the factors that regulate BRCA1 transcription could provide additional clues regarding its function.
It is interesting that the PRR encompasses a CpG dinucleotides reported by Mancini et al. (9) to be methylated in one case of sporadic breast cancer. The methylated cytosine was present in the putative CREB site present in the PRR (Fig. 3B). CREB proteins are known to mediate hormone stimulation of a variety of genes (25,26), and BRCA1 is known to be indirectly responsive to estrogen stimulation (27), prompting speculation of a regulatory role of CREB in BRCA1 transcription. However, treatment of cells with forskolin (a reagent that stimulates post-translational activation of CREB by phosphorylation; Ref. 28) did not show any effect on the activities of transiently transfected BRCA1 promoter (data not shown). In addition, forskolin treatment and cotransfection of a CREB-binding protein (a CREB coactivator; Refs. 29 and 30) expression plasmid elicited no response from the BRCA1 promoter (data not   5-7). In addition, 50-fold excess of sheared salmon sperm DNA was also used as a nonspecific competitor (lane 8). Specific competition was performed by the addition of 50-fold excess of unlabeled PRR-binding oligonucleotide (lane 9). B, DNA-protein UV cross-linking experiments with PRR probe and MCF-7 nuclear extracts. The protein-DNA complexes are indicated (arrows). The free PRR probe is at the bottom of the lanes. In lane 3, 0.5 g of double-stranded oligonucleotide (DS Oligo) containing the binding site for the p53 protein was added. C, comparison of protein affinities of PRR and random probes in UV cross-linking assays. All the reactions contain 3 g of poly(dI⅐dC) and 0.5 g of annealed double-stranded oligonucleotide containing the binding site for the C/EBP proteins. shown). Attempts to identify CREB proteins in the DNA-protein complex, either by supershift assays or immunoprecipitation (of cross-linked DNA-protein complexes) with CREB antibodies, were not successful (data not shown).
The composition of the PRR (21 of the first 22 bases are pyrimidines on the sense strand) provides possible hints regarding mechanisms involved in transcriptional regulation of BRCA1. Previous studies have described the tendency for such Py-Pu domains to form triplex DNA, which influence transcription (31,32). These tracts have been reported to be sensitive to S-1 nuclease digestion and are believed to influence the conformation of the chromatin assembly in the promoter region. In addition, a nuclear factor has been reported to bind a Py-Pu tract in the c-Ki-ras promoter (31). Therefore, it is possible that 1) the Py-Pu domain may alter the chromatin structure of the BRCA1 promoter region and 2) it may also be involved in specific recognition and binding by transcription factors.
There is no additional information available at present regarding the factors binding the PRR. Studies are being initiated to definitively identify and characterize the binding proteins in order to investigate their effects on BRCA1 transcription and their potential role in breast cancer.