BRCA1 augments transcription by the NF-kappaB transcription factor by binding to the Rel domain of the p65/RelA subunit.

BRCA1 is a tumor suppressor gene mutated in cases of hereditary breast and ovarian cancer. BRCA1 protein is involved in apoptosis and growth/tumor suppression. In this study, we present evidence that p65/RelA, one of the two subunits of the transcription factor NF-kappaB, binds to the BRCA1 protein. Treatment of 293T cells with the cytokine tumor necrosis factor-alpha induces an interaction between endogenous p65/RelA and BRCA1. GST-protein affinity assay experiments reveal that the Rel homology domain of the p65/RelA subunit of NF-kappaB interacts with multiple sites within the N-terminal region of BRCA1. Transient transfection of BRCA1 significantly enhances the ability of the tumor necrosis factor-alpha or interleukin-1beta to activate transcription from the promoters of NF-kappaB target genes. Mutation of the NF-kappaB-binding sites in the NF-kappaB reporter blocks the effect of BRCA1 on transcription. Also the ability of BRCA1 to activate NF-kappaB target genes is inhibited by a super-stable inhibitor of NF-kappaB and by the chemical inhibitor SN-50. These data indicate that BRCA1 acts as a co-activator with NF-kappaB. In addition, we show that cells infected with an adenovirus expressing BRCA1 up-regulate the endogenous expression of NF-kappaB target genes Fas and interferon-beta. Together, this information suggests that BRCA1 may play a role in cell life-death decisions following cell stress by modulation of the activity of NF-kappaB.

BRCA1 is a tumor suppressor gene involved in 50% of hereditary breast cancers and 80% of families with hereditary breast/ovarian cancers (1,2). The BRCA1 gene encodes a nuclear protein of 1863 residues, which contains two main motifs as follows: a zinc-binding RING finger domain at the N terminus, which mediates protein-protein interactions (3,4); and two tandem repeats of BRCT in the C terminus (5,6). The latter domain is involved in a variety of processes such as DNA repair, cell cycle control, and transcriptional regulation (7). The RING finger motif mediates heterodimerization with BARD1, and together the pair exhibits ubiquitin ligase activity (8).
A considerable body of data supports a role for BRCA1 in gene transcription. First, it was shown in both yeast and mam-malian cells that a GAL4-BRCA1 fusion protein activates transcription of target genes, whereas tumor-derived missense mutants in the C terminus of BRCA1 failed to do so (9). Second, the C-terminal domain of BRCA1 binds to the RNA helicase, a component of the RNA polymerase II holoenzyme, as well as the p300/CBP 1 co-activator (10 -12). In addition, chromatinmodifying proteins, such as the SWI/SNF-related complex, bind to the N terminus of BRCA1 (13). Third, BRCA1 is able to bind transcription factors, such as p53, ATF1, STAT-1, and the Jun family of proteins, to co-activate transcription (14 -17). BRCA1 also binds to Myc to inhibit transcription (18). Finally, BRCA1 was shown to regulate cell cycle progression by activating the p21 waf1/Cip1 promoter in cells that express wild type and mutant p53 (17,19). BRCA1 also was found to transactivate the expression of p27 kip1 , a member of the universal cyclindependent kinase inhibitor family (20). Additionally, BRCA1 appears to regulate transcriptionally the expression of GADD45, a protein that is involved in G 2 arrest. Cells that are null for BRCA1 or are deficient in GADD45 have a G 2 /M cell cycle checkpoint defect as well as genomic instability (21)(22)(23). Recently, it was shown that BRCA1 is essential for activating the Chk1 kinase, which represses the expression of cyclin B1 and induces G 2 /M arrest (24).
In this study, we show that BRCA1 interacts with another transcription factor, p65/RelA. p65/RelA is a member of the NF-B family of transcription factors, which consists of five members: p65/RelA, c-Rel, RelB, p50/p105, and p52/p100. The p105 and p100 proteins are processed into smaller, active ones (25)(26)(27). All of the members have a conserved N-terminal region of ϳ300 amino acids termed the Rel homology domain (RHD). The DNA-binding site, dimerization domain, nuclear localization signal, and IB-binding site reside within this domain. Three of the family members (p65/RelA, c-Rel, and RelB) have a transactivation domain in the C terminus (25,26). The NF-B transcription factor is found as an inactive homodimer or heterodimer within the cytoplasm. The most abundant form is the p65/p50 heterodimer. The dimer associates through the Rel homology domain with an inhibitory molecule, IB␣, a member of the IB protein family (26,28). The inactive NF-B-IB complex also associates with the catalytic subunit of cAMPdependent protein kinase, whose activity is inhibited by its association with the NF-B-IB complex (29). NF-B is activated by exogenous stimuli, such as oxidative (H 2 O 2 ) or radia-tion stress (UV and ␥-radiation), inflammatory cytokines (TNF␣ or IL-1␤), T cells activation, and growth factor stimulation (28,30,31). Upon activation, IB␣ is phosphorylated by the IB kinase complex, leading to the ubiquitination of IB␣ and its subsequent degradation by the 26 S proteosome (32). Once phosphorylated by cAMP-dependent protein kinase on serine 276, the NF-B dimer translocates to the nucleus (33,34), binds to sequence-specific promoter elements, and activates the transcription of target genes that are involved in immune and inflammatory reactions, anti-and pro-apoptotic processes, and cell cycle regulation (35).
In the present study, we show by both in vitro and in vivo assays that the p65/RelA interacts through the Rel homology domain with BRCA1. The association between these two molecules enhances the transcriptional activation of NF-B target genes, such as Fas and interferon ␤ (IFN␤). This process is dependent upon the phosphorylation of p65/RelA at serine 276 and the presence of NF-B-binding sites in the reporter gene. The transcriptional effect can be diminished by a super-stable inhibitor IB⌬N, a chemical inhibitor of NF-B, BARD1, and a BRCA1 RING domain mutant.
Luciferase Assay-Human 293T cells and MCF-7 cells were grown at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). The cells were seeded at a density of 3 ϫ 10 5 cells/well in a 12-well dish 1 day prior to transfection and transfected by Lipo-fectAMINE Plus (Qiagen, Valencia, CA) according to manufacturer's recommendation. Transfections were performed with a total amount of 1 g/well DNA, consisting of 30 -100 ng of reporter, 5 ng of tk-Renilla (Promega, Madison, WI), 50 -100 ng of pcr-3 BRCA1 or on a molar basis 25-50 ng of empty vector and various amounts of pBluescript (Stratagene, La Jolla, CA). 10 ng/ml TNF␣ (Calbiochem) or 2.5 ng/ml IL-1␤ (Invitrogen) were added for 12 h prior to lysis. Cells were harvested 48 h post-transfection by detergent-mediated lysis (Promega Dual luciferase lysis buffer), and 20 l of cell lysate were assayed for luciferase activity (Dual Luciferase, Promega, Madison, WI).
GST Protein Affinity Assay-Glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli strain BL-21. Expression was induced with 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside for 4 h, 37°C. The bacteria were lysed in phosphate-buffered saline, containing 3% Triton X-100 and protease inhibitors (Roche Applied Science), and sonicated. GST fusion proteins were isolated from the supernatant by incubation (15 h, 4°C) with glutathione-agarose beads (Amersham Biosciences). The beads were collected by centrifugation and extensively washed in lysis buffer. To quantify the amount of protein bound to the agarose, an aliquot of beads was boiled in 1ϫ SDS loading buffer (62.5 mM Tris, pH 6.9, 10% glycerol, 2% SDS, 5% ␤-mercaptoethanol), separated by electrophoresis through a 10% polyacrylamide gel, Coomassie-stained, and compared with known quantities of bovine serum albumin. Equal amounts of GST fusion proteins (10 -15 g) immobilized on glutathione-agarose beads were used for each in vitro protein affinity assay. To reduce nonspecific binding, GST fusion proteins were preincubated (30 min, 25°C) in 3% bovine serum albumin/NETN buffer (20 mM Tris, pH 8.0, 10 mM NaCl, 0.2% Nonidet P-40) supplemented with protease inhibitors. Binding assays were performed with 20 l of in vitro transcribed/translated (Promega, Madison, WI) [ 35 S]methioninelabeled protein (1 h, 25°C) in 1 ml of NETN. Beads were collected by centrifugation, washed three times in NETN buffer, and boiled in 1ϫ SDS loading buffer. Bound proteins were resolved by 12% SDS-PAGE. The gel was stained with Coomassie Blue, soaked in AMPLIFY (Amersham Biosciences), dried, and exposed to film (12-72 h, Ϫ80°C).
For endogenous immunoprecipitations, cellular extracts were lysed in 1% Nonidet P-40 lysis buffer and incubated with polyclonal anti-BRCA1 antibodies (Santa Cruz Biotechnology) or IgG as a control. Western blotting for detection of endogenous BRCA1 and endogenous p65/RelA was performed with mouse monoclonal anti-BRCA1 (AB1; Oncogene, Boston) and anti-p65 antibody (F-6; Santa Cruz Biotechnology), respectively. Detection of IB␣ in the cytoplasm after TNF␣ treatment was performed with an anti-IB␣ antibody (Santa Cruz Biotechnology).
Subcellular Fractionation-Subcellular fractionation was described previously (36). Briefly, cells were rinsed twice in ice-cold phosphatebuffered saline, pH 7.4, and gently resuspended in lysis buffer B (10 mM Tris, pH 8.4, 140 mM NaCl, 1.5 mM MgCl 2 , 0.5% Nonidet P-40, 1 mM dithiothreitol, and complete protease inhibitors (Roche Applied Science). The lysate was centrifuged at 1000 ϫ g for 3 min at 4°C, and the supernatant was saved as the cytoplasmic fraction. The pellets were resuspended in lysis buffer B, and a one-tenth volume of the detergent (3.3% (w/v) sodium deoxycholate and 6.6% (v/v) Tween 40) was added under slow vortexing, and the suspension was incubated on ice for 5 min. Nuclei were pelleted by centrifugation at 1000 ϫ g for 3 min at 4°C, and the supernatant (post-nuclear fraction) was saved and added to the cytoplasmic fraction. Nuclei were rinsed once in lysis buffer B. For Western blot analysis the nuclear and the cytoplasmic fractions were lysed in RIPA buffer supplemented with complete protease inhibitor (Roche Applied Science) on ice. A 50-g aliquot of each fraction was analyzed. This protocol yielded intact nuclei as determined by the presence of RNApolII (Santa Cruz Biotechnology) without significant cytoplasmic contamination as determined by the absence of ␤-actin (Sigma).
Reverse Transcription and Real Time PCR-MCF-7 cells were infected with an adenovirus containing BRCA1 or GFP (multiplicity of infection ϭ 30). Forty eight hours after infection cells were treated (6 h, 37°C) with TNF␣ (10 ng/ml). RNA from treated and untreated cells was isolated using the Qiagen RNeasy kit (Qiagen, Valencia, CA) and DNase-treated, and then reverse transcription was performed according to instructions for Superscript II, RNase H-reverse transcriptase (Invitrogen). Quantitative real time PCRs were performed in triplicate using 2 l of cDNA product from the reverse transcription reaction. The PCR contained 0.5 units of AmpliTaq Gold, 1ϫ Syber buffer, 2 mM MgCl 2 , 1 mM dNTP with dUTP (PE Biosystems, Warrington, UK), and 0.4 M of each 5Ј and 3Ј primer. Reactions were performed with an initial hot start incubation at 95°C for 10 min, followed by denaturation at 95°C for 30 s, annealing at various temperatures depending upon the primer pairs for 30 s, and extension at 72°C for 30 s. Forty amplification cycles were performed. The data were collected on an ABI Prism 7700 (PerkinElmer Life Sciences) and analyzed using the Sequence Detector version 1.7 program. The PCR end products were checked on 1.8% agarose gel. The primers pairs used for amplification of specific NF-B target genes are as follows: IFN␤ 5Ј-CACGACAGCTCTTTCCA-TGA-3Ј and 3Ј-AGCCAGTGCTCGATGAATCT-5Ј; Fas 5Ј-GCACAGTC-AATGGGGATGAACC-3Ј and 3Ј-GCACTTGGTATTCTGGGTCCG-5Ј; actin 5Ј-GCCCAGAGCAAGAGAGGTAT-3Ј and 3Ј-GGCCATCTCTTGC-TCGAAGT-5Ј.

BRCA1 Activates NF-B Target Promoters-Cytotoxic stress
and DNA damage activate the NF-B transcription factor, leading to cell cycle arrest, apoptosis, or DNA repair (30,31). Furthermore, it was shown that BRCA1 is also involved in apoptosis or DNA repair following DNA damage (21,37). In order to establish whether BRCA1 could modulate the transcriptional activity of NF-B, BRCA1 was co-transfected into 293T or MCF-7 cells with luciferase reporter genes linked to the promoters of NF-B target genes: IgK, IFN␤, and Fas. Thirty six hours after transfection cells were treated with TNF␣, a cytokine activator of NF-B. Control cells were transfected with equimolar amounts of empty expression vector and/or were mock-induced. Treatment of cells with TNF␣ in the absence of BRCA1 induced a 2-3-fold increase in the reporter activity ( Fig. 1A and data not shown). Addition of BRCA1 to TNF␣-treated cells induced a further 3-fold increase in reporter activity. Similar results were also observed with the human immunodeficiency virus promoter and a construct containing multimerized NF-B-binding sites in 293T cells (data not shown). Addition of the IL-1␤ cytokine to the co-transfected cells had the same effect on reporter activity (data not shown).
Because treatment of transfected cells with TNF␣ induces other transcription factors in addition to NF-B (38), subsequent experiments were performed to determine whether the increase in reporter activity was due to the specific activation of NF-B by TNF␣. For this purpose, we used a truncated version (Fas 0.4 kb) of the above-mentioned Fas promoter that only contains two NF-B-binding sites and a mutant form of this short promoter (Fas-mut 1,2), in which both NF-B sites were mutated (36). Cells transfected with the wild-type promoter showed a 4-fold increase in transcriptional activity in the presence of TNF␣ and an almost 8-fold increase in the presence of BRCA1. The mutant Fas promoter was not stimulated by addition of TNF␣, and co-transfection of BRCA1 did not enhance the transcriptional activity (Fig. 1B). Next, a deletion mutant of IB␣ (IB␣⌬N) was used as a super-stable inhibitor of NF-B. This inhibitor lacks two phosphorylation sites that are important for targeting IB for degradation, as a result, IB␣⌬N fails to dissociate from NF-B and blocks the translocation of NF-B to the nucleus after treatment of the cells with TNF␣ (Fig. 1C, compare lanes 1 and 2 to lanes 3 and 4). In the presence of IB␣⌬N, the ability of BRCA1 to augment transcription in the presence of TNF was reduced by 40% (Fig. 1D). IB␣⌬N had no affect on the localization of BRCA1 (Fig. 1C). Similar results were observed when transfected cells were treated with SN-50 (Calbiochem), a peptide that competes with the binding of NF-B to the nuclear membrane and thus blocks NF-B translocation into the nucleus (Fig. 1E). Collectively, these results indicate that the effect of BRCA1 on these reporter genes is mediated by NF-B.
Association of BRCA1 with the Rel Homology Domain of p65/RelA-To determine whether BRCA1 binds to the p65/ RelA subunit of NF-B in response to treatment with TNF␣, we performed endogenous co-immunoprecipitation experiments. 293T cells were seeded at low concentration on 10-cm 2 plates and 24 h later treated with TNF␣, whereas control cells were left untreated. Lysates from these cells were subjected to immunoprecipitation with either rabbit polyclonal anti-BRCA1 antibody or with IgG as a control. Western blot analysis performed with a mouse monoclonal anti-BRCA1 antibody revealed that endogenous BRCA1 was immunoprecipitated from treated and untreated cells by the BRCA1 antibody but not with the control IgG (Fig. 2). In contrast, the p65/RelA subunit was only co-immunoprecipitated with the BRCA1 antibody from cells treated with TNF␣ (Fig. 2). The endogenous expression of BRCA1 and p65/RelA is shown by Western blot analysis (Fig. 2). The co-immunoprecipitation of BRCA1 and p65/RelA was also observed in MCF-7 cells (data not shown). As a marker for the translocation of p65/RelA into the nucleus, the reduction of IB␣ levels was demonstrated after treatment with TNF␣ (Fig. 2). The p65/RelA subunit of NF-B consists of two main regions, the RHD located at the N terminus (aa 1-332) and the transactivation domain located at the C terminus of the molecule (aa 299 -550) (Fig. 3A). To identify which region of p65/RelA interacts with BRCA1, full-length p65/RelA and each individual domain of the molecule were HA-tagged (Fig. 3A) and expressed in 293T cells in the absence or presence of FLAG-BRCA1. The cells were treated with TNF␣, and lysates were immunoprecipitated with a FLAG antibody, and precipitated proteins were immunoblotted with FLAG antibody to detect BRCA1 and an HA-antibody to detect p65. As shown, fulllength p65/RelA and RHD were co-immunoprecipitated with BRCA1 in TNF␣-treated cells (Fig. 3B, lanes 4 and 6). In contrast, the fragment containing only amino acids 299 -550 of p65/RelA failed to associate with BRCA1 (Fig. 3B, lane 8), even though this protein was expressed in the cells (Fig. 3C, lanes 7  and 8). Taken together, these results indicate that NF-B interacts through the Rel homology domain with BRCA1.
Identification of the p65-binding Region within the BRCA1 Molecule-To determine which region of the BRCA1 molecule binds to p65/RelA, six fragments of BRCA1 (1-304, 260 -553, 502-802, 758 -1064, 1005-1313, and 1314 -1863) were expressed as glutathione S-transferase (GST) fusion proteins and used for in vitro protein affinity assays with three fragments of p65/RelA metabolically labeled with [ 35 S]methionine (Fig. 4A). Equivalent levels of GST fusion proteins were used in each protein affinity assay (data not shown). The GST-BRCA1 fragments 1-304, 260 -553, and 502-802 bound to the full-length p65/RelA protein as well as to the 1-332 p65/RelA fragment. p65/RelA bound most strongly to GST-BRCA1 fragment 260 -553 (Fig. 4A). A very weak binding was observed with the GST-BRCA1-(1314 -1863) fragment. In accordance with our earlier results, the 299 -550 p65/RelA did not bind to any of the BRCA1 fragments (Fig. 4A, bottom  panel). The inverse experiment was also performed. Specifically, the 1-332 p65/RelA fragment was expressed as a GST fusion protein and used in an in vitro protein affinity assay with four fragments of BRCA1 (1-802, 1-260, 261-553, and 554 -802) metabolically labeled with [ 35 S]methionine. All the BRCA1 fragments bound to GST-(1-332)-p65/RelA but not to GST alone (Fig. 4B). These data demonstrate that a broad, N-terminal region of BRCA1 is important for binding to the Rel homology domain of p65/RelA.
The RING finger domain, located in the N terminus of BRCA1, is important for protein-protein interactions. It is characterized by the presence of a conserved C 3 HC 4 sequence, which coordinates the binding of two zinc atoms. Missense mutations in BRCA1 occur, with varying frequencies, at four out of seven cysteine residues of the BRCA1 RING domain (cysteine 29, 34, 61, and 64). Mutation of cysteine 61 to glycine (C61G) was shown to disrupt the zinc binding and formation of BRCA1 N-terminal homodimers in vitro (39). In order to determine whether mutation in the RING finger domain influences the transcriptional effect of BRCA1 on NF-B target genes, 293T cells were transfected with two concentrations (100 or 150 ng/ml) of either wild type BRCA1 or BRCA1 mutated at cys- , and with either pcr-3 (50 ng) or pcr-3-BRCA1 (100 ng). Thirty six hours post-transfection, cells were treated with TNF␣ (10 ng/ml). Luciferase measurement was performed as above. C, Western blot (WB) of nuclear and cytoplasmic fractions from TNF␣-induced cells transfected with FLAG-BRCA1 (9 g/plate) and either IB␣⌬N or control (Cont) vector (3 g/plate). Detection of ␤-actin and RNA polII in the cytoplasmic and nuclear fraction, respectively, demonstrates purity of fractionation. D, 293T cells were transfected with IgK promoter (100 ng), tk-Renilla (5 ng), pcr-3 (25 ng), or pcr-3-BRCA1 (50 ng) in the absence or presence of super-stable IB␣ (IB␣⌬N) (50 ng). Thirty six hours post-transfection, cells were treated with TNF␣ (10 ng/ml). Luciferase measurement was performed as above. E, 293T cells were transfected with IgK promoter (100 ng), tk-Renilla (5 ng), and with either pcr-3 (25 ng) or pcr-3-BRCA1 (50 ng). Thirty six hours after transfection, the cells were treated with SN-50 (20 M) or mock-treated. One hour later, all cells were treated with TNF␣ (10 ng/ml). All of the above experiments were performed at least three times with similar results being obtained. teine 64 or cysteine 61. The cysteine mutants were 30 and 50% less active than the wild type BRCA1 transfected at the same concentration (Fig. 5A). The wild-type BRCA1 and mutant forms were expressed at comparable levels at both concentrations (Fig. 5B). BARD1 Inhibits Transcriptional Activation by BRCA1-BARD1 is a RING finger protein that binds to the N-terminal domain of BRCA1, including the RING finger and stabilizes the molecule (40). Because BARD1 and NF-B both bind the Nterminal portion of BRCA1, we determined whether BARD1 could affect the functional interaction between BRCA1 and NF-B. BARD1 was co-transfected into 293T cells with or without BRCA1. BRCA1 induced a 4-fold increase in reporter activity when cells were treated with TNF␣. However, BARD1 blocked the ability of BRCA1 to augment transcription by 50% (Fig. 5C), even though the level of BRCA1 was elevated in cells co-transfected with BARD1 (Fig. 5D). BARD1 had no significant effect on the ability of TNF␣ to activate the reporter gene in the absence of BRCA1 (Fig. 5C). A truncated form of BARD1, which lacks the RING finger domain, did not inhibit the transcriptional activity of BRCA1 (data not shown). To determine whether BARD1 blocked co-activation by BRCA1 by inhibiting the formation of a BRCA1-p65 complex, HA-p65/RelA and FLAG-BRCA1 were expressed in 293T cells in the absence or presence of BARD1. In this triple transfection experiment,

FIG. 2. BRCA1 associates with the p65/RelA subunit in vivo.
Lysates from 293T cells treated and untreated with TNF␣ were immunoprecipitated (IP) with either a polyclonal BRCA1 antibody or control IgG. The immunoblot was probed for the presence of BRCA1 and p65/ RelA (top panel, lanes 1-4). Western blots (WB) were performed on cells lysates to detect the presence of BRCA1, the p65/RelA subunit, and IB␣ (bottom panel).

FIG. 3. Identification of the BRCA1 binding region of p65/RelA.
A, schematic representation of p65/RelA fragments. Full-length p65/ RelA-(1-550) and two fragments of p65, the RHD (aa 1-332) and the C-terminal (aa 299 -550). B, 293T cells were transfected with either empty vector (lanes 1, 3, 5, and 7) or with FLAG-BRCA1 (lanes 2, 4, 6, and 8) and with the pCDNA vector expressing one of the p65 fragments, 1-550 (lanes 3 and 4); 1-332 (lanes 5 and 6); or 299 -550 (lanes 7 and 8). Co-immunoprecipitations (IP) were performed with an anti-FLAG antibody, and the immunoblot was probed with FLAG antibody to detect BRCA1 and anti-HA to detect p65/RelA. C, Western blot (WB) analysis was performed on the lysate samples described above (250 g of protein was applied for the detection of FLAG-BRCA1 and 25 g of protein was applied for the detection of the HA-p65/RelA fragments). The blot was probed with mouse monoclonal anti-FLAG and mouse monoclonal anti-HA. , were used in a protein affinity assay with 35 S-labeled BRCA1 fragments (aa 1-802, 1-260, 261-553, and 554 -802). Bound proteins were separated by 12% SDS-PAGE and exposed to film. Input represents 4% of in vitro transcribed/translated protein. BARD1 did not decrease the level of expression of BRCA1 in transfected cells (Fig. 5E, lanes 2 and 6) nor did it alter the subcellular localization of BRCA1 or p65 (data not shown). We consistently noted that p65/RelA expression decreased expression of BRCA1 in transfected cells, an effect possibly due to promoter or competition or squelching due to the overexpression of p65/RelA. Despite this, several important pieces of information could be determined from the experiment. When p65/RelA alone was transfected into cells, it could not be detected in BARD1 precipitates (Fig. 5E, lane 3). When cells were transfected with HA-p65/RelA and BARD1, HA-p65/RelA was detected in the BARD1 immunoprecipitates (Fig. 5E, lane 7). Curiously, a similar level of HA-p65 was also immunoprecipitated in the presence of FLAG-BRCA1 using BARD1 antibodies. This presumably occurred through endogenous BARD1 present in the cell (Fig. 5E, lane 4) and may be due to the mutual stabilization of BRCA1 and BARD1. Importantly, when BARD1 and BRCA1 were expressed together along with p65, the amount of p65 found in BARD1 immunoprecipitates was strongly enhanced. Similar results were obtained when BRCA1 was used to immunoprecipitate the proteins (data not shown). Therefore, the ability of BARD1 to inhibit trans-activation of TNF␣-mediated transcription by BRCA1 could not be explained by blocking the formation of the BRCA1-p65-RelA complex. On the contrary, BARD1 and BRCA1 together more readily interacted with p65 than alone.
Mutation of Serine 276 on the p65/RelA Subunit Abolishes the Effect of BRCA1-The p65/RelA subunit is phosphorylated by cAMP-dependent protein kinase at serine 276 in the Rel homology domain. This phosphorylation event is important for the binding of CBP. In order to determine whether the phosphorylation of the Rel homology domain is also important for the enhancement of the transcriptional activity by BRCA1, 293T cells were co-transfected with p65/RelA, p65/RelA (S276A) mutant, or empty vector in the presence or absence of BRCA1. In the presence of TNF␣ and wild-type p65/RelA, there is a 3-fold enhancement in the transcriptional activity induced by BRCA1. Transfection of the mutated form of p65/RelA was found to abolish this effect (Fig. 6A). To verify if this mutated form also influences the physical interaction between BRCA1 and p65/RelA, we performed a co-immunoprecipitation experiment. The mutant p65/RelA (S276A) was HA-tagged and expressed in 293T cells in the absence or presence of FLAG-BRCA1. Lysates from cells treated with TNF␣ were immunoprecipitated with anti-FLAG antibody. Precipitated proteins were analyzed by Western blot with anti-FLAG antibody to detect BRCA1 and an anti-HA antibody to detect mutated p65/RelA. As shown, mutant p65/RelA was immunoprecipitated only in the presence of BRCA1 (Fig. 6B, top panel). This suggests that phosphorylation at serine 276 is not required for the physical interaction between BRCA1 and p65/ RelA but is important for the enhancement of the transcriptional activity induced by BRCA1.
It was shown previously (11) that CBP also binds to BRCA1. Therefore, we next determined whether co-expression of CBP and BRCA1 would have a greater effect on transcription of NF-B target genes than either one alone. Whereas BRCA1 stimulated transcription from the reporter gene by nearly a factor of 3 and CBP stimulated transcription by 2.5-fold, the combination of BRCA1 plus CBP led to a 10-fold increase in transcriptional activity (Fig. 6C). Co-transfection of BRCA1 with a deletion mutant of CBP devoid of the histone acetyltransferase domain (CBP⌬HAT) resulted in significantly less stimulation of transcription. BRCA1 appeared to stabilize CBP, and in the presence of BRCA1, CBP and CBP⌬HAT were expressed at equivalent levels (Fig. 6D, lanes 4 and 6). In contrast, expression of CBP had no effect on the level of expression of BRCA1 (Fig. 6D, lanes 2, 4, and 6). We next wondered whether BRCA1, CBP, and p65/RelA could be found as a complex. For this purpose, 293T cells were co-transfected with HA-p65 and CBP in the absence or presence of FLAG-BRCA1. The cells were treated with TNF␣; lysates were immunoprecipitated with FLAG antibody, and precipitated proteins were immunoblotted with FLAG antibody to detect BRCA1, HA antibody to detect p65/RelA, and CBP antibody to detect CBP. CBP or p65/RelA alone could be co-precipitated with BRCA1 (Fig. 6E, lanes 4 and 7, respectively) and also when all three proteins were expressed, both HA-p65 and CBP could be coimmunoprecipitated with FLAG-BRCA1 (Fig. 6E, lane 8). Immunoprecipitation with a CBP antibody also precipitated both BRCA1 and p65 (data not shown). Together, these results suggest that CBP, by complexing with the activated and phosphorylated form of NF-B as well as with BRCA1, may mediate the transcriptional effects of BRCA1.

BRCA1 Stimulates Expression of Endogenous NF-B Target
Genes-Because BRCA1 enhanced the TNF␣-induced activity of IFN␤ and Fas reporter genes, we determined whether BRCA1 could affect expression of endogenous NF-B target genes. RNA was isolated from MCF-7 cells infected with an adenovirus expressing BRCA1 or GFP and treated with TNF␣. Quantitative real time PCR analysis revealed that BRCA1 significantly augmented the expression of IFN␤ and Fas  4, 7, and 8). Co-immunoprecipitations were performed with an anti-BARD1 antibody, and the immunoblot was probed with FLAG antibody to detect BRCA1, anti-HA to detect p65/RelA, and anti-BARD1 to detect BARD1. Western blot (WB) analysis was performed on the lysate samples described above. The blot was probed with anti-FLAG, anti-HA, and anti-BARD1 to detect BARD1. mRNA in infected cells above the increase mediated by TNF␣ alone (Fig. 7). Importantly, these results were in agreement with those obtained in reporter experiments and suggest that the ability of BRCA1 to interact with the NF-B system has relevance in vivo.

DISCUSSION
In this study, we present data demonstrating the interaction of the p65/RelA subunit of NF-B with the N-terminal domain of BRCA1. By both in vitro binding assays and co-immunoprecipitation analysis, we demonstrate that the Rel homology domain of the p65/RelA subunit, but not the C-terminal domain which contains the transactivation domain, interacts with the N-terminal region of BRCA1 (aa 1-802). This result was confirmed by a GST protein affinity assay, which further revealed that the major site of interaction on BRCA1 is between amino acids 260 and 553. The interaction between BRCA1 and the p65/RelA was also detected by co-immunoprecipitation of endogenous proteins in 293T cells, as well as in MCF-7 cells. The p50 subunit of NF-B, which also contains a Rel homology domain, interacts similarly with BRCA1 (data not shown). Tumor-associated BRCA1 RING finger mutants were partially defective for trans-activation, suggesting that the RING finger motif has a role in the functional interaction between BRCA1 and p65/RelA. BRCA1 can be found complexed with RNA polymerase II holoenzyme (41), an interaction dependent on the N-terminal portion of the protein including the RING finger. Furthermore, interaction of BRCA1 and CBP also requires the N terminus of BRCA1 (11). Point mutants of BRCA1 are known to preserve the interaction with BARD1 while disabling the ubiquitin ligase activity of BRCA1 (42,43). This suggests a link between the transcriptional activity of BRCA1 and ubiquitination and is consistent with emerging data indicating a link between transcriptional activation, ubiquitination, and degradation of transcription factors (reviewed in Ref. 44). An increasing number of ubiquitin-protein isopeptide ligases like Rsp5/ and with either pcr-3 (50 ng) or pcr-3-BRCA1 (100 ng). Thirty six hours after transfection the cells were induced with TNF␣ (10 ng/ml) for 12 h. This experiment was performed three times in triplicate (ϮS.D.). D, Western blot (WB) analysis was performed on the lysate samples described above to detect FLAG-BRCA1 (lanes 2, 4, and 6) and wild type CBP (lanes 3 and 4) or ⌬HAT CBP (lanes 5 and 6). E, 293T cells were transfected with empty vector (lanes 1, 2, 5, and 6), FLAG-BRCA1 (lanes 3, 4, 7, and 8), HA-p65 (lanes 5-8), or CBP (lanes 2, 4, 6, and 8). Co-immunoprecipitations were performed with an anti-FLAG antibody, and the immunoblot was probed with anti-FLAG antibody to detect BRCA1, anti-HA to detect p65, and anti-CBP to detect CBP. Western blot analysis was performed on the lysate samples described above. hPRF1 (45) and E6-AP (46), which can now include BRCA1, have been identified as transcriptional co-activators. What proteins of the transcription complex might be modified by BRCA1 is unknown.
The importance of the N-terminal region of BRCA1 for transcriptional activity was also highlighted by the ability of BARD1 to inhibit trans-activation by BRCA1. BARD1 and BRCA1 interact in their N terminus in a region immediately adjacent to each RING finger (47). The RING fingers of the proteins then together form a structure with considerable ubiquitin ligase activity (40). BARD1 expression, although generally increasing BRCA1 protein accumulation within the cell, actually inhibited the ability of BRCA1 to activate transcription through NF-B. The N terminus of BARD1 including the RING finger and sequences required for heterodimerization with BRCA1 was required for this effect. BARD1 did not change the subcellular localization of BRCA1 or p65/RelA and did not inhibit the formation of the BRCA1-p65 complex. On the contrary BRCA1, BARD1, and p65 readily interacted when co-expressed. This suggests that the transcriptional function of BRCA1 is altered when complexed by BARD1. This may reflect the ability of BARD1 to bind with BRCA1 in nuclear dots which might represent sites of DNA repair rather than transcription (48). The reduced transcriptional function of BRCA1 when co-expressed with BARD1 might also be related to the fact that BARD1 enhances the ubiquitin ligase activity of BRCA1 in vitro (43,49), potentially directing the activity of the complex to histones, particularly H2AX, found at sites of DNA damage. Alternatively, BARD1 might inhibit transcriptional activity of BRCA1 by association with the polymerase II holoenzyme, inhibiting polyadenylation though interaction with the CstF factor (50).
NF-B is primarily found in the cytoplasm in a complex with IB␣ and the catalytic subunit of protein kinase A. Upon activation by the cytokines TNF␣ or IL-1␤, p65/RelA is phosphorylated on serine 276 by protein kinase A and is translocated to the nucleus (55,56). This phosphorylation facilitates the docking of the transcriptional co-activator CBP, which enhances the ability of NF-B to activate transcription (33,34). It has been shown that the KIX domain of p300/CBP interacts with both the N-and the C-terminal domains of BRCA1 (11). We demonstrated by co-immunoprecipitation that BRCA1 can complex with both p65/RelA and CBP and cooperates with these proteins to activate NF-B target genes. Mutation of serine 276 to alanine in the p65/RelA subunit abrogates the ability of BRCA1 to enhance transcription, even though the interaction between p65 (S273A) and BRCA1 is not disrupted. This reduction in the transcriptional activity may be due to the inability of CBP to bind to the unphosphorylated form of p65/RelA. An analogous type of complex interaction occurs with another BRCA1-binding protein, ATF1. Like p65, the DNA-binding C-terminal domain of ATF1 is required for interaction with BRCA1. The N-terminal KID domain of ATF1, which is also phosphorylated by protein kinase A, mediates an interaction between ATF1 and CBP (14). The phosphorylated form of ATF1 is important for its transcriptional activity but is not important for the physical interaction between BRCA1 and ATF1 (14). This theme is found in yet another system. Stimulation of cells by another cytokine, IFN␥, leads to serine phosphorylation of STAT-1␣ and ultimately activation of IFN␥-inducible genes. Phosphorylation at Ser-727 in the C-terminal region of STAT-1␣ enhances transcription by recruiting p300/CBP and also BRCA1. BRCA1 acts in concert with STAT-1 to differentially regulate a subset of IFN␥ target genes (16, 51) involved in growth control. Similarly, BRCA1 may only activate a subset of NF-B genes. How specificity in BRCA1-dependent gene regulation is achieved remains unclear.
Apart from binding to co-activators, such as CBP/p300 or to transcription factors, such as ATF1 and p65/RelA, BRCA1 also binds to RNA polymerase II through its C-terminal domain (10,12). Based on these multiple interactions, we propose a model whereby BRCA1 functions as a scaffold protein by tethering together the various transcriptional elements (Fig. 8). In the absence of BRCA1 (Fig. 8A), CBP binds to the p65/RelA subunit and to the TATA box-binding protein and TFIIB, which binds to RNA polymerase II (52-54) and co-activates the transcription of NF-B target genes. Addition of BRCA1, which interacts physically with p65/RelA, CBP, and RNA polymerase II as demonstrated in Fig. 8A, enhances the transcriptional activation of NF-B target genes (Fig. 8B).
Both BRCA1 and NF-B have been reported to be involved in pro-apoptotic functions in various cell types. We show BRCA1 significantly increases the transcriptional activity of NF-B target promoters, such as those of IgK, IFN␤, and Fas, above the activity observed by stimulation by TNF␣ alone. This activity is inhibited by a super-stable inhibitor of NF-B, as well as by a chemical inhibitor of NF-B. The mutated form of the Fas gene could not be activated by BRCA1, demonstrating that BRCA1 is exerting its effect through NF-B. The potential physiological relevance of this finding was heightened by our result showing that overexpression of BRCA1 increased expression of the endogenous FAS and IFN␤ genes above the levels seen with TNF␣ treatment alone. The FAS receptor along with FASL activate a pathway leading to apoptosis. IFN␤ is a cytokine that can promote apoptosis in response to viral infection (51). Hence, through the induction of NF-B targets genes BRCA1 might induce an antiviral and anti-proliferative state in the cell.
In multiple systems, NF-B has been implicated in the apoptotic response. In T cells, topoisomerase poisons and UV irradiation activate the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, which leads to the induction of the NF-B transcription factor. NF-B activates FIG. 8. Model for BRCA1 as an NF-B co-activator. A, in the absence of BRCA1, activated NF-B translocates to the nucleus where it binds to target sequences in responsive genes. CBP binds to NF-B, TATA box-binding protein (TBP), and TFIIB, which also bind to the RNA polymerase II holoenzyme. B, BRCA1 may act as a scaffold protein, stabilizing the complex between NF-B, CBP, and RNA polymerase II holoenzyme, thereby enhancing transcriptional activity. FASL, which in turn binds to its receptor, FAS/CD95, thus resulting in apoptosis (55,56). In addition, in neuronal cells glutamate induces cell death by activating the NF-B transcription factor (57). Overexpression of a dominant-negative p65/RelA protein inhibits apoptosis induced by serum starvation in HEK 293 cells (58). Moreover, incubation of osteoblastic mc3T3-E1 cells with TNF␣ induces apoptosis and stimulates nuclear translocation of NF-B. Treatment with an anti-TNF␣ antibody prevents apoptosis and decreases NF-B activation, indicating that activation of NF-B through TNF␣ may be an important apoptotic signaling pathway in osteoblasts (59,60).
BRCA1 has also been implicated in the SAPK/JNK pathway leading to apoptosis (21). Thangaraju et al. (37) reported that overexpression of BRCA1 in breast and ovarian cancer cell lines induces expression of FAS and FASL, ultimately leading to apoptosis after serum withdrawal through the activation of the SAPK/JNK pathway. Because both NF-B and BRCA1 were shown to activate the expression of FAS through the JNK pathway, it is possible that BRCA1 is functioning as a transcriptional co-activator with NF-B in this system.
NF-B has also been linked to the prevention of apoptosis. NF-B is often constitutively active in breast cancer cells lines and diverse solid tumor-derived cell lines (61)(62)(63). NF-B can induce expression of anti-apoptotic genes, such as inhibitor of apoptosis protein (IAP) family and superoxide dismutase, and genes that contribute to proliferation, such as c-MYC and BCL-X L (64). Therefore, persistent activation of NF-B could provide a mechanism that protects cancer cells from apoptosis. In breast cancers where the tumor suppressor BRCA1 is mutated, the balance between pro-apoptotic and anti-apoptotic pathways may be shifted toward the latter. Future studies will be aimed at determining whether BRCA1 acts as a co-activator of only pro-apoptotic genes or as a general activator of all NF-B target genes.