BRCA1 Regulates the Interferon (cid:1) -mediated Apoptotic Response*

BRCA1 is a tumor suppressor gene implicated in transcriptional regulation. We have generated cell lines with inducible expression of BRCA1 as a tool to identify downstream targets that may be important mediators of BRCA1 function. Oligonucleotide array-based expression profiling identified 11 previously described interferon regulated genes that were up-regulated following inducible expression of BRCA1. Northern blot analysis revealed that a subset of the identified targets including IRF-7, MxA, and ISG-54 were synergistically up-regu-lated by BRCA1 in the presence of interferon (cid:1) (IFN- (cid:1) ) but not interferons (cid:2) or (cid:3) . Importantly, IFN- (cid:1) -mediated induction of IRF-7 and MxA was attenuated in the BRCA1 mutant cell line HCC1937, an effect that was rescued following reconstitution of exogenous wild type BRCA1 in these cells. Furthermore, reconstituted BRCA1 sensitized HCC1937 cells to IFN- (cid:1) -induced apoptotic cell death. overexpression of exogenous BRCA1, including the stress- and DNA dam-age-inducible GADD45 gene These findings suggest that BRCA1 functions as either a co-activator or co-repressor of transcription, an effect that may involve its ability to recruit both the basal transcription machinery and proteins implicated in chromatin remodeling including the histone deacetylases

BRCA1 is a tumor suppressor gene implicated in transcriptional regulation. We have generated cell lines with inducible expression of BRCA1 as a tool to identify downstream targets that may be important mediators of BRCA1 function. Oligonucleotide array-based expression profiling identified 11 previously described interferon regulated genes that were up-regulated following inducible expression of BRCA1. Northern blot analysis revealed that a subset of the identified targets including IRF-7, MxA, and ISG-54 were synergistically up-regulated by BRCA1 in the presence of interferon ␥ (IFN-␥) but not interferons ␣ or ␤. Importantly, IFN-␥-mediated induction of IRF-7 and MxA was attenuated in the BRCA1 mutant cell line HCC1937, an effect that was rescued following reconstitution of exogenous wild type BRCA1 in these cells. Furthermore, reconstituted BRCA1 sensitized HCC1937 cells to IFN-␥-induced apoptotic cell death. This study identifies BRCA1 as a component of the IFN-␥-regulated signaling pathway and suggests that BRCA1 may play a role in the regulation of IFN-␥-mediated apoptosis.
BRCA1 encodes a tumor suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer (1). Germline mutations of BRCA1 are found in half of breast-ovarian cancer pedigrees and in ϳ10% of women with early onset breast cancer, irrespective of family history (2). BRCA1 has been implicated in a number of important cellular functions including DNA damage repair, transcriptional regulation, cell cycle control, and, more recently, ubiquitination (3). The exact mechanism through which BRCA1 inactivation may lead to malignant transformation, however, remains to be defined.
A large body of evidence has accumulated over the last few years demonstrating a role for BRCA1 in DNA damage repair and in particular repair of double-strand breaks. It was initially observed that BRCA1 co-localized in nuclear foci during the S phase with RAD51 (4), the mammalian homologue of bacterial recA, which is involved in homologous recombination and the repair of double-strand breaks in DNA following ionizing radiation. Subsequently, it has been shown that BRCA1 is also a component of the RAD50-MRE11-NBS1 complex implicated in homologous recombination and nonhomologous end joining in response to DNA damage (5). Consistent with a potential role in the repair of double-strand breaks, treatment of cultured cells with ionizing radiation leads to BRCA1 hyperphosphorylation, an effect that is mediated in part by the ATM (6) and CHIT2 kinases (7). Genetic studies support a role for BRCA1 in the repair of double-strand breaks. Significantly, embryonic stem (ES) cells from BRCA1 knockout mice exhibit a defect in the repair of double-strand breaks by homologous recombination (8). Recently it has been reported that BRCA1 can bind to single-strand DNA in a non-sequence-specific manner with a high affinity for branched DNA structures (9). This has lead to the suggestion that BRCA1 is actively recruited to the sites of stalled replication forks following DNA damage where it can then recruit various proteins implicated in the repair of damaged DNA.
A role for BRCA1 in cell cycle checkpoint control has also been suggested following the observation that BRCA1 becomes hyperphosphorylated during the late G 1 and S phases and transiently dephosphorylated early after the M phase (10). Furthermore, overexpression of BRCA1 induces a G 1 /S arrest in human colon cancer cells (11), and genetic instability has been observed in BRCA1 exon 11 isoform-deficient cells resulting from a defective G 2 /M checkpoint and centrosome amplification (12). We have recently reported that inducible expression of BRCA1 can activate both the G 2 and mitotic checkpoints following treatment with taxol, suggesting a specific role for BRCA1 in the regulation of the G 2 /M checkpoint following disruption of the mitotic spindle (13).
Evidence has also accumulated over the last few years supporting a role for BRCA1 in transcriptional regulation. The C-terminal domain of BRCA1 is highly acidic and has been shown to mediate transcriptional activation when fused to a heterologous DNA-binding domain (14,15). Furthermore, BRCA1 co-purifies with RNA polymerase II holoenzyme complex through an association with RNA helicase A (16,17), suggesting that BRCA1 is a component of the core transcriptional machinery. A number of other reports have demonstrated that BRCA1 associates with a range of different transcription factors including p53, c-Myc, ATF1, and STAT1 and modulates their activity (18 -21). Consistent with a role in transcriptional regulation, we and others have identified a number of genes that are up-regulated following overexpression of exogenous BRCA1, including the stress-and DNA damage-inducible GADD45 gene (22,23). These findings suggest that BRCA1 functions as either a co-activator or co-repressor of transcription, an effect that may involve its ability to recruit both the basal transcription machinery and proteins implicated in chromatin remodeling including the histone deacetylases HDAC1 and HDAC2 and the SWI/SNF-related chromatin-remodeling complex (24,25).
In this study we report the identification by microarraybased expression profiling of a novel subset of BRCA1 target genes, (IRF-7, MxA, and ISG-54) that are synergistically upregulated following inducible expression of BRCA1 in the presence of IFN-␥. 1 Also we demonstrate that BRCA1 is required for IFN-␥-mediated induction of IRF-7 and MxA and show that BRCA1 sensitizes breast cancer cell lines to IFN-␥-mediated apoptosis. Taken together these data therefore suggest that BRCA1 may be an important component of the IFN-␥-regulated antiproliferative response.

EXPERIMENTAL PROCEDURES
Cell Lines-MBR62-bcl2 cells were grown as described previously (13). HCC1937 cells were grown in RPMI supplemented with 20% fetal calf serum, 1 mM sodium pyruvate, and 100 g/ml penicillin-streptomycin. T47D, MCF7, MDA435, and MDA468 cells were grown in RPMI supplemented with 10% fetal calf Serum, 1 mM sodium pyruvate, and 50 g/ml penicillin-streptomycin. HCC1-V and HCC-BRCA1 cells were generated by stable expression of the empty vector, Rc/CMV (Invitrogen), or Rc/CMV-BRCA1. Transfected HCC1937 cells were grown as described above with the addition of 0.2 mg/ml G418. The cells were grown in 5% CO 2 in a humidified incubator.
Oligonucleotide Array-based Expression Profiling-Oligonucleotide array-based expression profiling was carried out as described previously (22) with the following changes. Each RNA sample was hybridized in duplicate to the Affymetrix HG-U95A array containing 12,000 known full-length genes.
Cellular Proliferation, Colony Count, and Caspase 3 Cleavage Assays-To measure the inhibition of cellular proliferation, equal numbers of MBR62-bcl2 cells were split into 24-well plates and uninduced or induced to express BRCA1 in the presence or absence of IFN-␣ (500 units/ml), IFN-␤ (500 units/ml), or IFN-␥ (500 units/ml). The cell counts were carried out on a daily basis using a Coulter counter (Coulter Z2). The colony count assays were performed following Lipofectin (Invitrogen)-based transfection of MCF-7 and MDA435 breast cancer cells with mammalian expression constructs encoding MxA and IRF-7. The cells were transfected with the empty vector pCDNA3.1-V5-HIS-TOPO (Invitrogen) as a control. The transfected clones were selected in 0.5 mg/ml G418 for 3 weeks and then fixed with methanol for 10 min and stained with 0.5% crystal violet (BDH Chemicals). For caspase 3 cleavage assays, MBR62-bcl2 cells were uninduced or induced to express BRCA1 for 24 h in the presence of 100, 200, and 300 units/ml IFN-␥. Similarly HCC-V and HCC-BRCA1 cells were left untreated or were treated with 100 units/ml IFN-␥ for 24 h, and the protein lysates were extracted in RIPA buffer (50 M Hepes, pH 7.0, 150 mM NaCl, 0.1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). The proteins were separated on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane followed by immunoblotting using an anti-cleaved caspase 3 antibody that specifically recognizes the cleaved 17-and 19-kDa caspase 3 products.
Antibodies and Western Blot Analysis-Antibodies against BRCA1 include the rabbit polyclonal C-20 (Santa Cruz) and the mouse monoclonal AB1 (Calbiochem). Cleaved caspase 3 was detected using the rabbit polyclonal cleaved caspase 3 (Asp 175 ) antibody (Cell Signaling). The IRF-7 antibody was purchased from Santa Cruz Biotechnologies (catalog number sc-9083). ␤-Tubulin was detected using the monoclonal antibody T-4026 (Sigma).
Immunostaining-HCC-BRCA1 and HCC-V cells were grown to 60% confluence on coverslips and treated or untreated with 100 units of IFN-␥ for 72 h. The coverslips were fixed in 95% cold ethanol, washed three times in TBST for 5 min at room temperature, and incubated with anti-cleaved caspase 3 polyclonal antibody (Cell Signaling) diluted in TBST containing 5% bovine serum albumin overnight at 4°C. Following primary incubation, the coverslips were washed three times in TBST containing 5% bovine serum albumin followed by incubation with secondary fluorescein isothiocyanate-conjugated rabbit anti-goat IgG (Sigma) for 1 h at room temperature. The coverslips were washed three times in TBST followed by a single wash in TBS, counterstained with propidium iodide, and subsequently visualized on a LEICA DMLB fluorescent microscope.

Gene Expression Profiling to Identify BRCA1 Target
Genes-We generated an MDA435 breast cancer-derived cell line termed MBR62-bcl2 with tightly regulated tetracyclineinducible expression of BRCA1 and constitutive expression of bcl2. To evaluate the degree of BRCA1 induction in these cells, we carried out Northern and Western blot analysis. Time course Northern blot analysis revealed that BRCA1 expression levels were maximal for 24 h after tetracycline withdrawal, consistent with our previous observations (22) (Fig. 1A). Immunoprecipitation (IP)-Western blot analysis 24 h after tetracycline withdrawal when BRCA1 expression levels are highest demonstrated an ϳ5-fold induction above endogenous protein levels (Fig. 1A). This is well within the physiological range for BRCA1 overexpression observed in mouse models where BRCA1 expression increases ϳ10-fold during pregnancy and 1 The abbreviations used are: IFN, interferon; CMV, cytomegalovirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 1. Confirmation of target gene induction by Northern blot analysis.
A, Northern blot analysis illustrating inducible expression of BRCA1 in the MBR62-bcl2 cells. RNA was extracted at 12, 24, and 48 h after withdrawal of tetracycline and at 24 h in the presence of tetracycline as a control. The full-length exogenous message is labeled with an arrow in addition to two splice variants generated by activation of cryptic splice sites within the full-length construct (B) Immunoprecipitation (IP)-Western illustrating the degree of BRCA1 protein induction relative to endogenous levels in the MBR62-bcl2 cells, 24 h following tetracycline withdrawal when BRCA1 expression levels are highest. B, Northern blot analysis of total cellular RNA from MBR62-bcl2 cells grown in the presence (ϩtet) or absence (Ϫtet) of tetracycline for 24 h and hybridized with cDNA probes representing the identified BRCA1 target genes. GAPDH is included as a loading control. C, Western blot analysis of the time course of IRF-7 induction following inducible expression of BRCA1. The identical blot was reprobed with ␤-tubulin as a loading control. postlactional involution (26). Similarly in human cell lines, BRCA1 expression levels increase 5-10-fold, following treatment with estrogens or 1␣,25(OH) 2 D 3 (27)(28)(29). We used MBR62-bcl2 cells to search for endogenous genes whose expression levels might be altered in response to inducible expression of BRCA1. Total RNA was extracted 24 h after BRCA1 induction, biotinylated, and hybridized to the Affymetrix HG-U95A gene array representing 12,000 known transcripts and expressed sequence tags (22,30). A total of 32 genes were identified as being induced, and three were repressed following analysis of duplicate experiments. A total of 11 genes were identified as exhibiting 5-fold or greater induction and were further screened by Northern blot analysis (Table I). Surprisingly all of these 11 genes were previously described interferoninducible genes, suggesting a possible role for BRCA1 in mediating the IFN-regulated signaling pathways. Northern blot analysis confirmed induction of six of these genes including IRF-7, a transcription factor required for activation of a subset of IFN-␣ genes following viral infection (induced 6-fold) (31); MxA, a GTPase with activity against several RNA viruses (induced 8-fold) (32); ISG-54, an IFN-␣-inducible gene, implicated as a potential predictor of response to IFN-␣ in chronic myelogenous leukemia (induced 3-fold) (33); and TAP1, a transporter required for the major histocompatibility complex class I antigen presentation and a previously described p53 target gene (induced 2-fold) (34) (Table I and Fig. 1B). Either induction of the remaining five genes was not confirmed or the expression was not detectable by Northern blot analysis (Table  I). To determine the kinetics of induction of the identified targets by BRCA1, we carried out Western blot analysis. Increased expression of IRF-7 was observed as early as 6 h after inducible expression of BRCA1 and was maintained up to 72 h, suggesting that IRF-7 represents an immediate downstream target of BRCA1 (Fig. 1C).
To investigate the possibility that BRCA1-mediated induction of the identified targets was an indirect effect, we examined the ability of BRCA1 to induce expression of IFN-␣, IFN-␤, and IFN-␥ in the MBR62-bcl2 cells. Semi-quantative reverse transcription-PCR analysis failed to show induction of any of the three transcripts following inducible expression of BRCA1, suggesting that the ability of BRCA1 to induce expression of the targets does not occur through this indirect mechanism (data not shown). One of our previously identified BRCA1 targets, EGR1, was also induced in the current screen, although to a lesser extent (2-fold) than previously observed. Interestingly, we failed to identify GADD45 in this screen, even though we have previously demonstrated that GADD45 is clearly induced by BRCA1 in these cells (13).  (Fig. 2), suggesting a specific interaction between BRCA1 and IFN-␥ in the regulation of a subset of the identified target genes. In contrast, TAP1, which was responsive to IFN-␤ and IFN-␥, failed to exhibit synergy following induction of BRCA1 ( Fig. 2A). We calculated the average increase in expression for IRF-7, MxA, and ISG-54 relative to the GAPDH controls by densitometric analysis. IRF-7 expression was induced on average 6-fold by BRCA1 alone, 6-fold by IFN-␥ alone, and 23-fold by BRCA1 in the presence of IFN-␥ (Fig. 2). MxA expression was induced on average 8-fold by BRCA1, 17-fold by IFN-␥, and 44-fold by BRCA1 following treatment with IFN-␥ (Fig. 2). Similarly ISG-54 was induced on average 3-fold by BRCA1, 2.5-fold by IFN-␥, and 12-fold by BRCA1 in the presence of IFN-␥ (Fig. 2), indicating a synergistic induction of IRF-7, MxA, and ISG-54 by BRCA1 in the presence of IFN-␥. In contrast no such synergistic up-regulation was observed for TAP1, which was induced 2-fold by BRCA1 alone, 5-fold by IFN-␥ alone, and 6-fold by BRCA1 in the presence of IFN-␥, suggesting that TAP1 may be independently regulated by BRCA1 and IFN-␥ ( Fig. 2A).
BRCA1 Is Required for IFN-␥-mediated Induction of IRF-7 and MxA-To establish the potential role played by BRCA1 in mediating the IFN-␥-regulated signaling pathway, we examined the ability of IFN-␥ to induce expression of IRF-7 in a panel of breast cancer cell lines. These included the MDA468 and T47D cells, which express endogenous wild type BRCA1; the MBR62-bcl2 cells, which are induced to express exogenous BRCA1; and finally the HCC1937 cell line, which expresses a single copy of a C-terminally truncated BRCA1 protein product (35). Northern blot analysis confirmed IFN-␥-mediated induction of IRF-7 in the breast cancer cell lines expressing wild type BRCA1 or MBR62-bcl2 cells induced to express exogenous BRCA1 but not in the BRCA1 mutant HCC1937 cell line (Fig.  3A). To determine whether mutation of BRCA1 was the reason for the failure of IFN-␥ to induce IRF-7, we reconstituted BRCA1 expression in these cells using a CMV-driven wild type BRCA1 expression construct. To confirm expression of exogenous BRCA1 in the HCC1937 cells, we carried out Northern blot and reverse transcription-PCR analysis. We failed to de-

BRCA1 Regulates IFN-␥-mediated Apoptosis
tect the 5.6-kb exogenous BRCA1 transcript in the reconstituted cells by Northern blot analysis, indicating extremely low levels of exogenous BRCA1 expression in these cells (data not shown). However, we were able to confirm expression by reverse transcription-PCR using one primer complimentary to BRCA1 and a second primer designed against the poly(A) sequence encoded in the Rc/CMV vector (Fig. 3B). Reconstitution of BRCA1 in the HCC1937 cells (HCC-BRCA1) resulted in a significant induction of both IRF-7 and MxA following treat-ment with 100 units of IFN-␥ (Fig. 3C). Densitometric analysis revealed that MxA and IRF-7 expression levels were induced 1.3-and 1.5-fold, respectively, in HCC1937 reconstituted with empty vector (HCC-V) following stimulation with 100 units of IFN-␥. In contrast IFN-␥ stimulation increased MxA and IRF-7 expression ϳ7and 5-fold, respectively, following reconstitution with wild type exogenous BRCA1, suggesting that BRCA1 is required for IFN-␥-mediated induction of MxA and IRF-7 in these cells (Fig. 3C). BRCA1 Enhances IFN-␥-induced Apoptosis-We carried out cellular proliferation assays to evaluate the effect of interferon treatment on the growth of MBR62-bcl2 cells in the presence and absence of BRCA1 induction. Growth curves evaluating the effect of interferon treatment on MBR62-bcl2 cells over time demonstrated a synergistic inhibition of cellular proliferation when BRCA1 was expressed in the presence of 500 units of IFN-␥ (Fig. 4A).
In contrast to that observed for IFN-␥, IFN-␤ treatment failed to synergize with BRCA1, giving rise to a marked cytostatic effect in both the presence and absence of exogenous BRCA1 expression. IFN-␣ treatment did result in a mild differential phenotype, although it was much less marked that that observed for IFN-␥ (Fig. 4A). To determine the mechanistic basis of the observed growth inhibition, we evaluated the effect of IFN-␥ treatment on cell cycle profile and cell death in the presence and absence of BRCA1 induction. Fluorescenceactivated cell sorter analysis failed to identify any perturbations in the cell cycle profile following inducible expression of BRCA1 in the presence of IFN-␥ (data not shown), suggesting that the observed inhibition of proliferation was due to cell death.
To determine whether the observed cell death resulted from apoptosis, MBR62-bcl2 cells were induced to express BRCA1 in the presence of increasing concentrations of IFN-␥ for 24 h followed by caspase 3 cleavage assay. The cleaved 17-and 19-kDa caspase 3 products, which are an early hallmark of apoptosis, were only observed when BRCA1 was expressed in the presence of IFN-␥. Furthermore a dose-responsive increase in cleaved caspase 3 was observed when BRCA1 was expressed in the presence of 100, 200, or 300 units of IFN-␥, confirming that the observed inhibition of cellular growth was due to the activation of an apoptotic response to the combined effect of inducible BRCA1 expression in the presence of IFN-␥ (Fig. 4B). To gain some insight into which of the potential target genes might be mediating this apoptotic response, we carried out colony count assays following transfection of both MDA435 (from which the MBR62-bcl2 cells were derived) and MCF-7 breast cancer cells with expression constructs encoding fulllength IRF-7 or MxA. Constitutive expression of IRF-7 in these cells dramatically inhibited cell growth compared with cells transfected with vector only or MxA, suggesting that the synergistic induction of IRF-7 following inducible expression of BRCA1 in the presence of IFN-␥ may contribute to the observed apoptotic cell death (Fig. 5).
Reconstitution of Wild Type BRCA1 Sensitizes HCC1937 Cells to IFN-␥-induced Apoptosis-Having demonstrated that reconstitution of wild type BRCA1 in the HCC1937 cells dramatically enhances IFN-␥-mediated induction of IRF-7 and MxA (Fig. 3), we evaluated the effect of IFN-␥ on the proliferation of the BRCA1 reconstituted HCC-BRCA1 cells relative to the vector-transfected control HCC-V cells. Growth curves evaluating the effect of 100 units of IFN-␥ on both HCC-BRCA1 and HCC-V cells over time demonstrated an increased sensitivity of the HCC-BRCA1 cells to the antiproliferative effects of IFN-␥ compared with vector-transfected control HCC-V cells (Fig. 6A). To determine whether this increase in sensitivity reflected an increase in IFN-␥-mediated apoptosis in the HCC-BRCA1 cells, we carried out Western blot analysis to compare the degree of caspase 3 cleavage in the two cell lines following IFN-␥ treatment. Reconstitution of the HCC1937 cells with BRCA1 elevated the background levels of cleaved caspase 3 relative to vector-transfected controls, consistent with our previous observations that BRCA1 alone can induce apoptosis (22). Furthermore, caspase 3 cleavage was significantly increased in the HCC-BRCA1 cells compared with the HCC-V cell line following treatment with 100 units of IFN-␥, indicating an increased sensitivity to IFN-␥-induced apoptosis (Fig. 6B). To confirm these studies in vitro, we carried out indirect immunofluorescence in HCC-BRCA1 and HCC-V cells, using an antibody that recognizes cleaved caspase 3. Consistent with our biochemical data, IFN-␥ stimulation resulted in a significant increase in detectable cleaved caspase 3 levels in HCC-BRCA1 cells compared with HCC-V cells, suggesting a dramatic increase in apoptosis (Fig. 6C).

DISCUSSION
In this study we have utilized a breast cancer-derived BRCA1-inducible cell line to identify novel targets of BRCA1 by oligonucleotide array-based expression profiling. We feel that the modest overexpression of BRCA1 achieved in this model acts as a good surrogate for activation of BRCA1 as previously described in mouse models (26) and that inducible expression provides an acute intervention not readily available in studying stable transfectants or transiently reconstituted BRCA1 negative cells. Using this approach we have identified a novel subset of genes including IRF-7, MxA, and ISG-54 that are synergistically up-regulated by BRCA1 in the presence of IFN-␥. Furthermore, we demonstrated that BRCA1 sensitized both the MBR62-bcl2 and HCC1937 cells to IFN-␥-mediated apoptosis. The physiological significance of these findings was underscored by the observation that IFN-␥-mediated induction of IRF-7 and MxA was abrogated in the BRCA1 mutant cell line HCC1937, an effect that could be recovered by exogenous expression of wild type BRCA1.
We previously utilized an osteosarcoma-derived cell line with inducible expression of BRCA1 to identify BRCA1 regulated targets using a prototype oligonucleotide arrays (22). This study, although providing novel information on BRCA1 function, was limited by the complexity of the array (6800 genes and expressed sequence tags) and the origin of the cell line model system used. In the current study we felt that using a breast cancer-derived inducible model to screen a more representative oligonucleotide array (12,000 genes) might lead to the identification of additional novel targets that may represent important downstream effectors of BRCA1. We identified a total of 32 genes that were up-regulated and three genes that were down-regulated following inducible expression of BRCA1. We failed to identify GADD45 by oligonucleotide array, which had represented a major BRCA1 target in our initial screen, even though we can clearly demonstrate BRCA1-mediated induction of GADD45 by Northern blot analysis in these cells (13) and have confirmed that GADD45 was present on the U95A array. We did, however, confirm induction of EGR1, which we had also previously identified as a BRCA1 target (data not shown). The reasons for these inconsistencies are unclear and are likely to reflect variation in the quality of the RNA probes used in the two separate screens or inconsistencies between the different oligonucleotide arrays.
The observations reported here support a previous study demonstrating that BRCA1 and STAT1 cooperate to regulate the cyclin-dependent kinase inhibitor p21/WAF1, an effect that was mediated by a physical interaction between BRCA1 and STAT1 (21). In the current study we were unable to demonstrate induction of p21 by BRCA1 alone or following treatment with IFN-␥ (data not shown). Furthermore, we failed to observe G 1 /S arrest in these cells following BRCA1 induction in the presence of IFN-␥ but rather observed a dramatic apoptotic cell death phenotype. The conflicting data regarding p21 may reflect tissue-specific differences because of the use of different cell lines in each study or the fact that the MBR62-bcl2 cells are p53 mutant (13), whereas the 2fTGH, U3A, and G8 cell lines utilized by Ouichi et al. (21) are all p53 wild type. These data also support a number of other studies that demonstrate a role for BRCA1 in transcriptional regulation and that suggest that BRCA1 may act as a co-activator or co-repressor for a number of different transcription factors (18 -21).
The exact mechanism through which BRCA1 and IFN-␥ cooperate to induce the diverse range of genes identified in this study remains to be defined and may reflect indirect effects of BRCA1 or indicate that BRCA1 may be present in various STAT1-associated complexes in the nucleus. The genes identified as being synergistically up-regulated by BRCA1 in the presence of IFN-␥, including IRF-7 and MxA, contain interferon-stimulated response elements within their promoters, which is typically recognized by a STAT1/STAT2/IRF-9 (ISGF3) complex (36,37). In contrast, the p21 promoter contains a ␥-activated sequence element, which is specifically recognized by STAT1 homodimer containing complexes (21). These observations therefore raise the possibility that BRCA1 may also associate with the STAT1/STAT2/IRF-9 (ISGF3) complex, although this remains to be demonstrated. Similar studies have provided potential models through which BRCA1 may regulate GADD45 expression. BRCA1 has been shown to regulate GADD45 expression through its ability to interact with ZBRK1, a novel sequence-specific DNA binding zinc finger protein. ZBRK1 was shown to mediate BRCA1-dependent transcriptional repression of GADD45 through a ZBRK1 consensus site within intron 3 of GADD45. It was hypothesized that increased levels of BRCA1 or alterations in BRCA1 phosphorylation status following DNA damage may relieve this transcriptional repression through competitive displacement of rate-limiting co-factors within the transcriptional repressor complex or by dissociation of BRCA1 from ZBRK1 and leading to GADD45 induction (38).
Whatever the mechanisms underlying BRCA1 target gene regulation, there appears to be a link to activation of stress and DNA damage response pathways. It is interesting to note, therefore, that interferon-inducible genes are also activated by a variety of other cellular stress signals. STAT1-mediated transcriptional activation is enhanced by various DNA-damaging agents including UV irradiation, an effect that is dependent on p38/MAPK-induced phosphorylation on Ser 727 (39,40). Interestingly, it has also been demonstrated that BRCA1 preferentially associates with STAT1 when phosphorylated on Ser 727 , suggesting that BRCA1 may be involved in modulating STAT1 activity following DNA damage (21). Therefore it is interesting to speculate on the exact role BRCA1 may play in the IFN-␥ regulated signaling pathway and the functional consequences of disrupting this pathway. We have demonstrated that BRCA1 and STAT1 cooperate to induce expression of a subset of IFN-␥-regulated genes. These genes, including IRF-7, may be important effectors of IFN-␥-and/or BRCA1-mediated growth suppression in response to viral infection or following DNA damage. It has been suggested that IRF-7 may function as a tumor suppressor gene based on the observation that lack of IRF-7 expression in the 2fTGH fibrosarcoma cell line is due to methylation of the IRF-7 promoter and that IRF-7 is activated through the c-Jun N-terminal kinase/stress-activated protein kinase stress response pathway in response to various DNAdamaging chemotherapeutic agents (36,41). Our data support a role for IRF-7 in mediating growth suppression and suggest that specific genes such as IRF-7 may be coordinately regulated in response to different stimuli such as IFN-␥ or through a BRCA1-dependent DNA damage response pathway. Recently it has been demonstrated that RAG2-and STAT1-deficient mice exhibit a specific susceptibility to spontaneous mammary gland carcinomas, suggesting that lymphocytes and INF-␥ cooperate to protect against these tumors (42). This raises the intriguing possibility that in addition to regulating conventional growth suppressor pathways in response to DNA damage, tumor suppressor genes such as BRCA1 may also be utilized to regulate an immune surveillance pathway that functions in parallel to suppress tumor formation. Consequently inactivation of BRCA1 in breast epithelial cells may disrupt the natural tumor suppressor function of the immune system, leading to tumor formation.