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[Cancer Research 65, 6557-6567, August 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Cyclin D1 Antagonizes BRCA1 Repression of Estrogen Receptor {alpha} Activity

Chenguang Wang1, Saijun Fan1, Zhiping Li1, Maofu Fu1, Mahadev Rao1, Yongxian Ma1, Michael P. Lisanti2, Chris Albanese1, Benita S. Katzenellenbogen3, Peter J. Kushner4, Barbara Weber5, Eliot M. Rosen1 and Richard G. Pestell1

1 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, District of Columbia; 2 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; 3 Departments of Molecular and Integrative Physiology and Cell and Structural Biology, University of Illinois and College of Medicine, Urbana, Illinois; 4 Metabolic Research Unit, University of California-San Francisco School of Medicine, San Francisco, California; and 5 Department of Molecular Genetics, University of Pennsylvania, Philadelphia, Pennsylvania

Requests for reprints: Richard G. Pestell, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Research Building, Room E501, 3970 Reservoir Road Northwest, Box 571468, Washington, DC 20007. Phone: 202-687-2110; Fax: 202-687-6402; E-mail: pestell{at}georgetown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor {alpha} (ER{alpha})–dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ER{alpha} binding. Cyclin D1 and BRCA1 were both capable of binding ER{alpha} in a common region of the ER{alpha} hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ER{alpha} and for rescue of BRCA1-mediated ER{alpha} transcriptional repression. In chromatin immunoprecipitation assays, 17ß-estradiol (E2) enhanced ER{alpha} and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ER{alpha} recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ER{alpha} recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ER{alpha} recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ER{alpha} repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Germ line mutations in the BRCA1 tumor suppressor gene predispose women to ovarian and breast cancer and are associated with the development of prostate cancer in men (1, 2). In members of high-risk families, BRCA1 mutations increase the risk of breast cancer to 80% to 90% by age 70 years. The BRCA1 gene product, a nuclear polypeptide of 220 kDa, colocalizes in nuclear foci with RAD51, BRCA2, and RAD50 (3), consistent with a role for BRCA1 in DNA damage repair. In support of this model, BRCA1 is hyperphosphorylated in response to ionizing radiation by ATM, ATR, and hCds1/Chk2 (4, 5). In addition, protein complexes that regulate repair of DNA double-strand breaks, including hRAD50-hMre11-NBS1, MSH2, MSH6, and proliferating cell nuclear antigen, functionally interact with BRCA1 (3, 6). Furthermore, BRCA1-deficient cells are hypersensitive to ionizing radiation and exhibit defects in both repair of chromosomal double-strand breaks by homologous recombination and transcription-coupled repair processes (3, 7). The observation that BRCA1 associates with components of the DNA damage repair complexes in a temporally and spatially coordinated manner supports a model in which BRCA1 functions as a scaffold protein organizing DNA damage sensors, thereby coordinating the repair of damaged DNA (8).

Consistent with the genetic evidence that BRCA1 functions as a tumor suppressor, overexpression of wild-type (WT) BRCA1 inhibited colony formation and tumor growth in vivo, and antisense BRCA1 accelerated growth of breast cancer cell lines (9, 10). It has been proposed that BRCA1 may regulate expression and/or function of a select subset of genes and that mutation of BRCA1 may contribute to tumorigenesis by altering expression of these genes. In this context, roles for BRCA1 have been proposed as both a general and a selective transcriptional regulator. Components of the RNA polymerase II complex contain BRCA1 (10) and BRCA1 contains an acidic COOH-terminal activation domain sensitive to cancer-associated mutations (11). Although BRCA1 can associate with the RNA polymerase II, recent studies have shown that BRCA1 modulates transcription of only a subset of genes likely through interaction with specific binding proteins. Thus, BRCA1 has little or no effect on transcription controlled by Jun, Fos, Gal4, or USF (6, 12). In contrast, BRCA1 directly binds a subset of transcription factors, including p53, Myc, and ZBRK1, which may contribute to the regulation of a subset of genes and the cell cycle checkpoint control function of BRCA1.

One model proposed to explain the tissue-restrictive pattern of BRCA1-mediated tumor suppression implicates estrogen function in either initiation or progression of tumorigenesis (13). BRCA1 has been hypothesized to function as a tissue-specific tumor suppressor, repressing estrogen receptor (ER) signaling particularly during the early stages of breast tumorigenesis and dedifferentiation. It was considered unlikely that BRCA1 repression of ER{alpha} function contributed to the later stages of breast tumorigenesis when ER{alpha} expression was frequently reduced (14). In support of this model, BRCA1 was shown to inhibit the activity of the hormone-bound ER{alpha} in cultured breast cancer cell lines (12). In subsequent studies, BRCA1 was shown to physically interact in vitro with the ER{alpha} to repress ligand-dependent gene expression in breast cancer epithelial cells (15). BRCA1 also inhibited the basal activity of ER{alpha} in fibroblasts (16) and repressed a subset of estrogen-responsive genes in cultured 293 embryonal kidney cells (17), emphasizing the importance of cell type–specific regulators in determining BRCA1 function and the selectivity of BRCA1-dependent gene regulation. Together, these studies evidence the importance of understanding the mechanisms by which BRCA1 regulates ER{alpha} activity in breast and prostate epithelial cells.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein (pRb) tumor suppressor. Cyclin D1 is overexpressed in 30% to 40% of human breast cancers and is associated with poor prognosis in ER{alpha}-positive cases (18, 19). Evidence for a biologically relevant role for cyclin D1 in breast tumorigenesis includes findings that mammary gland–targeted cyclin D1 overexpression induced mammary tumors in transgenic mice (20), cyclin D1 antisense blocked ErbB2-induced mammary tumor growth in vivo (21), and mice deleted of the cyclin D1 gene were resistant to ErbB2- or Ras-induced mammary tumorigenesis (22). Given that BRCA1 function is compromised by mutations or reduced expression in human breast cancer and that cyclin D1 is a candidate mammary oncogene, we examined the possibility that cyclin D1 may antagonize BRCA1. In this report, we show that cyclin D1 and BRCA1 form distinct complexes with ER{alpha} in breast cancer cells. Cyclin D1 antagonizes BRCA1 repression of estrogen-responsive genes. Cyclin D1 overcomes BRCA1 repression of ER{alpha}-dependent gene transcription and competes with BRCA1 for ER{alpha} binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human embryonic kidney 293T, DU-145, and MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% FCS, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin in humidified atmosphere containing 5% CO2 at 37°C. Cells were seeded onto tissue culture dishes containing phenol red–free DMEM supplemented with 5% charcoal/dextran–treated fetal bovine serum and cultured for 48 hours before all experimental treatments with hormone.

Plasmids, transfections, and reporter assays. The expression vectors pCMV-cyclin D1 pCMV-cyclin D1-KE, CMV-cyclin D1-GH, pCMV-cyclin E, pCMV-cyclin A, Rous sarcoma virus (RSV)-Renilla luciferase reporter, and CMV-Luc were described previously (23). The human cyclin D1 mutants were derived by PCR-directed amplification using sequence-specific primers and cloned into pRC/CMV. The WT BRCA1 expression plasmid was generated by cloning the BRCA1 cDNA into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). The ER{alpha} expression constructions in pCI-Neo (Promega, Madison, WI) and glutathione S-transferase (GST) fusion proteins were derived from pHEGO. The reporter plasmid estrogen response element (ERE)2-Luc contains two copies of the vitellogenin A2 estrogen-responsive enhancer (12). DNA transfection and luciferase assays were done as described previously (12). Cells were transfected using LipofectAMINE Plus and the medium was changed after 3 hours. Luciferase activity was determined after 48 hours. A ß-gal reporter plasmid was included to control for transfection efficiency. The fold effect was determined by comparison with the effect of the empty expression vector cassette (pcDNA3).

Western blot and Northern blot. The antibodies used in Western blot analysis were BRCA1 (MS13, a gift from Dr. R. Scully, Harvard Institute of Medicine, Boston, MA), BRCA1 (C-20), ER{alpha} (H-184), and pS2 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were visualized by the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). The abundance of immunoreactive protein was quantified using a computing densitometer (ImageQuant version 1.11, Molecular Dynamics, Sunnyvale, CA). Northern blot analysis was done using a full-length pS2 cDNA clone from American Type Culture Collection (cDNA collection).

Immunostaining. The primary antibodies were Ab-3 to cyclin D1, H-184 to ER{alpha}, and monoclonal antibodies MS110, AP16, and MS13 (generous gifts from Dr. R. Scully) to BRCA1. The secondary antibodies were goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 568 (red; Molecular Probes, Eugene, OR). MCF-7 cells were cultured on glass coverslip or glass bottom microwells (MatTek Corp., Ashland, MA). The cells were fixed for 8 minutes at 37°C with freshly prepared 3.7% paraformaldehyde solution with 0.18% Triton X-100. The fixation was followed by two washes with PBS. Specimens were blocked with 1% bovine serum albumin in PBS at 37°C for 30 minutes. Cells were incubated with primary antibody diluted with blocking solution at room temperature for 60 minutes, rinsed, and then incubated with secondary antibody conjugated with Alexa Fluor using the same conditions as the primary antibody binding reaction. For confocal microscopy, cells were fixed and permeabilized as described previously (24). Monoclonal BRCA1 antibodies were used at 1:30 dilution of the hybridoma culture supernatant. Images were collected using an Olympus (Melville, NY) IX-70 laser scanning microscope with objective lenses (x60 with oil). Images were collected through the specimens every 2 µm in the vertical plane and overlaid to generated focus composite images and stored as TIFF files. Figures were assembled from the TIFF files using Adobe Photoshop software.

Protein-protein interaction assays in vitro and in cultured cells. In vitro protein-protein interactions were done as described (23). In vitro [35S]methionine-labeled proteins were prepared by coupled transcription-translation with a TNT-coupled reticulocyte lysate kit (Promega) using plasmid DNA (1.0 µg) in a total of 50 µL. GST fusion proteins were prepared from Escherichia coli cells (23). In vitro–translated protein (15 µL of In vitro–translated products) was added to 5 µg GST fusion protein of GST as control in 225 µL binding buffer [50 mmol/L Tris-HCl, 120 mmol/L NaCl, 1 mmol/L DTT, 0.5% NP40, 1 mmol/L EDTA, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 µg/mL pepstatin] and rotated for 2 hours at 4°C. Glutathione-Sepharose bead slurry (50 µL) was added and the mixture was rotated for a further 30 minutes at 4°C. Beads were washed five times with binding buffer (1 mL), and binding buffer (30 µL) was added after the final wash. Sepharose beads were washed five times with lysis buffer and boiled in SDS sample buffer, and released proteins were resolved by SDS-PAGE. The gel was fixed, dried, and subjected to autoradiography.

Immunoprecipitation/Western blot analysis was done in HEK 293T cells. Transfected cells were treated for 24 hours with 10 nmol/L 17ß-estradiol (E2) or vehicle as control. Cells were rinsed with PBS, harvested by scraping, pelleted, and lysed in buffer [50 mmol/L HEPES (pH 7.2), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.1% Tween 20, 0.1 mmol/L PMSF, 2.5 µg/mL leupeptin, 0.1 mmol/L sodium orthovanadate]. Extracts were cleared by centrifugation and further precleared by rocking at 4°C with washed protein A-Sepharose (Roche Molecular Biochemicals, Indianapolis, IN). Precleared extract (500 µg) was immunoprecipitated with 1 µg cyclin D1 antibody (DCS-11, NeoMarkers Lab Vision Corp., Fremont, CA), BRCA1 antibody or equivalent amounts of appropriate control IgG (Santa Cruz Biotechnology), and protein A agarose (50 µL) for 6 hours to overnight at 4°C. Agarose beads were washed five times with lysis buffer and boiled in SDS sample buffer, and released proteins were resolved by 10% SDS-PAGE. The gel was transferred to nitrocellulose, and Western blotting was done.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) analysis was done following a protocol provided by Upstate Biotechnology (Charlottesville, VA) under modified conditions. MCF-7 cells (1 x 106) were grown in DMEM with 10% charcoal/dextran–stripped serum for 3 days. Cells were treated with E2 for the time points as indicated in the figure legends (45 minutes, 1 hour, and 12 hours). On E2 (100 nmol/L) stimulation, the cells were cross-linked by adding 1.1% formaldehyde buffer containing 100 mmol/L NaCl, 1 mmol/L EDTA-Na (pH 8.0), 0.5 mmol/L EGTA-Na, and Tris-HCl (pH 8.0) directly to culture medium for 10 minutes at 37°C. The medium was aspirated, cells were washed thrice using ice-cold PBS containing 10 mmol/L DTT and protease inhibitors and lysed by warm 1% SDS lysis buffer and incubated for 10 minutes on ice. The cell lysates were sonicated to shear DNA to lengths between 200 and 1,000 bp and the samples were diluted to 10-fold in ChIP dilution buffer. To reduce nonspecific background, the cell pellet suspension was precleared with 60 µL salmon sperm DNA/protein A agarose-50% slurry (Upstate Biotechnology) for 2 hours at 4°C with agitation. Chromatin solutions were precipitated overnight at 4°C using 6 µg anti-ER{alpha} (H-184, Santa Cruz Biotechnology) with rotation. For a negative control, rabbit IgG was immunoprecipitated by incubating the supernatant fraction for 1 hour at 4°C with rotation. Salmon sperm DNA/protein A agarose-50% slurry (60 µL) was added for 2 hours at 4°C with rotation to collect the antibody/histone complex and washed extensively following the manufacturer's protocol. Input and immunoprecipitated chromatin were incubated at 65°C overnight to reverse cross-links. After proteinase K digestion for 1 hour, DNA was extracted using a Qiagen (Valencia, CA) spin column kit. Precipitated DNAs were analyzed by PCR. In separate experiments, MCF-7 cells were transiently transfected with plasmid DNA encoding cyclin D1, BRCA1, or empty vector and ChIP analysis was conducted for the endogenous pS2 promoter (25 cycles of PCR). The following primers were used for PCR of the pS2 promoter: sense 5'-GGCCATCTCTCACTATGAATCACTTCTGC-3' and antisense 5'-GCAGAAGTGATTCATAGTGAGAGATGGCC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BRCA1 repression of estrogen receptor {alpha} transcriptional activity is selectively reversed by cyclin D1. We examined the possibility that cyclin D1 may regulate the ability of BRCA1 to inhibit hormone-bound ER{alpha} activity (12). We first assessed the specificity of BRCA1-mediated regulation of E2-dependent ER{alpha} gene function. ER{alpha}-positive T47D breast cancer cells were transfected with either BRCA1 or control vector and treated with E2 or vehicle for 24 hours. BRCA1 protein levels were increased 3- to 4-fold in transfected cells compared with vector (ref. 25; data not shown), a change within the physiologic range that occurs during cell cycle transition. ER{alpha} activity measured as E2-stimulated activation of the estrogen-responsive reporter ERE-TK-Luc was inhibited by expression of BRCA1 (Fig. 1A). Transcriptional repression was selective, as BRCA1 did not inhibit either the activity of E2F-Luc or Sp1-Luc reporters or the transcriptional activity of Gal4-c-Jun or Gal4-E2F-1 (ref. 12; data not shown). Cyclin D1 did not affect activity of the liganded ER{alpha} but substantially reversed the BRCA1-mediated repression of ER{alpha}, increasing ERE-TK-Luc activity >200-fold (Fig. 1A, note log scale). In contrast, expression of other G1- and S-phase regulatory cyclins (cyclin A, E, D2, or D3), from the same CMV expression plasmid, did not affect the BRCA1 inhibition of liganded ER{alpha} activity (Fig. 1A). Cyclin D1 at the same vector doses did not affect the activity of several different viral luciferase reporter constructions (RSV and CMV) in either the presence or the absence of E2 in the same cells (data not shown), indicating the effect was specific for the ERE reporter gene. We assessed mutants of cyclin D1, defective in their ability to bind either cyclin-dependent kinase (cdk) 4/cdk6 (cyclin D1-KE), pRb protein (cyclin D1-GH), or ER{alpha} coactivator SRC-1 (cyclin D1 L254/255A; ref. 26). These mutants, which were expressed equally to WT cyclin D1 (27), conveyed a similar rescue function to WT cyclin D1 (Fig. 1B). We had the same observation in ER{alpha}-deficient DU-145 prostate cancer cells (Fig. 1C and D).



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  		Figure 1. BRCA1-mediated repression of liganded ER{alpha} activity is rescued by cyclin D1. The effects of cyclins expressed from an identical promoter (pRC/CMV) were compared for rescue of BRCA1-mediated repression of ERE2-Luc reporter activity in breast cancer epithelial cell T47D (A and B) and prostate cancer epithelial cell DU-145 (C and D). WT and mutant cyclin D1 (CD1) expression plasmids [cyclin D1 defective in binding to cdks (CD1 KE) and SRC-1 (CD1 LALA)] were compared for rescue of BRCA1 repression of ER{alpha} activity in T47D (B) and DU-145 (D) cells. Expression plasmid for ER{alpha} was cotransfected. Columns, mean of quadruplicate determinations throughout; bars, SE. Data are expressed as relative luciferase activity. C and D, DU-145 cells were cotransfected with the ER{alpha} expression plasmid.

 
Cyclin D1 overcomes BRCA1-mediated estrogen receptor {alpha} activation function-2 activity. We next assessed whether the regulation of ER{alpha} activity by cyclin D1 in the presence of BRCA1 required the DNA-binding domain of ER{alpha}. The COOH-terminal activation function (AF)-2 domain of the ER{alpha} linked to the heterologous Gal4 DNA-binding domain was induced by E2 in the human ER{alpha}-positive MCF-7 breast cancer cells and this activity was inhibited by >90% on expression of BRCA1 (Fig. 2A, note log scale). Cyclin D1 reversed BRCA1 repression of Gal4-ER AF2, suggesting that BRCA1 repression of ER{alpha} activity is not dependent on the ER{alpha} DNA-binding function. ER{alpha} contains two transactivation domains regulating ligand-independent and ligand-dependent gene transcription. AF1 consists of A and B domains, which are in the NH2 terminus of ER{alpha}. We examined whether AF1 contributes to BRCA1 repression of ER{alpha} activation. Figure 2B shows that ER{alpha} amino acids 1 to 282 (AF1) activated ERE-TK-Luc reporter in the absence of ligand and overexpression of BRCA1 could not repress AF1-mediated ER{alpha} activation. Taken together, these studies suggest BRCA1 repression of ER{alpha} is independent of the AF1 and DNA-binding domain. Further, the ligand-binding domain is sufficient for BRCA1 repression and cyclin D1 enhances ER{alpha} function through either AF1 domain (28, 29) or AF2 by antagonizing BRCA1 repression.



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  		Figure 2. Cyclin D1 overcomes BRCA1 repression of AF2-mediated ER{alpha} transactivity and ER{alpha}-mediated gene expression. A, the ER{alpha} AF2 domain linked to the Gal4 DNA-binding domain was assessed using a heterologous Gal4 DNA-binding site luciferase reporter, (UAS)5-E1B-TATA-Luc, in MCF-7 cells. The effect of cyclin D1 on BRCA1-mediated repression of liganded ER{alpha} activity is shown compared with equal amounts of empty expression vector cassette (pRC/CMV). E2 (10–6 mol/L) was added as indicated. B, the ERE2-Luc reporter was assessed using expression vectors for the ER{alpha} AF1 (amino acids 1-282) expression vector. Northern blot (C) or Western analysis (D) for pS2 expression in ER{alpha}-positive T47D cells transfected with BRCA1 or empty vector control and either treated with E2 (10–6 mol/L) or vehicle for 24 hours.

 
Cyclin D1 rescues BRCA1 repression of 17ß-estradiol–induced gene expression. The pS2 gene is induced by estrogen in breast cancer cells (30). To determine whether BRCA1 regulated pS2 expression, T47D cells were transfected with BRCA1 and pS2, gene expression was measured by Northern blot analysis. The 4-fold induction of pS2 gene expression by E2 was abrogated by BRCA1 expression (Fig. 2C). Western blot analysis showed that cyclin D1 reversed the BRCA1-mediated repression of pS2 abundance in T47D cells (Fig. 2D).

Cyclin D1 and BRCA1 physically associate with estrogen receptor {alpha} through similar domains. To investigate the mechanisms by which cyclin D1 antagonized BRCA1 repression of ER{alpha} activity, we examined the possibility that cyclin D1 may complex with ER{alpha} in vivo under physiologic conditions. Immunoprecipitation with the cyclin D1–specific antibody showed that cyclin D1 associated specifically with ER{alpha} in MCF-7 cells (Fig. 3A), consistent with previous in vitro studies (28). T47D cells were subjected to immunoprecipitation with a BRCA1-specific antibody and Western blotting for ER{alpha} was done (Fig. 3B). ER{alpha} was coprecipitated with BRCA1, whereas control IgG did not precipitate ER{alpha} from an equivalent amount of cell lysate. We next determined the domain of ER{alpha} required for binding to cyclin D1. A series of mutant ER{alpha} expression plasmids were assessed in cultured 293T cells for expression by ER{alpha} Western blotting (Fig. 3C). Equal amounts of protein extract from the transfected cells were then subjected to immunoprecipitation with the cyclin D1 antibody and the immunoprecipitated product was subjected to ER{alpha} Western blotting. The experiments were conducted on at least three separate occasions with similar results. ER{alpha} was identified in the cyclin D1 immunoprecipitation. The abundance of ER{alpha} in the cyclin D1 immunoprecipitate was not reproducibly significantly changed by the addition of ligand (Fig. 3D, cyclin D1 immunoprecipitation). The ER{alpha} mutants with sequential deletion of the COOH terminus from N595 to N378 were efficiently coimmunoprecipitated with cyclin D1 by the cyclin D1 antibody. However, the ER{alpha} mutant 1 to 282 was not coimmunoprecipitated by the cyclin D1 immunoprecipitation, although this mutant was readily detected by direct Western blot analysis (Fig. 3D). These studies show that cyclin D1 binding to the ER{alpha} in cultured cells requires ER{alpha} residues 282 to 378.



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  		Figure 3. ER{alpha} associates with endogenous cyclin D1 and BRCA1 in breast cancer cell lines. A, MCF-7 cell lysates were immunoprecipitated using the cyclin D1 antibody and subjected to Western blotting for either ER{alpha} and cyclin D1 as indicated. Comparison is shown with Western blotting of MCF-7 cell lysate. B, the ER{alpha}-positive T47D and ER{alpha}-negative DU-145 cell extracts were subjected to BRCA1 immunoprecipitation with sequential Western blotting for BRCA1, ER{alpha}. Equal amounts of protein were incubated with the control IgG and the immunoprecipitation was subjected to identical Western analysis. C, expression plasmids of human ER{alpha}. D, expression plasmids encoding either ER{alpha} WT or mutant were transfected into 293T cells and subjected to either Western blotting for ER{alpha} or cyclin D1 immunoprecipitation with subsequent ER{alpha} Western blotting. Although the ER{alpha} antibody detects the 1 to 282 protein in the transfected cells, the ER{alpha} 1 to 282 mutant was not identified in the cyclin D1 immunoprecipitation. E, in vitro–translated ER{alpha} and truncated mutant protein was incubated with beads coated with GST-cyclin D1 and the results of the ER{alpha} pull-down are shown. The ER{alpha} domain from 282 to 378 is required for binding to cyclin D1.

 
GST pull-down experiments were next conducted using in vitro–translated, 35S-labeled ER{alpha} incubated with equal amounts of GST-cyclin D1 fusion protein. Approximately 14% of the ER{alpha} input bound to GST-cyclin D1 (Fig. 3E). Deletion of the ER{alpha} residues 282 to 378 again abrogated binding of the ER{alpha} to cyclin D1.

Cyclin D1 competes with BRCA1 for estrogen receptor {alpha} binding. To investigate the mechanisms by which cyclin D1 rescued BRCA1 repression of liganded ER{alpha} activity, we determined the domains of the ER{alpha} required for binding to cyclin D1. As shown in Fig. 4A, five fragments that cover the full length of ER{alpha} were fused with GST. These GST fusion proteins were assessed for their binding capacity to cyclin D1 and BRCA1 (Fig. 4B and C). Equal amounts of GST fusion protein were incubated with the in vitro–translated, 35S-labeled cyclin D1. The ER{alpha} 338 to 379 associated efficiently with cyclin D1. GST pull-down experiments were next conducted using 35S-labeled BRCA1 amino acids 1 to 302, which was identified previously as the minimal region required for binding to ER{alpha} (15). The same domain of ER{alpha} (338-379) required for binding to cyclin D1 was also sufficient for binding to BRCA1 (Fig. 4C). Requirement of the same region of ER{alpha} for cyclin D1 or BRCA1 binding raised the possibility that cyclin D1 and BRCA1 may compete for binding to ER{alpha}. We examined this possibility by conducting ER{alpha} pull-down experiments with either cyclin D1 (Fig. 4D) or BRCA1 (Fig. 4E). We found that preincubation with increasing amounts of BRCA1 decreased cyclin D1 binding to ER{alpha}. Furthermore, preincubation with cyclin D1 reduced BRCA1 association with ER{alpha} (Fig. 4E). These results show that cyclin D1 has the ability to inhibit BRCA1 association with ER{alpha}, providing one potential mechanism for antagonism.



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  		Figure 4. Cyclin D1 competes with BRCA1 for ER{alpha} binding. A, schematic map of ER{alpha}. Five fragments that cover the full length of ER{alpha} were constructed as GST fusion protein. B, in vitro–translated, 35S-labeled cyclin D1 was incubated with either beads coated with GST-ER{alpha} fragments or equal amounts of GST protein showing the minimal sequence requirement of ER{alpha} for physical association between ER{alpha} and cyclin D1. C, in vitro–translated, 35S-labeled NH2-terminal of BRCA1 that is required for ER{alpha} binding was incubated with either beads coated with GST-ER{alpha} fragments or equal amounts of GST protein showing the same region of ER{alpha} required for cyclin D1 binding is also sufficient for BRCA1 binding. D, increased dose of BRCA1 proteins were incubated with GST-ER{alpha} (amino acids 338-379) before 35S-labeled cyclin D1 addition. Preoccupied ER{alpha} with BRCA1 prevents cyclin D1 from binding. E, in GST-ER{alpha} (amino acids 338-379) and 35S-labeled BRCA1 (amino acids 1-302) experiment, preincubation of cyclin D1 with ER{alpha} decreases BRCA1 binding.

 
17ß-Estradiol facilitates formation of an active estrogen receptor {alpha} complex in vivo. As cyclin D1 and BRCA1 were both capable of binding ER{alpha}, we reasoned that distinct complexes may form between these proteins in vivo serving to physically segregate their interactions and functional activities. BRCA1 localizes to discrete nuclear dots during S phase in MCF-7 cells (10). BRCA1 functions as a cell cycle checkpoint protein through its BRCT domain and BRCA1 associates with RNA polymerase II holoenzyme (10), suggesting that BRCA1 might be directly involved in transcriptional regulation. We examined the possibility that E2 may alter the BRCA1/ER{alpha} association using immunofluorescent staining to view the dynamic chances of BRCA1 in response to E2. Cells were grown in the absence of E2 for 48 hours followed by E2 treatment. Monoclonal antibody against BRCA1 (MS110, AP16, and MS13) and polyclonal ER{alpha} antibody (H-184) were used in this study. In the absence of E2, most cells expressed ER{alpha} and BRCA1 to a comparable level (Fig. 5A, green for BRCA1 and red for ER{alpha}). Colocalization of BRCA1 and ER{alpha} (yellow) was observed in nuclear dots. E2 treatment for 1 hour reduced BRCA1 and ER{alpha} colocalization (Fig. 5B). Approximately 50% of the cells maintained relatively strong expression of ER{alpha} (red) with reduced colocalization of BRCA1 in the nucleus. In ~50% of the remaining cells, BRCA1 dots remained (Fig. 5B, green), but costaining of ER{alpha} was substantially reduced. Thus, the addition of ligand reduces ER{alpha}/BRCA1 colocalized dots. These studies do not necessarily reflect activity of the ER{alpha} within the local chromatin structure of an ERE; therefore, ChIP assays were conducted.



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  		Figure 5. BRCA1 and ER{alpha} colocalize in MCF-7 cells. A, confocal microscopy for BRCA1 (green) and ER{alpha} (red) in MCF-7 cell shows colocalization (yellow) in nuclear dots. The addition of E2 (10–8 mol/L) reduces ER{alpha} colocalization with BRCA1 (B). Approximately 50% cells maintain BRCA1 in nuclear dots (green). The minority of which contains ER{alpha}. The remaining cells contain predominantly nuclear ER{alpha} (red) without colocalizing BRCA1.

 
Cyclin D1 and BRCA1 form functionally distinct complexes with estrogen receptor {alpha} in the context of an estrogen-responsive gene promoter. It has been reported that ER{alpha} and p160 coactivators are recruited to the promoters of endogenous estrogen-responsive target genes contributing to transcriptional activation of ER{alpha} (31). Given the evidence that BRCA1 repressed ER{alpha} transactivation and estrogen-induced pS2 gene expression, we examined the recruitment of BRCA1 to the endogenous estrogen-responsive target gene pS2 in the context of its local chromatin structure using ChIP assays. The estrogen-responsive MCF-7 cell line was grown in the absence of estrogen for 3 days and then treated with either E2 or vehicle control for 1 hour. ChIP assays were done to determine the endogenous transcriptional complexes present on the pS2 promoter. E2 increased ER{alpha} recruitment to the pS2 gene promoter (Fig. 6). BRCA1 occupancy on the pS2 gene promoter was also observed and E2 significantly decreased BRCA1 recruitment to the pS2 gene promoter (Fig. 6A). To examine the possibility that cyclin D1 may antagonize BRCA1 function at an estrogen-responsive promoter through inhibiting BRCA1 recruitment, we expressed cyclin D1 in MCF-7 cells and assessed BRCA1 occupancy by ChIP assay (Fig. 6B). Cyclin D1 expression reduced the amount of BRCA1 recruited to the pS2 promoter in both basal and E2-induced states. BRCA1 transfection increased BRCA1 occupancy at the promoter ~3-fold. Cyclin D1 expression reduced the effect of BRCA1 expression on BRCA1 occupancy at the pS2 promoter.



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  		Figure 6. Cyclin D1 inhibits BRCA1 recruitment to an endogenous estrogen-responsive gene in the content of local chromatin. A, ChIP assays were conducted on MCF-7 cells treated with E2 for 45 minutes and immunoprecipitation was conducted with antibodies against ER{alpha} or BRCA1 as indicated. The final DNA extractions were amplified using pairs of primers to the ERE region of the estrogen-responsive pS2 gene. B and C, MCF-7 cells were transfected with expression vectors encoding cyclin D1, BRCA1, or BRCA1 and cyclin D1. ChIP assays were conducted for the endogenous pS2 promoter using antibody directed to BRCA1. E2 treatment was for 45 minutes (B), 1 hour, or 12 hours (C) as indicated.

 
E2 induces ER{alpha} to the ERE with cyclical kinetics (31). To examine the role of cyclin D1 and BRCA1 in regulating the kinetics of ER{alpha} recruitment to the ERE in the context of its local chromatin structure, ChIP assays were conducted. The pS2 promoter was used as a target of ER{alpha}. Cells were treated with E2 for 0, 1, or 12 hours (Fig. 6C). These studies showed that ER is recruited to an ERE in the presence of ligand within 1 hour (Fig. 6C). Cyclin D1 enhanced ligand-induced recruitment of ER{alpha} to the ERE. BRCA1 expression reduced ER{alpha} recruitment in MCF-7 cells at both 1 and 12 hours of E2 treatment. The coexpression of cyclin D1 and BRCA1 resulted in the relative increase of ER{alpha} recruitment at an ERE compared with the effect of BRCA1 alone (Fig. 6C). Compared with the effect of BRCA1, coexpression of cyclin D1 enhanced ER{alpha} recruitment to the ERE after 12 hours of ligand treatment. As cyclin D1 increases in response to E2 treatment in ER{alpha}-positive breast cancer cells after 6 to 12 hours (32), the inhibition of BRCA1 occupancy at an ERE may contribute to the physiologic mechanism by which E2-induced cyclin D1 expression antagonizes BRCA1 repression of ER{alpha} function in the context of the local chromatin structure. The finding that cyclin D1 enhances ER{alpha} recruitment to an ERE in the presence of E2 and reduces the effect of BRCA1 to inhibit ER recruitment to an ERE may contribute to the ability of cyclin D1 to overcome BRCA1 repression at an ERE.

The ChIP assays for BRCA1 confirmed previous findings that BRCA1 occupies an ERE and that E2 reduces BRCA1 recruitment (ref. 16; Fig. 6C). Herein, BRCA1 expression enhanced BRCA1 recruitment to the ERE. The expression of cyclin D1 reduced BRCA1 recruitment to the ERE at 0 hour but did not significantly alter the abundance of BRCA1 at an ERE at 1 and 12 hours of E2 treatment.

The ChIP assays of cyclin D1 at the ERE showed that E2 enhanced cyclin D1 recruitment to an ERE at 1 hour and that forced expression of cyclin D1 enhanced cyclin D1 presence at the ERE. The expression of BRCA1 reduced the ligand-induced recruitment of cyclin D1 to the ERE at 1 hour. Together, these studies suggest that cyclin D1, BRCA1, and ER{alpha} are recruited to an ERE in breast epithelial cells and that the presence of E2 induces dynamic changes in the presence of these proteins with distinct kinetics. E2 treatment (1 hour) is associated with the recruitment of ER{alpha}, increased presence of cyclin D1, and reduced BRCA1. The expression of BRCA1 reduces the recruitment of cyclin D1 at 1 hour and reduces ER{alpha} recruitment at 12 hours. These findings are consistent with the model in which BRCA1 reduces ER{alpha} occupancy at an ERE. In addition, these studies show that coexpression of cyclin D1 reduces the ability of BRCA1 to inhibit ER{alpha} recruitment in the presence of E2 at 12 hours to an ERE. These findings are consistent with the model in which BRCA1 reduces ER{alpha} occupancy at an ERE in the presence of ligand and that cyclin D1 antagonizes this function, particularly at 12 hours.

Mapping functional domains of cyclin D1. The domains of cyclin D1 required for binding to ER{alpha} were assessed using expression plasmids encoding additional mutants of cyclin D1 (Fig. 7A). In vitro–translated [35S]cyclin D1 and COOH-terminal truncated mutants were incubated with GST-ER{alpha} 282 to 420. Deletion of residues 142 to 178 of cyclin D1 completely abolished binding to ER{alpha} (Fig. 7A). The domains of cyclin D1 required for antagonism of BRCA1-mediated repression of ER{alpha} activity was next determined using expression plasmids encoding mutants of cyclin D1. The expression of mutant cyclin D1 proteins was at least equal to that of the WT cyclin D1 expression vector (27). Deletion of cyclin D1 residues 142 to 178 abolished functional antagonism of BRCA1 to <1% of WT cyclin D1. None of the NH2-terminal cyclin D1 mutants provided BRCA1 rescue function in DU-145 cells (Fig. 7B) or T47D cells (data not shown). (In T47D cells, the rescue function of cyclin D1 1 to 142 was <3% of WT cyclin D1.) Thus, the domain of cyclin D1 between 142 and 178, which is predicted to form a helix-loop-helix (HLH) structure (27), is necessary for antagonism of BRCA1 repression of ER{alpha} activity.



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  		Figure 7. Mapping of domains of cyclin D1 required for antagonism of BRCA1-mediated ER{alpha} repression. A, in vitro–translated, 35S-labeled cyclin D1 full-length and truncated mutant protein was incubated with either beads coated with GST-ER{alpha} fragments (amino acids 280-420) or equal amounts of GST protein showing the sequence requirement of cyclin D1 for physical association between ER{alpha} and cyclin D1. B, cyclin D1 mutant expression plasmids were assessed for rescue of BRCA1-mediated repression of ERE2-Luc reporter activity. Deletion of the residues between 142 and 178 abolished rescue of BRCA1-mediated ER{alpha} repression. Data are expressed as relative luciferase activity. DU-145 cells were cotransfected with the ER{alpha} expression plasmid.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current studies provide the first evidence that cyclin D1 can directly regulate a transcriptional function of BRCA1. Herein, BRCA1 inhibited E2-induced endogenous gene expression at levels of BRCA1 that are within the physiologic range. BRCA1 and ER{alpha} colocalized in nuclear dots. The addition of E2 reduced BRCA1/ER{alpha} colocalization. In the context of the local chromatin structure by ChIP assay, BRCA1 binding to the estrogen-responsive pS2 gene was reduced by E2 or cyclin D1 overexpression. As cyclin D1 abundance is induced by diverse mitogenic and oncogenic signals (21, 33, 34), the antagonism by cyclin D1 of BRCA1 function at the level of the liganded ER{alpha} may contribute to signal transduction cross-talk between oncogenic signals and BRCA1 function.

The direct association between BRCA1 and ER{alpha} as shown herein provides a physical means by which BRCA1 may be tethered to select transcriptional regulatory loci. In the current studies, BRCA1 and ER{alpha} were physically associated using in vitro GST pull-down and coimmunoprecipitation assays (Figs. 3 and 4). The direct binding of BRCA1 to ER{alpha} may serve to select BRCA1 genomic targets as shown herein for the pS2 gene. BRCA1 interacts with general transcriptional regulators, including p300/CBP (35), corepressors (CtIP/CtBP and HDAC complexes; refs. 36, 37), and the BRG1 subunit of the SWI/SNF complex (35, 38), yet regulates expression of a relatively specific subset of transcription factors and target genes. BRCA1 directly binds a subset of transcription factors, including p53, Myc, and ZBRK1, without affecting transcription controlled by Jun, Fos, Gal4, or USF (6, 12). The selective regulation of a subset of genes by BRCA1 likely contributes to the diverse role of BRCA1 including cell cycle checkpoint control function (39, 40). The ability of BRCA1 to repress ER{alpha} signaling is not limited to one or a few estrogen-responsive genes, such as pS2. Previously, we showed that in MCF-7 breast cancer cells overexpression of BRCA1 blocks the E2-inducible expression of ~90% of the E2-responsive transcriptosome. Thus, as determined by DNA microarray analysis and confirmed by semiquantitative and quantitative reverse transcription-PCR analysis, exogenous BRCA1 blocked the ability of E2 to alter the expression of the vast majority of E2-responsive genes in MCF-7 cells (41).

The current findings extend previous studies in which E2 reduced BRCA1 binding to an ERE (16) by firstly demonstrating the kinetics of this process, secondly by demonstrating the effect of cyclin D1 expression on ER{alpha} recruitment, and thirdly by characterizing the effect of cyclin D1 to inhibit BRCA1 recruitment in the basal state and after 12 hours of E2 treatment. The ChIP studies herein show that cyclin D1 antagonizes BRCA1 inhibition of ER{alpha} occupancy at an ERE in the context of its local chromatin structure. This model is compatible with the identification of a ternary complex between cyclin D1/ER{alpha} and cyclin D1 by immunoprecipitation herein and suggests cyclin D1 regulates function of proteins in the complex at the local promoter level. The differences between ChIP and immunoprecipitation studies typically relate to the relatively small fraction of the target protein (in this case, the ER{alpha}) that is recruited to the promoter in the context of its local chromatin structure. Frequently, proteins identified in immunoprecipitation/Western analysis are not the same as those present within the transcriptional complex; alternatively, the dynamics of the entire pool of interacting proteins (identified by immunoprecipitation/Western blotting) is not the same as the dynamics of the specific pools of proteins present at the relevant promoter sites.

It is important to note that BRCA1 levels that antagonized ER{alpha} signaling herein are consistent with changes in BRCA1 levels that occur during physiologic and pathologic perturbations. BRCA1 levels change 8-fold during cell cycle transition and BRCA1 expression is high in proliferating mammary epithelial cells undergoing differentiation during puberty and pregnancy (42). Thus, BRCA1 is highly expressed in specific windows of time that may be most pertinent to the BRCA1 tumor suppressor function. The mechanism by which cyclin D1 antagonized BRCA1 function is quite distinct from our previous studies of p300 function (25). p300 exhibits a "rescue" function that is quite distinct from its ER{alpha} coactivator function. BRCA1 down-regulates p300 and exogenous p300 rescues BRCA1 repression of ER{alpha}. The difference between the rescue and coactivator function was shown by the finding that the CH3 domain of p300 was both necessary and sufficient to rescue repression of ER{alpha} activity by BRCA1, whereas its coactivator function requires other domains (e.g., histone acetyl transferase domain and SRC-1-binding domain) that are not required for rescue. Assessment of several other ER{alpha} coactivators (SRC-1 and PCAF) failed to rescue BRCA1 repression. Clearly, the coactivation and rescue functions are separable structurally and functionally.

The domain of cyclin D1 required to antagonize BRCA1 repression at an ERE is distinct from the previously described cdk-, pRb-, or SRC-1-binding residue, requiring a domain between residues 141 and 178, which was required for ER{alpha} binding, and is predicted to form a HLH-like structure (27). The domain of cyclin D1 required for reversal of BRCA1 repression of the ligand-bound ER{alpha} correlated with the domain of cyclin D1 required for ER{alpha} binding. Cyclin D1 inhibited BRCA1 association and ER{alpha} in vitro and reduced BRCA1 recruitment to endogenous estrogen-responsive gene using ChIP assays. These studies suggest the physical association of cyclin D1 with ER{alpha} may exclude BRCA1 association with ER{alpha} and thereby contribute to activation at an ERE. BRCA1 binds strongly to an ER{alpha} region (amino acids 338-379) and much more weakly to a second site (amino acids 420-595). Cyclin D1 interacts with the ER{alpha} at the major site of interaction with BRCA1, consistent with a model in which cyclin D1 may exclude physical access of BRCA1 to the ER{alpha}. Alternatively, the interaction between cyclin D1 and ER{alpha} may alter the conformation of BRCA1 bound to the ER{alpha} and/or change binding of other BRCA1-associated proteins (Fig. 8). These two models remain nonmutually exclusive and compatible with the ChIP data, indicating reduced association of BRCA1 at an ERE in the presence of coexpressed cyclin D1 after 12 hours of E2 treatment. As distinct clusters of genes are induced by E2 with distinct temporal kinetics by genome-wide analysis, the identification of the genes regulated by E2 at 12 hours and repressed by BRCA1 may be of considerable interest.



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  		Figure 8. Hypothetical model by which cyclin D1 antagonizes BRCA1 function through competing with BRCA1 for ER{alpha} binding. A, summary of data describing the physical association between ER{alpha} and BRCA1, ER{alpha}, and cyclin D1. B, schematic representation of a hypothetical model in which cyclin D1 antagonizes BRCA1 repression of ER{alpha} activity through competing with the BRCA1/ER{alpha} complex.

 
It is important to consider the current findings in the context of recent epidemiologic data. On one hand, immunohistochemical and microarray analyses of human breast tumors harboring BRCA1 mutations are usually ER negative (43–45). Further, BRCA1 mutant breast cancers have a basal epithelial phenotype unlike ER-positive tumors (46). In contrast, prophylactic oophorectomy significantly reduces the risk of breast cancer in BRCA1 carriers (50% overall and as high as 75% if done at an early age; refs. 47, 48). This is prima facie evidence that ovarian hormones contribute to breast cancer risk in BRCA1 carriers. Secondly, in the general female population, early first pregnancy (which is associated with very high circulating levels of E2 and progesterone) reduces the risk for breast cancer. However, in female carriers of BRCA1 mutations, early first pregnancy increases breast cancer risk, providing further evidence that the hormonal milieu influences the development of breast cancer in these women (49). Thirdly, studies by Zheng et al. (16) established that inactivation of BRCA1 (by gene deletion or antisense) causes ligand-independent activation of ER. Finally, although BRCA1 mutant cancers are usually ER negative, sporadic breast cancers are usually ER positive. Multiple studies indicate that 30% to 40% of sporadic breast cancers show absent or reduced expression of BRCA1 (50–52) and 46% of sporadic breast cancers show loss of one BRCA1 allele (i.e., these cancers may be haploinsufficient for BRCA1; ref. 53). Together, these findings suggest that BRCA1 regulation of ER{alpha} function may be important to both BRCA1 carriers and sporadic breast cancers, which are known to exhibit a high incidence of ER positivity.

Cyclin D1, which is overexpressed in human breast cancer, is both necessary and sufficient for mammary tumorigenesis in transgenic mice (20, 22). The BRCA1 protein is WT in the majority of human breast cancers, and inhibition of BRCA1 activity by cyclin D1 would be predicted to provide both a growth advantage imparted by cyclin D1 overexpression and an important activation through enhancement of liganded ER{alpha} activity. Cyclin D1 overexpression is a common early event during mammary tumorigenesis and the loss of ER{alpha} expression in BRCA1-associated tumors is likely to represent a relatively late event (14). Like cyclin D1 overexpression, BRCA1 mutations found in human breast cancer fail to repress liganded ER{alpha} activity (12). The prevailing view in cancer cell biology is that two general classes of cancer genes exist (54). The first class of genes regulates genomic stability and includes antimutators and DNA repair proteins. The second class of genes is cell cycle control genes involved in cellular proliferation and tumor growth, including cyclins, cdk inhibitors, and pRb. BRCA1 plays an essential role in the maintenance of genomic integrity during DNA damage repair (6, 55) functioning as a sensor of DNA damage by physically interacting with and thereby serving to recruit components of the DNA repair apparatus. The finding in the current studies that the product of the cell cycle control gene cyclin D1 physically interacts with BRCA1 and antagonizes BRCA1 repression function provides evidence that BRCA1 functions as a tumor suppressor by physically interacting with both classes of cancer-promoting genes.


   Acknowledgments
 
Grant support: Susan Komen Breast Cancer Foundation, BCTR0504227 (C. Wang) Breast Cancer Alliance, Inc., grants R01CA70896, RO1CA75503, RO1CA86072, and RO1CA86071 (R.G. Pestell), RO3AG20337 (C. Albanese), RO1CA18119 (B.S. Katzenellenbogen), RO1ES109169, RO1CA82599, and RO1CA80000; and U.S. Army Breast Cancer Research Program grant DAMD17-99-1-89254 (E.M. Rosen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. P. Chambon for the ER{alpha} constructs.


   Footnotes
 
Note: C. Wang and S. Fan contributed equally to the work.

Received 2/14/05. Accepted 5/13/05.


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 Materials and Methods
 Results
 Discussion
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