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Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês

10.1074/jbc.m501375200

1083-351X

Manish Kumar Tripathi, Smita Misra, Sheetal V. Khedkar, Nalo Hamilton, Charletha V. Irvin-Wilson, Chakradhari Sharan, Linda Sealy, Gautam Chaudhuri,

DNA Repair Mechanisms

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the –701 to –921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression. The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the –701 to –921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression. BRCA2 is a tumor suppressor protein with diverse functions (1Turner N. Tutt A. Ashworth A. Nat. Rev. Cancer. 2004; 4: 814-819Crossref PubMed Scopus (1359) Google Scholar, 2Scully R. Xie A. J. Mammary Gland Biol. Neoplasia. 2004; 9: 237-246Crossref PubMed Scopus (12) Google Scholar, 3Keen J.C. Davidson N.E. Cancer. 2003; 97: 825-833Crossref PubMed Scopus (163) Google Scholar). BRCA2 deficiency has been attributed as the cause for many cases of breast, ovarian, and pancreatic carcinoma (1Turner N. Tutt A. Ashworth A. Nat. Rev. Cancer. 2004; 4: 814-819Crossref PubMed Scopus (1359) Google Scholar, 2Scully R. Xie A. J. Mammary Gland Biol. Neoplasia. 2004; 9: 237-246Crossref PubMed Scopus (12) Google Scholar, 3Keen J.C. Davidson N.E. Cancer. 2003; 97: 825-833Crossref PubMed Scopus (163) Google Scholar). BRCA2 deficiency may be caused by the molecular defects in the BRCA2 gene or due to sporadic reasons. The BRCA2 gene is not expressed in non-dividing cells, and the rate of expression of this protein is increased with the rate of cell proliferation (4Chodosh L.A. J. Mammary Gland Biol. Neoplasia. 1998; 3: 389-402Crossref PubMed Scopus (30) Google Scholar, 5Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar, 6Narod S.A. Foulkes W.D. Nat. Rev. Cancer. 2004; 4: 665-676Crossref PubMed Scopus (740) Google Scholar). This growth-dependent turn-on/turn-off mechanism of this gene is not well understood. Any number of environmental cues may influence the turn-on event and may initiate a molecular domino effect leading to DNA damage and oncogenesis. Although undue expression of BRCA2 protein in non-dividing cells may initiate apoptosis, the failure of the cell to produce the appropriate amount of BRCA2 corresponding to the growth rate of the cell may also be detrimental. Since the majority of the breast cancer cases are sporadic (1Turner N. Tutt A. Ashworth A. Nat. Rev. Cancer. 2004; 4: 814-819Crossref PubMed Scopus (1359) Google Scholar, 2Scully R. Xie A. J. Mammary Gland Biol. Neoplasia. 2004; 9: 237-246Crossref PubMed Scopus (12) Google Scholar, 3Keen J.C. Davidson N.E. Cancer. 2003; 97: 825-833Crossref PubMed Scopus (163) Google Scholar, 4Chodosh L.A. J. Mammary Gland Biol. Neoplasia. 1998; 3: 389-402Crossref PubMed Scopus (30) Google Scholar, 5Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar, 6Narod S.A. Foulkes W.D. Nat. Rev. Cancer. 2004; 4: 665-676Crossref PubMed Scopus (740) Google Scholar), such cancers may be initiated as a result of the transient absence of BRCA2 during the proliferative stages of the breast cells. As alluded to above, the BRCA2 gene is stringently regulated during the cell cycle (7Bertwistle D. Swift S. Marston N.J. Jackson L.E. Crossland S. Crompton M.R. Marshall C.J. Ashworth A. Cancer Res. 1997; 57: 5485-5488PubMed Google Scholar, 8Su L.K. Wang S.C. Qi Y. Luo W. Hung M.C. Lin S.H. Oncogene. 1998; 17: 2377-2381Crossref PubMed Scopus (20) Google Scholar, 9Davis P.L. Miron A. Andersen L.M. Iglehart J.D. Marks J.R. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar, 10Wu K. Jiang S.W. Thangaraju M. Wu G. Couch F.J. J. Biol. Chem. 2000; 275: 35548-35556Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Wu K. Jiang S.W. Couch F.J. J. Biol. Chem. 2003; 278: 15652-15660Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). To understand how BRCA2 gene expression is regulated in human mammary epithelial cells, the regulatory DNA sequence elements around the proximal upstream region of the gene are under extensive study (8Su L.K. Wang S.C. Qi Y. Luo W. Hung M.C. Lin S.H. Oncogene. 1998; 17: 2377-2381Crossref PubMed Scopus (20) Google Scholar, 9Davis P.L. Miron A. Andersen L.M. Iglehart J.D. Marks J.R. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar, 10Wu K. Jiang S.W. Thangaraju M. Wu G. Couch F.J. J. Biol. Chem. 2000; 275: 35548-35556Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Wu K. Jiang S.W. Couch F.J. J. Biol. Chem. 2003; 278: 15652-15660Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar). Examination of the minimal promoter sequence has revealed several canonical elements for the binding of transcription factors including an E-box, E2F, and Ets recognition motifs (9Davis P.L. Miron A. Andersen L.M. Iglehart J.D. Marks J.R. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar). Antibodies to candidate transcription factors used in supershift experiments revealed specific interactions between the BRCA2 promoter and the basic region/helix-loop-helix containing USF-1 and -2 proteins and Elf-1, an Ets domain protein. Myc-Max or Max-Max dimers were reported not to bind this E-box sequence (9Davis P.L. Miron A. Andersen L.M. Iglehart J.D. Marks J.R. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar). Analysis of the –144 to –59 region identified a putative NFκB binding site (10Wu K. Jiang S.W. Thangaraju M. Wu G. Couch F.J. J. Biol. Chem. 2000; 275: 35548-35556Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Thus, NFκB and upstream stimulatory facter regulate BRCA2 expression through the BRCA2 promoter (10Wu K. Jiang S.W. Thangaraju M. Wu G. Couch F.J. J. Biol. Chem. 2000; 275: 35548-35556Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). P53 was found to repress the expression of BRCA2 promoter activity (11Wu K. Jiang S.W. Couch F.J. J. Biol. Chem. 2003; 278: 15652-15660Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We previously reported a 221-bp silencer sequence located at 700 bp upstream of the BRCA2 gene transcription start site (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar) (see Fig. 1A). We report here a mechanism for the negative regulation of the BRCA2 gene expression by this silencer. We show evidence that a unique E2-box sequence surrounded by Alu sequences is responsible for the function of the silencer. Our chromatin immunoprecipitation data suggest that the E2-box may mediate the silencing by recruiting the zinc finger suppressor protein SLUG, which most likely then recruits CtBP-1 and HDAC-1 1The abbreviations used are: HDAC-1, histone deacetylase-1; CtBP-1, C-terminal-binding protein-1; ACR1, Alu-co-repressor 1; HMEC, human mammary epithelial cells; siRNA, small interfering RNA; Dox, doxycycline; RT, reverse transcriptase; SBP, silencer-binding protein; ChIP, chromatin immunoprecipitation; TSA, trichostatin A. to result in deacetylation of the acetylated histones at the BRCA2 gene promoter. Histone deacetylation then probably causes the inhibition of BRCA2 gene expression. Cell Culture—We used a series of commercially available lines of human breast cells including HMEC (Clonetics, purchased through Fisher), MDA-MB-231, MCF-7, MDA-MB-468, and BT-549. All cells, other than the HMEC cells, were purchased from American Type Culture Collection (ATCC, Manassas, VA). HMEC cells were grown in medium purchased from Clonetics under their recommended conditions. Other cells were maintained and grown in ATCC recommended media and conditions, as described before (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar, 13Tripathi M.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 2005; 328: 43-48Crossref PubMed Scopus (18) Google Scholar). Resting or non-dividing cells are defined as the cells that are in a 100% confluent state of growth in a culture flask or the cells that are arrested mostly at the G0 phase by serum starvation. Serum starvation of the cells to arrest them at the G0 phase was done for 60–80 h following established protocols (9Davis P.L. Miron A. Andersen L.M. Iglehart J.D. Marks J.R. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar). The population of non-dividing cells was determined by flow cytometry using propidium iodide staining methods (14Spector D.L. Goldman R.D. Leinwand L.A. Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar). More than 88% of the cells used as non-dividing cells were at the G0/G1 phase. Dividing cells are the cells from S/G2 phase (>82%) of cell growth. Promoter/Silencer Constructs and Luciferase Assay—Human BRCA2 promoter (–187 to +301) was amplified with the primer set PF1, 5′-GCGAGAAGAGAACACACA-3′/PR1, 5′-GCAGAGAAAAGGCAA-3′. The 497-bp fragment was initially cloned into pCRII-Topo vector (Invitrogen). The recombinant plasmid with the insert having the forward orientation with respect to the T7 RNA polymerase promoter was selected. The insert was cut out of the plasmid with BamHI/XhoI and subcloned into the BglII/XhoI site at the multiple cloning site of pGL3-Basic plasmid (Promega). This gave us the BRCA2 promoter vector, pGL3-P. The 221-bp silencer DNA (–701 to –921) was amplified using the primer set SF1, 5′-GCAAGATGGGCCGGGTGT-3′/SR1, 5′-CCA GGGTGTGGTTCTC-3′ and initially cloned into pCRII-Topo vector. The recombinant plasmid clone with insert in the reverse orientation with respect to T7 RNA polymerase promoter was selected. The silencer sequence was digested out of the recombinant plasmid with KpnI/XhoI and subcloned at the KpnI/XhoI sites of the pGL3-P vector. This gave us the promoter-silencer vector, pGL3-PS. Transient transfections were performed in six-well plates using Lipofectamine 2000 transfection reagent (Invitrogen) with 1 μg of pGL3-P or pGL3-PS and 0.1 μg of pRL-TK-Renilla luciferase control vector (Promega). Protein lysates were prepared from the cells, and luciferase activities were measured as described previously (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar). Firefly luciferase activity was normalized with respect to Renilla luciferase activity and presented as a ratio (relative light units). Protein contents of the extract, when needed, were determined using RC-DC reagents and protocol from Bio-Rad. Site-directed Mutagenesis—PCR-based site-directed mutagenesis (the QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA) technique was used for the generation of reporter gene constructs with E2-box mutations following the manufacturer's instructions. The E2-box element was mutated from 5′-CACCTG-3′ to 5′-AACCTA-3′ (sense strand). Such mutations are known to ablate the function of the E2-box (15Hajra K.M. Chen D.Y. Fearon E.R. Cancer Res. 2002; 62: 1613-1618PubMed Google Scholar). DNA Affinity Pull-down of Silencer-binding Proteins—We purified the silencer-binding proteins by DNA affinity chromatography following the protocol provided by Dynal Biotech, using biotin-tagged 221-bp BRCA2 silencer fragment employing Dynabeads M-280-streptavidin beads (Dynal Biotech). The biotin-labeled silencer fragment was obtained by PCR using 5′-biotin-labeled primers. The proteins in BT-549 or MDA-MB-231 cells were biosynthetically labeled with [35S]methionine, and the nuclear extract was isolated following standard protocols (14Spector D.L. Goldman R.D. Leinwand L.A. Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar). The biotin-tagged silencer DNA was bound to Dynabeads M280 following the manufacturer's instructions (Dynal Biotech). The nuclear protein was incubated for 1 h at 25 °C with biotinylated silencer DNA bound to Dynabeads M280 streptavidin in protein binding buffer (80 mm NaCl, 50 mm Tris-HCL, pH 7.5, 4% (v/v) glycerol, 5 mm MgCl2, 0.25 mg/ml poly(dI-dC), 2.5 mm EDTA, 30 μm mutant double-strand oligonucleotide, 1 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol). The magnetic beads were washed three times with protein binding buffer in 80 mm NaCl containing excess poly(dI-dC) and mutant (at the E2-box) duplex oligonucleotide competitor DNA. The fractions were eluted with elution buffer (0.5 m NaCl, 50 mm Tris-HCL, pH 7.5, 4%(v/v) glycerol, 5 mm MgCl2, 0.25 mg/ml poly(dI-dC), 2.5 mm EDTA, 30 μm mutant double-strand oligonucleotide, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol) and were stored at –80 °C. Silencer-binding proteins (SBPs) were electrophoresed on a 10% Tris-glycine SDS-polyacrylamide gel. The gel was fixed, dried, and exposed to x-ray film at –80 °C with intensifying screen. Expression of Recombinant SLUG in MDA-MB-468 Cells—Human SLUG coding sequence was amplified from RNA isolated from BT-549 cells using N-terminal primer (5′-GACGGATCCATGCCGCGCTCCTTCCTG-3′) and the C-terminal primer (5′-CGTCGACTCACTTATCGTCGTCATCCTTGTAATCGTGTGCTACACAGC-3′). The sequence-verified amplified cDNA (831 bp) was digested with BamHI/SalI and was cloned at the BamHI/SalI sites at the multiple cloning site of pRevTet-TRE plasmid (BD Biosciences). PT67 packaging cells were transfected with either the pRevTet-ON plasmid (BD Biosciences) or the pRevTet-TRE-SLUG plasmid to collect the recombinant retrovirus. PT67 cells were grown to ∼50% confluency in 100-mm culture dishes and then transfected with 4 μg of plasmid DNA using Lipofectamine 2000 (Invitrogen). The culture medium was replaced at 24 h after transfection, and retrovirus-containing medium was collected at 48 h after transfection and filtered through a 0.45-μm filter. For long term storage, the retrovirus-containing medium was frozen in liquid nitrogen and stored at –80 °C. MDA-MB-468 cells were grown to ∼40–50% confluency in 100-mm culture dishes and then transduced first by retrovirus containing pRevTet-ON plasmid by the addition of 1:1 retrovirus-containing medium and growth medium. The culture medium was replaced after 24 h with cell growth medium. Medium containing 1 mg/ml Geneticin was added to select stable cell population at 48 h. This Geneticin-resistant cell population was further transduced by pRe-vTRE-SLUG plasmid containing virus as described above, and the stable cell line was selected further with 500 μg/ml hygromycin B. For all the experiments, this double-resistant cell line was maintained in 100 μg/ml of Geneticin and hygromycin B. Expression of SLUG was induced by doxycycline (1 μg/ml). Knock-down of SLUG Gene Expression in BT-549 Cells—The anti-human SLUG siRNAs were designed using the Invitrogen site software and custom-synthesized from Invitrogen. The nucleotide sequences of the siRNA pair (SR) and its respective control (SRC) are as follows (the number indicates the location of the sequence in the SLUG coding region): SR661 5′-GCAUUUGC AGACAGGUCAA-3′/5′-UUGAACUGUCUGCA AAUGC-3′; SRC661: 5′-GCAACGUACAGUG GUUCAA-3′/5′-UUGAACCACUGUACGUUGC-3′. We used Trans-messenger reagent kit (Qiagen) and protocol for the transfection of the BT-549 cells with the siRNAs. RNA was prepared by TRIZOL reagent and DNase I treated before RT-PCR analysis. End Point RT-PCR Analysis—Total RNA was isolated from cells and analyzed for specific transcripts by RT-PCR analysis (13Tripathi M.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 2005; 328: 43-48Crossref PubMed Scopus (18) Google Scholar). β-Actin was used as a loading control. Total RNAs (5 μg) were treated with DNase I (Promega) and reverse-transcribed with Superscript II (Invitrogen). The resulting cDNA was used to carry out PCR amplification of BRCA2 (225 bp; 5′-GTACAGGAAACAAGCTTCTGA-3′ and 5′-GACTAACAGGTGGAGGTAAAG-3′); SLUG (807 bp; 5′-CCTGGTCAAGAAGCATTTCAACG-3′ and 5′-CCCCAAAGATGAGGAGTATCCG-3′); and β-actin (353 bp; 5′-GCTCGTCGTCGACAACGGCTC-3′ and 5′-CAAACATGATCTGGGTCATCTTCTC-3′). Real-time RT-PCR Analysis—Quantitation of BRCA2 and SLUG transcripts were performed using a TaqMan assay (Applied Biosystems) as described before (13Tripathi M.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 2005; 328: 43-48Crossref PubMed Scopus (18) Google Scholar). 18 S rRNA was used as an endogenous control (product 4319413E). All PCR reactions were performed in triplicate for each sample and were repeated three times. All experimental data were expressed as the mean ± S.E. A one-way analysis of variance, a two-way repeated measure analysis of variance, and Student's t test were used to determine the significance of the difference (16Campbell M.J. Machin D. Medical Statistics: A Commonsense Approach. Second Ed. John Wiley & Sons, Inc., New York1994Google Scholar). Western Blotting—SLUG and actin protein levels in cell lysates were analyzed by immunoblotting. We amplified the coding sequence of human SLUG and cloned in pET100D/Topo vector (Invitrogen), expressed the His6-tagged SLUG in Escherichia coli (DE3), and purified the recombinant protein using nickel-nitrilotriacetic acid agarose following the supplier's instructions (Qiagen). Anti-SLUG antiserum was custom-developed (Antibody Solutions, Mountain View, CA) and purified. Proteins from cell lysate were analyzed by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane, as described before (13Tripathi M.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 2005; 328: 43-48Crossref PubMed Scopus (18) Google Scholar). Membranes were blotted with anti-SLUG antibody or anti-actin antibody (Sigma) and then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Signals were visualized using the SuperSignal chemiluminescent detection system (Pierce). Electerophoretic Mobility Shift Assay—Double-stranded DNA containing the 221-bp silencer fragment was labeled with [γ-32P]ATP and used in electrophoretic mobility shift assay (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar). Double-stranded DNA probes were purified from the reaction mixture using a Bio-Gel P-100 column (Bio-Rad), incubated with whole cell extract from breast cells, and separated on 5% polyacrylamide gels as described previously (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar, 17Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Third Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Supershift assay using anti-SLUG antibodies (made in our laboratory) was also performed (17Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Third Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Immuno-pull-down of SLUG to remove SLUG from the purified protein preparations was done using Dynabead-Protein A following supplier-provided protocol (Dynal Biotech, Brown Deer, WI). Chromatin Immunoprecipitation Assays—To test the in vivo binding of SLUG, CtBP-1, and HDAC-1 to the silencer or the deacetylation of histones at the BRCA2 promoter during the silencing process, we performed chromatin immunoprecipitation assays (ChIP) using the standard protocol supplied by Upstate Biotechnology with their ChIP assay reagents (18Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8393-8399Crossref PubMed Scopus (147) Google Scholar, 19Orlando V. Trends Biochem. Sci. 2000; 25: 99-104Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). For immunoprecipitation, we used anti-SLUG antibody developed in our laboratory as described above. Following this, other antibodies were procured from Upstate Biotechnology: anti-CtBP-1 (rabbit polyclonal IgG, catalog number 07-306); anti-HDAC-1, clone 2E10 (mouse monoclonal IgG, catalog number 05-614); anti-acetyl histone H3 (rabbit polyclonal, catalog number 06-599); and anti-acetyl histone H4 (rabbit polyclonal, catalog number 06-866). PCR was performed with 10 μl of DNA, and Taq DNA polymerase, using 5′-32P-labeled primers SF1/SR1 to amplify the 221 bp of the silencer or PF1/PR1 to amplify the 497-bp BRCA2 gene promoter. The gel was dried and exposed to x-ray film to visualize the amplified band of expected size. Human BRCA2 Gene Silencer Is Differentially Active in Various Breast Cells and at Different Cell Growth Stages—The 221-bp silencer sequence at the upstream (–701 to –921) of the human BRCA2 gene has several interesting structural features. It has two Alu sequences (AluI and AluII) each of 55 bp separated by 81-bp inter-Alu sequences (Fig. 1, A and B). This inter-Alu sequence houses an E2-box sequence (5′-CACCTG-3′) as well as a 63-bp patch overlapping the E2-box that has >83% identity with the footprint of a RNA polymerase III transcriptional regulator ACR1 (20Kropotov A. Sedova V. Ivanov V. Sazeeva N. Tomilin A. Krutilina R. Oei S.L. Griesenbeck J. Buchlow G. Tomilin N. Eur. J. Biochem. 1999; 260: 336-346Crossref PubMed Scopus (56) Google Scholar) (Fig. 1, A and B). The Alu sequences have ∼82% identity at the nucleotide level (Fig. 1C). We have shown previously that the entire 221-bp sequences are essential for the function of the silencer (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar). We further verified that this silencer is not only active with heterologous promoter, such as SV40 promoter (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar), but also active with its endogenous BRCA2 gene promoter (Fig. 1D). This functionality of the silencer is not only demonstrated in a transient transfection assay but also in stably transfected BT-549 cells (data not shown). One of the interesting features of this silencer is that it is active in certain breast cancer cells (Fig. 1D) and only in the non-dividing stages (G0/G1 phases) of the cell (Fig. 1E). Thus, the activity of the silencer is 8–10-fold higher in resting or predominantly G0-arrested BT-549 cells than in the dividing cells (Fig. 1E). The cell cycle stage-dependent activity of the silencer may be relevant because BRCA2 mRNA and protein are expressed significantly only in the dividing cells with optimum levels in the S/G2 phase cells (9Davis P.L. Miron A. Andersen L.M. Iglehart J.D. Marks J.R. Oncogene. 1999; 18: 6000-6012Crossref PubMed Scopus (50) Google Scholar, 10Wu K. Jiang S.W. Thangaraju M. Wu G. Couch F.J. J. Biol. Chem. 2000; 275: 35548-35556Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The silencer is also active in the non-dividing HMEC cells (data not shown), suggesting that the activity of the silencer may not be specific for certain cancer cells only. The E2-box in the Silencer Is Essential for Its Function— Since the E2-box is involved in many transcriptional repression processes, we checked whether the E2-box sequence in the BRCA2 silencer is necessary for its function. When the E2-box sequence was mutated from 5′-CACCTG-3′ to 5′-AACCTA-3′, the function of the silencer is abolished in the cells where it is functional (Fig. 1, D and E). The silencer is not active in the MCF-7 and the MDA-MB-468 cells. Thus, there are no significant differences in the activity of the promoter whether or not wild-type or mutated silencer is present (Fig. 1D). On the other hand, the silencer is significantly active in the BT-549 and MDA-MB-231 cells (Fig. 1D). Mutation of the E2-box of the silencer ablated its activity (Fig. 1D) in these cells. The essentiality of the E2-box for the silencer function was context-dependent. Thus, replacement of any of the Alu elements from the silencer with unrelated sequence of similar size ablated the function of the silencer although the E2-box remained unaltered (data not shown). We previously reported that the silencer DNA forms protein-DNA complexes with the nuclear proteins from MDA-MB-231 cells (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar). These complexes were not formed with nuclear proteins from the breast cells where the silencer is inactive. Oligonucleotide sequence containing the wild-type E2-box could successfully compete the binding of the protein to the silencer DNA probe, but that containing the mutated E2-box could not (12Sharan C. Hamilton N. Parl A.K. Singh P.K. Chaudhuri G. Biochem. Biophys. Res. Commun. 1999; 265: 285-290Crossref PubMed Scopus (30) Google Scholar). All these data strongly suggest that the E2-box sequence within its context in the BRCA2 silencer is necessary for the binding of proteins to it and thus for its function. We also have verified the essentiality of the E2-box in BT-549 cells stably transfected with the wild type or the mutated silencer-containing luciferase constructs (data not shown). Silencer Binds with SLUG from Breast Cell Nuclear Extract—Since the E2-box of the silencer is essential for the binding of the nuclear proteins as well as for the function of the silencer, our hypothesis was that the E2-box-binding proteins regulate the function of the silencer. We had noted that the silencer failed to function in the human breast cancer cells where the E2-box binding repressor protein SLUG is not expressed. For example, it has been reported that SLUG mRNAs are below detection levels in the MCF-7 and MDA-MB-468 cells, whereas the BT-549 and MDA-MB-231 cells express plenty of SLUG mRNA (15Hajra K.M. Chen D.Y. Fearon E.R. Cancer Res. 2002; 62: 1613-1618PubMed Google Scholar). Our RT-PCR data further verified these observations (Fig. 2A). We experimented with the nuclear extracts from different human breast cells to verify whether it indeed binds to SLUG. These pull-down experiments show the purification of a 29-kDa protein from the nuclear extracts of the cells where the silencer is functional, e.g. BT-549 and MDA-MB-231, whereas it did not purify any such protein from MDA-MB-468 cells where the silencer is not active (Fig. 2B). Immunoblot analysis with anti-SLUG antibodies revealed that the affinity-purified protein band contains SLUG (Fig. 2C). An electrophoretic mobility shift assay with the purified SBP showed that the protein specifically binds to the wild-type silencer DNA but not the E2-box-mutated silencer DNA (Fig. 2D). The formation of the protein-DNA complex was competed out with unlabeled wild-type si