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

Abstract

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 that a E2-box sequence by is for the of the silencer. immunoprecipitation that the E2-box may the silencing by recruiting the suppressor protein SLUG, which and used histone C-terminal-binding human mammary epithelial small interfering silencer-binding trichostatin A. to result in of the at the BRCA2 gene the inhibition of BRCA2 gene expression. used a of of human breast cells including through and cells, the cells, were from cells were in from under cells were and in and as (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, Chaudhuri G. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). or non-dividing cells are as the cells that are in a of growth in a or the cells that are at the by of the cells to at the was for (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 of non-dividing cells was by L.A. Scholar). of the cells used as non-dividing cells were at the cells are the cells from of cell and BRCA2 promoter to was with the The was The with the the with to the RNA promoter was The was of the with and the site at the site of This the BRCA2 promoter The 221-bp silencer DNA to was the and The with in the with to RNA promoter was The silencer sequence was of the with and at the of the This the were in with of or and of were from the cells, and were as 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). activity was with to activity and as a of the were and from was used for the of gene with E2-box the The E2-box was from to are to the of the E2-box Cancer Res. 2002; Google Scholar). DNA of purified the silencer-binding proteins by DNA the by 221-bp BRCA2 silencer The silencer was by The proteins in BT-549 or cells were with and the nuclear was L.A. Scholar). The silencer DNA was to the The nuclear protein was for at with silencer DNA to in protein binding and The were with protein binding in containing and the The were with and were at proteins were a The was and to at with of SLUG in MDA-MB-468 SLUG sequence was from RNA from BT-549 cells and the The was with and was at the at the site of cells were with the or the to the cells were to in and with of DNA The was at and was at and through a the was in and at MDA-MB-468 cells were to in and by containing by the of and growth The was with cell growth containing was to cell at This cell was further by containing as above, and the cell was further with the this cell was in of and of SLUG was by of SLUG in BT-549 SLUG were the site and from The of the and its are as number the of the sequence in the SLUG We used and for the of the BT-549 cells with the RNA was by and RNA was from cells and for specific by analysis Chaudhuri G. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). was used as a were with and with The was used to of BRCA2 and SLUG and and and of BRCA2 and SLUG were a assay as Chaudhuri G. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). was used as an were in for and were were expressed as the analysis of a analysis of and were used to the of the D. Scholar). and protein levels in cell were by We the sequence of human SLUG and in expressed the SLUG in and purified the protein the was and from cell were by and to as Chaudhuri G. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). were with anti-SLUG antibody or antibody and with were the DNA containing the 221-bp silencer was with and used in 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). DNA were purified from the a with cell from breast cells, and as 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, J. Scholar). Supershift assay anti-SLUG in was also J. Scholar). of SLUG to SLUG from the purified protein was Chromatin the in binding of SLUG, and to the silencer or the of at the BRCA2 promoter during the silencing immunoprecipitation assays the by with assay Cell. Biol. 1999; PubMed Scopus Google Scholar, Biochem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). used anti-SLUG antibody in as were from number number histone number and histone number was with of and DNA to the bp of the silencer or to the BRCA2 gene The was and to to the of BRCA2 in and at 221-bp silencer sequence at the upstream to of the human BRCA2 gene has several It has and of bp by and This sequence an E2-box sequence as well as a the E2-box that has identity with the of a RNA regulator A. N. A. R. J. G. N. J. Biochem. 1999; PubMed Scopus Google Scholar) and The identity at the We previously that the 221-bp are essential for the 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 that this silencer is not with such as 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), also with its BRCA2 gene promoter This of the silencer is not in a transient assay also in BT-549 cells not of the of this silencer is that is in breast cancer cells and in the non-dividing stages of the cell Thus, the activity of the silencer is in or BT-549 cells in the dividing cells The cell cycle stage-dependent activity of the silencer may be BRCA2 mRNA and protein are expressed in the dividing cells with levels in the 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 in the non-dividing cells not suggesting that the activity of the silencer may not be specific for cancer cells The E2-box in the for Since the E2-box is involved in many whether the E2-box sequence in the BRCA2 silencer is for its function. the E2-box sequence was from to the of the silencer is in the cells is and The silencer is not in the MCF-7 and the MDA-MB-468 cells. Thus, are in the activity of the promoter whether or not or silencer is the the silencer is in the BT-549 and cells of the E2-box of the silencer its activity in cells. The of the E2-box for the silencer was Thus, of of the elements from the silencer with sequence of similar the of the silencer the E2-box not We previously reported that the silencer DNA with the nuclear proteins from 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). were not with nuclear proteins from the breast cells the silencer is sequence containing the E2-box the binding of the protein to the silencer DNA that containing the E2-box 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). that the E2-box sequence its in the BRCA2 silencer is for the binding of proteins to and thus for its function. We also the of the E2-box in BT-549 cells with the or the not with SLUG from the E2-box of the silencer is essential for the binding of the nuclear proteins as well as for the of the was that the E2-box-binding proteins regulate the of the silencer. We that the silencer to in the human breast cancer cells the E2-box binding repressor protein SLUG is not has been reported that SLUG are levels in the MCF-7 and MDA-MB-468 cells, the BT-549 and cells express of SLUG mRNA Cancer Res. 2002; Google Scholar). further We with the nuclear extracts from human breast cells to whether to SLUG. experiments show the of a 29-kDa protein from the nuclear extracts of the cells the silencer is BT-549 and not such protein from MDA-MB-468 cells the silencer is not analysis with anti-SLUG revealed that the affinity-purified protein SLUG assay with the purified SBP that the protein to the silencer DNA not the silencer DNA The of the was with silencer was a supershift of the with anti-SLUG antibody the of SLUG in the The of the with the of in the whether the of the 221-bp silencer sequence to in the of MDA-MB-468 cells is due to the absence of SLUG, SLUG from a promoter and whether the silencer in the of the analysis of the SLUG mRNA levels in the and cells that is a in the mRNA The of SLUG mRNA is also increased of the of the analysis with the nuclear proteins from the or MDA-MB-468 cells also the of the SLUG protein in the cells The of SLUG expression was also by analysis the also revealed that the of SLUG mRNA in the non-dividing MDA-MB-468 cells is with the in the BRCA2 mRNA levels Although the proteins of the nuclear from the MDA-MB-468 cells do not bind to the silencer DNA in was a of the silencer with the proteins of nuclear from the MDA-MB-468 cells The that is with the nuclear from MDA-MB-468 cells SLUG, as was revealed by supershift assay anti-SLUG not We thus that the expression of SLUG protein in the MDA-MB-468 cells. that the inhibition of BRCA2 mRNA in the MDA-MB-468 cells is due to the activation of the BRCA2 silencer in cells. Although is in the activity of the BRCA2 gene promoter in the or cells, its activity was reduced in the of the silencer sequence was in the not the the silencer sequence of SLUG in BT-549 cells increased silencer activity several and in the expression of BRCA2 as revealed by analysis not To further the between SLUG gene expression and the of BRCA2 silencer in human breast cells, SLUG expression in the BT-549 cells SLUG analysis of the SLUG mRNA levels in the or SLUG BT-549 cells a in the SLUG mRNA in the The in the SLUG protein in the SLUG cells was from the analysis of the BRCA2 silencer sequence in SLUG non-dividing BT-549 cells a in the of the silencer activity and of BRCA2 mRNA in the BT-549 cells the that the transcription SLUG expression of the human BRCA2 gene this identified silencer sequence at –701 to –921 upstream of the BRCA2 gene start SLUG and at the is to repress the expression of a gene by binding to the E2-box and recruiting the protein and the histone K. 2000; PubMed Scopus Google Scholar, Nat. Rev. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar). To whether SLUG is at the BRCA2 gene silencer the cell a immunoprecipitation This analysis revealed that not SLUG also the and the histone are at the silencer in and BT-549 cells, to the silencing and are not in the in MDA-MB-468 cells SLUG gene expression The of the of at the promoter is the of the the DNA PubMed Scopus Google Scholar, J.R. A. Marks R. 1999; PubMed Scopus Google Scholar). We whether the of SLUG expression in the MDA-MB-468 cells the of at the BRCA2 assay that is at the silencer and may be involved in the of at the BRCA2 promoter We further the involvement of HDAC in the histone at the BRCA2 promoter of the MDA-MB-468 cells the general HDAC trichostatin is a specific of PubMed Scopus Google Scholar, J.R. A. Marks R. 1999; PubMed Scopus Google Scholar). It is to transcription through and thus in cause increased in the promoter The assay the of at the BRCA2 gene promoter with or with is in Fig. the absence of SLUG protein was and the promoter is to and The histone not with the of the the of SLUG expression was with the of and the of the histone of the BRCA2 further suggesting the involvement of HDAC in the We also assays with the MDA-MB-468 cells with the which has the silencer sequence of the silencer As found of the silencer DNA from the SLUG antibody SLUG was by not Although the E2-box was in the are of the silencer in the cell that the assay To whether the silencer the and the of of suggesting that the silencer not bind to SLUG in the of the from the from DNA the silencer as To whether HDAC activity is involved in the of the silencer was to the silencer assays inhibited the of BRCA2 promoter activity by the silencer in BT-549 and cells, was effect the promoter activity by the silencer sequence in of in MDA-MB-468 cells BRCA2 a of this protein in the of of the cells including cells. human mammary epithelial cells, the expression of BRCA2 protein appears to be regulated by a of and that are and of The study for mechanism for the regulation of BRCA2 gene expression. the of study of the transcription of the human BRCA2 an silencer that the of the BRCA2 gene promoter in the cells and the cells express the transcription SLUG. We to the of SLUG in BRCA2 gene expression SLUG bind to DNA that the E2-box sequence K. 2000; PubMed Scopus Google Scholar, Nat. Rev. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar). It is as of the of tumor cell Y. J. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Mammary Gland Biol. Neoplasia. 2004; 9: PubMed Scopus Google Scholar). of this of are in the of and as well as in the of epithelial K. 2000; PubMed Scopus Google Scholar, Nat. Rev. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar). of SLUG protein in epithelial cells to of expression of such as and Y. J. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Mammary Gland Biol. Neoplasia. 2004; 9: PubMed Scopus Google Scholar, M. Cell. Biol. 2004; PubMed Scopus Google Scholar). SLUG a highly region at the of the protein containing of the K. 2000; PubMed Scopus Google Scholar, Nat. Rev. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar). The interactions with The of SLUG the domain K. 2000; PubMed Scopus Google Scholar, Nat. Rev. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar) that may the of SLUG. It is that SLUG a which in and that to gene Y. J. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Mammary Gland Biol. Neoplasia. 2004; 9: PubMed Scopus Google Scholar). We previously reported activity of the BRCA2 gene silencer in human breast cell (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). of SLUG of cells revealed a between the SLUG gene expression in a cell and the activity of the silencer in that MCF-7 cells do not express SLUG, and the silencer is not in cells. a of MCF-7 cells, found activity of the silencer with 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). a of MCF-7 cells from revealed that the silencer is inactive in the MCF-7 cells. of the silencer in the MCF-7 cells may also this negative regulatory was not identified previously by the human BRCA2 gene promoter (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) as used the MCF-7 cells. of the of the human BRCA2 silencer is the of the for the (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). are by RNA and this transcription is regulated by many RNA transcription factors A. N. A. R. J. G. N. J. Biochem. 1999; PubMed Scopus Google Scholar). of factors is an protein A. N. A. R. J. G. N. J. Biochem. 1999; PubMed Scopus Google Scholar). Although the human BRCA2 silencer has the sequence Thus, this silencer may not be by RNA the the sequence the E2-box and the binding protein may bind to and the of the E2-box from by SLUG repressor protein. from the transcription may also bind to the sequence of the A. N. A. R. J. G. N. J. Biochem. 1999; PubMed Scopus Google Scholar, Google Scholar) and has the to to the It is that many silencer of several human were found to J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of cell and The by which are not the of the BRCA2 gene silencer is to be of the be the specific of SLUG at the E2-box by to be Although here that SLUG to the BRCA2 gene silencer to the silencing many proteins the for binding to the E2-box many proteins to the of basic transcription of the of and the of repressor proteins K. 2000; PubMed Scopus Google Scholar, Nat. Rev. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar, A. L.A. C. G. D. K. Cell. PubMed Scopus Google Scholar). thus the to bind the E2-box of the silencer and with SLUG. The of the SLUG for BRCA2 gene expression be that SLUG binding to the silencer E2-box of the BRCA2 gene in the growth of the breast cells in the of a silencing that the expression of the gene by histone at the promoter region Although do not at proteins such as or may the E2-box in the in the dividing cells, the binding of SLUG and BRCA2 gene expression. study not the of of proteins or HDAC in the silencing process. is also to histone to silencing Y. J. G. J.R. Y. Y. 2003; PubMed Scopus Google Scholar). We not that for the regulation of BRCA2 gene expression. of the of the silencing thus further of the silencing and the of of the BRCA2 gene at the of the cell is of the BRCA2 in the cell may to cell the of the BRCA2 gene the cell is dividing to appropriate of cell growth The regulatory of BRCA2 thus including SLUG expression and SLUG are for the of its expression. breast cancer cells that do not express SLUG may to regulate the expression of BRCA2 gene at the non-dividing expression of BRCA2 be in of the of the in the of SLUG at the E2-box of the silencer may a mechanism of BRCA2 gene expression regulation at the and to understand the of of the sporadic breast and cancer We and J. for the during the of this study and for the