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• W2068065167 abstract "Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation assays and mammary-specific genes as models, we show here that extracellular matrix molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the β- and γ-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both β- and γ-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. Chromatin immunoprecipitation analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Co-immunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, CCAAT/enhancer-binding protein β, and glucocorticoid receptor. Thus, extracellular matrix- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors. Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation assays and mammary-specific genes as models, we show here that extracellular matrix molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the β- and γ-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both β- and γ-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. Chromatin immunoprecipitation analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Co-immunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, CCAAT/enhancer-binding protein β, and glucocorticoid receptor. Thus, extracellular matrix- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors. Differentiated function of mammary epithelial cells is regulated by signals from both ECM 2The abbreviations used are: ECM, extracellular matrix; ChIP, chromatin immunoprecipitation; co-IP, co-immunoprecipitation; GR, glucocorticoid receptor; TSA, trichostatin A; MEC, mammary epithelial cell; DN-Brg1, dominant negative Brg1; STAT, signal transducers and activators of transcription; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AcH4, acetylated histone H4; C/EBP, CCAAT/enhancer-binding protein; lrECM, laminin-rich ECM. and lactogenic hormones (1Streuli C.H. Schmidhauser C. Bailey N. Yurchenco P. Skubitz A.P. Roskelley C. Bissell M.J. J. Cell Biol. 1995; 129: 591-603Crossref PubMed Scopus (342) Google Scholar, 2Streuli C.H. Bailey N. Bissell M.J. J. Cell Biol. 1991; 115: 1383-1395Crossref PubMed Scopus (532) Google Scholar, 3Roskelley C.D. Desprez P.Y. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12378-12382Crossref PubMed Scopus (409) Google Scholar). The gene encoding the milk protein, β-casein, has been used widely as a marker for functional differentiation of MECs. We and others have shown that in both primary mouse mammary epithelial cells and immortalized mammary epithelial cell lines (4Brinkmann V. Foroutan H. Sachs M. Weidner K.M. Birchmeier W. J. Cell Biol. 1995; 131 (6 Pt 1): 1573-1586Crossref PubMed Scopus (296) Google Scholar, 5Reichmann E. Ball R. Groner B. Friis R.R. J. Cell Biol. 1989; 108: 1127-1138Crossref PubMed Scopus (141) Google Scholar, 6Schmidhauser C. Bissell M.J. Myers C.A. Casperson G.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9118-9122Crossref PubMed Scopus (185) Google Scholar), transcription of β-casein requires signals from both laminin-111 (previously referred to as laminin-1) and prolactin (1Streuli C.H. Schmidhauser C. Bailey N. Yurchenco P. Skubitz A.P. Roskelley C. Bissell M.J. J. Cell Biol. 1995; 129: 591-603Crossref PubMed Scopus (342) Google Scholar, 2Streuli C.H. Bailey N. Bissell M.J. J. Cell Biol. 1991; 115: 1383-1395Crossref PubMed Scopus (532) Google Scholar, 7Rosen J.M. Wyszomierski S.L. Hadsell D. Annu. Rev. Nutr. 1999; 19: 407-436Crossref PubMed Scopus (176) Google Scholar, 8Roskelley C.D. Srebrow A. Bissell M.J. Curr. Opin. Cell Biol. 1995; 7: 736-747Crossref PubMed Scopus (351) Google Scholar, 9Lee E.Y. Lee W.H. Kaetzel C.S. Parry G. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1419-1423Crossref PubMed Scopus (204) Google Scholar, 10Weir M.L. Oppizzi M.L. Henry M.D. Onishi A. Campbell K.P. Bissell M.J. Muschler J.L. J. Cell Sci. 2006; 119: 4047-4058Crossref PubMed Scopus (83) Google Scholar). A number of transcription factors, including STAT5, C/EBPβ, and GR, have been shown to be involved in this process (reviewed in Ref. 7Rosen J.M. Wyszomierski S.L. Hadsell D. Annu. Rev. Nutr. 1999; 19: 407-436Crossref PubMed Scopus (176) Google Scholar). Modulation of chromatin structure by histone modifications and ATP-dependent remodeling has been implicated in cell differentiation and transcriptional control of tissue-specific and inducible genes (11Hsiao P.W. Deroo B.J. Archer T.K. Biochem. Cell Biol. 2002; 80: 343-351Crossref PubMed Scopus (30) Google Scholar, 12Muller C. Leutz A. Curr. Opin. Genet. Dev. 2001; 11: 167-174Crossref PubMed Scopus (108) Google Scholar, 13Farkas G. Leibovitch B.A. Elgin S.C. Gene (Amst.). 2000; 253: 117-136Crossref PubMed Scopus (73) Google Scholar). Histone-modifying enzymes are believed to be recruited to promoter regions through their association with transcription factors and are critical for tissue-specific gene expression and functional differentiation of specific cell types (14de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar, 15Chan H.M. La Thangue N.B. J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar). Histone acetylation is a dynamic process and is regulated by histone acetyltransferases and histone deacetylases (16Sun J.M. Spencer V.A. Chen H.Y. Li L. Davie J.R. Methods. 2003; 31: 12-23Crossref PubMed Scopus (37) Google Scholar). The mapping of global histone acetylation patterns has demonstrated that chromatin accessibility and gene expression are correlated with histone hyperacetylation of promoters and other cis-elements (17Roh T.Y. Cuddapah S. Zhao K. Genes Dev. 2005; 19: 542-552Crossref PubMed Scopus (373) Google Scholar, 18Bernstein B.E. Humphrey E.L. Erlich R.L. Schneider R. Bouman P. Liu J.S. Kouzarides T. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8695-8700Crossref PubMed Scopus (601) Google Scholar). The p300 histone acetyltransferase cooperates with STAT5 to enhance exogenous β-casein promoter activity in COS cells, indicating that histone acetylation may play a role in β-casein transcription (19Pfitzner E. Jahne R. Wissler M. Stoecklin E. Groner B. Mol. Endocrinol. 1998; 12: 1582-1593Crossref PubMed Google Scholar). Trichostatin A (TSA), an inhibitor of histone deacetylase, was shown to activate the bovine casein ECM response element (BCE-1) in an ECM-independent fashion in a mouse epithelial cell line in tissue culture plastic, but surprisingly the same treatment inhibited the endogenous β-casein transcription (20Myers C.A. Schmidhauser C. Mellentin-Michelotti J. Fragoso G. Roskelley C.D. Casperson G. Mossi R. Pujuguet P. Hager G. Bissell M.J. Mol. Cell. Biol. 1998; 18: 2184-2195Crossref PubMed Scopus (91) Google Scholar, 21Pujuguet P. Radisky D. Levy D. Lacza C. Bissell M.J. J. Cell. Biochem. 2001; 83: 660-670Crossref PubMed Scopus (22) Google Scholar). Therefore, the role of histone acetylation in mammary-specific gene transcription has not been elucidated. ATP-dependent chromatin remodeling SWI/SNF complexes are involved in cellular differentiation and tissue-specific transcription (14de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar, 22Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar, 23Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Mammalian cells contain at least two SWI/SNF-like complexes that share a number of subunits but are distinguished from one another by their ATPase subunits, Brg1 and Brm1 (24Fry C.J. Peterson C.L. Curr. Biol. 2001; 11: R185-R197Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Introducing a dominant negative Brg1 (DN-Brg1) or Brm1 into mouse NIH-3T3 fibroblasts completely abrogated MyoD-mediated muscle differentiation, and failure to induce transcription of the muscle-specific myogenin gene was correlated with inhibition of chromatin remodeling in the promoter region (14de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar). To date, two different mechanisms have been described for recruiting SWI/SNF complexes to tissue-specific genes. Transcription factors, such as GR and C/EBPβ, have been shown in mammalian cells to recruit the SWI/SNF complex to cis-elements to activate specific gene transcription (23Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 25Muller W.G. Walker D. Hager G.L. McNally J.G. J. Cell Biol. 2001; 154: 33-48Crossref PubMed Scopus (145) Google Scholar, 26Muchardt C. Yaniv M. EMBO J. 1993; 12: 4279-4290Crossref PubMed Scopus (523) Google Scholar, 27Fryer C.J. Archer T.K. Nature. 1998; 393: 88-91Crossref PubMed Scopus (411) Google Scholar). Alternatively, the ATPase subunits of SWI/SNF contain bromodomains, which can bind directly to acetylated histone tails in vitro (28Martens J.A. Winston F. Curr. Opin. Genet. Dev. 2003; 13: 136-142Crossref PubMed Scopus (298) Google Scholar, 29Dhalluin C. Carlson J.E. Zeng L. He C. Aggarwal A.K. Zhou M.M. Nature. 1999; 399: 491-496Crossref PubMed Scopus (1320) Google Scholar). Thus, acetylated histones in a particular chromatin region may contribute to the recruitment of SWI/SNF complexes to specific genes. Using the β- and γ-casein genes as models, here we investigate how ECM and prolactin regulate the activity of STAT5 and C/EBPβ, and we elucidate the roles of histone acetylation and ATP-dependent chromatin remodeling in expression of these mammary-specific genes. The findings from this study indicate that the precise regulation of mammary-specific gene transcription depends not only on transcription factor activation and histone modifications but also on ATP-dependent chromatin remodeling. Reagents and Antibodies—Antibodies against acetylated H4 and acetylated H3 were from Upstate Biotechnology, Inc. The H3 antibody was from Abcam. The STAT5 antibody was from R & D Systems, and those against C/EBPβ, RNA polymerase II, GR, and Brg1 were from Santa Cruz Biotechnology. Anti-FLAG antibody (M2) was from Sigma. Protein A-agarose beads were obtained from Upstate Biotechnology, Inc. Phosphatase inhibitor mixture and protease inhibitor mixture were from Calbiochem. Cell Culture and Transfections—EpH4 cells were derived from IM-2 cells, originally isolated from the mammary tissue of a mid-pregnant mouse (4Brinkmann V. Foroutan H. Sachs M. Weidner K.M. Birchmeier W. J. Cell Biol. 1995; 131 (6 Pt 1): 1573-1586Crossref PubMed Scopus (296) Google Scholar, 5Reichmann E. Ball R. Groner B. Friis R.R. J. Cell Biol. 1989; 108: 1127-1138Crossref PubMed Scopus (141) Google Scholar). EpH4 cells were maintained in growth medium consisting of Dulbecco’s modified Eagle’s medium/F-12 (UCSF cell culture facility) supplemented with 2% fetal bovine serum (Invitrogen), 50 μg/ml gentamycin (UCSF cell culture facility), and 5 μg/ml insulin (Sigma). The cells were plated at a density of 10,000/cm2 in growth medium and allowed to attach for 16-24 h. The cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 5 μg/ml insulin and 1 μg/ml hydrocortisone (Sigma) (GIH medium). In other experiments, 3 μg/ml prolactin, 2% laminin-rich ECM (lrECM; Matrigel®, BD Biosciences), 3 μg/ml prolactin plus 2% lrECM (or 100 μg/ml laminin-111 (Trevigen)) were added to the GIH medium (1Streuli C.H. Schmidhauser C. Bailey N. Yurchenco P. Skubitz A.P. Roskelley C. Bissell M.J. J. Cell Biol. 1995; 129: 591-603Crossref PubMed Scopus (342) Google Scholar). EpH4 cells were seeded onto 35-mm dishes and transfected using Lipofectamine 2000 (Invitrogen) with the following plasmids: 3 μg of DN-Brg1 plasmid (14de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar) (a kind gift from Dr. Anthony N. Imbalzano, University of Massachusetts Medical School), 1 μg of pTet-tak, which encodes the tet-VP16 regulator, and 0.4 μg of pNeo plasmid. Twenty-four hours after transfection, 400 μg/ml geneticin (Sigma) was added to the media, which were changed every 48 h for 10 days. The resulting stably transfected clones were washed twice with growth medium and incubated in the presence or absence of 0.5 μg/ml tetracycline for 4 days. Positive clones expressing a 200-kDa FLAG tag protein in the absence, but not presence of, tetracycline were identified by Western blot analysis. Promoter Reporter Plasmid Construction and Luciferase Assays—A 340-bp DNA fragment containing the β-casein promoter region was amplified from mouse genomic DNA using the following primer sequences: forward primer, 5′-CGA GGT ACC TTC ATA ACT GAG GTT AAA GCC-3′; reverse primer, 5′-CAG AAG CTT GTC CTA TCA GAC TCT GTG AC-3′. PCR product was digested with HindIII and KpnI and subsequently cloned into a reporter vector pGL3 (Promega). EpH4 cells were co-transfected with pGL-casein and pNeo plasmid (1:10). Stably transfected cells were isolated by G418 selection and cultured in GIH medium for 2 days in the presence of different inducers. Following induction, equal amounts of cell lysates were assayed for luciferase activity. Western Blot and Co-immunoprecipitation (co-IP)—Western blot experiments were performed as previously described (30Muschler J. Lochter A. Roskelley C.D. Yurchenco P. Bissell M.J. Mol. Biol. Cell. 1999; 10: 2817-2828Crossref PubMed Scopus (100) Google Scholar). Total and nuclear protein were extracted from EpH4 cells or nuclei using radioimmunoprecipitation buffer (50 mm Tris, pH 7.4, 30 mm NaCl, 1% (v/v) Nonidet P-40, 1% (w/v) deoxycholate, 0.1% (w/v) SDS, protease inhibitor mixture, and phosphatase inhibitor mixture). After sonication, insoluble material was removed by centrifugation at 15,000 × g for 10 min. Proteins (20 μg) from each sample were subjected to SDS gel electrophoresis and then transferred to nitrocellulose membrane (Schleicher & Schuell). The membrane was subsequently blocked in TBST buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 0.1% (v/v) Tween 20) containing 5% Carnation nonfat dried milk and incubated in blocking buffer containing primary antibody. All of the blots were further incubated in blocking buffer containing horseradish peroxidase-conjugated secondary antibodies and subjected to ECL using the SuperSignal chemiluminescent substrate (Pierce). Cells transfected with DN-Brg1 were cultured in the presence or absence of tetracycline for 2 days in GIH medium containing prolactin and lrECM, and the nuclei were isolated using a nucleus isolation kit (Sigma). The nuclei were resuspended and sonicated in lysis buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 0.5% Triton X-100, protease inhibitor mixture) and centrifuged at 15,000 × g for 10 min. After centrifugation, 40 μl of agarose beads conjugated to an anti-FLAG M2 antibody were added to the supernatant of each sample and incubated with shaking at 4 °C for 4 h The agarose beads were washed with rinsing buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.05% Triton X-100). Agarose-associated protein complexes were eluted using SDS loading buffer and analyzed by Western blot. RT-PCR and Real Time PCR—Total RNA was extracted from cells with TRIzol reagent (Invitrogen). cDNA was synthesized using Superscript first strand synthesis kit (Invitrogen) from 1-μg RNA samples. One microliter of cDNA was used as a template for PCR and real time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified and used as a loading control. The following primers were used to amplify β-casein, γ-casein, lactoferrin, and GAPDH cDNA sequences: forward primer of the β-casein gene 5′-GCT CAG GCT CAA ACC ATC TC-3′ and reverse primer 5′-TGT GGA AGG AAG GGT GCT AC-3′; forward primer of the γ-casein gene: 5′-CCC AGG AGT CTT CCT TTT CC-3′ and reverse primer 5′-GGA AAC CAC GAA GAA ACC AA-3′; forward primer of the lactoferrin gene: 5′-AGT GAG GAG AAG CGC AAG TGT G-3′ and reverse primer 5′-AGC CCC AGT GTA GCC TTG GTA T-3′; and forward primer for GAPDH gene 5′CCC CTG GCC AAG GTC ATC CAT GAC-3′ and reverse primer 5′CAT ACC AGG AAA TGA GCT TGA CAA AG-3′. Quantitative real time PCR analysis was performed with the Lightcycler System (Roche Applied Science) using the Lightcycler FastStart DNA Master SYBR Green I kit (Roche Applied Science) (31Novaro V. Roskelley C.D. Bissell M.J. J. Cell Sci. 2003; 116: 2975-2986Crossref PubMed Scopus (78) Google Scholar). The following Lightcycler PCR amplification protocol was used: 95 °C for 10 min (initial denaturation) and 45 amplification cycles (95 °C for 5 s, 60 °C for 10 s, and 72 °C for 5 s). Amplification was followed by melting curve analysis to verify the presence of a single PCR product. Chromatin Immunoprecipitation (ChIP)—The ChIP assay was performed based on the Upstate Biotechnology, Inc. ChIP protocol (32Nelson E.A. Walker S.R. Alvarez J.V. Frank D.A. J. Biol. Chem. 2004; 279: 54724-54730Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) with a few modifications. Cellular components were cross-linked by adding formaldehyde to a final concentration of 1% and incubated at room temperature for 10 min. The cross-linking reaction was stopped by adding glycine to a final concentration of 125 mm. The nuclei were isolated with a nucleus isolation kit and resuspended in ChIP lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.0) containing protease inhibitor mixture. The nuclei were then sonicated to shear DNA to lengths between 200 and 1000 bp. The sonicated lysates were diluted to an A260 of 2 units/ml with ChIP dilution buffer and incubated with 60 μl of protein A-conjugated agarose beads to reduce nonspecific binding. Primary antibodies were added to the precleared supernatant fraction and incubated from 5 h to overnight at 4 °C with rotation. Protein A-conjugated agarose beads (50 μl) were then added to the samples for 1 h, and the protein-DNA complexes were eluted from the protein A-agarose by incubation in 250 μl of elution buffer (1% SDS, 0.1 m NaHCO3). Protein-DNA cross-links were reversed by heating at 65 °C for 5 h. The immunoprecipitated DNA was phenol/chloroform extracted and ethanol-precipitated in the presence of 15 μg of linear polyacrylamide, an inert carrier. The isolated DNA was then analyzed by semi-quantitative PCR using the following primers: β-casein promoter forward primer 5′-GTC CTC TCA CTT GGC TGG AG-3′ and reverse primer 5′-GTG GAG GAC AAG AGA GGA GGT-3′; amylase promoter forward primer 5′-TCA GTT GTA ATT CTC CTT GTA CGG-3′ and reverse primer 5′-CCT CCC ATC TGA AGT ATG TGG GTC-3′; and γ-casein promoter forward primer 5′-AAA CAG GTG AGT CTG CCT TCA-3′ and reverse primer 5′-CCA AAT GGA AGA CGA GAG GA-3′. Statistics—All of the data analysis was performed using Sigma Plot. The bar graphs represent the means ± S.E. Expression of mammary-specific genes depends on signals from both lactogenic hormones and ECM molecules; for β-casein, the relevant ECM molecule is laminin-111 (2Streuli C.H. Bailey N. Bissell M.J. J. Cell Biol. 1991; 115: 1383-1395Crossref PubMed Scopus (532) Google Scholar, 9Lee E.Y. Lee W.H. Kaetzel C.S. Parry G. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1419-1423Crossref PubMed Scopus (204) Google Scholar, 10Weir M.L. Oppizzi M.L. Henry M.D. Onishi A. Campbell K.P. Bissell M.J. Muschler J.L. J. Cell Sci. 2006; 119: 4047-4058Crossref PubMed Scopus (83) Google Scholar). Using EpH4, an epithelial cell line derived from normal mouse mammary gland (4Brinkmann V. Foroutan H. Sachs M. Weidner K.M. Birchmeier W. J. Cell Biol. 1995; 131 (6 Pt 1): 1573-1586Crossref PubMed Scopus (296) Google Scholar, 5Reichmann E. Ball R. Groner B. Friis R.R. J. Cell Biol. 1989; 108: 1127-1138Crossref PubMed Scopus (141) Google Scholar), we observed that both β- and γ-casein mRNA levels were highly up-regulated in response to prolactin and lrECM treatment (Fig. 1A); expression correlated with significant changes in cellular morphology as shown previously for β-casein (3Roskelley C.D. Desprez P.Y. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12378-12382Crossref PubMed Scopus (409) Google Scholar). Furthermore, consistent with previous studies (1Streuli C.H. Schmidhauser C. Bailey N. Yurchenco P. Skubitz A.P. Roskelley C. Bissell M.J. J. Cell Biol. 1995; 129: 591-603Crossref PubMed Scopus (342) Google Scholar, 30Muschler J. Lochter A. Roskelley C.D. Yurchenco P. Bissell M.J. Mol. Biol. Cell. 1999; 10: 2817-2828Crossref PubMed Scopus (100) Google Scholar), we established that laminin-111 was indeed the lrECM constituent that induced β-casein expression in EpH4 cells (data not shown). The mouse β-casein promoter contains binding sites for STAT5 and C/EBPβ, and half-sites of the palindromic glucocorticoid response element (33Wyszomierski S.L. Rosen J.M. Mol. Endocrinol. 2001; 15: 228-240Crossref PubMed Scopus (88) Google Scholar, 34Lechner J. Welte T. Tomasi J.K. Bruno P. Cairns C. Gustafsson J. Doppler W. J. Biol. Chem. 1997; 272: 20954-20960Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 35Schmitt-Ney M. Doppler W. Ball R.K. Groner B. Mol. Cell. Biol. 1991; 11: 3745-3755Crossref PubMed Google Scholar). To determine whether ECM and prolactin directly induce transcriptional activation of the β-casein promoter, we amplified and cloned the promoter region from -340 to -1 into a luciferase reporter vector and stably transfected the reporter plasmid into EpH4 cells. Luciferase activity was dramatically induced in the transfected cells after treatment with lrECM and prolactin (Fig. 1B), indicating that the β-casein promoter is transcriptionally activated in these cells. Consistent with the PCR results, neither prolactin nor lrECM alone could appreciably enhance promoter activity. STAT5- and C/EBPβ-binding sites were also identified in the bovine β-casein ECM response element, BCE-1 (20Myers C.A. Schmidhauser C. Mellentin-Michelotti J. Fragoso G. Roskelley C.D. Casperson G. Mossi R. Pujuguet P. Hager G. Bissell M.J. Mol. Cell. Biol. 1998; 18: 2184-2195Crossref PubMed Scopus (91) Google Scholar). To determine whether these two factors regulate ECM- and prolactin-induced expression of the endogenous β-casein gene, total and nuclear lysates of EpH4 cells were analyzed by Western blotting. Although the total level of STAT5 did not change, the levels of phosphorylated STAT5 and its nuclear translocation increased after combined treatment with lrECM and prolactin. However, neither treatment alone could induce these changes (Fig. 1, C and D). Total cell and nuclear levels of C/EBPβ remained unchanged after the treatments (Fig. 1, C and D). To determine whether STAT5 and C/EBPβ become associated with the β-casein promoter after ECM and prolactin treatment, we performed ChIP assays. Addition of these two ligands significantly increased the association of STAT5 and C/EBPβ with the β-casein promoter, whereas the interaction between the promoter and GR remained at the control level (Fig. 2A). The promoter of the β-amylase gene, which is not expressed in mammary epithelial cells, was included as a negative control and was not detected in any of the ChIP samples (data not shown). Thus, exposure of EpH4 cells to lrECM and prolactin increases both STAT5 levels in the nucleus and the binding of this factor and C/EBPβ to the β-casein promoter. We also found that treatment with prolactin and lrECM moderately enhanced the binding of Brg1, the ATPase subunit of SWI/SNF complex, to the β-casein promoter in EpH4 and primary mammary epithelial cells (Fig. 2A and supplemental Fig S1). Analysis of the DNA immunoprecipitated with a RNA polymerase II antibody showed an increased association of this protein with the β-casein promoter in response to treatment with lrECM and prolactin (Fig. 2A). Binding sites of STAT5 and C/EBPβ were identified in promoters of other milk protein genes, such as γ-casein (36Kolb A.F. Biochim. Biophys. Acta. 2002; 1579: 101-116Crossref PubMed Scopus (15) Google Scholar). We asked whether γ-casein was regulated similarly to β-casein. The association of these factors with the γ-casein promoter was determined by ChIP assays. We found that treatment with prolactin and lrECM enhanced binding of STAT5 and C/EBPβ and increased Brg1 and RNA polymerase II levels in the promoter region of γ-casein gene (Fig. 2B). The mouse casein genes cluster at a single gene locus on chromosome 5 in this order: α, β, γ, δ, and κ (37Rijnkels M. Wheeler D.A. de Boer H.A. Pieper F.R. Mamm. Genome. 1997; 8: 9-15Crossref PubMed Scopus (37) Google Scholar), and the expression of casein genes is coordinately regulated during pregnancy and lactation (38Hobbs A.A. Richards D.A. Kessler D.J. Rosen J.M. J. Biol. Chem. 1982; 257: 3598-3605Abstract Full Text PDF PubMed Google Scholar). Thus, the binding of these transcription factors and the chromatin remodeling complex together appears to activate the entire gene locus. We showed above that treatment with lrECM and prolactin induced the recruitment of transcription factors and the SWI/SNF complex to the β-casein promoter. To determine whether ECM and prolactin control these events separately or cooperatively, we performed ChIP analysis after cells were treated either singly or with both agents. We found that STAT5 bound to the β-casein promoter in cells treated with both lrECM and prolactin, but treatment with either component alone failed to induce appreciable binding (Fig. 2C). These results are consistent with the Western data showing that nuclear translocation of STAT5 depends on both the ECM and hormonal signals (Fig. 1D). Combined lrECM and prolactin treatment also induced binding of C/EBPβ to the β-casein promoter (Fig. 2C). Recruitment of Brg1 and RNA polymerase II in the β-casein promoter required also both lrECM and prolactin (Fig. 2C). These results establish that ECM cooperates with prolactin to induce the binding of transcription factors as well as the transcriptional machinery to the β-casein promoter. Previously, we showed that treatment with histone deacetylase inhibitors could partially substitute for lrECM in activating a stably integrated bovine ECM response element (BCE-1) in a mammary epithelial cell line (CID-9), suggesting that histone acetylation may play a role in transcriptional regulation of this enhancer (20Myers C.A. Schmidhauser C. Mellentin-Michelotti J. Fragoso G. Roskelley C.D. Casperson G. Mossi R. Pujuguet P. Hager G. Bissell M.J. Mol. Cell. Biol. 1998; 18: 2184-2195Crossref PubMed Scopus (91) Google Scholar). Surprisingly, however, the same treatment was later shown to inhibit transcriptional activation of the endogenous β-casein gene (21Pujuguet P. Radisky D. Levy D. Lacza C. Bissell M.J. J. Cell. Biochem. 2001; 83: 660-670Crossref PubMed Scopus (22) Google Scholar). Here we sought to determine whether histone acetylation is involved in transcriptional regulation of the endogenous β-casein gene. ChIP assays using antibodies against acetylated histone H3 and H4 demonstrated enhanced histone acetylation in the β-casein promoter, but not the β-amylase promoter, in response to treatment with lrECM and prolactin (Fig. 3A). In addition, neither lrECM nor prolactin alone induced histone acetylation in the β-casein promoter (data not shown), confirming that the cooperation between the two signals is important. To determine whether the increase of acetylated histone in the β-casein promoter was sufficient to induce transcription of the endogenous gene, EpH4 cells were treated with TSA in the presence or absence of ECM and prolactin. ChIP data showed that the levels of acetylated histone H4 (AcH4) appreciably increased in the β-casein promoter (Fig. 3B). Quantitative PCR showed, however, that the level of β-casein mRNA was increased by only 1.6-fold in undifferentiated cells after TSA treatment; the levels of both total and phosphorylated STAT5, C/EBPβ, and GR did not change (Fig. 3, C and D). In the functionally differentiated cells that were cultured with prolactin and lrECM, TSA treatment significantly suppressed the induction of β-casein expression. Western blot analysis showed that phosphorylated STAT5 levels decreased in TSA-treated cells, suggesting that this inhibition may be due to an indirect effect of TSA on STAT5 phosphorylation (Fig. 3, C and D). These results now clarify previous contradictions and indicate that histone acetylation alone is not sufficient to induce transcription of the endogenous β-casein gene above the basal level. A point mutation in the ATP-binding site" @default.
• W2068065167 created "2016-06-24" @default.
• W2068065167 creator A5039002471 @default.
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• W2068065167 title "Extracellular Matrix-regulated Gene Expression Requires Cooperation of SWI/SNF and Transcription Factors" @default.
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