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Originally published In Press as doi:10.1074/jbc.M610316200 on March 26, 2007

J. Biol. Chem., Vol. 282, Issue 20, 14992-14999, May 18, 2007
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Extracellular Matrix-regulated Gene Expression Requires Cooperation of SWI/SNF and Transcription Factors*Formula

Ren Xu, Virginia A. Spencer, and Mina J. Bissell1

From the Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720

Received for publication, November 6, 2006 , and in revised form, January 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 beta- and {gamma}-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both beta- and {gamma}-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 beta, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiated function of mammary epithelial cells is regulated by signals from both ECM2 and lactogenic hormones (1-3). The gene encoding the milk protein, beta-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 (4-6), transcription of beta-casein requires signals from both laminin-111 (previously referred to as laminin-1) and prolactin (1, 2, 7-10). A number of transcription factors, including STAT5, C/EBPbeta, and GR, have been shown to be involved in this process (reviewed in Ref. 7).

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 (11-13). 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 (14, 15). Histone acetylation is a dynamic process and is regulated by histone acetyltransferases and histone deacetylases (16). 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 (17, 18). The p300 histone acetyltransferase cooperates with STAT5 to enhance exogenous beta-casein promoter activity in COS cells, indicating that histone acetylation may play a role in beta-casein transcription (19). 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 beta-casein transcription (20, 21). 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 (14, 22, 23). 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 (24). 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 (14). To date, two different mechanisms have been described for recruiting SWI/SNF complexes to tissue-specific genes. Transcription factors, such as GR and C/EBPbeta, have been shown in mammalian cells to recruit the SWI/SNF complex to cis-elements to activate specific gene transcription (23, 25-27). Alternatively, the ATPase subunits of SWI/SNF contain bromodomains, which can bind directly to acetylated histone tails in vitro (28, 29). Thus, acetylated histones in a particular chromatin region may contribute to the recruitment of SWI/SNF complexes to specific genes.

Using the beta- and {gamma}-casein genes as models, here we investigate how ECM and prolactin regulate the activity of STAT5 and C/EBPbeta, 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Antibodies against acetylated H4 and acetylated H3 were from Upstate%20Biotechnology">Upstate Biotechnology, Inc. The H3 antibody was from Abcam. The STAT5 antibody was from R & D Systems, and those against C/EBPbeta, 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%20Biotechnology">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 (4, 5). 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 (1).

EpH4 cells were seeded onto 35-mm dishes and transfected using Lipofectamine 2000 (Invitrogen) with the following plasmids: 3 µg of DN-Brg1 plasmid (14) (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 beta-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 (30). 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 x 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 x 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 beta-casein, {gamma}-casein, lactoferrin, and GAPDH cDNA sequences: forward primer of the beta-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 {gamma}-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) (31). 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%20Biotechnology">Upstate Biotechnology, Inc. ChIP protocol (32) 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: beta-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 {gamma}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of mammary-specific genes depends on signals from both lactogenic hormones and ECM molecules; for beta-casein, the relevant ECM molecule is laminin-111 (2, 9, 10). Using EpH4, an epithelial cell line derived from normal mouse mammary gland (4, 5), we observed that both beta- and {gamma}-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 beta-casein (3). Furthermore, consistent with previous studies (1, 30), we established that laminin-111 was indeed the lrECM constituent that induced beta-casein expression in EpH4 cells (data not shown).

The mouse beta-casein promoter contains binding sites for STAT5 and C/EBPbeta, and half-sites of the palindromic glucocorticoid response element (33-35). To determine whether ECM and prolactin directly induce transcriptional activation of the beta-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 beta-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/EBPbeta-binding sites were also identified in the bovine beta-casein ECM response element, BCE-1 (20). To determine whether these two factors regulate ECM- and prolactin-induced expression of the endogenous beta-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/EBPbeta remained unchanged after the treatments (Fig. 1, C and D).


Figure 1
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  		FIGURE 1.
STAT5 phosphorylation and nuclear translocation are activated in EpH4 cells in response to ECM and prolactin treatment. A, beta- and {gamma}-casein expression was determined by RT-PCR in EpH4 cells cultured for 2 days in GIH (Dulbecco's modified Eagle's medium/F-12 supplemented with 5 µg/ml insulin and 1 µg/ml hydrocortisone). Media alone (Ctrl), GIH + 3 µg/µl prolactin (PRL), GIH + 2% lrECM (ECM), and GIH + 3 µg/µl prolactin + 2% lrECM (ECM+PRL). GAPDH cDNA was used as a loading control. B, thebeta-casein promoter was cloned into pGL3 luciferase vector and stably transfected into EpH4 cells. The beta-casein promoter activity was determined by luciferase assays. C and D, transcription factor levels in the cell lysates (C) and nuclear lysates (D) of EpH4 cells were determined by Western blotting.

 
To determine whether STAT5 and C/EBPbeta become associated with the beta-casein promoter after ECM and prolactin treatment, we performed ChIP assays. Addition of these two ligands significantly increased the association of STAT5 and C/EBPbeta with the beta-casein promoter, whereas the interaction between the promoter and GR remained at the control level (Fig. 2A). The promoter of the beta-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/EBPbeta to the beta-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 beta-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 beta-casein promoter in response to treatment with lrECM and prolactin (Fig. 2A).


Figure 2
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  		FIGURE 2.
Binding of transcription factors and the SWI/SNF complex to the beta-casein promoter is regulated by ECM and prolactin. A and B, ChIP assays followed by PCR analysis to detect the binding of STAT5, C/EBPbeta, GR, Brg1, and RNA Polymerase II (Pol II) in the beta-casein (A; n = 4) and {gamma}-casein (B; n = 2) promoters. The PCR results were quantified by AlphaEaseFC software, and the values of bound DNA were normalized to input DNA. Fold enrichments were determined by dividing the normalized values from treated cells by that of untreated cells. *, p < 0.05. Ctrl, control. C, quantification of ChIP results on the beta-casein promoter in EpH4 cells treated with prolactin (PRL), lrECM (ECM), or prolactin plus lrECM (PRL+ECM). The graph displays the means of two experiments.

 
Binding sites of STAT5 and C/EBPbeta were identified in promoters of other milk protein genes, such as {gamma}-casein (36). We asked whether {gamma}-casein was regulated similarly to beta-casein. The association of these factors with the {gamma}-casein promoter was determined by ChIP assays. We found that treatment with prolactin and lrECM enhanced binding of STAT5 and C/EBPbeta and increased Brg1 and RNA polymerase II levels in the promoter region of {gamma}-casein gene (Fig. 2B). The mouse casein genes cluster at a single gene locus on chromosome 5 in this order: {alpha}, beta, {gamma}, {delta}, and {kappa} (37), and the expression of casein genes is coordinately regulated during pregnancy and lactation (38). 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 beta-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 beta-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/EBPbeta to the beta-casein promoter (Fig. 2C). Recruitment of Brg1 and RNA polymerase II in the beta-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 beta-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 (20). Surprisingly, however, the same treatment was later shown to inhibit transcriptional activation of the endogenous beta-casein gene (21). Here we sought to determine whether histone acetylation is involved in transcriptional regulation of the endogenous beta-casein gene. ChIP assays using antibodies against acetylated histone H3 and H4 demonstrated enhanced histone acetylation in the beta-casein promoter, but not the beta-amylase promoter, in response to treatment with lrECM and prolactin (Fig. 3A). In addition, neither lrECM nor prolactin alone induced histone acetylation in the beta-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 beta-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 beta-casein promoter (Fig. 3B). Quantitative PCR showed, however, that the level of beta-casein mRNA was increased by only 1.6-fold in undifferentiated cells after TSA treatment; the levels of both total and phosphorylated STAT5, C/EBPbeta, 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 beta-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 beta-casein gene above the basal level.


Figure 3
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  		FIGURE 3.
Histone acetylation contributes to but is not sufficient to induce beta-casein expression. A, the levels of total histone H3, as well as acetylated histone H4 and H3 in the beta-casein promoter were measured by ChIP analysis. The beta-amylase promoter was used as a control. *, p < 0.05 (n = 3). B, the levels of AcH4 in the beta-casein promoter were determined by ChIP assays in control and TSA (80 nM)-treated cells. C, the beta-casein mRNA levels in undifferentiated EpH4 cells (in GIH medium) and differentiated cells (in GIH plus prolactin and lrECM) were measured by quantitative RT-PCR after TSA treatment. The graph displays averages ± S.E. *, p < 0.05; ***, p < 0.01 (n = 4). D, protein levels of phosphorylated STAT5, total cell STAT5, C/EBPbeta, and GR were analyzed by Western blotting after TSA treatment. Ctrl, control.

 
A point mutation in the ATP-binding site of Brg1 was shown to abolish its ATPase activity and produce a dominant negative effect on the chromatin-remodeling function of the SWI/SNF complex (39). To examine whether this ATPase was indeed the additional factor required for beta-casein expression, we generated a mammary cell line that conditionally expressed FLAG-tagged DN-Brg1 under the control of a tetracycline-repressible transactivator. Withdrawal of tetracycline from culture medium for 2 days caused a dramatic induction of DN-Brg1 expression (Fig. 4A) and a significant repression in beta- and {gamma}-casein transcription (Fig. 4, B and C), but lactoferrin and GAPDH transcription remained unchanged. Thus, the ATPase activity of SWI/SNF is necessary for transcriptional activation of casein genes. The binding of STAT5 and C/EBPbeta, as well as the levels of AcH4 in the beta-casein promoter in ECM- and prolactin-treated cells did not change significantly in response to DN-Brg1 expression (Fig. 4D). Western blot analysis revealed that DN-Brg1 expression also did not affect the nuclear levels of STAT5, C/EBPbeta, and GR (data not shown). These results rule out the possibility that expression of DN-Brg1 inhibited beta-casein transcription indirectly by repressing the activity of transcription factors and suggest that transcription factor binding and histone acetylation in the beta-casein promoter are events that take place upstream of Brg1 ATPase activity and are not dependent on SWI/SNF. In contrast, recruitment of RNA polymerase II to the beta-casein promoter was inhibited by DN-Brg1 expression, suggesting that its function in mediating transcription of casein genes occurs downstream of SWI/SNF and therefore is dependent on ATP-dependent chromatin remodeling (Fig. 4D).

Formation of the SWI/SNF complex was shown to occur independently of its ATPase activity in NIH 3T3 cells (40, 41). We asked whether the SWI/SNF complex interacts with STAT5, C/EBPbeta, and GR and whether the ATPase activity of Brg1 is necessary for this interaction in mammary epithelial cells. Protein complexes from tet+ (no DN-Brg1 expression) and tet-(with DN-Brg1 expression) cells were immunoprecipitated with agarose beads conjugated with anti-FLAG M2 antibody. The immunoprecipitated protein complexes were analyzed by Western blot using antibodies against STAT5, C/EBPbeta, GR, and lamin B. STAT5 co-immunoprecipitated with the DN-Brg1 in the tet-cells, but it was absent from the immunoprecipitate of tet+ cells (Fig. 5A). We also detected interactions between DN-Brg1 and GR, and DN-Brg1 and C/EBPbeta in lysates from the tet-cells but not tet+ cells (Fig. 5A). However, lamin B was not enriched in the co-IP samples from tet-cells compared with control (tet+) cells, suggesting that the interaction between STAT5, C/EBPbeta, GR, and the SWI/SNF complex is specific (Fig. 5A). That SWI/SNF is associated with GR and C/EBPbeta has been shown (23, 25), but the interaction between SWI/SNF and STAT5 has not been reported previously. To confirm the co-IP results in DN-Brg1-expressing cells, we performed a co-IP experiment using the parental cells. The results showed that endogenous wild type SWI/SNF was bound to STAT5 in lrECM- and prolactin-treated EpH4 cells but not in control cells (Fig. 5B). Therefore, the SWI/SNF chromatin remodeling complex may be recruited to the beta-casein promoter by STAT5, C/EBPbeta, and/or GR. Several other milk proteins, including {alpha}-casein, {gamma}-casein, whey acidic protein, and beta-lactoglobulin, have been shown to be regulated by ECM and lactogenic hormones (7, 42-44). The promoter or enhancer elements of these genes contain binding sites for STAT5 and GR (7, 36, 45). Interestingly, DN-Brg1 expression inhibited {gamma}-casein transcription significantly but had no detectable effect on transcription of the lactoferrin gene (Fig. 4B). These data are consistent with the finding that the expression of lactoferrin is not dependent on the cooperation of ECM and prolactin signals in MECs and that basal transcriptional regulation may be different for lactoferrin expression (46). These results indicate that transcription factors such as STAT5 determine recruitment of the SWI/SNF complex by binding to specific promoters to allow the expression of milk protein genes.


Figure 4
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  		FIGURE 4.
DN-Brg1 expression in EpH4 cells suppresses transcription of thebeta-casein gene. A, Western blot analysis of DN-Brg1 expression in stably transfected EpH4 cells. B and C, RT-PCR (B) and quantitative PCR (C) analysis of the levels ofbeta- and{gamma}-casein genes in DN-Brg1-expressing (tet-) and nonexpressing (tet+) cells. The graph displays averages ± S.E. ***, p < 0.01 (n = 4). D, ChIP assays showing the levels of AcH4 and the binding activity of STAT5 and C/EBPbeta in the beta-casein promoter in DN-Brg1-expressing cells. The graph displays the means of three experiments. *, p < 0.05.

 


Figure 5
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  		FIGURE 5.
STAT5, C/EBPbeta, and GR interact with DN-Brg1 in EpH4 cells. A, interaction between transcription factors and DN-Brg1 was determined by co-IP analysis. Total lysates before immunoprecipitation (IP) were used as input control. B, interaction between endogenous Brg1 and STAT5 was detected by co-IP analysis. C, model displaying how exposure of mammary epithelial cells to ECM and prolactin may induce the recruitment of transcription factors and chromatin remodeling enzymes to the beta-casein promoter and how aberrations in SWI/SNF function interfere with RNA polymerase II recruitment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
By necessity, lactation is a tightly regulated process during mammary gland development. Extracellular and intracellular signals must be coordinated precisely and rapidly to ensure that milk is produced and delivered in a timely fashion after parturition and during suckling. Using mammary-specific casein genes as markers, we show that laminin and prolactin regulate casein expression by inducing binding of STAT5 and C/EBPbeta to their promoters and that both histone acetylation and ATP-dependent chromatin remodeling are involved in this process (Fig. 5C).

Previous studies have shown that ECM cooperates with prolactin to control beta-lactoglobulin expression by activating the JAK2/STAT5 signal transduction pathway to induce STAT5 phosphorylation and its nuclear translocation in primary MECs (42, 47). Here we demonstrate that laminin and prolactin cooperatively regulate the binding of STAT5 to the beta-casein promoter. Upon the addition of prolactin and lrECM, C/EBPbeta becomes bound to the beta-casein promoter. How these extracellular signals regulate C/EBPbeta activity still remains to be determined, but one report showed that the nuclear levels of C/EBPbeta in primary rabbit mammary epithelial cells increased when these cells were plated on a collagen gel (48). We did not observe a significant change in the nuclear level of C/EBPbeta in EpH4 cells in response to laminin and prolactin treatment. Indeed a luciferase reporter gene fused to a C/EBPbeta response element was not activated by laminin and prolactin (data not shown), suggesting that C/EBPbeta DNA binding activity to the beta-casein promoter is enhanced selectively. The binding sites of STAT5, C/EBPbeta, and GR are in close proximity to one another within the beta-casein promoter, and our co-IP experiments showed that these three factors are all associated with the SWI/SNF complex. It has been reported that STAT5 cooperates with C/EBPbeta to regulate beta-casein promoter activity and that this cooperation is mediated by GR (33). Therefore, the factors most likely form a protein complex with chromatin remodeling enzymes on the beta-casein promoter, and the binding of STAT5 may enhance the interaction of C/EBPbeta with the promoter.

The tight link between eukaryotic gene transcription and histone acetylation is now firmly established (49, 50). Using foot-printing analysis, previously we showed that whereas binding of transcription factors was not sufficient to activate the BCE-1 element in another mouse epithelial cell line, the enhancer element could be activated in the absence of ECM upon TSA treatment (20). Surprisingly, however, the expression of the endogenous beta-casein was inhibited by the same treatment (21). Using ChIP assays, which can directly detect the level of histone acetylation in a specific chromatin region, we demonstrated here that histone acetylation was indeed involved in transcriptional activation of the endogenous beta-casein gene in functional MECs, which is consistent with a recent study in HC11 cells (51). We show that TSA treatment only slightly increased the basal level of beta-casein transcription in undifferentiated cells, indicating that histone acetylation contributes to, but is not sufficient for, induction of endogenous mammary-specific gene transcription. The controversial effects of TSA on the activity of endogenous beta-casein gene and exogenous BCE-1 element may be due to differences in the nuclear environment surrounding these sequences and the manner with which each sequence is packaged into chromatin. Analysis of global histone acetylation patterns has demonstrated that gene expression is correlated with histone hyperacetylation in specific chromatin regions (17, 18). However, treatment with histone deacetylase inhibitors only induced expression of less than 3% of genes in cultured cells (52, 53). These findings imply that transcription of other tissue-specific genes and inducible genes may require different types of chromatin remodeling.

ATP-dependent chromatin remodeling conducted by the SWI/SNF complex increases nucleosome mobility and may uncover a core promoter for assembly of a preinitiation complex (54). Studies with ATPase-deficient Brg1 have demonstrated that the ATPase activity of SWI/SNF is required for expression of tissue-specific genes in muscle and adipose tissues (14, 55, 56). However, whether the expression of mammary-specific genes depends on SWI/SNF function was not addressed previously. The ChIP assays show that the SWI/SNF complex is associated with the beta-casein promoter in EpH4 cells upon transcriptional activation. This association is detected also in primary mammary epithelial cells (supplemental Fig. S1). Furthermore, the ATPase activity of SWI/SNF is necessary for transcription of the beta-casein gene. DN-Brg1 expression leads to a reduction in binding of RNA polymerase II, but not the levels of STAT5, C/EBPbeta, and AcH4 in the promoter region. Together with the data generated from TSA experiments, these results suggest that chromatin remodeling induced by histone acetylation is not sufficient for assembly or stabilization of the RNA transcriptional machinery on the beta-casein promoter and that this process depends on ATP-dependent chromatin remodeling (Fig. 5C). Such precise regulation from extracellular signals to chromatin structure is most likely fundamental to mammary gland development and function to ensure control of milk protein gene expression during lactation, a process that is vital to the survival of the offspring.


   FOOTNOTES
 
* This work was supported by U. S. Department of Energy, Office of Biological and Environmental Research Grant DE-AC03-76SF00098, a distinguished fellow award by the same Office, U. S. Department of Defense Breast Cancer Research Program Innovator Award DAMD17-02-1-0438 (to M. J. B.), as well as NCI, National Institutes of Health Grant CA057621-13 (to Z. W. and M. J. B.), and in part by Department of Defense Breast Cancer Research Program Postdoctoral Fellowships DAMD17-02-1-0441 (to R. X.) and W81XWH0410581 (to V. A. S.) and a postdoctoral fellowship from the Canadian Institute for Health Research (to V. A. S.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., MS 977R225A, Berkeley, CA 94720. Tel.: 510-486-4365; Fax: 510-486-5586; E-mail: MJBissell{at}lbl.gov.

2 The 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. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Anthony N. Imbalzano for providing the DN-Brg1 plasmid. We thank Drs. Zendra E. Zehner, Paraic Kenny, Derek C. Radisky, Celeste Nelson, and other members of Bissell laboratory for critical reading of the manuscript. We thank Hui Zhang for help in isolation of the primary mammary epithelial cells.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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