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Molecular and Cellular Biology, April 2004, p. 3347-3358, Vol. 24, No. 8
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.8.3347-3358.2004

Reconstitution of Glucocorticoid Receptor-Dependent Transcription In Vivo

Kevin W. Trotter and Trevor K. Archer*

Chromatin and Gene Expression Section, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received 2 December 2003/ Returned for modification 5 January 2004/ Accepted 27 January 2004


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ABSTRACT
 
We developed a model system to study glucocorticoid receptor (GR)-mediated chromatin remodeling by the BRG1 complex. Introduction of the BRG1 ATPase into the SW-13 cell line initiates the formation of a functional remodeling complex. This complex is able to induce transcriptional activation from a transiently transfected promoter with wild-type and chromatin-remodeling-deficient BRG1 mutants, suggesting that the complex possesses a coactivator function independent from remodeling. Transactivation from a chromatin template requires the BRG1 remodeling function, which induces regions of hypersensitivity and transcription factor loading onto the integrated MMTV promoter. We report that BRG1 remodeling activity is required for GR-mediated transactivation and that this activity cannot be replaced by other ATP-dependent remodeling proteins. Further characterization of the BRG1-associated factors (BAFs) present in these cells (for example, the expression of BAF250 but not BAF180) reveals that the BAF complex rather than the polybromo-associated BAF complex is the necessary and sufficient chromatin-remodeling component with which the receptor functions in vivo. These results in conjunction with previous findings demonstrate that the GR functions with multiple forms of the SWI/SNF complex in vivo.


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INTRODUCTION
 
The assembly of eukaryotic DNA into nucleosomes tends to have a repressive effect on gene transcription (64). This chromatin structure inhibits transcription by blocking access of the required components of the transcriptional machinery and gene-specific activators to recognition sequences within target promoters (22, 28). The structural changes in chromatin that accompany transcriptional activation often require multiprotein complexes that manipulate nucleosomal architecture (48, 59). There are at least two main classes of complexes that disrupt chromatin structure: one covalently modifies histones by altering acetylation, methylation, phosphorylation, and/or ubiquitination patterns, while the other uses ATP hydrolysis to disrupt histone-DNA interactions (6, 30, 38, 45, 50, 61, 69).

A number of multiprotein complexes have been identified that utilize ATP to alter the arrangement of chromatin (21, 48, 60). These remodeling complexes tend to increase the accessibility of nucleosomal DNA to nucleases or other DNA-binding proteins (18). Remodeling complexes, such as SWI/SNF, RSC, ACF, CHRAC, and NURF, have been shown to alter the relationship between DNA and histone octamers within nucleosomes by a mechanism that is not completely understood (8, 38). These complexes can be classified into at least three major families based on subunit composition and the content of their central ATPase which can include Saccharomyces cerevisiae Swi-2/Snf-2, Drosophila Brahma and ISWI, or human BRG1 and hBrm (57). All of these complexes have been shown to remodel the nucleosomal structure in vitro; however, their activities appear to be biochemically distinct (29).

The SWI/SNF remodeling complex was originally identified in yeast by mutations in mating-type switching (SWI) and sucrose fermentation (SNF) (67), and its function is highly conserved in eukaryotes, with homologous proteins found in Drosophila and humans (45). The SWI/SNF family of nucleosomal remodeling complexes has been shown to play important roles in gene expression by altering local chromatin structures and facilitating the binding of transcription factors to sequence-specific DNA (36, 39, 40). In mammals, the SWI/SNF complex is highly divergent and present in multiple forms (51, 65, 66). Human SWI/SNF is a large multiprotein complex that possesses either BRG1 or hBrm, two ATPases related to yeast SWI2/SNF2 and STH1 (35, 37, 40, 46), and is comprised of about 10 BRG1-associated factors (BAFs), most of which are orthologous to those found in yeast SWI/SNF and RSC (51, 68).

Multiple mammalian SWI/SNF complexes that have diverse subunit compositions have been identified although BRG1 (or hBrm), BAF170, BAF155, and BAF47 represent the core components (66). Reconstitution assays have shown that BRG1 alone is sufficient to stimulate nucleosomal remodeling, albeit at reduced efficiency when compared to the complete SWI/SNF complex. However, the addition of the core components reconstituted chromatin remodeling near levels resembling that for the complete purified complex (54). Thus, the role of the BAF proteins within the remodeling complex may be to target or stabilize nucleosomes in a particular conformation that is favorable for BRG1 remodeling activity.

It has been shown in mammalian cells that the glucocorticoid receptor (GR) targets the SWI/SNF complex to chromatin promoters that contain glucocorticoid response elements, resulting in disruption of the local nucleosomal structure (24). The mouse mammary tumor virus (MMTV) promoter has proved to be a well-developed system to study the processes involved in transcriptional activation by steroid hormone receptors. When stably integrated into chromatin, the promoter acquires a highly organized structure where the long terminal repeat (LTR) is complexed into a phased array of six nucleosomes with hormone response elements and transcription factor binding sites falling within the region occupied by the second nucleosome, nucleosome B (Nuc-B) (4, 55). The hormone response elements found within this region allow for strong promoter activation by glucocorticoids where ligand-bound GR mediates transcription through mechanisms involving chromatin remodeling (3, 55). These features make the MMTV promoter an ideal system for the study of steroid hormone receptor-dependent chromatin remodeling by the SWI/SNF complex.

In vitro studies have revealed a requirement for BRG1 recruitment to the MMTV promoter for glucocorticoid-mediated transcription. The GR has been shown to stimulate BRG1 chromatin remodeling by using partially purified complexes from HeLa cell extracts (53). On MMTV LTR DNA reconstituted into nucleosomal arrays with Drosophila embryo extracts, purified GR required ATP and HeLa nuclear extract to induce chromatin remodeling (23). In yeast, SWI/SNF stimulated transcription from assembled chromatin templates (63).

The SW-13 cell line, derived from a small-cell carcinoma of the adrenal cortex, was used to study GR-mediated chromatin remodeling of the MMTV promoter by BRG1. These cells lack a functional SWI/SNF remodeling complex due to the absence of BRG1 and hBrm proteins while they express the BAF proteins required for complex formation (15, 20, 46). In this study, we describe the establishment of a model system that takes advantage of the SW-13 cell line to study the requirement for chromatin remodeling in GR-mediated transactivation of the MMTV promoter in vivo. Due to the absence of BRG1 and hBrm, the contribution of BRG1 in GR-induced chromatin remodeling can be assessed without interference from the endogenous protein. Previous studies of human and mouse breast cancer cells demonstrated that GR-mediated transcription requires the recruitment of the BRG1 chromatin-remodeling complex (24). These assays make a compelling, but not absolute, case for the BRG1 complex in hormone action. We show that the BRG1 complex is required for GR-mediated chromatin remodeling and the transcriptional activation of a stably maintained MMTV promoter. In the absence of BRG1, the GR is unable to activate transcription from the MMTV promoter despite the presence of other ATPases such as ISWI (29) and Mi-2 (49). Secondly, both ATPase-deficient and wild-type BRG1 can stimulate transcription from a transiently transfected DNA, revealing a previously unsuspected coactivator function independent of chromatin remodeling. In contrast, only the native protein containing ATPase activity stimulated transcription from the chromatin template. Interestingly, the absence of the BAF180 subunit in our model suggests that the SWI/SNF remodeling complex, instead of the polybromo-associated BAF (PBAF) complex, is sufficient for GR-mediated activation in vivo. Finally, we demonstrate the importance of BRG1 remodeling activity on selected endogenous GR-responsive promoters within the SW-13 genome, suggesting that the dependence of BRG1 activity is not unique to the MMTV promoter.


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MATERIALS AND METHODS
 
Cell culture. SW-13 human adrenal carcinoma cells were maintained in normal growth medium containing Dulbecco's modified Eagle high-glucose medium supplemented with 10% fetal bovine serum, 100 U of penicillin-streptomycin per ml, and 2 mM L-glutamine. T47D/A1-2 cells were cultured according to a standard protocol (5) and MCF-7 cells were maintained in accordance with American Type Culture Collection specifications.

Transient transfection and luciferase assay. Transfections were carried out in serum-free Dulbecco's modified Eagle high-glucose medium by using Lipofectamine Plus according to the manufacturer's instructions. Transient transfections were carried out with the pMMTV-LTR reporter and/or pcDNA3.1(-).rat-GR, pBJ5 (empty vector), pBJ5.hBRG1, pBJ5.hBRG1-K798R, or pBJ5.hBRG1-{Delta}E7 expression vectors. Concentrations of BRG1 expression plasmids were optimized to yield equal protein expression as determined by Western blot analysis. Transfection efficiencies were greater than 50% for all assays. At 10 h posttransfection, cells were treated with dexamethasone (Dex; 10-7 M) or an equal volume of vehicle (100% ethanol) for 16 h in normal growth medium. Following treatment, cells were assayed for luciferase activity as previously described (7). Relative light units (RLU) were normalized to the total protein measured.

Immunoblotting. Cells from subconfluent cultures were washed and scraped into phosphate-buffered saline and pelleted by centrifugation. For immunoblotting whole-cell lysates were prepared by using buffer X (24) containing mammalian protease cocktail (Sigma). For nuclear extracts, cells were lysed in NE-PER extraction reagent (Pierce) according to the manufacturer's protocol. Protein concentration was determined by a Bradford assay. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride membrane, and blocked in Tris-buffered saline (0.15 M NaCl, 0.05 M Tris-HCl [pH 8.0], 0.05% [vol/vol] Tween 20) containing either 10% nonfat dry milk or 5% normal donkey serum. Western blot analysis was performed as described previously (58) by using the indicated antibodies at recommended dilutions. To remove bound antibody, membranes were incubated in Restore Western blot stripping buffer (Pierce) according to the manufacturer's protocol.

IP. SW-13 cells were transfected as described above. Twenty-four hours posttransfection, cells were treated either with Dex (10-7 M) or an equal volume of vehicle (100% ethanol) for 5 h, followed by homogenization in ice-cold immunoprecipitation (IP) buffer K (250 mM NaCl, 5 mM HEPES [pH 7.0], 0.1% NP-40) containing a protease inhibitor cocktail (Sigma). Preincubation of whole-cell extracts with protein G-agarose beads (Sigma) was performed to exclude nonspecific binding. Volumes containing 0.5 mg of protein from whole-cell lysates were incubated with 10 µg of anti-BRG1, -BAF170, or -BAF155 antibodies (Santa Cruz) overnight at 4°C in IP buffer K. Antibody complexes were collected by using G-coupled agarose beads (Sigma), followed by four washes with buffer K and two washes with 50 mM Tris-HCl, pH 8.0. Samples were boiled in SDS-loading buffer containing 2-mercaptoethanol and subjected to SDS-PAGE. Coprecipitated proteins were identified by immunoblotting with the indicated antibodies.

Establishment of stable cell lines. To create cell lines that stably express GR and contain integrated MMTV reporter, SW-13 cells were cotransfected with pMMTV-LTR-luciferase and pcDNA3.1(-).rat-GR expression vectors at a 10:1 ratio by using Lipofectamine Plus reagent. After 24 h, cells were collected and grown in normal growth medium supplemented with 500 µg of Geneticin per ml. Stable clones were picked, tested for stable MMTV-LTR integration and expression of GR by a transient transfection assay with the pBJ5.hBRG1 expression plasmid, treated with Dex or vehicle, and analyzed for the presence of luciferase. Western blot analysis using anti-GR antibodies also confirmed the stable expression of GR in picked clones. Once generated, cell lines were maintained in selection media containing G148.

MNase analysis of chromatin structure. Nuclei were isolated and subjected to micrococcal nuclease (MNase) digestion as previously described (2). For Southern blot analysis, 20 µg of MNase-treated DNA was digested to completion with PstI and XbaI, separated on 1.5% agarose, and transferred to Hybond N+ membrane (Amersham-Pharmacia). Control genomic DNA was prepared by proteinase K treatment, followed by MNase digestion with 1 U of MNase per ml and then digestion with restriction enzymes. Fragments (at the indicated positions) corresponding to MMTV-LTR PstI (-1311)/ClaI (-865) and SstI (-105)/XbaI (+190) were radiolabeled and used for hybridizations. Membranes were hybridized overnight with the indicated probe. Fragment analysis was performed by using ImageQuant analysis software.

In vivo chromatin analysis and transcription factor loading. SW-13 MG2-13 cells were transfected with the pBJ5, pBJ5.hBRG1, or pBJ5.hBRG1-K798R expression vectors. Cells were treated with Dex (10-7 M) or vehicle for 1 h. Nuclei were isolated, digested with restriction endonucleases, or subjected to exonuclease III footprinting analysis as previously described (2). For footprinting analysis, HaeIII was used as the in vivo entry site enzyme. Genomic DNA was then purified by phenol-chloroform extraction and digested to completion with HaeIII to provide an internal standard for assessing the extent of in vivo cleavage and to confirm that equal amounts of DNA template were used for all reactions. Samples were analyzed by using linear Taq polymerase amplification with a 32P-labeled oligonucleotide (5'-TCTGGAAAGTGAAGGATAAAGTGACGA-3') specific for the MMTV promoter (oligo22). Purified products were analyzed on polyacrylamide denaturing gels. Quantification was performed by using ImageQuant analysis software comparing Dex-induced nuclear factor 1 (NF1) binding in the presence of BRG1 to empty vector and the K798R mutant.

Chromatin IP assay. SW-13/MG2-13 cells were transiently transfected with BRG1 or pBJ5 vector, followed by treatment with Dex (10-7 M) or vehicle for 1 h. Cell monolayers were fixed for 20 min at 37°C with 1% formaldehyde and washed twice with phosphate-buffered saline. Nuclei were isolated as previously described (2). Nuclei were lysed in SDS lysis buffer (Upstate) containing a protease inhibitor cocktail (Sigma) and sonicated to obtain DNA lengths between 200 and 1,500 bp as described previously (47). Soluble lysates were centrifuged at 10,000 x g for 10 min at 4°C and precleared by incubation for 1 h at 4°C with protein A agarose. IPs were carried out by incubating 10 µg of anti-BRG1, anti-GR (Santa Cruz), or normal immunoglobulin G (IgG) with 1.0 ml of sheared nuclear lysate overnight at 4°C. Immunoprecipitates were collected by protein A-agarose and washed in low-salt followed by high-salt buffers (Upstate). After final washes in 0.25 M LiCl and TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), immunocomplexes were eluted, reverse cross-linked, and digested with proteinase K. Eluted DNA was recovered by phenol-chloroform extraction and ethanol precipitation and resuspended in 50 µl of sterile water. Immunoprecipitated DNA and sheared inputs (5 µl) were subjected to 30 cycles of PCR by using 32P-labeled primer pairs specific for the MMTV promoter region -197 to +84 (5'-TTAAGTAAGTTTTTGGTTACAAACT-3' and 5'-TCTGGAAAGTGAAGGATAAAGTGACGA-3') or luciferase reporter region 1591 to 1785 (5'-CGTTGTTGTTTTGGAGCAC-3' and 5'-CTACAATTTGGACTTTCCGC-3'). Amplified products were resolved on polyacrylamide gels, exposed to a propidium iodide screen and analyzed by ImageQuant analysis software.

Reverse transcription (RT)-PCR. Total RNA was isolated from SW-13/MG2-13 cells transfected overnight with the pBJ5, BRG1, or BRG1-K798R expression vector. At 24 h posttransfection, cells were treated with Dex (10-7M) or vehicle for 4 h, followed by the extraction of total RNA by using RNeasy (QIAGEN). DNase-treated total RNA (2 µg) was reverse transcribed into single-stranded cDNA by using oligo(dT)12-18 primers and SuperScript II (Invitrogen). An equal amount of cDNA from each experimental condition was amplified by PCR with Taq polymerase and 32P-labeled gene-specific oligonucleotides. Amplified products were resolved on denaturing polyacrylamide gels and analyzed with ImageQuant analysis software. Optimized conditions for PCR amplification and gene-specific primer sequences are available upon request.


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RESULTS
 
Expression profile of GR and chromatin remodeling proteins present in the SW-13 cell line. While SW-13 cells were known to be deficient in BRG1 and hBrm protein expression (15, 20, 46), the presence of the GR and other cofactors was unreported. We therefore compared the expression patterns of these proteins in SW-13 cells with those in two human breast cancer lines, T47D/A1-2 and MCF-7. In contrast to T47D/A1-2 and MCF-7 cells, GR protein was not detected in the SW-13 cell line (Fig. 1A). Western blot analysis using nuclear extracts and antibodies specific for BRG1, hBrm, Mi-2, and ISWI confirmed the lack of endogenous BRG1 and hBrm proteins while detecting ATP-dependent chromatin-remodeling proteins Mi-2 and ISWI (29, 62) (Fig. 1B). Further analysis revealed the presence of a number of BAF proteins including BAF250, BAF170, BAF155, BAF60a, BAF57, and BAF47 in all three of the cell lines (Fig. 1C). Interestingly, SW-13 cells do not appear to express the BAF180 subunit (Fig. 1C). This differential expression of BAF250 and BAF180 observed in SW-13 and T47D/A1-2 cells suggests that were BRG1 expression restored in SW-13 cells only the SWI/SNF remodeling complex and not the PBAF complex would be present (25, 43, 51).



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  		FIG. 1. Profile of GR, chromatin remodeling, and BAF proteins in the SW-13 cell line. (A) Western blot analysis comparing GR expression in T47D/A1-2, MCF-7, and SW-13 cells. Total cell extracts were analyzed by Western blotting with antibodies specific for GR or ß-tubulin (loading control). (B) Western blot analysis comparing expression of chromatin-remodeling proteins in SW-13 cells. Nuclear extracts were subjected to SDS-PAGE and immunoblotted with indicated antibodies. (C) Differential expression of BAF proteins in SW-13 cells. Nuclear extracts were analyzed by Western blotting with antibodies specific for the individual BAF proteins.

Wild-type and mutant BRG1 proteins have similar effects on GR activity at transiently transfected templates. We next performed a series of promoter-driven assays with a reporter plasmid containing the MMTV promoter LTR fused to luciferase transiently introduced into SW-13 cells along with an expression construct for rat GR. The transient template was only slightly transcriptionally active in cells expressing GR upon treatment with Dex compared to transcription in the reporter alone (Fig. 2, lanes 1 and 2). However, coexpression of GR with BRG1 resulted in a marked increase in transcription from the reporter in a hormone-dependent fashion (Fig. 2, lane 4).



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  		FIG. 2. Wild-type and ATPase mutant BRG1 proteins have similar effects on GR activity at transiently transfected DNA. An MMTV reporter plasmid was transiently introduced into SW-13 cells with and without GR along with expression vectors for BRG1, BRG1-K798R, or BRG1-{Delta}E7. Cells were harvested after hormone treatment and assayed for luciferase activity. RLU were normalized to the total protein measured. The induction of MMTV luciferase activity is indicated as induction (n-fold) relative to that for vehicle-treated cells. Protein expression for transfected plasmids was determined by Western blot analysis using antibodies directed against GR or BRG1. Data are presented as the means of triplicate values obtained from a representative experiment that was independently repeated three times.

To test whether GR-mediated transcription from the transient template requires a functional ATPase domain, a mutant BRG1 construct was utilized in our assay that contains a point mutation of lysine to arginine at the conserved amino acid 798 within the ATPase domain (BRG1-K798R). Also tested was a BRG1 mutant exhibiting a 115-amino-acid deletion within the retinoblastoma (Rb) binding pocket (BRG1-{Delta}E7). Both mutants have previously been characterized and described as chromatin-remodeling deficient due to impaired ATPase activity or disrupted Rb binding (20, 37, 41). As shown in Fig. 2 (lanes 5 and 6), expression of the BRG1 mutants also resulted in an enhanced transactivation from the transient promoter. The resulting induction levels from the mutant proteins were comparable to the level observed with wild-type BRG1 when compared to reporter, GR, or BRG1 alone. Western blot analysis showed that the amount of transfected DNA resulted in the equal expression of both wild-type and mutant BRG1 protein (Fig. 2). Therefore, induction levels observed in the presence of the BRG1 proteins did not result from disproportionate protein expression. The increased activity at the transient promoter observed with the mutant proteins suggests that BRG1 may possess an intrinsic coactivator function that enhances transcription independent of ATP-dependent chromatin remodeling.

Characterization of a human SW-13 cell line stably transfected with an MMTV luciferase reporter and a GR expression vector. Previous analysis of GR-mediated transactivation of transiently transfected reporters has demonstrated that these conditions do not fully recapitulate GR activation from chromatin templates (4, 24). To examine GR-dependent chromatin remodeling by the BRG1 complex, the MMTV-LTR reporter was stably transfected into SW-13 cells along with a GR expression plasmid. Selected clones were transfected with a BRG1 expression plasmid, treated with Dex, and assayed for luciferase activity. All clones showed hormone-induced luciferase activity, indicating stable MMTV integration, with the highest level of Dex-induced activity observed in clone SW-13/MG2-13 (Fig. 3A). To examine GR expression, a Western blot analysis was performed with total cell extracts prepared from each clone. Each stable line tested positive for the expression of GR (Fig. 3B). Clone MG2-13 was selected for further characterization and used in studies involving hormone-dependent remodeling by the BRG1 complex.



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  		FIG. 3. Characterization of a stable SW-13 clone that contains integrated MMTV reporter and stably expressed GR. (A) Luciferase screen of MG2 clones stably transfected with MMTV-LTR and a GR expression vector. Clones were transfected with a BRG1 expression construct, treated with Dex or vehicle, and assayed for luciferase activity. RLU were normalized to the total protein measured. (B) Western blot screening to determine stable GR expression in picked clones. A Western blot analysis was performed on total cell extract by using anti-GR-specific antibody. Extract from SW-13 wild-type (wt) cells was used as a negative control. (C) The MMTV promoter is organized into a regular array of nucleosomes within the SW-13 genome. Nuclei (lanes 1 to 6) and genomic DNA (lane 7) from SW-13 clone MG2-13 were digested with 0 (lane 1), 1 (lane 7), or 5 to 100 (lanes 2 to 6) U of MNase per ml. DNA fragments were analyzed by Southern blot analysis with radiolabeled fragments complementary to the 5' (P1) or 3' (P2) boundaries of the MMTV promoter. All sizes are indicated in base pairs.

The MMTV promoter is organized into a regular phased array of nucleosomes within the SW-13 genome. Previous analysis has shown that the MMTV promoter is reproducibly organized into a phased array of six nucleosomes in vivo when it is stably maintained as chromatin (47, 55). To ensure that the MMTV-LTR was similarly organized in clone MG-13, we performed MNase digestion of SW-13/MG2-13 genomic DNA in conjunction with Southern blot analysis and reiterative primer extension. Regularly positioned nucleosomes were observed over the entire region from -1311 to +190 (Fig. 3C). Deproteinized genomic DNA treated with MNase failed to produce this banding array (Fig. 3C, lane 7), indicating that the observed in vivo digestion pattern resulted from bound nucleosomes. The nucleosomal spacing over the MMTV promoter, determined by Southern blotting with radiolabeled MMTV fragments (P1 and P2), was ~195 ± 5 bp, suggesting that the integrated promoter is arranged in a phased array of nucleosomes.

To confirm the nucleosomal spacing observed by Southern analysis, high resolution mapping of nucleosomal borders was performed by using MNase. This footprint identified a protected region extending from approximately bp -75 to -221 and a region sensitive to MNase treatment between bp -35 and -75 (data not shown). This protected region maps to the previously reported Nuc-B region located within the promoter and is similar to the regions found in other cell lines harboring an integrated MMTV promoter (47).

BRG1 mutants are unable to activate transcription from the chromatin promoter. SW-13/MG2-13 cells were transfected with wild-type or mutant BRG1 expression plasmids, treated with Dex, and assayed for luciferase activity. In cells expressing BRG1, MMTV luciferase activity was significantly induced upon Dex treatment. Activity levels observed in treated cells expressing the mutant constructs were equivalent to levels in control cells transfected with empty vector (see Fig. 6), suggesting that BRG1 ATPase function, along with a native Rb binding pocket, is required for transactivation from the chromatin promoter. Immunoblotting revealed equal protein expression from each transfection construct, and GR levels remained unchanged upon transfection (Fig. 4). Taken together, these results suggest that the BRG1 complex is required for GR-mediated remodeling of the chromatin promoter. Interestingly, other ATP-dependent remodeling proteins present in SW-13 cells, Mi-2 and ISWI, do not appear to substitute for BRG1 activity in this assay.



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  		FIG. 6. The SWI/SNF remodeling complex physically associates with GR and with the MMTV promoter in vivo. (A) The SWI/SNF remodeling complex associates with GR in a hormone-dependent manner within the SW-13 clone MG2-13. Shown is the coimmunoprecipitation of BAF155 or BRG1 from hormone-treated SW-13 MG2-13 cells expressing BRG1. IPs were performed on whole-cell extracts from Dex- or vehicle-treated cells transfected with empty vector (-BRG1) or a BRG1 expression plasmid (+BRG1). Immunoprecipitates were analyzed by immunoblotting with antibodies specific for BRG1, BAF250, BAF155, BAF170, and GR. IP with normal IgG was used to control for nonspecific interactions. (B) GR binding at the MMTV promoter is increased in the presence of BRG1. MG2-13 cells were transfected as above and chromatin was prepared by sonication from Dex- or vehicle-treated cells prior to formaldehyde cross-linking. Nuclear extracts were incubated with antibodies to GR, BRG1, or control IgG by using protein A-agarose as an absorbent. Purified immunoprecipitated DNA fragments were analyzed by PCR with 32P-labeled primers covering the MMTV promoter (position -199 to +84) or downstream luciferase sequence (position +1591 to +1795; control). Amplified products were resolved by electrophoresis on 6% denaturing polyacrylamide gels and analyzed as above. Ab, antibody.



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  		FIG. 4. Only wild-type BRG1 showed enhanced transcription from the chromatin promoter. (A) SW-13 MG2-13 cells were transiently transfected with pBJ5 (empty vector), BRG1, BRG1-K798R, or BRG1-{Delta}E7 expression plasmids. Cells were treated with Dex, harvested, and assayed for luciferase activity. RLU were normalized to the total protein measured. (B) Induction of MMTV luciferase activity is indicated as the induction (n-fold) relative to control cells. Protein expression from transfected plasmids was monitored by Western blot analysis using antibodies specific for GR and BRG1. Data are presented as the means of triplicate values obtained from a representative experiment that was independently repeated three times. K798R, BRG1-K798R; {Delta}E7, BRG1-{Delta}E7.

BRG1 induces GR-mediated chromatin remodeling of and transcription factor loading onto the integrated MMTV promoter. It has been demonstrated that the nucleosomal organization of the MMTV promoter undergoes a conformational change upon glucocorticoid treatment, inducing the Nuc-B region to become hypersensitive to restriction endonucleases (42). To determine if BRG1 remodeling is required for glucocorticoid-induced regions of hypersensitivity within the integrated promoter, restriction enzyme hypersensitivity assays were performed with SW-13/MG2-13 cells and various restriction endonucleases. MG2-13 cells were transfected with empty vector or the BRG1 or BRG1-K798R expression plasmid followed by Dex treatment. Nuclei were isolated and digested with restriction enzymes, which cleave within the Nuc-B region of the promoter (Fig. 5A). Compared with control groups, Dex treatment resulted in substantially elevated cleavage by restriction enzymes only in the presence of wild-type BRG1 protein (Fig. 5B). GR-mediated cleavage of treated cells expressing the remodeling mutant failed to induce enzyme-accessible regions within the MMTV promoter. Western blot analysis revealed that expression levels were equivalent between transfection groups and that Dex treatment had no effect on protein expression (data not shown). These results suggest that BRG1 protein must be present and contain ATPase activity for GR-mediated chromatin remodeling to take place, making the promoter hypersensitive to nuclease attack.



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  		FIG. 5. BRG1 induces GR-mediated restriction enzyme hypersensitivity in and transcription factor loading onto the integrated MMTV promoter. (A) Schematic of the proximal portion of the MMTV promoter indicating its chromatin structure, restriction endonuclease cleavage sites, binding site for NF1, and the position of the oligonucleotide used for PCR analysis (oligo22). (B) BRG1 induces GR-mediated disruption of the stably integrated MMTV promoter. MG2-13 cells were transfected with pBJ5 (empty vector), BRG1, or BRG1-K798R (K798R). Cells were treated with Dex, followed by nucleus isolation and in vivo digestion with SstI, MboI, or AflII. After purification, all samples were digested to completion with HaeIII and then subjected to reiterative primer extension. Purified extension products were resolved on 6% denaturing polyacrylamide gels and exposed to a phosphorimager screen. Quantitative analysis was performed with ImageQuant analysis software. Quantitation data are presented as the means of values obtained from three independently repeated experiments. (C) Exonuclease III footprint of NF1 binding on integrated MMTV promoter. MG2-13 cells were transfected with pBJ5, BRG1, or BRG1-K798R (K798R) constructs. Cells were treated with Dex, followed by nucleus isolation and digestion with HaeIII and exonuclease III. Samples were then subjected to reiterative primer extension and analyzed as described for panel B. The arrows indicate the exonuclease III boundary generated by NF1 binding and the cleavage site for HaeIII. Lane G, G sequencing track for position reference; lane M, molecular weight marker ({Phi}X, phiX174).

To determine if the BRG1 complex is required for hormone-induced transcription factor loading onto the integrated promoter, we utilized an in vivo exonuclease III footprinting assay (10, 42). SW-13/MG2-13 cells transfected with empty vector or the BRG1 or BRG1-K798R expression plasmid were used to investigate the effect BRG1 has on transcription factor binding onto the stably maintained promoter. In this assay, binding at the NF1 site was not observed in nonstimulated, vehicle-treated cells (Fig. 5C, lanes 1, 3, and 5). However, treatment with Dex resulted in increased NF1 protection in cells expressing wild-type BRG1 (Fig. 5C, lane 4). A fourfold increase in transcription factor binding was observed in treated cells expressing the wild-type BRG1 compared to the levels of treated groups transfected with empty vector or the remodeling mutant. Finally, because BRG1 is introduced via transient transfection with an efficiency of ~50%, this increase represents a minimal estimate of the enhanced transcription factor binding observed. The ability of BRG1 to permit hormone-dependent hypersensitivity and transcription factor binding within the nucleosome-assembled promoter strongly argues for the requirement of BRG1 in GR-mediated transcription in vivo.

The SWI/SNF remodeling complex physically associates with GR and the MMTV promoter in a hormone-dependent manner. Because T47D/A1-2 cells express BAF180 instead of BAF250 (Fig. 1C), the remodeling complex in these cells is most likely the PBAF complex (43, 51). In order to determine if ligand-bound GR interacts with the SWI/SNF complex in SW 13/MG2-13 cells, which express BAF250 but not BAF180 (Fig. 1C), IPs were performed from cells expressing BRG1. Total protein extracts from hormone-treated cells transfected with empty vector or a BRG1 expression plasmid were immunoprecipitated with antibodies specific for the BRG1 or BAF155 proteins. BRG1 interacts with the GR, and this association is significantly enhanced in the presence of glucocorticoids (Fig. 6A, compare lanes 12 to 16). Hormone-dependent GR binding was also observed in treated cells expressing BRG1 when they were immunoprecipitated with BAF155 antibodies (Fig. 6A, compare lanes 11 to 15). These data are consistent with previous observations that demonstrate that BAF250 is capable of interacting directly with the GR in vitro, which may aid the recruitment of the remodeling complex to the target promoter (34, 51). Binding was not observed in the treated group transfected with empty expression vector or when extracts were immunoprecipitated with normal immunoglobulin G (IgG) (Fig. 6A). These data suggest that, similar to the GR-PBAF hormone-dependent association seen in T47D/A1-2 cells (BAF180+), the GR interacts with the SWI/SNF complex in SW-13/MG2-13 cells (BAF250+) in a hormone-dependent manner.

To determine if GR and the BRG1 complex bind to the MMTV promoter in vivo, we performed chromatin IP assays using SW-13/MG2-13 cells with and without the expression of BRG1 protein. Cross-linked DNA fragments produced by sonication were immunoprecipitated with GR- or BRG1-specific antibodies and analyzed by PCR using MMTV primers spanning the glucocorticoid response element/Nuc-B region of the MMTV promoter. Occupancy of the promoter by GR was observed in a hormone-dependent manner, independent of BRG1 expression (Fig. 6B, lanes 5 and 6). In the presence of BRG1, the promoter sequence was significantly enriched when immunoprecipitated with both anti-GR and anti-BRG1 antibodies compared to IPs with normal IgG and the control fragment (Fig. 6B, lanes 5 to 8). These results indicate that the BRG1 complex and GR associate with the integrated promoter in living cells. Interestingly, our data suggest that GR binding to MMTV promoter does not require the remodeling complex although a slight increase in GR and promoter binding is observed in the presence of the remodeling complex. When purified immunocomplexes were analyzed by PCR with primers for a region of the luciferase gene ~2,000 bp downstream from transcription start site, no association of either GR or BRG1 was detected, indicating the specificity of GR and BRG1 recruitment to the MMTV promoter. These data suggest that ligand-induced binding of GR to the chromatin promoter may lead to recruitment of the BRG1 remodeling complex followed by transcriptional activation.

BRG1 enhances GR induction of endogenous genes within the SW-13 genome of MG2-13 cells. To identify glucocorticoid- responsive genes that require SWI/SNF activity, we performed RT-PCR analysis using cDNA produced from SW-13/MG2-13 cells transfected with empty vector (pBJ5), BRG1, or BRG1-K798R expression constructs, followed by treatment with vehicle or Dex and primers specific for glucocorticoid-responsive genes: 11-ß-HSD type 2 (14), GOS-8 (32), p21 (9), p53 (13), and I{kappa}B{alpha} (17) genes. Consistent with the activation of the MMTV promoter, we have identified endogenous promoters within the SW-13 genome that show enhanced GR-mediated transactivation in the presence of BRG1. Hormone treatment of cells expressing wild-type BRG1 led to a significant increase in 11-ß-HSD type 2 and p21 message in comparison to that for cells expressing mutant BRG1 protein (Fig. 7A). GR activation of I{kappa}B{alpha} is independent of BRG1 function (Fig. 7B), which is indicative of the previously described open chromatin architecture of the promoter (17), whereas message levels for p53 and GOS-8 remained unchanged. These findings suggest that BRG1 may be necessary for optimal gene transcription through chromatin-remodeling and/or coactivator activity at selected endogenous promoters (64).



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  		FIG. 7. BRG1 enhances GR-mediated transactivation of endogenous glucocorticoid-responsive genes with MG2-13 cells. (A) SW-13/MG2-13 cells were transfected with pBJ5 (empty vector), BRG1, or BRG1-K798R (K798R). Cells were treated with Dex (10-7 M; +) or vehicle (-) for 4 h, followed by RNA isolation. Total RNA was used for RT-PCR analysis with oligonucleotides specific for glucocorticoid-induced genes. (B) Quantitation data are presented as the means of values obtained from three independently repeated experiments. In measuring increases (n-fold) in activation, amplified products were normalized to ß2-microglobulin to correct for loading discrepancies before quantitative analysis was performed with ImageQuant analysis software.


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DISCUSSION
 
To delineate the molecular mechanisms involved in hormone-dependent transcriptional initiation, we developed an in vivo system to study BRG1 activity at glucocorticoid-responsive promoters. Using the SW-13 cell model, we have established a requirement for BRG1 chromatin-remodeling activity for GR-mediated transcriptional activation of the MMTV promoter within chromatin. Upon addition of the hormone, the GR recruits the remodeling complex to the MMTV promoter, where alterations in local nucleosomal structure, binding of transcription factors, and gene transcription follow (31). Similarly, a number of endogenous glucocorticoid-regulated genes within the SW-13 genome become responsive upon introduction of a functional BRG1 complex. Use of this model system reveals a number of distinct characteristics of the BRG1 chromatin-remodeling complex and permits dissection of the steps involved in gene transactivation.

Two distinct human SWI/SNF complexes have been reported, BAF and PBAF, that differ with respect to subunit composition. Both are reported to contain the following core components and accessory subunits: BAF170, BAF155, BAF60a, BAF57, BAF57, BAF53, BAF47, and actin (57). However, the BAF complex contains BAF250 and excludes BAF180, which harbors structural motifs characteristic of three components of the yeast Ras protein (43). In contrast, the PBAF complex includes BAF180 but not BAF250. This raises the interesting possibility that due to subunit composition, the two remodeling complexes are likely to share structural similarities but may possess functional or targeting differences. Western blot analysis has revealed the presence of BAF250 and the absence of BAF180 in SW-13 cells, suggesting that the introduction of BRG1 should result in the assembly of a BAF complex.

Recent in vitro biochemical purification of the PBAF and BAF complexes demonstrated that the PBAF but not the BAF complex would permit ligand-dependent transactivation by nuclear hormone receptors (43). These experiments with the VDR (vitamin D receptor), PPAR (peroxisome proliferator-activated receptor), and RXR (retinoid X receptor) also demonstrated that the ISWI-containing chromatin-remodeling complex ACF (ATP-utilizing chromatin assembly factor) failed to potentiate transcription in this system. Previous studies of human T47D cells that express BAF180 but not BAF250 have shown that transcription and chromatin remodeling mediated by both the progesterone receptor (PR) and GR are BRG1 dependent and mediated by the native PBAF complex (24, 51). The data presented here establish that the reconstituted BAF complex in SW-13 cells can mediate GR-dependent transcription from the MMTV promoter and a select number of endogenous promoters. Whether these differences represent the differences in experimental systems (in vitro versus in vivo) or between the members of the receptor superfamily (nuclear receptors versus steroid receptors) is unclear. However, the availability of T47D cells with the PBAF complex and of SW-13 cells, upon BRG1 introduction, with the BAF remodeling complex suggests that the GR may functionally participate with multiple forms of SWI/SNF remodeling complexes and will allow for a more informative comparison between the complexes expressed in the two human cell lines.

IP studies in SW-13 cells have shown that transient expression of BRG1 reconstitutes the complete remodeling complex. While theses cells contain no detectable BRG1, it has been reported that BAF-like complexes exist (46, 52). Coimmunoprecipitation studies have revealed the existence of a BAF170/BAF155 complex in SW-13 cells. Antibodies specific for BAF170 and BAF155 were able to pull down BAF155 and BAF170 protein, respectively, as well as BAF250 in the absence of BRG1 protein. The introduction of BRG1 leads to the formation of a complex containing the core subunits BAF170 and BAF155 and the central ATPase (Fig. 6A). Consistent with previous reports, these finding suggest that the expression of BRG1 in SW-13 cells leads to the assembly of the complete SWI/SNF-BAF complex (20, 54).

We examined transactivation from the MMTV promoter in SW-13 cells transiently transfected with an MMTV-reporter and GR, BRG1, or the chromatin-remodeling-deficient mutants BRG1-K798R or BRG1-{Delta}E7. In this assay, concentrations of BRG1 and mutant expression plasmids were transfected into cells to achieve equal levels of protein expression when compared by immunoblotting. In the presence of Dex, the degree of stimulation at the transient template was much greater when BRG1 and GR were coexpressed than when the proteins were expressed individually. Interestingly, a similar enhancement of induction was observed in cells expressing GR and the BRG1 mutants. In this assay, the transiently transfected MMTV promoter exists in an accessible structure where GR-mediated transcriptional activation does not require a remodeling step (3). Therefore, promoter activation by the chromatin-remodeling mutants suggests that transient templates do not require remodeling due to their constitutively open conformation.

The ligand-induced transcriptional activity in the presence of both wild-type and mutant BRG1 suggests that the reconstituted BRG1 complex may contain intrinsic coactivator activity independent of its ability to remodel chromatin. This activity may be an inherent property of BRG1 or may be recruited by the remodeling complex through direct or indirect interactions. A recent report suggests that the putative nuclear receptor coactivator TMF/ARA160 associates with the highly conserved N-terminal region of BRG1 (44). Among factors that bind BRG1, yeast two-hybrid screening identified the TMF/ARA160-BRG1 interaction. Originally isolated as a human immunodeficiency virus type 1 TATA modulator factor (hence, TMF) (26), this cofactor has been characterized as a coactivator (ARA160) for the androgen receptor (AR) (33). Ligand-dependent binding of this coactivator to the AR was observed both with in vitro pull-downs and with mammalian two-hybrid assays. The TMF/ARA160 coactivator was also shown to enhance transcriptional activity from the MMTV promoter in the presence of GR, AR, and PR in a ligand-dependent manner (44).

Recent studies have revealed that there is an ordered recruitment of chromatin-remodeling complexes and coactivators to the promoters of a variety of genes including those encoding the HO endonuclease, PHO80, {alpha}1-antitrypsin, beta interferon, and cathepsin D (1, 11, 12, 27, 56). An examination of chromatin remodeling by the BRG1 complex now provides a picture of the events involved in glucocorticoid-mediated gene activation. We showed that BRG1 expression in SW-13 cells induces the formation of the BAF remodeling complex (Fig. 1). We observed binding of the GR to the chromatin promoter in the absence of BRG1 (Fig. 6B). This interaction suggests that the receptor may recruit the SWI/SNF complex after binding to the promoter and induce transcriptional activation through chromatin-remodeling and/or coactivator activity. The association between GR and the remodeling complex has been described as occurring at least in part through an interaction with the BAF250 subunit and more recently via the BAF60a subunit (34, 51). Thus, it is intriguing that no interaction with these BAFs is seen in the absence of BRG1 (Fig. 6A), suggesting that the complex of the BAFs minus BRG1 is organized in a conformation that obscures the GR interaction surfaces. Alternatively, the presence of BRG1 in the SW-13 cells may facilitate and/or stabilize GR interactions with these BAFs in vivo. Further biochemical characterization of the complex of BAFs that forms in the absence of BRG1 and its comparison to a full BRG1-containing complex, as well as a molecular description of the GR-interacting surfaces, will be necessary to resolve this issue.

The data establish that the in vivo transcriptional activity of the BRG1 complex is dependent on a functional BRG1 ATPase domain, given that ATPase mutants were unable to remodel or initiate gene transcription from the chromatin promoter. The results also indicate that the chromatin-remodeling activity of BRG1 cannot be replaced by other ATP-dependent chromatin-remodeling proteins expressed in this cell type. SW-13 cells express the proteins ISWI and Mi-2 (Fig. 1B) involved in ATP-dependent chromatin remodeling. In the absence of a functional BRG1, ligand-bound GR was unable to potentiate a response from (Fig. 4) or alter the structure of the integrated promoter (Fig. 5), suggesting that ISWI and Mi-2 are unable to remodel the MMTV promoter under our experimental conditions. This finding is in contrast to published reports of in vitro assays that implicate an ATP-dependent chromatin-remodeling ISWI-containing complex in transactivation by the PR (19). However, it is supported by recent in vivo studies of SW-13 cells which found that the PR requires a functional BRG1 complex to activate transcription, as seen with the GR (K. W. Trotter and T. K. Archer, unpublished results). Indeed, another in vitro transcription study demonstrated that human ISWI-containing remodeling complex ACF failed to potentiate transcription from chromatin templates by the VDR and RXR (43). These disparate findings may be a consequence of the intrinsic disparities found between the in vitro assays and the in vivo system employed. Together, these finding suggest that, at least in vivo, different ATP-dependent chromatin-remodeling complexes, although similar in function, are not interchangeable in supporting transcription. However, with a better understanding of the processes involved in GR-mediated chromatin remodeling of the MMTV promoter by both BAF and PBAF complexes, the potential mechanisms involved in transcription initiation will emerge (16).


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ACKNOWLEDGMENTS
 
We thank Maureen Bunger, Parthiba Hebbar, H. Karimi Kinyamu, and members of the Archer laboratory for critical reading of the manuscript; Adam Brothers for help in the initial screening of cell lines; Paul Wade (Emory University) and Weidong Wang (NIA/NIH) for the ISWI and BAF250 antibodies, respectively; and Robert Kingston (MGH/Harvard University) and Keith Yamamoto (UCSF) for various expression plasmids used in the study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Dr., P. O. Box 12233 (MD E4-06), Research Triangle Park, NC 27709. Phone: (919) 316-4565. Fax: (919) 316-4566. E-mail: archer1{at}niehs.nih.gov. Back


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Molecular and Cellular Biology, April 2004, p. 3347-3358, Vol. 24, No. 8
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.8.3347-3358.2004




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