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Department of Cell Biology,1 Center for Environmental Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio,3 The Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina2
Received 14 June 2004/ Returned for modification 26 July 2004/ Accepted 29 November 2004
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ABSTRACT |
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INTRODUCTION |
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The AR is activated by ligand binding to induce transactivation potential. Testosterone, the most prevalent AR ligand in the sera, is converted to dihydrotestosterone (DHT) in the prostate through the action of 5-alpha reductase. DHT serves as a high-affinity ligand for the AR and stimulates a cascade of events that initiate AR activation (6, 16, 52). Initially, AR is released from heat shock proteins, undergoes homodimerization, and rapidly translocates to the nucleus, where it is targeted to androgen response elements (AREs) located within AR target gene promoters/enhancers. Once activated by ligand, AR recruits a host of cofactors that facilitate the formation of an active transcriptional complex (23, 59). In general, these cofactors act to release DNA from chromatin through either covalent modification of histones or remodeling of chromatin structure. Such modifications foster a state of open chromatin, allowing recruitment of basal transcription machinery and RNA polymerase II (27, 46, 62, 68).
Several classes of AR coactivators promote histone modifications. Members of the p160 coactivator family (including SRC1, SRC2/TIF2/GRIP1, and SRC3/RAC3/AIB1) serve as platforms for the recruitment of histone acetyltransferases (HATs), such as CBP/p300 or p/CAF. SRC1 and SRC3 also harbor intrinsic HAT activity, which likely contributes to their action as coactivators (12, 63, 67). Acetylation of core histones by the recruited HATs results in a displacement or loosening of DNA from the nucleosome, thus facilitating the recruitment of the subsequent cofactors and the basal transcription machinery. In addition, p/CAF has been shown to acetylate the AR itself, an event shown to be essential for AR activity (20). Thus, through their ability to modify both AR and core histones, the p160 coactivators play pivotal roles in AR activation. The importance of such coactivator regulation in prostate cancer is apparent, since aberrant expression of both SRC1 and TIF2 is associated with relapse after therapeutic intervention (21, 24). Many additional AR coactivators have been identified whose actions are less well described. For example, ARA70 and ARA55, in addition to enhancing AR activity, are able to increase the androgenic activity of anti-androgens such as hydroxyflutamide (22, 44). Recently, ARA70 expression was shown to be enhanced in the neoplastic prostate compared to benign tissue, thus further implicating a role for this coactivator in prostate cancer progression (29). Lastly, the E2 conjugating enzyme, Ubc9, has been shown to bind directly to and activate the AR, independent of its SUMO-1 conjugation function (51). The coordinate actions of these coactivators are thought to play pivotal roles in AR activation and likely influence tissue specific AR activity. The contribution of these coactivators to histone acetylation and cooperation with the p160 family has yet to be thoroughly examined.
Although the p160 coactivators initiate histone modifications through acetylation, it has been shown that several nuclear receptors also attract complexes that enzymatically regulate chromatin structure. Several classes of chromatin remodeling complexes have been described, including WINAC, NuRD, ISWI, and SWI/SNF (4, 39, 46, 70, 72). ARIP4 has been described as a SWI/SNF-like chromatin remodeling enzyme and was shown to modulate AR activity (57, 61). In general, chromatin remodeling complexes utilize the hydrolysis of ATP to disrupt histone-DNA association, thus altering chromatin structure to foster a state of either closed chromatin (resulting in gene repression) or open chromatin (resulting in enhanced gene transcription). Although the precise mechanisms of SWI/SNF action in vivo have yet to be elucidated, associated activities in vitro include alterations in nucleosome structure, nucleosome sliding, dinucleosome formation, and histone octamer transfer (17, 46). Such alterations in chromatin structure can be distinguished from histone modifications and are not dependent on histone acetylation (30). Moreover, SWI/SNF action has been shown to contribute to the dynamic activity of nuclear receptors, facilitating rapid mobilization of the receptors after SWI/SNF recruitment (18). The activity of several nuclear receptors, including the estrogen receptor (ER) and glucocorticoid receptor (GR), has been shown to be enhanced by the action of SWI/SNF (3, 10, 14, 18, 19, 31, 45, 50, 72, 74). SWI/SNF represents a class of large, 2-MDa chromatin remodeling complexes that contain a central ATPase (either BRM/SNF2
or BRG1/SNF2ß) and a variable series of associated subunits termed BRG1-associated factors (BAFs). SWI/SNF exists as a multitude of biochemically diverse complexes, and although the function of each specialized complex remains unclear, it has been speculated that variances in combinatorial assembly dictate diverse biological outcomes. Supporting this view, it has been shown that BRG1 and BRM demonstrate specificity for protein-protein interaction and likely govern unique gene targets (35). Although some redundancies do exist, BRG1 is predominantly found in cell types undergoing proliferation or self-renewal, whereas BRM expression is increased after cellular differentiation. Interestingly, BRM was recently shown to be expressed in normal human prostatic epithelia, whereas BRG1 could not be detected in this tissue (53). We and others have shown that the AR requires SWI/SNF function for activity, and our studies highlighted a specific role for BRM complexes in AR regulation (30, 32, 43). However, the mechanism by which BRM specifically regulates AR activity remained undetermined.
It is speculated that the BAF subunits largely contribute to SWI/SNF specificity (47). Subunits commonly associated with the SWI/SNF complex include BAF155, BAF170, INI1/SNF5, BAF250, BAF60, and BAF57 (46, 71, 72). Several of these subunits have been shown to harbor critical functions. For example, the loss of the INI1/SNF5 subunit gives rise to rhabdoid tumors in humans and can promote tumorigenesis, thus identifying INI1/SNF5 as a tumor suppressor (69). Subsequent studies demonstrated that reintroduction of INI1/SNF5 into human tumor cell lines deficient in this subunit induced cell cycle arrest (5). Other subunits, including BAF250, BAF60a, and BAF57, have been shown to directly interact with the GR and to promote GR activation (28, 47). Lastly, the BAF57 subunit has been shown to interact with both the ER and the p160 coactivators. Although BAF57 function was dispensable for ER activation, BAF57 was essential for SRC1 to modulate the ER (3). Collectively, these observations implicate SWI/SNF subunits as critical regulators of nuclear receptor-SWI/SNF interactions.
BAF57 contains DNA-binding capability through its high-mobility-group (HMG) motif and a kinesin-like coiled-coil domain and is suggested to play an architectural role in the formation of higher-order protein complexes. Although these properties of BAF57 are dispensable for SWI/SNF chromatin remodeling activity (71), it is hypothesized that BAF57 may lend specificity in targeting to the SWI/SNF complex. This contention was supported by observations in transgenic mice, wherein mutations in the HMG domain of BAF57 inhibited specific SWI/SNF target gene regulation (9).
We show here that BAF57 is expressed in the AR-positive luminal epithelial layer of the prostate and that BAF57 strongly regulates AR function. We show that BAF57 directly binds to AR and is recruited to AR regulatory regions in a ligand-dependent fashion. Using both exogenous and endogenous prostate-specific AR targets, we show that AR requires BAF57 function for robust transcriptional response. We also demonstrate that this function of BAF57 requires SWI/SNF ATPase function, firmly linking BAF57 action to alterations in nucleosome structure. Similar to what was observed with ER, BAF57 also facilitated p160-mediated AR enhancement, and a similar profile was observed with Ubc9. Interestingly, ARA70 and ARA55 maintained activity in the absence of BAF57 but were augmented by BAF57 for AR activation. Thus, BAF57 exhibited coactivator specific modulation of AR activity. Given the observed necessity of BAF57 for AR target gene transcription and coactivator function, we examined the requirement of BAF57 and SWI/SNF for proliferation in AR-dependent prostate cancer cell growth. Strikingly, inhibition of either BAF57 or BRM function significantly attenuated cellular proliferation in this cell type. Collectively, these data establish a firm requirement of BAF57 for AR and coactivator function. Moreover, these data implicate BRM and BAF57 as putative therapeutic targets in prostate cancer therapy.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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46-408) was received from Ollie Janne (University of University of Helsinki) (49). pCMV-p16ink4a was previously described (40). pBabe-WTBAF57-FLAG and pBabe-BAF57N-FLAG were constructed by PCR amplification of each gene from the pBJ5 vector by using primers that added BamHI (5') and EcoRI/FLAG (3'). The PCR product was digested and inserted into the pBabe vector (which also encodes the puromycin resistance marker). The pHTP-BRMi construct uses a 64-mer double-stranded oligonucleotide to generate a shRNA targeting nucleotides 3388 to 3408 of the BRM mRNA. The fragment (or a scrambled control of the siRNA oligonucleotide sequence) was cloned into the pHTP-siRNA expression vector (kindly provided by William Reed, University of North Carolina at Chapel Hill) by using BglII and XhoI overhangs. The vector includes a puromycin resistance marker and places the expression oligonucleotide under the control of the human H1 promoter. pGreenLantern (Gibco) encodes the green fluorescent protein (GFP). Immunohistochemistry. Prostates were resected from 9-week-old FVB/N mice. Individual lobes (ventral, dorsolateral, and anterior) were isolated, fixed by immersion in 10% neutral-buffered formalin, and subsequently embedded in paraffin. Blocks were sectioned (5 µm), mounted on slides, deparaffinized in xylene, and rehydrated in ethanol solutions. After antigen retrieval by boiling for 20 min in Antigen Unmasking Solution (Vector Laboratories), the sections were incubated for 15 min in peroxidase blocking reagent (Dako Cytomation) to quench endogenous peroxidase. The sections were then blocked for 20 min in diluted normal goat serum (Rabbit IgG Vectastain ABC Kit; Vector Laboratories), followed by incubation with anti-BAF57c rabbit polyclonal antibody (1:1,000 dilution in phosphate-buffered saline [PBS] solution) overnight at 4°C. After incubation for 30 min with anti-rabbit immunoglobulin G (IgG) biotinylated secondary antibody solution (Rabbit IgG Vectastain ABC Kit; Vector Laboratories), sections were incubated in ABC reagent for another 30 min. Antibodies were visualized with DAB Chromogen (Dako Cytomation), and nuclei were counterstained with hematoxylin.
Tissue culture and treatment. BT459 cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, Ga.), 2 mM L-glutamine, and 100 U of penicillin-streptomycin (Mediatech, Herndon, Va.)/ml. For reporter analyses, BT549 cells were cultured in phenol red-free RPMI medium and 10% charcoal dextran-treated FBS (CDT; Atlanta Biologicals). LNCaP cells received from the ATCC were cultured in improved minimal essential medium (IMEM) supplemented with 5% heat inactivated FBS, 2 mM L-glutamine, and 100 U of penicillin-streptomycin/ml. LNCaP reporter assays were performed in phenol red-free IMEM supplemented with 5% CDT. MCF7, CV1, and SW13 cells were obtained from the ATCC and maintained in Dulbecco modified Eagle medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U of penicillin-streptomycin/ml. Reporter analysis in these cell lines was carried out in phenol red-free medium containing 10% CDT.
LB1-luc cells were generated from the LNCaP cell line by cotransfection of the pARR2-Luc reporter construct (43) and the pBabe-PURO vector (which confers puromycin resistance). After selection in puromycin, individual colonies were isolated and screened for retention of ARR2 reporter activity. The resulting LB1-Luc cell line was tested by seeding 105 cells into a 12-well dish with IMEM medium containing 5% CDT. Cells were cultured in this condition of steroid deprivation for 3 days and were then stimulated with the indicated concentrations of DHT (Sigma). Cells were harvested and measured for luciferase activity corrected against total protein (as determined via the Bio-Rad-DC assay [Bio-Rad]).
Transfection and reporter analysis.
MCF7, CV1, and SW13 cells were seeded in Dulbecco modified Eagle medium containing 10% CDT. Cells were transfected the following day using the BES-calcium phosphate protocol as previously described (8). Cells were transfected with 0.5 µg of pARR2-Luc, 0.5 µg of pSG5AR, 0.25 µg of pTK-Renilla luciferase, 2.25 µg of pBabe-WTBAF57-FLAG, 1 µg of pCG-hBRM, or parental vector (to a total of 4.5 µg) as indicated. Posttransfection, cells were treated with 0.1% ethanol (EtOH) vehicle or 0.1 nM DHT for 24 h. We used 0.1 nM DHT since this is the optimal mitogenic dose for prostate cancer cells (0.1 to 1.0 nM DHT) (37). Cells were then harvested, lysed, and assayed for luciferase activity using the Dual Luciferase Assay System (Promega). For BT549 reporter analyses, cells were seeded in six-well dishes with RPMI containing 10% CDT. Cells were transfected the following day using the FuGENE6 (Roche Molecular Biochemicals) method as recommended by the manufacturer. Transfections consisted of 0.5 µg of pARR2-Luc reporter, 0.5 µg of pSG5AR, and 2.25 µg of pBabe-BAF57-FLAG (unless otherwise indicated), and/or 1 µg of coactivator (pCR3.1-SRC1, pCMV-TIF2, pFLAG-Ubc9, pSG5ARA70, or pSG5ARA55) or 1 µg of pCG-dnBRM or pSG5ARA70N2 as indicated. For each transfection, 0.25 µg of pTK-Renilla luciferase was used to normalize for transfection efficiency. Total plasmid DNA was brought to 4.5 µg with the parental (empty) vector. After transfection, fresh medium was added and cells were treated with either 0.1% EtOH or 0.1 nM DHT for 24 h. After stimulation, cells were harvested and lysed, and reporter analyses were carried out by using the Dual Luciferase Assay Reporter System (Promega). LNCaP reporter analysis was carried out in six-well dishes with IMEM containing 5% CDT. Cells were transfected by using the Lipofectin (Invitrogen) transfection reagent as recommended by the manufacturer. LNCaP cells were transfected with 1 µg of pARR2-Luc reporter and 2.25 µg of pBabe-BAF57N-FLAG or parental vector (to 3.5 µg of total DNA). Next, 0.25 µg of pTK-Renilla luciferase was used to normalize for transfection efficiency. After transfection, cells were stimulated with 1 nM DHT. After 48 h, cells were harvested, and the Dual Luciferase Assay Reporter System was used to measure relative luciferase activity. Reporter assays were performed from at least in six independent transfections for each condition. Averages and standard deviations are shown. Statistical analysis was performed by using the Student t test, with a P value of <0.05 indicating statistical significance. Transfection method was chosen based on the optimal method for each cell line (determined empirically using pTK-Renilla luciferase). Where indicated, parallel transfections were performed in which the internal control plasmid was replaced by pGreenLantern, and cells were harvested to monitor relative AR expression levels via immunoblot.
Immunoblotting. For protein analysis, cells were harvested by trypsinization (for mouse prostate, individual lobes were microdissected and ground), and lysis was carried out in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris [pH 8.0]) supplemented with protease inhibitors and phenylmethylsulfonyl fluoride (PMSF). After brief sonication and clarification, equal protein was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to an Immobilon-P membrane (Millipore). The membrane was immunoblotted with the indicated antibodies by standard techniques and visualized by chemiluminescence (Western Lightning; Perkin-Elmer Life Sciences). The antibodies used were generated against BAF57c (a gift from Weidong Wang), AR (N-20; Santa Cruz), GFP (B2; Santa Cruz), and CDK4 (H-22; Santa Cruz).
Immunoblotting to verify BRM knockdown via small interfering RNA (siRNA) was carried out by transfection with 7 µg of either pHTP-vector or pHTP-BRMi, along with cotransfection of 1 µg of pEGFP H2B-GFP into LNCaP cells. Posttransfection, cells were selected by using 0.625 µg of puromycin (Sigma)/ml. Cells were simultaneously harvested after >90% of the population was H2B-GFP positive (ca. 3 days). Pellets were rinsed in cold phosphate-buffered saline (PBS) and resuspended in buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol [DTT], 0.5 mM PMSF, and 0.5 mg of leupeptin and 1 mg of aprotinin/ml), followed by incubation on ice for 20 min. Next, 6.5 µl of 10% NP-40 was then added, and the pellets were vortexed and centrifuged for 5 min at 4°C. The supernatant was removed, and the pellet was resuspended in 50 µl of cold buffer C (20 mM HEPES [pH 7.9], 400 mM NaCl, 15 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 0.5 mg of leupeptin and 1 mg of aprotinin/ml). Equal amounts of protein were subjected to SDS-7.5% PAGE and transferred to an Immobilon-P membrane (Millipore). Immunoblotting was performed with the indicated antibodies by standard techniques. Antibodies used were generated against BRM (courtesy of Moshe Yaniv) and actin (Sigma).
GST pull-down. AR protein was produced and radiolabeled with [35S]methionine by using the TnT-Coupled Reticulocyte Lysate system (Promega) for in vitro transcription and translation according to the manufacturer's specifications and the NEG-772 Easytag Express protein labeling mix (Perkin-Elmer Life Sciences). Glutathione S-transferase (GST) or GST-BAF57 was transformed into BL21 bacteria and induced by using 0.1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 3 to 5 h. After centrifugation, bacteria were washed one time in STE (150 mM NaCl, 25 mM Tris [pH 8.0], and 1 mM EDTA) and then resuspended in 3 ml of STE. Pellets were lysed upon the addition of 0.1 mg of lysozyme/ml and 1.5% Sarkosyl, sonicated, and clarified by centrifugation. The lysate was then incubated with 300 µl of glutathione-agarose beads at 4°C overnight. Beads were washed four times in PBS and then incubated with the in vitro-translated protein for 3 h at 4°C. Reactions were performed in 500 µl of NETN (20 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 0.5% NP-40) supplemented with PMSF and protease inhibitors and in the presence of either 0.1% EtOH or 10 nM DHT, as indicated. "Input" (3% of total) and "bound" fractions were separated by SDS-10% PAGE, stained with Coomassie blue, and subjected to fluorography by using Fluoro-Hance (Research Products International Corp., Mount Prospect, Ill.). Labeled protein was detected by autoradiography and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).
Coimmunoprecipitation. Immunoprecipitations were performed as previously described (28). LNCaP cells were harvested, washed in PBS, and lysed in immunoprecipitation buffer (IP buffer; 20 mM HEPES [pH 7.9], 250 mM NaCl, 10% [vol/vol] glycerol, 0.1% [vol/vol] Tween 20, 0.2 mM EDTA, 2 mM DTT) supplemented with protease inhibitors, 0.5 mM PMSF, and 100 ng of leupeptin and aprotinin per ml. After sonication and clarification, protein was quantified by using the Bio-Rad DC protein assay kit. A total of 1 mg of protein was used for each immunoprecipitation reaction. Lysate was precleared with 1% preimmune rabbit serum and 20 µl of protein-A Sepharose (Amersham) in 0.5 ml of IP buffer for 1 h at 4°C. Anti-AR, anti-BAF57, and preimmune serum (negative control) were incubated with the lysate overnight at 4°C. Then, 20 µl of protein-A Sepharose was added for a 1-h incubation. Beads were subjected to five washes in IP buffer, resuspended in 2x SDS-PAGE buffer, and boiled to denature proteins. Samples were then subjected to SDS-PAGE and immunoblot analysis with antibody generated against AR (AR-N20; Santa Cruz).
ChIP.
LNCaP or LB1-Luc cells were seeded into 15-cm dishes in phenol red-free IMEM supplemented with 5% CDT. After 4 days of incubation, cells were stimulated with either 0.1% EtOH or 10 nM DHT for 60 min. A higher than physiological dose was utilized for chromatin immunoprecipitation (ChIP) analysis (consistent with the literature) in order to enhance AR on target promoters (59). Cells were then treated with 1% formaldehyde diluted in PBS to allow for protein cross-linking to DNA, followed by quenching with 0.125 M glycine. Cells were then washed with ice-cold PBS, treated with trypsin, and pelleted. Pellets were washed in PBS containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 100 ng of leupeptin and aprotinin per ml. Cells were lysed in cell lysis buffer (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40, 0.5 mM PMSF, and 100 ng of leupeptin and aprotinin per ml) for 10 min. Nuclei were lysed in 400 µl of nucleus lysis buffer (1% sodium dodecyl sulfate [SDS], 50 mM Tris-Cl [pH 8.1], 10 mM EDTA, 0.5 mM PMSF, 100 ng of leupeptin and aprotinin per ml) for 10 min, and sonication was performed to shear DNA. Immunoprecipitations were carried out with 80 µl of lysate and antibodies to AR (N-20; Santa Cruz), acetylated histone H4 (catalog no. 06-866; Upstate Biotechnology), BAF57c (specific for C terminus), BAF57h (specific for HMG domain; a gift from Weidong Wang) (71), and a control IgG antibody (preimmune sera). Chromatin and antibody were incubated in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris [pH 8.0]) for 1.5 h at 4°C, at which time protein-A Sepharose (Amersham) was added for an additional incubation. Beads were washed three times with super-RIPA (RIPA buffer plus 300 mM NaCl), once with RIPA buffer, and once with TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA), with 10-min rotations between each wash at room temperature. Next, 300 µl of ChIP extraction buffer (1% SDS, 0.1 M NaHCO3) was added, along with 20 µl of 5 M NaCl and RNase A. Cross-linking was reversed by incubation at 65°C (4 h up to overnight). DNA was purified by using the QIAquick PCR purification kit (Qiagen). PCR amplification was performed on recovered DNA products as well as input chromatin (10% of input for Fig. 6A and C; 1% of input in Fig. 6B) in the presence of [-32P]dCTP, which allows accurate quantification of the PCR products over a broad range of DNA concentrations (60). To monitor recruitment to the prostate specific antigen (PSA) enhancer, PCR was performed by using G/H primers, as previously described (59). These primers are specific for a region containing an androgen responsive element (AREIII) contained within the enhancer region of the PSA gene. For PCR, the parameters were as follows: 94°C for 5 min; followed by 28 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and finally 72°C for 10 min. To monitor recruitment to the probasin promoter, the primers 5'-CATCTACCATTCCAGTTAAG (forward) and 5'-CCAGCGGTTCCATCC (reverse) were used. PCR parameters were 94°C for 5 min; followed by 40 cycles of 94°C for 45 s, 49°C for 45 s, and 72°C for 45 s. PCR products were separated on a 6% polyacrylamide gel and visualized with a PhosphorImager.
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N-FLAG, or dominant-negative AR (pSG5AR46-408, positive control). After transfection, cells were allowed to recover for ca. 24 h. Transfected cells were selected by using 0.625 µg of puromycin (Sigma)/ml. Selection was carried out until >90% of cells were H2B-GFP positive (ca. 3 days). RNA was isolated by using the TRIzol Reagent (Gibco) as recommended by the manufacturer. To quantify expression, reverse transcription-PCR (RT-PCR) was performed with amplification of the cDNA performed in the presence of [32P]dCTP to quantify expression over a large linear range, as previously described (60). Primers were used for PSA (primer pair 5'-CTTGTAGCCTCTCGTGGCAG-3' and 5'-GACCTTCATAGCATCCGTGAG-3') and GAPDH (loading control; primer pair 5'-CCACCCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3'). PCR conditions were as follows: 94°C for 2 min; followed by 23 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s; and finally 72°C for 10 min. PCRs were separated by PAGE, and signal was quantified on a PhosphorImager. For comparison, maximal AR activity (PSA and GAPDH) in the presence of vector was set to 100. Representative data from two independent experiments are shown.
BrdU incorporation.
LNCaP cells were seeded onto poly-L-lysine coated coverslips, followed by transfection with Lipofectin, as described above. Cells were transfected with 0.5 µg of H2B-GFP, along with 4.5 µg of one of the following expression plasmids: pBabe-PURO, pBabe-BAF57N-FLAG, dominant-negative AR (pSG5AR46-408), pHTP vector, pHTP-scrambled, pHTP-BRMi, or pCMV-p16ink4a. Posttransfection (3 days for the pBabe-PURO/BAF57N/dnAR experiment or 4 days for the pHTP-vector/pHTP-BRMi/pCMV-p16ink4a experiment), bromodeoxyuridine (BrdU; Amersham Biosciences) labeling reagent was added, followed by incubation for 16 h. Detection of BrdU by indirect immunofluorescence was performed as previously described (41). A minimum of 150 H2B-GFP transfected cells per sample were counted for BrdU incorporation. The results are plotted as the percent inhibition versus vector control, and the average of at least three replicates from two independent experiments is shown.
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RESULTS |
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N, was utilized to suppress endogenous function. BAF57N lacks the proline-rich region and the HMG domain (N terminus) and has been shown to block BAF57 activity and downregulate endogenous BAF57 expression (via immunoblot analysis) in the thymocytes of BAF57N transgenic mice (9). Importantly, this allele has been shown to retain association with SWI/SNF. For these experiments, LNCaP cells were transfected in the absence of ligand with pARR2-Luc and either pBabe-BAF57N or vector control to monitor the impact on endogenous AR activity (Fig. 4A). As expected, ligand induced robust activation of the pARR2-Luc target gene by endogenous AR in the presence of empty vector (28-fold over basal activity). In contrast, there was a significant reduction in endogenous AR activation of the reporter upon transfection of pBabe-BAF57N (an 
2-fold reduction in activity), thereby demonstrating the importance of BAF57 for endogenous AR function. Furthermore, endogenous AR levels remained constant upon transfection of vector control and pBabe-BAF57N relative to control (CDK4) (Fig. 4B). Transient introduction of pBabe-BAF57N into LNCaP cells demonstrated no effect on BAF57 expression levels (data not shown), thus indicating that in this system, BAF57-N functions through its defined dominant-negative capabilities. Ectopic expression of wild-type BAF57 has been previously demonstrated to also act in a dominant-negative fashion in cell lines containing endogenous BAF57, presumably by titrating endogenous complexes (3). Combined, our observations indicate that inhibition of BAF57 activity reduces AR function.
To verify this hypothesis, the effect of BAF57N on an endogenous AR target gene (PSA) was examined. For these experiments, LNCaP cells were transfected with pBabe-puro (encoding a puromycin resistance marker), pEGFP H2B-GFP (to monitor transfection and/or selection), and either vector control, pBabe-BAF57N, or pSG5-AR46-408 (dominant-negative AR) as a positive control. After rapid selection with puromycin, RNA was isolated, and RT-PCR amplification was performed in the presence of [32P]dCTP to examine PSA mRNA expression (Fig. 4C). As expected, dnAR significantly reduced PSA mRNA expression, relative to vector control (upper panel, compare lanes 1 and 3). Strikingly, BAF57-N similarly attenuated PSA expression (upper panel, compare lanes 1 and 2). Quantification of these data indicate that BAF57-N inhibits PSA expression similar to dominant-negative AR (lower panel). Together, these data confirm that disruption of BAF57 function inhibits AR activity on both exogenous and endogenous AR targets.
SWI/SNF ATPase activity is required for BAF57 function. Although these data indicate a requirement of BAF57 for AR transactivation potential, the necessity of SWI/SNF ATPase activity for BAF57 function has not been demonstrated. This point is particularly salient since BAF57 can bind DNA independent of the SWI/SNF complex (71). Therefore, we initially tested the ability of BAF57 to promote AR function in coordination with the preferred core ATPase, BRM, in BT549 cells. Although null for BAF57, BT549 cells have been shown previously to express the BRM ATPase (13). Ectopic expression of dominant-negative BRM (pCG-dnBRM) was utilized to examine the impact of the core ATPase in the absence of BAF57. This allele of BRM has been previously described (45) and encodes an ATPase defective mutant protein which can still assemble into SWI/SNF but fails to remodel chromatin. Consistent with our previous observations (Fig. 3D), ligand induced a similar fold induction (i.e., eightfold) over basal activity (EtOH control) (Fig. 5A). Moreover, dnBRM failed to promote receptor activity and slightly inhibited residual AR activity. This may be indicative of the ability of SWI/SNF to regulate AR activity even in the absence of BAF57 (albeit inefficiently). As expected, BAF57 restored AR activation (Fig. 5A). However, BAF57-mediated AR activation was significantly reduced upon expression of dnBRM, thus indicating that BAF57 requires SWI/SNF ATPase function to efficiently promote AR activity. Parallel experiments with GFP expression as an internal control demonstrated that expression of dnBRM had no impact on AR protein levels (Fig. 5B). To verify the requirement of BAF57 for SWI/SNF function, SW13 cells were utilized. These cells are null for both BRG1 and BRM and thus lack SWI/SNF activity. However, these cells do express BAF57 (13). Consistent with our published findings (43), SW13 cells failed to support significant AR activity in reporter assays (Fig. 5C); however, expression of BRM restored AR function. Moreover, BAF57 failed to promote AR activity in this model system, thus demonstrating that BAF57 action is dependent on a functional SWI/SNF complex.
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BAF57 functions cooperatively with p160 HAT coactivators to activate transcription of AR target genes. A role for BAF57 has been previously established in p160 HAT coactivator mediated function, using ER as a model (3). Interestingly, p160 coactivators have been shown to target histones H3 and H4 for acetylation (38, 42, 64). Since BAF57 recruitment was observed when histones were acetylated (Fig. 6), we analyzed the impact of BAF57 in coordination with p160 coactivators (Fig. 7) to enhance AR activity. For these experiments, reporter analyses were performed in BT549 cells as in Fig. 3. As shown, SRC1 expression failed to generate significant AR activity in the presence of ligand compared to vector control. As expected, BAF57 restored significant AR activity, a finding consistent with our previous results (Fig. 3D). However, the combination of SRC1 and BAF57 led to markedly enhanced AR activity (10.3-fold over SRC1 alone and 2.1-fold over BAF57 alone) (Fig. 7A). Interestingly, unlike SRC1 alone, TIF2 by itself appeared to significantly enhance AR activity similar to BAF57 alone (Fig. 7B). However, a similar profile to SRC1 was observed upon cotransfection of TIF2 and BAF57 (3.2-fold versus TIF2 alone and 2.5-fold versus BAF57 alone). These data indicate that BAF57 may cooperate with p160 coactivators to enhance AR activity.
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N) into LNCaP cells. This approach was utilized since attempts to target BAF57 using siRNA technology have not yet proven successful (data not shown). This experiment was performed by transfection of LNCaP cells with pEGFP-H2B-GFP (to mark transfected cells) and either parental vector, pBabe-BAF57N, or pSG5AR46-408 (dnAR). As expected, dnAR attenuated LNCaP cell proliferation (34% inhibition) (Fig. 9A, right panel). Representative data from two independent experiments are shown in Fig. 9A (left panel) and demonstrate that cells transfected with pBabe-BAF57N were dramatically inhibited for BrdU incorporation. The percent inhibition of BrdU incorporation over vector control was measured (Fig. 9A, right panel). Interestingly, inhibition of BAF57 slows proliferation of LNCaP cells (35% inhibition) over vector control in a manner similar to the positive control, dnAR.
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DISCUSSION |
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Although the SWI/SNF complexes are widely expressed, their function results in a multitude of diverse biological outcomes. For example, association of SWI/SNF with the GR is required for GR-mediated transcriptional activation (45, 74). In contrast, both core ATPase subunits (BRG1 and BRM) associate with the retinoblastoma tumor suppressor protein, RB, to regulate RB-dependent transcriptional repression of E2F target genes. This mechanism of repressive action is controversial in that it has been demonstrated to occur through direct interaction between the ATPase and RB (15, 54, 65, 66, 75), as well as through upregulation of the cdk inhibitor p21cip1 (36). This function of RB is essential for its antiproliferative function, and loss of SWI/SNF association renders all tested cells resistant to RB-mediated growth arrest (54, 66). Thus, SWI/SNF can confer either transcriptional activation or repression. Specificity for SWI/SNF action is purported to be governed by the biochemical diversity and plasticity of SWI/SNF subunit composition, wherein selected subunits likely regulate tissue or cell-type-specific protein-protein interactions.
Targeted deletion of the core ATPases in mouse model systems first revealed nonredundancies in SWI/SNF action. Deletion of BRG1 is an embryonic lethal event (7), whereas BRM–/– mice are viable and demonstrate increased body weight (55). Intriguingly, BRM-null mice exhibited slightly reduced testicular weight, indicating a putative defect in androgen-dependent development (55). A specificity for the ATPase has also been shown biochemically, wherein BRG1 was shown to preferentially regulate selected zinc finger proteins, whereas BRM interacted preferentially with members of the Notch pathway (35). Variations in cell type expression may partially underlie specificity of ATPase action, since BRG1 is primarily expressed in proliferating or self-renewing cells, and BRM protein levels are enriched in differentiated cell types. Differentiated prostatic epithelia and prostatic adenocarcinoma cells require AR for survival and, consistent with the role of BRM in such cell types, we have shown that the AR is preferentially activated by BRM-containing SWI/SNF complexes (43). Furthermore, in support of this observation, normal human prostate epithelia were recently shown to express the BRM ATPase; in contrast, BRG1 was not detected (53). Thus, the core ATPase lends some specificity to SWI/SNF modulation of the AR. However, no direct interaction between the AR and BRM was observed in vitro (data not shown), thus indicating that SWI/SNF action must be either indirect or through association with alternate SWI/SNF subunits.
The ability of SWI/SNF subunits to direct the complex to sequence-specific transcriptional complexes and thus regulate discrete biological processes has strong precedent. For example, loss of the SNF5 subunit is associated with human rhabdoid tumorigenesis, and SNF5–/– mice develop lymphomas or rhabdoid tumors with complete penetrance (56). SNF5 tumor suppressor activity is thought to occur in part through the cell cycle inhibitor p16ink4a (5, 48). The BAF250 subunit, which contains intrinsic DNA-binding capabilities through an AT-rich DNA-binding domain, directs SWI/SNF activity to the GR (47). Thus, BAF250 may utilize both DNA and protein-binding functions in combination to confer specificity of action. In contrast, BAF60a likely utilizes two distinct protein-binding surfaces (the first for GR and BRG1 and the second for BAF155 and BAF170) to regulate GR specificity (28). BAF60a has also been shown to function as to discriminately activate a subset of Fos/Jun dimers, thus governing specificity of AP-1 action (33). Lastly, BAF57 harbors multiple activities that likely assist in the regulation of sequence-specific DNA-binding factors (2, 3, 71).
BAF57 is composed of an N-terminal HMG DNA-binding domain and a kinesin-like coiled-coil region. Given this structure and its ability to bind four-way junction DNA, BAF57 has been suggested as a potential targeting subunit of the SWI/SNF complex that regulates selected target genes (71). This hypothesis has been tested with the HMG allele of BAF57, which does not disrupt SWI/SNF complex formation or SWI/SNF recruitment but rather lacks DNA-binding potential. Transgenic mice expressing this allele in T cells demonstrate a specific derepression of the CD4 locus (9). We report here that BAF57 interacts directly with the AR and activates AR function through SWI/SNF. Although AR can bind BAF57 independent of ligand (similar to what was reported for GR [28]), BAF57 was recruited to AR targets in a ligand-dependent manner. Since we have also observed that BAF57 resides predominantly in the nucleus (data not shown), interaction between AR and BAF57 is hypothesized to occur in this cellular compartment. Mechanisms regulating the AR-BAF57 interaction will be the subject of future studies. The ER has also been shown to interact with BAF57, but ER binding is enhanced by ligand, thus indicating a disparate mode of interaction. In support of this view, ER was shown to retain transactivation capacity in BAF57 null cells (3), whereas AR activity was markedly reduced by BAF57 loss or inhibition. The requirement of BAF57 for AR was apparent in both transient assays and upon analysis of PSA, an endogenous AR target gene. It is of interest that previous analyses of SWI/SNF effects on this AR target revealed that in transient assays with an ectopically expressed PSA regulatory region, the presence of the PSA enhancer relieved the requirement for SWI/SNF for AR function (43), thus indicating that DNA composition or architecture likely influences the necessity for SWI/SNF. Although such studies are useful for dissecting the mode of SWI/SNF action on discrete templates, the data shown herein establish a solid requirement for BAF57 and SWI/SNF action on prostate-specific AR targets in the context of chromatin.
Independent of nuclear receptor association, BAF57 has been shown to regulate coactivator function. Specifically, BAF57 has been shown to interact directly with the p160 coactivators and is required for the ability of these coactivators to modulate ER function (3). Similarly, we show that SRC1 maintains little AR coactivation function in the absence of BAF57 and cooperates with BAF57 to augment AR activity. In contrast to SRC1, TIF2 maintains activity in the absence of BAF57; however, the addition of BAF57 facilitates AR action similar to SRC1. Surprisingly, potentiation of AR by Ubc9 demonstrated a similar requirement, indicating that the BAF57 requirement extends beyond the p160 class of coactivators. Although the mechanism by which Ubc9 modulates AR remains elusive, these data put forth the hypothesis that Ubc9 may activate p160 function. In contrast, ARA70 and ARA55 potentiated AR gene transcription independent of BAF57 and actually synergized with BAF57 for AR activation. Thus, this class of coactivators maintain function independent of SWI/SNF. Collectively, these data indicate that, like the nuclear receptors, coactivators demonstrate discrete requirements for SWI/SNF function. Given the importance of selected coactivators in prostate cancer progression, knowledge of SWI/SNF requirement will be an important component of targeting coactivator function in cancer therapy.
Inhibition of AR activity is the main line of treatment for prostate cancer therapy, and recurrent, late-stage tumors are hallmarked by inappropriate restoration of AR activity (1, 34). We have shown that inhibition of either BRM or BAF57 significantly retards proliferation in prostate cancer cells. These observations again underscore the plasticity of the SWI/SNF requirement, since loss of SWI/SNF ATPase activity or selected subunits is often associated with growth advantage, such as is observed upon SNF5 loss or in response to RB upon loss of SWI/SNF ATPase activity (5, 54, 56, 66, 69). Together, these data implicate a specific response for BAF57 in AR-dependent growth and further identify the importance of the BAF subunits in SWI/SNF function.
In summary, these data reveal an essential role for BAF57 in regulating SWI/SNF modification of AR. Moreover, our studies demonstrate that BAF57 strongly influences the impact of coactivators on AR activation. These functions of BAF57 are critical, since inhibition of BAF57 or BRM function blocked proliferation of androgen-dependent prostate cancer cells. Together, these studies implicate BAF57 as a major regulator of AR and prostate cancer proliferation, thus identifying BAF57 as a valid target for potential therapeutic intervention.
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ACKNOWLEDGMENTS |
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This study was supported by DAMD 02-1-0037 (K.E.K.) and RO1CA099996 (K.E.K.). B.W., G.R., and E.H. are supported by grants CA91048 and CA102848. K.A.L. is supported by NIEHS training grant 2 T32 ES07250-16.
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FOOTNOTES |
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, P < 0.05. (B) LNCaP cells transfected with either vector (lane 1) or BAF57
) or presence of 1 µg of pSG5ARA70 (
). As described in Materials and Methods, all transfections were brought to a total 4.5 µg by using empty vector. Posttransfection, cells were treated with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and the luciferase activity was measured. The ligand-activated AR activity was set to 100 (data not shown), and the average relative luciferase activity and standard deviations are shown (P < 0.05). (B to D) BT549 cells (2 x 105) were transfected with 0.5 µg of pARR2-Luc reporter, 0.25 µg of pTK-Renilla luciferase, 0.5 µg of pSG5AR, and 2.25 µg of pBabe-WTBAF57, vector, or the indicated coactivator (pSG5ARA55 and pFLAG-Ubc9) or mutant (pcDNA3-ARA70N2) expression plasmid (1 µg). Posttransfection, cells were treated with 0.1% EtOH or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and luciferase activity was measured. The ligand-activated AR activity in the absence of BAF57 was set to 100, and the average relative luciferase activity and standard deviations are shown (P < 0.05).