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Molecular and Cellular Biology, July 2007, p. 4863-4875, Vol. 27, No. 13
0270-7306/07/$08.00+0     doi:10.1128/MCB.02144-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptional Activity of Androgen Receptor Is Modulated by Two RNA Splicing Factors, PSF and p54nrb

Xuesen Dong,¹ Joan Sweet,² John R. G. Challis,³ Theodore Brown,¹ and Stephen J. Lye^1*

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Canada M5G 1X5,¹ University Health Network, University of Toronto, 200 Elizabeth Street, Toronto, Canada M5G 2M9,² Department of Physiology, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Canada M5S 1A8³

Received 15 November 2006/ Returned for modification 16 January 2007/ Accepted 11 April 2007

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Nuclear receptors regulate gene activation or repression through dynamic interactions with coregulators. The interactions between nuclear receptors and RNA splicing factors link gene transcription initiation with pre-mRNA splicing, providing a coordinated control of the products of gene transcription. Here we report that two RNA splicing factors, PTB-associated splicing factor (PSF) and p54nrb, synergistically form protein complexes with the androgen receptor (AR) in a ligand-independent manner and inhibit its transcriptional activity. PSF does not affect AR protein stability, as in the case of the progesterone receptor, but impedes the interaction of AR with the androgen response element. Both splicing factors interact directly with mSin3A and attract mSin3A to the AR complex in a synergistic manner. The suppression of AR transcriptional activity by PSF and p54nrb is reversed by the inhibition of histone deacetylase activity. These data demonstrated that PSF and p54nrb complex with AR and play a key role in modulating AR-mediated gene transcription.

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Gene transcription requires coordination of histone modifications, ATPase-dependent chromatin remodeling, transcription initiation and elongation, and RNA processing. Although each of these biochemical reactions is accomplished by different protein complexes, communication between transcription factors and RNA splicing factors indicates cotranscriptional RNA splicing, performed by one "general gene expression machine" (44). Initiation of gene transcription is executed by a large number of proteins including DNA-bound transcription factors, general transcription machinery, and coregulators that possess a variety of enzymatic activities facilitating either transcriptional activation or repression (42, 52). The androgen receptor (AR) is a member of the nuclear receptor superfamily that functions as a ligand-dependent transcription factor to develop and maintain the male reproductive system. Altered AR signaling can affect male fertility and has been implicated in the onset and progression of prostate cancer (1, 18, 38, 60). Upon ligand binding, activated AR interacts with androgen response elements (AREs) in the regulatory region of target genes and with coregulators, including coactivators and corepressors (6, 19, 37). While agonist-bound AR recruits coactivator complexes containing histone acetyltransferase to unpack chromatin in a relaxed form (29), antagonist-bound AR associates with corepressors such as nuclear receptor corepressor (NCoR) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) (23, 25, 36). NCoR and SMRT not only contain multiple repression domains that transfer their active repression functions but also recruit histone deacetylases (HDACs) that induce chromatin condensation and gene repression (8, 24, 47, 48). NCoR has been isolated in several complexes including the Sin3/HDAC complex (66, 67). Sin3 was originally identified as a corepressor associated with the Mad family, is highly conserved across species, and exists as two isoforms, mSin3A and mSin3B, in mammals (3, 55). It was found that mSin3A forms a large multiprotein complex containing HDAC1, HDAC2, RbAP46/48, and a number of Sin-associated proteins including the recently identified SAP25 (14, 20, 33, 59, 66). While mSin3 provides the platform for the complex and HDAC1 and HDAC2 provide enzymatic activities, other components provide stabilization for interactions within the complex and between the complex and histones (21, 34, 66).

Increasing evidence indicates that RNA splicing factors are actively involved in gene transcription through interaction with transcriptional factors and/or the core general transcriptional machinery (5, 65). RNA splicing factors influence transcription through the direct association with the carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic polymerase II (Pol II) directly (15, 53). Splicing factors such as PGC-1, CoAA, and CAPER coactivate the transcriptional activity of nuclear receptors (2, 12, 43). The RNA splicing factors, PSF and p54nrb, share 71% identity within a central region that includes two RNA recognition motifs (RRMs). These factors can interact either directly (or indirectly through N-WASP) with the CTD of Pol II to facilitate the assembly of splicing complexes on the nascent RNA, thereby resulting in the stimulation of splicing and downstream RNA processing (53, 63). We have demonstrated that PSF corepresses several nuclear receptors including the progesterone receptor (PR) and the AR (11). PSF binds directly with DNA sequences containing an insulin-like growth factor response element in the p450 gene and represses its transcription (61). PSF and p54nrb have also been reported to inhibit transcription by recruiting HDACs through mSin3A to other nuclear receptors including steroidogenic factor 1, thyroid hormone receptors, and retinoid X receptors (41, 56). The mechanisms underlying these actions are unclear.

In addition to transcriptional modulation, PSF and p54nrb form multiple protein complexes. These different complexes perform several nuclear processes including pre-mRNA processing, nuclear retention of edited RNA, and DNA relaxation (58). In this study, we investigated the regulation of AR transactivation by PSF and p54nrb. We demonstrated that PSF and p54nrb interact with AR in a ligand-independent manner and corepress AR transactivation in several cell and promoter contexts, including the well-defined native AR target gene, prostate-specific antigen (PSA). Both PSF and p54nrb interact directly with mSin3A and mediate AR transactivation repression through recruitment of the HDAC complex. These findings indicate that in addition to their roles in facilitating RNA splicing, PSF and p54nrb function as corepressors of AR.

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Plasmids. All recombinant DNA work was performed according to standard procedures (54). His-PSF, pM-PSF, pM-PRB, gluthathione S-transferase (GST)-PSF deletion mutants, and pCEP-AR vectors were described previously (10, 11, 22). Flag-AR, Flag-p54nrb, and GST-p54nrb were kindly provided by Michael J. Weber (University of Virginia Health system), B. J. Blencowe (University of Toronto), and Michel Vincent (Laval Universite), respectively. The pSPmSin3A vector was generously provided by Robert N. Eisenman (Fred Hutchinson Cancer Research Center). The pcDNA AR was constructed by digestion of pCEP4-AR with BamHI and BglII and ligation to the BamHI in pcDNA3.1 (Invitrogen). Using His-PSF as a template, His-PSF-F was generated by PCR with the primers 5'-GGGGAATTCATGTCTCGGGATCGGTTCCGG-3' and 5'-GGGGTCGACTCAGCCAACATCAATCATTCTTACCATGTCACTTCCCATCATGG-3'. The PCR product was digested with EcoRI and SalI and ligated to pcDNA3.1HisC at the same sites. The pM-p54nrb was constructed by PCR using Flag-p54nrb as a template and ligated to the pM vector at BamHI and HindIII. The pSG5-mSin3A was cloned by digestion of pSP6-mSin3A with EcoRI and ligation to the pSG5 vector at the same site.

Cell culture and transfection. 293T and 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), and PC-3 and LNCaP cells were maintained in RPMI 1640 medium plus 10% fetal calf serum, as described previously (10, 32). For experiments involving steroid exposure, the medium was replaced with phenol red-free medium containing 10% charcoal-treated fetal bovine serum (HyClone). 293T or 293 cells were transfected with Exgen500 (MBI), while PC-3 and LNCaP cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were transfected 24 h before the addition of dihydrotestosterone (DHT) and/or sodium butyrate (NaBu) for 24 h. Luciferase (Luc) assays were performed using a Promega Luciferase Kit according to the manufacturer's instruction.

Coimmunoprecipitation and Western blotting. For immunoprecipitation, 293T cells were transfected with a total of 10 µg of expression vectors in 100-mm culture dishes. Cells were lysed in TNET buffer (50 mM Tris 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton) plus protease inhibitor cocktail. Cell lysates precleared with 5 µg of rabbit immunoglobulin G (IgG; Sigma) were incubated with specific antibodies overnight at 4°C, followed by the addition of 30 µl of protein A/G for another 2 h at 4°C. Resins were washed with TNET buffer and eluted with 1x Laemmli buffer, boiled, and centrifuged. The supernatant was separated by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis (SDS-8% PAGE), transferred to polyvinylidene difluoride membrane, and blotted by specific antibodies.

For immunoprecipitation of the endogenous AR, PSF, and p54nrb, LNCaP cells cultured in 150-mm dishes were lysed in TNET buffer. Cell lysates were then incubated with 5 µg of either AR antibody or rabbit IgG at 4°C overnight, followed by the addition of 30 µl of protein A/G Plus-agarose beads (Santa Cruz) for another 2 h at 4°C. Resins were washed with TNET buffer containing 250 mM NaCl and eluted with 1x Laemmli buffer. The eluted proteins along with the whole-cell extract were Western blotted by AR-, PSF-, and p54nrb-specific antibodies. Antibodies used were as follows: AR (N-20) and His tag (H-15) from Santa Cruz Biotechnology, PSF (B92) and Flag (M2) from Sigma, and p54nrb from BD Transduction Labs.

GST pull-down assay. A GST pull-down assay was performed as previously described (10). mSin3A was expressed in vitro using a TNT T7-coupled reticulocyte lysate system (Promega) with pSG5-mSin3A as the DNA template. The reticulocyte lysate containing [³⁵S]methionine-labeled protein was incubated with GST and its fusion proteins. Associated proteins were recovered in SDS sample buffer and separated on SDS-PAGE gels. Gels were treated with Enhancer (NEN), dried, and analyzed by autoradiography.

Construction of siRNA vector. An internet-based program (Ambion Inc.) was used to design small interfering RNAs (siRNAs). Oligonucleotide DNA sequences based on these targeting sequences were synthesized and were engineered to possess BamHI- and HindIII-compatible overhangs and ligated to pSilencer 2.0 vector (Ambion Inc.). The target sequence used for human PSF (accession no. NM_005066) was 5'-AACTTACACACAGCGATGTCG-3' and the control scrambled sequence was 5'-AAATTACACATAGCGATGGCG-3'. Both vectors, siPSF and scrPSF, respectively, were confirmed by nucleotide sequencing.

ChIP and re-ChIP assays. Chromatin immunoprecipitation and reimmunoprecipitation (ChIP and re-ChIP) assays were performed with modifications of a previously described by procedure (17, 57). In brief, LNCaP cells were grown in phenol red-free RPMI 1640 medium with 10% charcoal-stripped fetal calf serum for 2 days before exposure to DHT. Chromatin was cross-linked with 1% formaldehyde for 10 min at 37°C and sonicated in lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris, pH 8.0, plus protease inhibitor cocktail). After centrifugation, 10 µl of the supernatants was used as input, and the remaining lysate was subjected to a ChIP assay. In re-ChIP assays, complexes were eluted by incubation in re-ChIP buffer (0.5 mM dithiothreitol, 1% Triton, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris, pH 8.0) for 30 min and subjected again to a ChIP assay. The primers used to amplify AREs were the following: ARE I, 5'-TCTGCCTTTGTCCCCTAGAT-3' and 5'-AACCTTCATTCCCCAGGACT-3'; ARE III, 5'-CCTCCCAGGTTCAAGTGATT-3' and 5'-GCCTGTAATCCCAGCACTTT-3'; ARE X, 5'-CTGTGCTTGGAGTTTACCTGA-3' and 5'-GCAGAGGTTGCAGTGAGCC-3'.

Gel shift assay. Gel shift assays were performed as previously described (11). Flag-tagged AR, PSF, and p54nrb were purified from 293 cells stably transfected with expression vectors with 400 ng/ml G418 selection. The cell lysate was incubated with M2-Sepharose beads (Sigma) according to the manufacturer's protocol. Double-strand synthetic oligonucleotide probes containing a palindromic consensus ARE (5'-AGCTTAGAACACAGTGTTCTCTAGAG-3') were labeled with [-³²P]ATP. Binding reactions were performed in a total volume of 20 µl in 1x reaction buffer [5% glycerol, 5 mM dithiothreitol, 5 mM EDTA, 250 mM KCl, 100 mM HEPES (pH 7.5), 1 µg of poly(dI-dC), 25 mM MgCl₂, 1 mg/ml bovine serum albumin, 1 µg of salmon sperm DNA, 0.05% Triton X-100], 0.5 ng of labeled probe, 10 nM DHT, and purified proteins. The binding reaction was allowed to proceed for 20 min at room temperature (the supershift assay was performed by adding 1.5 to 5 µg of M2 antibody for an additional 45 min) before loading onto 5% nondenaturing polyacrylamide gels.

PSA analysis. Concentrations of PSA were determined by using an enzyme-linked immunosorbent assay (ELISA) with an Immulite 2500 PSA kit and the automatic immunometric analyzer Elecsys (Roche). Its detection limit is 0.005 ng/ml with a dynamic range up to 100 ng/ml. Briefly, culture medium was incubated with a biotinylated monoclonal PSA-specific antibody and a monoclonal PSA-specific antibody labeled with a ruthenium complex to form a sandwich complex. The complex was then bound to streptavidin-coated microparticles and captured onto the surface of the electrode. The unbound substances were removed with PreCell. Application of a voltage to the electrode induced a chemiluminescent emission, which was measured by a photomultiplier. Results were determined by a calibration curve, which was instrument-specifically generated by two-point calibration and a master curve provided via the reagent bar code. Other details for the ELISAs used have been described elsewhere (13, 32).

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PSF and p54nrb complex with AR. We previously demonstrated that when overexpressed, PSF represses transcriptional activity of several steroid receptors including PR, glucocorticoid receptor, and AR. In the case of PR, this involved a direct interaction between PSF and PR. To determine if PSF interacts with AR, 293T cells were transiently transfected with AR and/or His-tagged PSF expression constructs. Immunoprecipitation studies were performed on whole-cell lysates (WCLs) using AR- and His-specific antibodies. AR antibody coprecipitated AR and PSF (Fig. 1A). Likewise, His antibody precipitated both His-PSF and AR, demonstrating an in vivo interaction of AR/PSF. Since p54nrb heterodimerizes with PSF, we speculated that p54nrb is also present in the AR/PSF complex. To test this, 293T cells were transfected with AR, His-PSF, and Flag-p54nrb expression constructs. WCLs were immunoprecipitated with either AR or Flag antibodies, followed by Western blotting to detect the presence of AR, PSF, and p54nrb in the precipitate. Both PSF and p54nrb were present in AR complexes (Fig. 1B and C). To address whether endogenously expressed AR, PSF, and p54nrb form a complex, WCL from LNCaP cells was immunoprecipitated with AR antibody, followed by Western blotting with PSF- and p54nrb-specific antibodies. PSF and p54nrb were detected in the AR precipitate, irrespective of whether the cells were treated with DHT (Fig. 1D). To determine the regions of AR and p54nrb interaction, GST fusion proteins containing various domains of PSF were incubated with LNCaP cell lysates. Our data indicate that the RRM I and II regions of PSF mediate the interaction with both AR and p54nrb (Fig. 1E). In addition, a weak interaction between p54nrb and PSF residues 450 to 662 was observed. Thus, these data unequivocally demonstrated that PSF and p54nrb form a complex with AR.

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FIG. 1. AR forms a protein complex with PSF and p54nrb. (A, B, and C) 293T cells were transiently transfected with vectors encoding pcDNA3 AR, His-PSF, and Flag-p54nrb, as indicated. WCLs were immunoprecipitated with antibodies against AR, His, or Flag tag. The associated proteins were immunoblotted to detect existence of AR, His-PSF, and Flag-p54nrb in the precipitate. (D) Endogenous AR/PSF/p54nrb complex was immunoprecipitated with LNCaP cell lysate with AR antibody in the presence of vehicle or 10 nM DHT. Associations of PSF and p54nrb were detected with PSF or p54nrb antibody. (E) GST or GST-PSF fusion proteins were immobilized on glutathione beads and incubated with WCL from LNCaP cells. Association of AR and p54nrb was detected by AR and p54nrb antibody. IP, immunoprecipitation; WB, Western blotting.

AR/PSF complex interacts with human PSA gene. Since AR and PSF form a protein complex, we addressed whether the AR/PSF complex is present on AR target genes. For these studies, we chose the PSA gene as a model to perform ChIP in LNCaP cells. Figure 2A shows the location of the designed primers used to amplify ARE-containing regions (ARE I and ARE III) or a non-ARE region (ARE X) of the PSA gene promoter. LNCaP cells were maintained in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped serum for 48 h, after which cells were treated with vehicle or 10 nM DHT for 2 h. ChIP assays were performed by using AR or PSF antibodies or with rabbit IgG as a control. In the absence of DHT treatment, AR was detected at both ARE I and III but only at very low levels. AR recruitment was markedly enhanced by treatment with DHT (Fig. 2B). In contrast, PSF was detected at both ARE I and III in the absence of DHT treatment. Interestingly, 2-h DHT administration appeared to increase PSF recruitment to ARE I but decreased recruitment to ARE III. Neither PSF nor AR was detected at the control ARE X site. To further confirm that PSF and AR are in the same complex on AREs, we performed a re-ChIP assay. LNCaP chromatin was first precipitated with AR antibody, and the eluted DNA-protein complexes were reprecipitated with either AR or PSF antibody. We observed clearly that PSF is in the same complex with AR on both ARE I and III but not on ARE X (Fig. 2C).

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FIG. 2. AR and PSF recruitment on PSA gene. (A) Schematic diagram of the PSA gene regulatory regions. Boxes depict AREs. The numbers represent the positions upstream from the PSA transcription start site. Primers were used to amplify these corresponding DNA fragments. (B) LNCaP cells were treated with vehicle or 10 nM DHT for 2 h. Cross-linked chromatin was immunoprecipitated with antibodies as indicated. The precipitated DNA was amplified using primers to amplify ARE I, III, and X described in panel A. (C) Re-ChIP assay was performed using LNCaP cells treated with vehicle or 10 nM DHT for 2 h. Chromatin extracts were immunoprecipitated with AR antibody followed by reimmunoprecipitation with AR, PSF, or rabbit IgG antibody. The precipitated DNA was amplified using primers described in panel A. (D) Chromatin extracts from LNCaP cells treated with 10 nM DHT for 0 to 90 min were immunoprecipitated with AR or PSF antibody, followed by PCR using primers described in panel A. IP, immunoprecipitation.

AR recruitment to the PSA gene promoter following androgen treatment is time dependent. To further explore the differential recruitment of PSF to ARE I and III, we performed ChIP assays with AR or PSF antibody at time points ranging 0 to 90 min after DHT treatment. Consistent with published data, AR was recruited to the PSA promoter in a dynamic pattern (Fig. 2D). While PSF occupied both ARE I and III prior to AR binding, different recruitment patterns were observed. Association of PSF with the ARE I region was generally maintained after DHT treatment. In contrast, PSF association with the ARE III region decreased after 30 min and started to recover 75 min after treatment, a pattern opposite that of AR recruitment. Our data indicate that exogenous androgen is not required for binding of PSF to AREs in LNCaP cells; however, exposure to androgen nevertheless differentially modified PSF binding to ARE I and III. In addition, PSF and AR form a complex at ARE I and III.

PSF inhibits AR transcriptional activity. Transient transfection reporter gene assays were conducted in several mammalian cell lines to determine the functional significance of PSF on AR transactivation. 293T human embryonic kidney cells and PC-3 human prostate cancer cells do not express endogenous AR, whereas LNCaP prostate cancer cells express an activated AR as a result of a point mutation [T877A, yielding AR(T877A)] in the ligand binding domain (LBD). We used two androgen-responsive promoters in these experiments: an artificial promoter with three copies of the ARE (3xARE) and the steroid-responsive mouse mammary tumor virus (MMTV) promoter. To control for possible adverse effects associated with overexpression of PSF cDNA, cells were also transfected with PSF-F. PSF-F shares the same amino acid sequences with PSF through amino acid residue 662, followed by a unique 9-amino-acid sequence at its C terminus. PSF-F localizes to the nuclear membrane within the cytoplasm, whereas PSF immunostaining is restricted to the cell nucleus in a punctate pattern (data not shown). In both 293T and PC-3 cells, overexpression of AR in the presence of 10 nM DHT resulted in a significant increase in Luc activity driven by the 3xARE and MMTV promoters (Fig. 3A). Cotransfection of PSF, but not PSF-F, decreased AR-induced Luc activity by up to 70%. PSF was also shown to inhibit transactivational activity of the mutant AR(T877A) in LNCaP cells (Fig. 3B). In addition, cotransfection of PSF with AR in 293T cells resulted in a dose-dependent inhibition of DHT-activated AR transactivation, but no inhibitory effects were observed in the absence of DHT (Fig. 3C). This inhibition was also observed over a wide range of DHT concentrations, from 0.1 nM to 1 µM (Fig. 3D).

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FIG. 3. PSF inhibits transcriptional activity of AR. (A and B) 293T, PC-3, and LNCaP cells were transiently transfected with AR, PSF, or PSF-F expression vector and 3xARE-Luc or MMTV-Luc reporter gene for 24 h. Cells were treated with vehicle or 10 nM DHT for additional 24 h. 293T cells were transiently transfected with AR and increasing doses as indicated (C) or a constant dose (0.5 µg) (D) of PSF expression vector together with MMTV-Luc and treated with vehicle or DHT for 24 h. (E) 293T cells were transfected with siPSF or scrPSF for 48 h. PSF protein expression levels were detected by immunoblotting with PSF antibody, and -tubulin served as a loading control. (F) 293T cells were transiently transfected with siPSF or scrPSF together with an AR expression vector and MMTV-Luc for 48 h and then treated with vehicle or 10 nM DHT for an additional 24 h. Luc activities were normalized to ß-Gal. The means ± standard deviations from two independent experiments performed in triplicate are shown.

To further confirm the inhibitory effects of PSF on AR transactivation, we used an siRNA expression construct to target PSF. Transfection of this vector, but not a vector expressing a scrambled sequence, blocked 80% of endogenous PSF protein expression (Fig. 3E). Transfection of this siPSF vector increased DHT-induced Luc activity in a dose-dependent manner in both AR-transfected 293T cells (Fig. 3F) and in LNCaP cells (data not shown). These data provide evidence that endogenous PSF plays an important role in repressing AR transactivation.

p54nrb is a corepressor of AR. PSF has been demonstrated to heterodimerize with p54nrb, and our data indicate that p54nrb is found in AR/PSF complexes (Fig. 1). To characterize the functional effects of this interaction on AR transactivation, we transfected 293T and LNCaP cells with p54nrb and AR expression constructs and an MMTV-Luc reporter gene. Overexpression of p54nrb repressed AR activity in a dose-dependent fashion (Fig. 4A and B). To further confirm this inhibitory effect, we performed a mammalian one-hybrid assay, in which p54nrb cDNA was inserted into the pM vector to encode a fusion protein with the Gal4 DNA binding domain (DBD) at the N terminus of p54nrb (Fig. 4C). The pM-p54nrb was cotransfected with the reporter vector G5-Luc containing five Gal4 DBD response elements that are recognized by the fusion protein. Thus, the Luc activity reflects the impact of pM-p54nrb on transcriptional activity. We also constructed pM-PSF and pM-PR-B to serve as transcription-negative and -positive controls, respectively. Deletion mutations of PSF [PSF containing residues 150 to 707, 290 to 707, and 370 to 707, yielding PSF(150-707), PSF(290-707), and PSF(370-707), respectively] were included in this study, as the amino acid sequences of p54nrb are homologous to that of PSF(290-707). We observed that Gal4-dependent Luc activity mediated by pM-p54nrb was decreased compared to that obtained with the empty pM vector and was similar to that obtained with pM-PSF(290-707) (Fig. 4D). Furthermore, increasing doses of pM-p54nrb resulted in a proportional decrease of Luc activity (Fig. 4E). The expression levels of Gal4 DBD chimeras were confirmed by Western blotting of the transfected cell lysate with Gal4 DBD antibody (Fig. 4F and G). These data indicate that p54nrb possesses inhibitory function and represses AR-mediated transcription in different cell contexts.

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FIG. 4. AR's transcriptional activity is inhibited by p54nrb. (A and B) 293T and LNCaP cells were transiently transfected with AR, p54nrb, and MMTV-Luc vectors as indicated for 24 h and then treated with vehicle or 10 nM DHT for additional 24 h. (C) Schematic diagram describing vectors encoding Gal4 DBD fusion proteins with p54nrb or deletion mutants of PSF. 293T cells were transfected with pM, pM-PSF or its deletion mutants, pM-p54nrb, and pM-PRB (D) or with increasing doses of pM vectors without or with inserts of PSF, p54nrb, or PRB cDNA (E) together with G5-Luc reporter vector for 24 h. Luc activities were measured and normalized to ß-Gal activity. The means ± standard deviations from two independent experiments performed in triplicate are shown. (F and G) Expression of pM and its fusion proteins was confirmed by immunoblotting with Gal4 DBD antibody.

Mechanisms of PSF and p54nrb repression of AR transactivation. We along with others have previously reported that PSF represses nuclear receptor transactivation through mechanisms including receptor protein degradation, blockade of receptor binding to DNA, and recruitment of the HDAC complex through its interaction with the adaptor protein mSin3A (11, 41). In the present study, we observed that while overexpression of PSF reduced levels of PR isoform A protein (11), it did not reduce AR protein expression (Fig. 5A). Similarly, overexpression of p54nrb did not affect AR protein expression (Fig. 5B).

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FIG. 5. Mechanisms of PSF inhibition of AR transactivation. (A and B) 293T cells were transfected with expression vectors as indicated. AR, PSF, and p54nrb were detected by Western blotting with specific antibodies, and -tubulin served as a loading control. (C) Flag-tagged AR, PSF, or p54nrb protein was purified using M2-Sepharose beads with the lysate from 293 cells stably transfected with Flag-tagged expression vectors. Proteins were separated on SDS gels and stained with Coomassie blue. (D) Five micrograms of purified Flag-tagged proteins was subjected to Western blotting with M2 antibody to confirm their identities. (E) A gel shift assay was performed by incubating purified Flag-AR with -32P-labeled double-strand ARE oligonucleotide in the presence of 10 nM DHT, and the AR/ARE complex was separated by nondenatured PAGE for 2 h. Nonlabeled ARE (lanes 5 and 6) and increasing doses of M2 antibody (lanes 9 and 10) were used to confirm the identity of the components in the migration-shifted band. Purified PSF and p54nrb were added to the reaction mixture (lanes 13 to 16). (F) LNCaP cells were transiently transfected with mock (CMV-Flag, Sigma), Flag-PSF, or Flag-p54nrb expression vectors. WCLs were immunoblotted with antibodies against AR, PSF, p54nrb, and Flag tag to confirm overexpression of PSF and p54nrb. (G) LNCaP cells transfected as indicated in panel F were treated with vehicle or 10 nM DHT for 2 h. Chromatin extracts were used to perform ChIP assays with AR antibody and rabbit IgG as a control, followed by PCR using primers described in the legend of Fig. 2A. (H) 293T cells were transfected with AR and/or PSF expression vector and MMTV-Luc for 24 h before the addition of 0 to 5 mM NaBu and 10 nM DHT for an additional 24 h. (I) 293T cells were transfected with pM or pM-PSF vector together with G5-Luc for 24 h before the addition of 0 to 5 mM NaBu for an additional 24 h. Luc activities were measured and normalized by ß-Gal activity. The means ± standard deviations from two independent experiments performed in triplicate are shown.

We then tested whether PSF or p54nrb changes AR binding to DNA as detected by a gel shift assay. AR, PSF, or p54nrb was expressed in 293 cells that were stably transfected with Flag-tagged expression vectors and purified by using the M2 antibody-conjugated Sepharose beads. This procedure produced up to 80% purification of AR, PSF, and p54nrb proteins (Fig. 5C). Western blotting with M2 antibody confirmed the identity of the purified proteins (Fig. 5D). In the gel shift assay, AR protein was incubated with -³²P-labeled double-strand oligonucleotide containing a consensus ARE. One major migration-retarded band was observed, and its intensity increased proportionally to the AR protein input (Fig. 5E). The addition of 50- and 500-fold excess nonlabeled ARE resulted in disappearance of the retarded band, confirming the presence of ARE within the complex. M2 antibody induced the supershifted band, confirming the presence of AR within the complex. Addition of PSF but not p54nrb decreased this AR/ARE association. These data indicated that PSF but not p54nrb blocks AR binding to ARE in vitro.

To further confirm that PSF impairs AR binding to ARE in vivo, we performed ChIP assays using LNCaP cells transiently transfected with mock (CMV-Flag, where CMV is cytomegalovirus; Sigma), Flag-PSF, or Flag-p54nrb expression vectors. The protein expression levels of AR, PSF, p54nrb were confirmed by immunoblotting with antibodies as indicated in Fig. 5F. In LNCaP cells transfected with mock vector, we detected robust recruitments of AR to both ARE I and III regions of the PSA gene after a 2-h treatment of DHT. Overexpression of PSF reduces the associations of AR with AREs, consistent with the gel shift assay evidence that PSF blocks AR binding to ARE. Interestingly, while p54nrb has no impact on AR binding to ARE in the in vitro gel shift assay, our ChIP assay indicated that overexpression of p54nrb also decreases the associations of AR to AREs. It could be that overexpression of p54nrb strengthens the association of PSF with AR (as we demonstrated by subsequent coimmunoprecipitation) (see Fig. 6D), thus blocking AR binding to ARE in the ChIP assay. Thus, we conclude from these data that both PSF and p54nrb block AR binding to ARE in vivo.

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FIG. 6. Mechanisms of p54nrb inhibition of AR transactivation. (A) A GST pull-down assay was performed by incubating GST, GST-p54nrb, or GST-PSF with [35S]methionine-labeled mSin3A (top). GST or GST fusion proteins used in the assay were separated on SDS gels and stained with Coomassie blue (bottom). (B) 293T cells were transfected with pM or pM-p54nrb vector together with G5-Luc and treated with 0 to 5 mM NaBu for 24 h. (C) 293T cells were transfected with AR, MMTV-Luc, and p54nrb or mock expression vector for 24 h before the addition of 10 nM DHT and 0 to 5 mM NaBu for an additional 24 h. (D) 293T cells were transfected with expression vectors as shown, and the WCLs were immunoprecipitated (IP) with AR antibody, followed by immunoblotting using antibodies against Flag-PSF, Flag-p54nrb, and endogenous mSin3A. (E) Cells were transfected with AR, PSF, and/or p54nrb expression as indicated together with MMTV-Luc. Cells were treated with 10 nM DHT for 24 h. (G) 293T cells were transfected with expression vectors as indicated and G5-Luc for 24 h. Luc activities were measured and normalized to ß-Gal activity. The means ± standard deviations from two independent experiments performed in triplicate are shown. (H) LNCaP cells were maintained in phenol red-free medium with 10% charcoal-stripped serum for 48 h and then transfected with expression vectors as indicated. Cells were then treated with vehicle or 10 nM DHT for 4 days. PSA protein levels from the culture medium were measured by ELISA.

To demonstrate PSF-mediated repression through the mSin3A/HDAC complex, we used the HDAC inhibitor NaBu. This chemical does not cause cellular morphological change or cell toxicity at doses of 0.2 to 5 mM for 24 h. Luc activity was slightly increased by NaBu when 293T cells were transfected with AR vector and exposed to 10 nM DHT (Fig. 5H). Cotransfection of PSF vector resulted in decreased AR-mediated Luc activity, and this effect could be rescued in a dose-dependent manner by treating the cells with NaBu. Similarly, using a one-hybrid assay system, pM-PSF repressed Gal4-responsive promoter activity, and this effect was also reversed by increasing doses of NaBu (Fig. 5I). Together, data from these two systems indicate that the repressor activity of PSF involves recruitment of HDAC activity to the target gene.

P54nrb mediates direct interaction with mSin3A and synergizes with PSF in repressing AR. Our data demonstrate that p54nrb represses transcriptional activity as determined by a one-hybrid assay and a steroid receptor-MMTV reporter system. However, it is not clear whether the repressor activity of p54nrb is mediated through direct interaction with mSin3A or indirectly through the formation of a protein complex with PSF. To address this, we performed a GST pull-down assay, in which immobilized GST, GST-p54nrb, or PSF was incubated with in vitro translated ³⁵S-labeled mSin3A. We observed that both p54nrb and PSF interact directly with mSin3A (Fig. 6A). Furthermore, we showed that NaBu could reverse (i) p54nrb inhibition of Gal4 promoter activity (Fig. 6B) and (ii) p54nrb inhibition of AR-mediated MMTV promoter activity (Fig. 6C). Taken together, these data confirm that the repressor activity of p54nrb is mediated through direct binding to mSin3A and subsequent recruitment of HDAC to the complex. Immunoprecipitation analysis indicated that cotransfection of PSF and p54nrb resulted in considerably greater interaction with AR than that with either corepressor alone (Fig. 6D). Endogenously expressed mSin3A also exhibited a more intensive association with AR when both PSF and p54nrb were cotransfected. The functional consequences of this synergistic action were also explored by a one-hybrid assay and a steroid receptor-MMTV reporter system. Cotransfection of PSF and p54nrb resulted in a greater decrease of AR-mediated MMTV promoter activity than transfection of PSF or p54nrb alone (Fig. 6E). The repression effect of pM-PSF in the Gal4-responsive promoter was further enhanced by cotransfection with Flag-p54nrb; conversely, cotransfection of His-PSF induced a stronger inhibition by pM-p54nrb (Fig. 6F). However, the failure of His-PSF to further enhance repression by pM-PSF and of Flag-p54nrb to further enhance repression by pM-p54nrb argues against the formation of PSF or p54nrb homodimers. Finally, we showed that the repressor function of PSF and p54nrb is apparent in vivo. Stimulation of the prostate tumor cell line, LNCaP, with 10 nM DHT induced secretion of PSA, and this was inhibited by either PSF or p54nrb overexpression (Fig. 6G). Furthermore, as with the artificial promoter systems, the combination of PSF and p54nrb overexpression markedly enhanced this repression, consistent with the idea that PSF and p54nrb act as heterodimers. Thus, we conclude that the mechanisms by which p54nrb represses AR transactivation include (i) direct interaction with mSin3A/HDAC complexes and (ii) enhancement of the repressor activity of PSF.

PSF and p54nrb enhance the recruitments of mSin3A and HDAC1 to the PSA gene. In order to provide direct evidence that PSF and p54nrb recruit mSin3A and HDACs to the PSA gene, we performed a ChIP assay using LNCaP cells transfected with mock, Flag-PSF, or Flag-p54nrb expression vectors as shown in Fig. 5F. In mock-transfected LNCaP cells, we observed low but detectable levels of association of mSin3A and HDAC1 to both the ARE I and III regions of the PSA gene in the absence of DHT (Fig. 7). Treatment with DHT totally abolished these associations. Overexpression of either PSF or p54nrb enhanced the recruitment of mSin3A and HDAC1 to the AR-regulated promoter regions, providing evidence that these two splicing factors recruit the Sin3A/HDAC complex to AR complex on chromatin.

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FIG. 7. PSF and p54nrb enhance the recruitment of mSin3A and HDAC1 to the PSA gene. LNCaP cells transfected as indicated in the legend of Fig. 5F were treated with vehicle or 10 nM DHT for 2 h. Chromatin extracts were used to perform ChIP assays with mSin3A, HDCA1 antibody, or rabbit IgG as a control, followed by PCR using the primers described in the legend of Fig. 2A. CMV, cytomegalovirus.

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The AR modulates gene transcription through a dynamic exchange of coactivators and corepressors. Here, we demonstrate that two RNA splicing factors, PSF and p54nrb, interact with and inhibit AR transactivation. These data not only support previously observed transcription-coupled RNA splicing but also demonstrate that these two splicing factors function as corepressors of AR-mediated gene transcription. Current models of gene transcription suggest that transcription initiation/elongation and RNA splicing are tightly coupled. Evidence in support of this association includes protein-protein interaction data between transcription factors and RNA splicing factors. Thus, Pol II binds the RNA processing factors in a complex known as the "mRNA factory" (7). The CTD of Pol II not only facilitates the assembly of RNA splicing factors but also brings together consecutive exons to facilitate cotranscriptional splicing (64). Promoter structures and their binding transcription factors and cofactors influence RNA alternative splicing by controlling the rate of transcription initiation/elongation and by forming protein complexes with RNA splicing factors (16). On the other hand, splicing factors such as spliceosomal U snRNPs stimulate transcription elongation. Spliceosomal U RNPs form a complex with P-TEFb, which in turn phosphorylates the CTD of Pol II and switches Pol II from a nonprocessive to a processive form (16, 45, 46, 49). In addition, proteins such as PGC-1, CAPER, and (SF)3a p120 play dual roles, acting as both transcription coactivators and RNA splicing factors to coordinate transcription and splicing processes (12, 40, 43). Our data provide new insights into the association between transcription and splicing by showing that splicing factors can also act as transcriptional corepressors.

Our data indicate that PSF and p54nrb repress transactivation of AR through ligand-independent interactions, likely involving binding to the DBD of AR in a manner similar to that demonstrated for other nuclear receptors, such as PR and thyroid hormone receptors (11, 41). Our finding that PSF/p54nrb can repress transactivation of the mutant AR(T877A) (which changes the conformation of the LBD and broadens the specificity of ligand binding) further suggests that the LBD does not impact on PSF/p54nrb corepression of AR.

Our ChIP assay confirmed that the AR/PSF complex is present at both the ARE I and ARE III sites within the PSA gene. Overexpression of PSF and p54nrb enhanced the recruitment of the mSin3A/HDAC complex and, in turn, mediated histone deacetylation within the ARE I and III regions of the PSA gene. Our data suggest that the mechanism underlying the recruitment of the complexes to the AR is different from that of NCoR and SMRT. Thus, the recruitment of mSin3A/HDAC complex by NCoR and SMRT to the AR requires the presence of antagonist, whereas PSF/p54nrb can recruit this complex in a ligand-independent manner. Importantly, PSF occupies the ARE prior to detectable AR binding. Sin3A and HDACs have been reported to be recruited to chromatin in the absence of nuclear receptor ligand (35, 50). These observations suggest the existence of the mechanisms that prevent transcriptional leakage from the ligand-unbound AR, which is found within the nucleus (26, 30, 39).

Transcriptional activation of the PSA gene by AR involves a sequence of events including recruitment of AR and its coactivator complex to both ARE I and ARE III, thus bridging these two elements and inducing the looping of the promoter (62). Pol II is recruited in a sequential fashion, first to ARE III, then to the middle region, and finally to ARE I (62). Once transcription is initiated, Pol II moves along the gene, driving transcriptional elongation and cotranscriptional splicing. Our data suggest that in the absence of ligand, PSF occupies the AREs and forms protein complexes with mSin3A. This occupancy inhibits AR associations with both ARE I and III. In the presence of DHT, dissociation of PSF from ARE III permits AR/ARE III association and the initiation of gene transcription. The occupancy of PSF (a component of the spliceosome) on ARE I may facilitate the recruitment of the spliceosome complex to Pol II at the site of transcription initiation, enabling cotranscriptional splicing.

Our demonstration that p54nrb is a corepressor of AR contrasts with the previous report of Ishitami et al. (28). Our conclusion is based on the observations that (i) p54nrb interacts directly with mSin3A, (ii) p54nrb strengthens both the interaction with and corepression of AR by PSF, (iii) p54nrb represses transcription in multiple promoter contexts (MMTV, PSA, and Gal4 response promoter), (iv) the repressive effects of p54nrb can be reversed by the HDAC inhibitor NaBu, and (v) p54nrb blocks AR binding to AREs within the PSA gene in vivo. Furthermore, our own data (unpublished) as well as that of others (27, 56, 61) show that PSF/p54nrb can repress gene transcription mediated by other transcription factors. Nevertheless, PSF and p54nrb may also derepress gene transcription. Thus, cyclic AMP-induced dephosphorylation of PSF/p54nrb has been shown to result in the dissociation of these proteins from the mSin3A/HDAC complex (56). We speculate that phosphorylation of PSF and p54nrb may induce a conformational change in these proteins that modifies their corepressor activity. This possibility is currently under investigation in our laboratory.

Current models propose that transcription regulates splicing by controlling the rate of transcription and elongation; our data suggest that PSF/p54nrb may therefore impact transcriptional elongation. Slow elongation or an internal elongation pause favors the inclusion rather than the skipping of alternative exons (9, 31, 51). We hypothesize that PSF/p54nrb regulates the rate of transcription elongation through two possible mechanisms: (i) direct repression of AR-mediated gene transcription initiation and (ii) recruitment of the Sin3A/HDAC complex to deacetylate histones during transcription elongation. The observation that the inhibitor of histone deacetylation, trichostatin A, favors exon skipping (46) and that chromatin remodeling complex SWI/SNF regulates alternative splicing (4) supports the hypothesis that RNA splicing factors could recruit histone acetylase to modulate chromatin structure and, in turn, regulate the elongation rate and alternative splicing.

Dynamic exchange of coactivators and corepressors controls gene transcription and elongation. However, these processes must be coordinated with RNA processing to ensure smooth operation of the transcriptional machinery as it passes along the gene. The repressor activity of splicing factors such as PSF and p54nrb contributes to this smooth operation by slowing down both transcription initiation and elongation so that this can be coordinated with alternative splicing.

 
We thank Michael J. Weber, B. J. Blencowe, and Michel Vincent for providing AR and p54nrb expression vectors and Robert Eisenman for mSin3A constructs. We also thank Antoninus Soosaipillai from the E. P. Diamandis laboratory for technical assistance with the PSA ELISA assays, Harry P. Elsholtz for suggestions and comments on the manuscript, and members of the Lye laboratory, especially Celeste Yu, for technical assistance and fruitful discussion.

This work was supported by an operating grant from the Canadian Institutes of Health Research (MOP-42378) and the Prostate Cancer Foundation of Canada.

 
* Corresponding author. Mailing address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Canada M5G 1X5. Phone: (416) 586-4800, ext. 8640. Fax: (416) 586-8857. E-mail: lye{at}mshri.on.ca

Published ahead of print on 23 April 2007.

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Molecular and Cellular Biology, July 2007, p. 4863-4875, Vol. 27, No. 13
0270-7306/07/$08.00+0     doi:10.1128/MCB.02144-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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