5'TG3' Interacting Factor Interacts with Sin3A and Represses AR-Mediated Transcription

Manju Sharma and Zijie Sun

Departments of Surgery and Genetics, Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: Zijie Sun, Ph.D., Departments of Surgery and Genetics, R135, Edwards Building, Stanford University School of Medicine, Stanford, California 94305-5328. E-mail: zsun{at}stanford.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Like other nuclear receptors, the AR exerts its transcriptional function by binding to cis elements upstream of promoters and interacting with other transcriptional factors (e.g. activators, repressors, and modulators). Among them, histone acetyltransferases (HATs) and histone deacetylases (HDACs) play critical roles in altering the acetylation state of core histones, thereby regulating nuclear hormone receptor-mediated transcription. The nuclear receptor corepressor can repress the TR and RAR in the absence of ligand through either a Sin3A-dependent or -independent manner by recruiting HDACs. AR and some other steroid hormone receptors cannot silence transcription through a similar mechanism in that they are located in the cytoplasm as complexes with heat-shock proteins before exposure to ligand. It has been shown that AR can bind to p160/SRC, cAMP response element-binding protein-binding protein (CBP)/P300 and other coactivators to increase the AR-mediated transcription. However, the molecular mechanism for turning AR from transcriptionally active into silent states is unknown. In this study, we demonstrated that the transcription repressor, 5'TG3' interacting factor (TGIF), selectively represses AR-mediated transcription from several AR-responsive promoters. The repression is mediated through binding of TGIF to the DNA binding domain of AR and is trichostatin sensitive. We also identified a direct protein-protein interaction between TGIF and a transcription corepressor, Sin3A, which suggests a novel pathway for TGIF recruiting HDAC1 to the repression complex. These results provide fresh insight into understanding the mechanism for repressing AR-, and perhaps other steroid hormone receptor-, mediated transcriptions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RECENT STUDIES HAVE demonstrated that the acetylation state of core histones plays a critical role in regulating eukaryotic gene transcription (1, 2). Histone acetyltransferases (HATs) alter nucleosomal structure by acetylation of histone amino-terminal tails, which increases accessibility of promoters to the transcriptional machinery (3). The recruitment of HAT activity by a sequence-specific DNA-binding protein may be a general feature of transcriptional activation (1). In contrast, histone deacetylases (HDACs) play the opposite role in this process (2). HDACs facilitate transcriptional repression on specific promoters by binding to transcriptional repressors, leading to a more compact nucleosomal structure that results in decreased transcription factor accessibility to the promoter.

In recent years, numerous studies have shown that nuclear hormone receptors mediate specific gene transcription by directly or indirectly interacting with other transcriptional cofactors (4). Several nuclear receptor coactivators possessing intrinsic HAT activities have been identified in recent years, including cAMP response element-binding protein-binding protein (CBP)/P300 (5), the p160/SRC family (6, 7), and pCAF/GCN5 (8, 9). In the presence of ligand, these coactivators bind to the nuclear receptors and acetylate histone in chromatin to facilitate nuclear receptor-mediated, ligand-dependent transcription. The TR, the RAR, and other nuclear hormone receptors can function as potent transcription repressors in the absence of ligand (4). Recent studies on the mechanisms led to the identification of two related proteins, known as nuclear receptor corepressor (NCoR) and silencing mediator for RAR and TR (SMRT) (10, 11, 12). These two proteins mediated the repressive effect of unliganded nuclear receptors through Sin3A-dependent or -independent mechanisms to recruit HDACs in a multisubunit repressor complex leading to a more compact, transcriptionally repressed chromatin structure. Based on these observations, it has been suggested that recruitment of corepressors to the nuclear receptors may be a conversion point from transcription activation to repression.

AR belongs to the nuclear hormone receptor superfamily and plays a critical role in promoting normal and tumoral cell growth (13, 14). However, an important feature of the AR and some other steroid hormone receptors that distinguishes them from TR and RAR is that they are compartmentalized to the cytoplasm as complexes with heat-shock proteins (HSPs) before exposure to ligand (15, 16). Upon binding to ligand, AR dissociates from the HSPs and translocates into the nucleus, where it facilitates androgen-regulated transcription (17, 18). It has been shown that AR can bind to p160/SRC, CBP/P300, and other coactivators to increase AR-mediated transcription (19, 20). However, the molecular mechanism for converting AR from transcriptionally active into silent states is unknown. For the ER{alpha}, it has been suggested that the ubiquitin proteasome pathway is involved in down-regulation by protein degradation (21). However, the mechanism by which the nucleosome is repacked into an inactive state, and whether HDACs are involved in transcriptional repression of AR and other steroid hormone receptors, is still unclear.

5'TG3' interacting factor (TGIF) is a homeodomain transcription repressor that was previously shown to bind to the RXR response element and inhibit RXR-mediated transcription by competitive DNA binding to an overlapping site (22). Recently, it has been demonstrated that TGIF interacts with Smad2, the mediator of TGF-ß signaling, and represses TGF-ß-activated transcription (23). Repression of Smad2-mediated transcription by TGIF is mediated through recruitment of HDACs (23, 24). Identification of a protein-protein interaction between TGIF and HDACs suggested a molecular mechanism by which Smad2 switched transcriptional activation to repression by recruiting TGIF to compete with p300/CBP binding.

In this study, we demonstrated that TGIF selectively represses AR-mediated transcription on the androgen-induced promoters. The repression is mediated through binding of TGIF to the DNA binding domain (DBD) of AR and is trichostatin (TSA), a HDAC inhibitor, sensitive. The results provide the first line of evidence showing that AR-mediated transcription is regulated via the HDAC pathway. We also identified a protein-protein interaction between TGIF and a transcriptional corepressor, Sin3A. Interestingly, the region of TGIF that interacts with Sin3A is also responsible for the binding to HDAC1 (24). Using several biochemical approaches, we further demonstrated that Sin3A directly interacts with TGIF, indicating that it may bridge both TGIF and HDAC1 in a protein complex. This study suggests a novel mechanism for TGIF-mediated transcriptional repression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TGIF Represses Ligand-Dependent Activation of the AR
An earlier report showed that TGIF negatively regulated RXR-mediated transcription (22). TGIF is expressed in a number of human adult tissues including prostate and testis (22). We hypothesized that TGIF might also function to regulate other steroid hormone pathways, including androgen-dependent transcription. Plasmids capable of expressing AR, TGIF, and a luciferase reporter plasmid regulated by the androgen responsive elements (AREs) in the mouse mammary tumor virus (MMTV) long terminal repeat (MMTVpA3-Luc) vector were transfected into CV-1 cells. An approximately 30-fold induction of AR-mediated transcriptional activity was observed in the presence of 10 nM DHT (Fig. 1AGo). Cotransfection of TGIF expression construct showed a dosage-dependent repression of ligand-dependent AR activation. In contrast, the positive control, the known AR-coactivator ARA70 (25), brought about a doubling in DHT-dependent transcription. Thus, this result suggests that TGIF repressed transcription from an AR-dependent promoter.



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Figure 1. TGIF Represses AR-Mediated Transactivation

A, CV1 cells were transiently transfected with 100 ng of pMMTV-Luc, 25 ng of pSV40-ß-gal, 10 ng of pSV-hAR, and, as indicated, different amounts of pcDNA3-Flag-TGIF plasmids or 20 ng of pSG5-ARA70. White bars represent the absence of DHT, and black bars represent the addition of 10 nM DHT. Relative luciferase activity is reported and represented as mean ± SD (luciferase light units/ß-gal). B, The transient transfections were repeated in PC3 cells with a luciferase reporter driven by MMTV promoter or the 7-kb promoter fragment of human PSA. One hundred nanograms of luciferase reporters, 25 ng of pCMV-ß-gal, 10 ng of pCMV-hAR, and 10 ng of pcDNA3-Flag-TGIF plasmids per well were used in the experiments. C, LNCaP cells were transfected with 100 ng of pPSA-Luc, 25 ng of pCMV-ß-gal, and, where indicated, 10 or 20 ng of pcDNA3-Flag-TGIF constructs. No AR expression vector was used in this experiment.

 
To explore the biological significance of the TGIF-mediated repression further, we looked for a repression effect by TGIF in a prostate cancer cell line, PC3. As shown in Fig. 1BGo, TGIF reduced AR-mediated transcription on an MMTV-luc reporter by approximately 50% at TGIF/AR plasmid ratios of 1:1. The human prostate-specific antigen (PSA) gene is an AR-regulated target gene that has been widely used as a prostate-specific tumor marker (26, 27). To determine whether the repression of AR activity by TGIF could be reproduced on a natural AR-dependent promoter, transient transfections were repeated in PC3 cells with a luciferase reporter gene driven by the 7-kb PSA promoter (28). TGIF plasmid reduced AR-mediated transcription from the PSA promoter by 70% (Fig. 1BGo).

We next tested repression of AR-dependent transcription by TGIF in a physiologically relevant cellular context. Transient transfections were repeated in LNCaP, a human prostate cancer cell line that expresses endogenous AR protein. Repression of endogenous AR activity by TGIF is measured with a cotransfected PSA promoter. As seen in Fig. 1CGo, endogenous AR induces the PSA promoter approximately 25-fold in the presence of DHT. Overexpression of TGIF showed a dosage-dependent repression of AR activity. This result demonstrates that TGIF can repress endogenous AR protein-mediated transcription from the PSA promoter.

To ensure that the TGIF-mediated repression did not reflect toxic or other nonspecific effects of the cotransfected plasmids, luciferase expression in all experiments was normalized using ß-galactosidase (ß-gal) production from a cotransfected plasmid. We also examined the intracellular steady-state levels of AR protein in the above transfectants and found them to be similar, indicating that the TGIF-mediated repression was not due to reduced levels of expression of AR (data not shown).

TGIF Selectively Represses AR-Mediated Transcription
TGIF is expressed in most human tissues, and it also functions to repress transcription mediated by other activators such as Smad2, a transducer of TGFß signals. To assess whether TGIF represses other steroid hormone receptor activities, we tested GR and PRß along with AR, using similar experimental conditions. The MMTV promoter (MMTVpA3-Luc) containing the steroid hormone responsive elements for the respective receptors (29, 30) were cotransfected with AR, PRß, or GR expression vector into CV-1 cells. With the appropriate ligand, the three receptors showed ligand-dependent transcription from the MMTVpA3-Luc reporter vector (Fig. 2AGo). As we observed previously, TGIF showed a dosage-dependent repression of AR activity. However, TGIF only slightly repressed GR-mediated transcription when 10 ng of the plasmid were used, and has no repression of PRß activity (Fig. 2AGo). Based on these results, we conclude that TGIF mediated a specific transcriptional repression on AR under identical experimental conditions.



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Figure 2. TGIF Specifically Represses AR-Mediated Transcription

A, CV-1 cells were transiently transfected with 100 ng of pMMTV-Luc, 25 ng of pSV40-ß-gal, 10 ng of pSV-hAR, pSV-hGR, or pSV-hPRß, and as indicated, 5 or 10 ng of pcDNA3-Flag-TGIF. The corresponding ligands for each receptor were used in the experiments, including 10 nM DHT (AR), 10 nM dexamethasone (GR), and 10 nM progesterone (PR). White bars represent the absence of ligands, whereas black bars represent the addition of ligands. B, For each sample, 25 ng pSV-ß-gal, 100 ng luciferase reporter vectors containing the different hormone response elements for each receptors (pARE-luc, pVDRE-luc, pERE-luc, and pTRE-luc), and 10 ng of the corresponding receptor expression constructs were transfected into CV-1 cells, with or without 10 ng of pcDNA3-Flag-TGIF. The specific ligands to each receptor were added in the following concentrations: 10 nM DHT, 100 nM ß-E2 (ER), 10 nM T3 (TR), and 10 nM 1{alpha},25-dihydroxyvitamin D3 (VDR).

 
The specificity of TGIF repression was further investigated with other nuclear receptors. As shown in Fig. 2BGo, expression of AR produced about a 10-fold induction on a luciferase reporter driven by duplicated AREs in the presence of DHT. Induction was reduced by 70% by cotransfection of TGIF, which is consistent with the results shown in Fig. 1Go for both the MMTV and PSA promoters. In contrast, there was no repression by TGIF of ER-, TR-, and VDR-controlled promoters driven by the corresponding responsive elements (Fig. 2BGo). Taken together, our results suggest that TGIF selectively represses AR-mediated transcription.

Repression of AR Activity by TGIF Is Mediated Through HDAC Pathways
Although an earlier study showed that TGIF represses RXR-mediated transcription by competing the RXR binding to its target elements (22), the recent studies have shown that transcriptional repression by TGIF is mainly mediated through the HDAC pathway (23, 24). In this regard, we tested whether repression on AR is also through the HDAC pathway using a HDAC inhibitor, TSA (31). In transfected CV1 cells with both AR and TGIF expression plasmids, TGIF strongly repressed AR-mediated transcription (Fig. 3AGo). However, when the transfected cells were treated with TSA at 5 or 10 nM, the repression was partially or fully reversed, and high luciferase activity was observed compared with untreated cells (Fig. 3AGo).



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Figure 3. TGIF-Mediated Repression on AR-Mediated Transcription Is TSA Sensitive

A, Transient transfections were performed in CV-1 cells with 100 ng of pMMTV-Luc, 25 ng of pSV40-ß-gal, 10 ng of pSV-hAR, and 10 ng of pcDNA3-Flag-TGIF in the absence or presence of 5 or 10 nM TSA. B, The experiments were repeated with 10 ng of pcDNA3-I{kappa}Bß1 and other plasmids as described in A. After transfection, the cells were treated with 10 nM of TSA.

 
To ensure that the effect of TSA is specific to TGIF rather than a general effect on the basic transcription machinery, we repeated the experiment with the pcDNA3-I{kappa}Bß1 expression plasmid as a control, which was previously shown to repress transcription mediated by the AR and other nuclear receptors (32). As shown in Fig. 3BGo, cotransfection of AR and an equal amount of the TGIF or I{kappa}Bß1 expression construct driven by a cytomegalovirus (CMV) promoter reduced the ligand-dependent AR transcription. Addition of 10 nM TSA in the transfected cells can reverse TGIF-mediated repression, but showed no effect on the cells transfected with pcDNA3-I{kappa}Bß1. These results clearly indicate that TGIF-mediated repression is TSA sensitive and that the HDAC pathway is involved in the regulation process.

TGIF Interacts with AR Protein
Our results indicate that repression of AR activity by TGIF is mediated through the HDAC pathway. One possible mechanism for TGIF repressing AR on multiple AR-dependent promoters would be by a physical interaction with the AR. Therefore, a series of glutathione S-transferase (GST)-AR fusion proteins containing various functional domains were generated to assess possible interactions with TGIF (Fig. 4AGo). Binding of [35S]methionine-labeled full-length TGIF protein to GST-AR fusion proteins was analyzed by SDS-PAGE and detected by autoradiography. As seen in Fig. 4BGo, a specific retention of TGIF protein was observed when AR-DBD was present, indicating that TGIF was binding to a region of the AR in or near the DBD (Fig. 4BGo). Additional GST-AR fusion proteins were generated to determine whether the binding site was in the DBD or in the hinge regions flanking the DBD. The GST fusion proteins incorporating the amino-terminal (AR-5'DBD) or carboxyl-terminal hinge regions (AR-3'DBD) did not bind to TGIF (top panel, Fig. 4CGo). In contrast, the construct containing precisely the DBD (AR-C'DBD) showed a strong interaction with TGIF when similar amounts of the GST fusion proteins were used in the experiments (bottom panel, Fig. 4CGo). These results indicate that a physical protein-protein interaction occurs between AR and TGIF, and that the DBD of AR is responsible for the binding.



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Figure 4. TGIF Interacts with the DBD of AR in Vitro

A, The different portions of AR were fused to GST in pGEX-2TK vector and shown in the figure. B, In vitro-translated, 35S-labeled full-length TGIF was incubated with various GST-AR fusion proteins on beads, the beads were washed three times, and proteins were resolved by SDS-PAGE gel and visualized by autoradiography. C, Additional GST-AR fusion proteins containing the small fragments of the DBD were made, and the pull-down experiments were repeated (top panel). Ten microliters of the GST-AR DBD fusion proteins used in the above protein pull-down experiments were resolved in SDS-PAGE and stained with Coomassie blue (bottom panel).

 
TGIF Interacts with a Transcriptional Corepressor, Sin3A
A previous study showed that TGIF physically interacts with HDAC1 and negatively regulates Smad2 (23, 24). The class I HDAC proteins, including HDAC1–3, can target the transcription repression complexes through the transcriptional corepressor, Sin3A (2, 33). We therefore tested the possibility of Sin3A to interact with TGIF. Using the N-terminal fragment of Sin3A between amino acids 1 and 700 as "bait" in a yeast two-hybrid screening, we isolated 43 putative binding cDNA clones from a myeloid cDNA library (34). Seventeen of these clones perfectly matched the sequence encoding TGIF (22). Retransformation with GAL4 DBD alone or the bait plasmid containing either the N- or C-terminal fragment of Sin3A and the TGIF or pVP16 plasmid, respectively, were carried out to confirm the interaction. Cotransformants containing both the pGBT9-Sin3A-N and a plasmid in which the VP16 activation domain was fused to TGIF showed a strong interaction as evidenced by high expression of ß-gal reporter gene (Fig. 5BGo). These results verify that Sin3A specifically interacts with TGIF in yeast cells.



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Figure 5. TGIF Interacts with Sin3A in Vitro and in Vivo

A, Schematic representation of the full length of Sin3A protein. Numbers correspond to amino acid residues. B, Yeast strain PJ-69 4A was cotransformed with the plasmids containing GAL4-DBD (pGBT9) and VP16-AD (pVP16). Yeast colonies from the SD-Leu-Trp plates were cultured in SD-Leu-Trp medium. ß-Gal activities were measured using the Galacto-light Plus kit (Tropix Inc., Bedford, MA) and normalized by cell density (OD600). The data represent the mean ± SD of three independent colonies. C, Coimmunoprecipitation of TGIF and Sin3A proteins was performed in the transfected CV-1 cells, and total cell lysates were analyzed by Western blot to check the levels of expressed proteins (top and middle panels). Equal amounts of cell lysates were immunoprecipitated with normal mouse IgG or anti-Flag monoclonal antibody (F) at 4 C. The immune complex bound to protein-A Sepharose beads was then resolved by SDS-PAGE and analyzed by Western blot using anti-Sin3A polyclonal antibody (bottom panel). D, In vitro-translated Sin3A protein (35S-labeled) was incubated at 4 C for 2 h with an equal amounts of various GST-TGIF fusion proteins coupled to Sepharose beads (see Materials and Methods). The beads were washed, and proteins were resolved on 10% SDS-PAGE gel and analyzed by autoradiography. 35S-labeled Sin3A is marked in the figure.

 
To determine whether the interaction between Sin3A and TGIF occurs in vivo, we examined the protein-protein interaction by coimmunoprecipitation analysis. A Flag-tagged TGIF expression plasmid was transfected into CV1 cells. Both endogenous Sin3A and Flag-tagged TGIF proteins were detected in the transfected cells (Fig. 5CGo, top and middle panels). Whole-cell lysates were immunoprecipitated with normal mouse IgG or an anti-Flag monoclonal antibody. To increase the specificity of protein complexes precipitated by the Flag antibody, immunoprecipitates were eluted with the Flag peptide. Eluted protein complexes were then analyzed by Western blot using a Sin3A N-terminal antibody. As shown in the bottom panel of Fig. 5CGo, endogenous Sin3A protein was detected in anti-Flag immunoprecipitates from cells transfected with the Flag-TGIF plasmid (lane 4), but not in untransfected cells (lane 2) or when normal mouse IgG was used (lanes 1 and 3). These results demonstrate that interaction between the full-length Sin3A and TGIF proteins occurs in vivo.

To precisely map the interaction region of TGIF protein with Sin3A, GST pull-down experiments were carried out with a series of GST-TGIF fusion proteins (Fig. 5DGo). [35S]Methionine-labeled full-length Sin3A protein bound to GST-TGIF fusion proteins was analyzed by SDS-PAGE and detected by autoradiography. As shown in Fig. 5DGo, a strong retention of Sin3A protein was specifically observed for the samples with GST-TGIF protein containing amino acids 108–192. These results are consistent with those of the yeast two-hybrid and coimmunoprecipitation experiments and suggest that the region between 108 and 192 amino acid residues within TGIF is required for the interaction with Sin3A.

TGIF Directly Interacts with Sin3A
Previous studies showed that Sin3A functions as a corepressor through recruitment of HDAC factors to transcriptional complexes (10, 11, 12). An interaction between TGIF and HDAC1 was observed previously in coimmunprecipitation experiments using the overexpressed proteins (24). In this study, we have identified a protein-protein interaction between Sin3A and TGIF. Importantly, the interacting region of TGIF for both Sin3A and HDAC1 is mapped to amino acids 108–192 (24). Therefore, it is possible that the TGIF-HDAC1 interaction identified previously may be mediated through Sin3A protein. To test this model, we first examined the interaction between TGIF and HDAC1 or Sin3A using in vitro protein pull-down experiments. The truncated GST-TGIF proteins between amino acids 108 and 192 and the control constructs were incubated with in vitro-translated HDAC1 or Sin3A proteins. As observed previously, the GST-TGIF protein containing amino acids 108–192 showed a strong retention of in vitro-translated Sin3A protein (right panel, Fig. 6AGo). In contrast, in the identical experiment setting, no binding was detected between the GST-TGIF proteins and HDAC1 (left panel, Fig. 6AGo). The results from these experiments suggest that TGIF directly binds Sin3A but not HDAC1.



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Figure 6. TGIF-Recruiting HDAC1 Is Sin3A-Dependent

A, In vitro-translated, 35S-labeled full-length Sin3A or HDAC1 were incubated with equal amounts of the GST proteins on beads. The beads were washed three times, and proteins were resolved by SDS-PAGE gel and visualized by autoradiography. B, Both overexpressed Sin3A and Flag-tagged HDAC1 proteins were coimmunoprecipitated with anti-Sin3A and -Flag antibodies and were then analyzed by SDS-PAGE and transferred to two identical nitrocellulose membranes. One membrane was analyzed by Western blotting for detection of the protein expression (left panel). The other blot was renatured and then hybridized with a 32P-labeled TGIF protein fragment between amino acids 108–192. C, CV-1 cells were transiently cotransfected with Flag-tagged HDAC1 and T7-tagged TGIF. Equal amounts of cell lysate were immunoprecipitated with normal mouse IgG or anti-Flag monoclonal antibody at 4 C. The precipitated fractions with either normal IgG or flag antibody (IP) were then resolved by SDS-PAGE and analyzed by Western blot (WT) using anti-Flag antibody (top panel), anti-Sin3A antibody (middle panel), or anti-T7 antibody (bottom panel).

 
Although the results from the GST pull-down experiments suggest that TGIF may directly interact with Sin3A, it still remains possible that the interaction may be mediated indirectly by another protein in the programmed cell lysates that were used to synthesize Sin3A and HDAC1 proteins. For this reason, we performed a Far Western blotting assay, a potentially more specific method, to further confirm the direct interaction that we observed above. Equal amounts of both full-length Sin3A and HDAC1 proteins were precipitated with anti-Sin3A and -Flag (for HDAC1) antibodies from the whole-cell lysates and were run on SDS-PAGE and transferred to a nitrocellulose membrane (Fig. 6BGo). Blots were renatured for 48 h and probed with the 32P-labeled TGIF protein fragment (amino acids 108–192). As shown in Fig. 6BGo, a specific Sin3A band was labeled with the TGIF probe but not HDAC1. These data confirmed the above-described GST pull-down results and indicate that Sin3A protein directly interacts with TGIF.

To further test our hypothesis that Sin3A bridges TGIF and HDAC1 proteins in the same protein complex, we cotransfected both Flag-tagged HDAC1 and T7-tagged TGIF expression constructs into CV-1 cells. CV-1 cells were chosen because they contain detectable endogenous levels of Sin3A protein. A Flag-tag antibody was used for coimmunoprecipitations, and then the coimmunprecipitates with both normal IgG and Flag antibody were analyzed by Western blots with Flag, T7, and Sin3A antibodies. As shown in Fig. 6CGo, both Sin3A and TGIF proteins were detected in the complex with HDAC1. These results provide an additional line of evidence showing that Sin3A mediates the interaction between HDAC1 and TGIF proteins in the cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional activation by AR and other nuclear hormone receptors is a dynamic and complex process that requires recruitment of other transcriptional cofactors to assemble a transactivation complex on the target promoter. It is likely that the acetylation state of core histones plays an important role in the regulation of transcription (5, 35), and selective recruitment of histone acetylases and HDACs can convert transcriptionally active states into silent states. A number of HATs, including p300/CBP, PCAF, SRC-1, and ACTR, which possess intrinsic HAT activity, have been identified as transcriptional coactivators of nuclear hormone receptors. Transcriptional activation by TR, RAR, and other nuclear hormone receptors has been shown to be regulated by both HATs and HDACs (7, 36). Unbound TR, RAR, and other nuclear hormone receptors stay in a latent state in the nucleus by association with NCoR and SMRT (10, 11, 12). After binding to ligand, the receptors can be switched from repressed states into activated states by disassociation of repressors and association with transcriptional activators. Because most steroid hormone receptors are sequestered by HSPs in the cytoplasm, the mechanism by which transcriptional activation is switched to repression for AR and other steroid hormone receptors may be different from the other nuclear hormone receptors. Recently, it has been reported that the active transcriptional complex formed by ER{alpha} and its coactivator is down-regulated through the ubiquitin proteasome pathway (21). In addition, a recent report has demonstrated that upon binding an antagonist, tamoxifen, ER{alpha} recruits both NCoR and SMRT to form a transcriptional repressor complex (37). Although both reports have shed light on the negative regulation of ER-mediated transcription, it is unclear whether AR and other steroid hormone receptors are also regulated in the same manner. Moreover, there is still a gap in our knowledge regarding how an open, active nucleosome with an active transcriptional complex can turn into a compact, inactive form on AR- and other steroid hormone receptor-regulated promoters. In this report, we have identified the transcriptional repressor, TGIF, which functions as a bona fide repressor to specifically repress AR-mediated transcription. The repression by TGIF is mediated through a protein complex containing TGIF, Sin3A, and HDAC1 proteins, which provides evidence for the first time that the HDAC pathway is involved in AR-mediated transcription.

TGIF is a homeobox protein that contains multiple transcriptional repression domains (24). Although it was shown earlier that TGIF binds to RXR response element and interferes with RXR{alpha} DNA binding, recent evidence has suggested that TGIF repression is mediated through the HDAC pathway (23). In our transient transfection assays, we found that TGIF substantially represses AR-mediated transcription on several androgen-induced promoters. A protein-protein interaction between the DBD of AR and TGIF was demonstrated. The repression by TGIF was further determined to be TSA sensitive. In the process of probing the mechanism of TGIF-mediated repression, using several in vivo and in vitro approaches, we determined that TGIF physically interacts with a corepressor, Sin3A. Previous studies have shown that Sin3A is a component of transcription repression complexes and functions as a mediator between sequence-specific DNA binding repressors and HDAC proteins. Our results suggest a potential role for Sin3A in the TGIF-mediated repression.

Using a series of deletion mutants of both TGIF and Sin3A, we mapped the interaction regions of the two proteins. The interaction region of Sin3A with TGIF was mapped to the N-terminal region (Fig. 5AGo), which contains the paired amphipathic {alpha}-helix 1, 2, and 3 domains. This region was also previously shown to interact with the transcriptional repressors MAD and NCoR/SMRT (38, 39, 40), suggesting that a common mechanism may apply to TGIF and these transcriptional repressors. The binding region of TGIF with Sin3A was mapped to the middle portion of TGIF (amino acids 108–192), which is the exact same region that interacts with HDAC1 (24). Interestingly, in our GST pull-down experiments, we only detected the interaction between TGIF and Sin3A, but not HDAC1, under identical experimental conditions. One possibility is that the interaction between TGIF and HDAC1 is not direct and is possibly mediated through Sin3A. Using far Western blot, we confirmed that a direct interaction between TGIF and Sin3A occurs. Taken together, these results suggest that Sin3A is necessary for TGIF to recruit HDAC1 for the repression of AR-mediated transcription.

More than seven HDAC proteins have been identified. Based on their sequences, they can be divided into two classes: class I includes HDAC1–3, whereas class II includes HDAC4–6 (36, 41, 42). Several reports have shown that Sin3A appears to play a pivotal role in targeting the class I HDACs to transcriptional repressor complexes (10, 36). However, HDAC4, HDAC5, and an as yet unnamed new member of the class II HDACs are capable of interacting directly with the corepressors NCoR and SMRT to target various transcriptional complexes in a Sin3A-independent manner (43). Our finding that Sin3A allows HDAC1 to complex with TGIF is consistent with the previous observation and suggests that TGIF may function on AR in a Sin3A-dependent manner.

TGIF has been reported previously to be a corepressor of Smad2 protein. In this report, we show that TGIF interacts with the AR and represses AR-mediated transcription. However, we found that TGIF probably does not act broadly as a nuclear hormone corepressor, because it repressed AR-mediated transcription but not the activities of other nuclear hormone receptors, including PRß, ER{alpha}, TR, and VDR. In the context of the MMTV promoter, we showed that TGIF slightly inhibits GR activity. Because the DBD of AR, which is composed of two zinc fingers, was mapped as the interacting region with TGIF, it is likely that the selective repression by TGIF on AR and GR may be due to the close sequence similarities in the DBD regions of these proteins.

In this report, we demonstrated that TGIF interacts with the DBD of AR, facilitating binding of Sin3A and HDAC1 to form a repression complex resulting in deacetylation of the core histones. Although it is unclear whether other cellular factors are involved in this process, it is likely that TGIF can trigger this regulation. Repression of AR-mediated transcription by TGIF suggests a potentially significant mechanism to control androgen-induced cell growth, which may play a critical role in the growth and development of normal prostate tissue, and in the development and progression of prostate cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Cultures and Transient Transfections
A monkey kidney cell line, CV-1 (44), and a human prostate cell line, PC-3 (45), were maintained in RPMI 1640 medium supplemented with 5% FCS (HyClone Laboratories, Inc., Logan, UT). The AR-positive human prostate cancer cell line, LNCaP (46), was cultured in T medium (Life Technologies, Inc., Gaithersburg, MD) with 5% FCS.

Transient transfections for CV-1 were carried out by using a LipofectAMINE transfection kit (Life Technologies, Inc.) as described previously (47). About 200 ng of total plasmid DNA per well was used in the transfection. The total amount of DNA per dish was kept constant by adding pBluescript plasmid (Stratagene, La Jolla, CA). Approximately 16 h after transfection, the cells were washed and fed medium containing 5% charcoal-stripped (steroid hormone-free) FCS (HyClone Laboratories, Inc.) in the presence or absence of steroid hormones. LNCaP and PC3 cells were transfected with the LipofectAMINE 2000 reagent. Because we have observed that simian virus 40 (SV40) promoter is transcriptionally inactive in these prostate cancer cell lines, the expression vectors driven by a CMV promoter were used in the experiments. Luciferase activity was measured in relative light units as previously described (48, 49). The relative light units from individual transfections were normalized by measurement of ß-gal activity expressed from a cotransfected plasmid in the same samples. Individual transfection experiments were done in triplicate and the results are reported as mean luciferase/ß-gal (±SD) from representative experiments.

Plasmid Construction
The human TGIF cDNA was a kind gift of Dr. Roger Clerc (Roche, Basel, Switzerland). GST-TGIF constructs containing different portions of TGIF and the full-length TGIF expression vector were generated by PCR with specific primers and subcloned into the pGEX (Amersham Pharmacia Biotech, Piscataway, NJ) and pcDNA3-Flag vectors, respectively. For yeast two-hybrid screening, N-terminal (amino acids 1–700) and C-terminal segments (amino acids 700–1,219) of human Sin3A were generated by PCR and fused to the GAL4 DBD in the pGBT9 vector (CLONTECH Laboratories, Inc., Palo Alto, CA). Human HDAC1 expression vector (pBJ5.1-HDAC1) with a carboxyl-terminal Flag epitope tag was kindly provided by Dr. Edward Seto (University of South Florida, Tampa, FL).

The AR expression vector, pSV-hAR, was provided by Dr. Albert Brinkmann (Erasmus University, Rotterdam, The Netherlands). pMMTV-luc was provided by Dr. Richard Pestell (Albert Einstein College of Medicine, New York, NY). The pPSA-luc reporter plasmid was obtained from Dr. Belldegrun (UCLA, Los Angeles, CA) (28). pSV-ß-gal, an SV40 driven ß-gal reporter plasmid (Promega Corp., Madison, WI) was used in CV-1 cells as an internal control. The pSG5-ARA70 plasmid, containing the full-length ARA70 cDNA, and the reporter plasmid pARE-luc were the kind gifts of Dr. Chawnshang Chang (University of Rochester, Rochester, NY) (25). pCMV-VDR, pSV-hGR, and pVDRE-luc were provided by Dr. David Feldman (Stanford University, Stanford, CA). A human ER expression construct (pcDNA3-ER) and a luciferase reporter plasmid with three estrogen responsive elements were kindly provided by Dr. Myles Brown (Dana-Farber Cancer Institute, Boston, MA). A human TRß expression vector and a luciferase reporter controlled by two testosterone responsive elements were kindly provided by Dr. Anthony Hollenberg (Beth Israel Deaconess Medical Center, Boston, MA). The human PRß expression vector, pSV-hPRß, was a gift of Dr. Nancy L. Weigel (Baylor College of Medicine, Houston, TX). The human I{kappa}Bß1 expression vector, pcDNA3-I{kappa}Bß1, was kindly provided by Dr. David Moore (Baylor College of Medicine, Houston, TX).

Yeast Two-Hybrid Screen
The interaction between Sin3A and TGIF was examined in a yeast two-hybrid assay. pGBT9-Sin3A-N (amino acids 1–700), pGBT9-Sin3A-C (amino acids 700-1219), and a myeloid cDNA library (34) were used in the experiments with a modified yeast strain, PJ69-4A (50). The experimental conditions for yeast two-hybrid screenings were previously described (49). Transformants were selected on Sabouraud dextrose medium lacking adenine, leucine, and tryptophan. Specific interactions were measured by the production of adenine and ß-gal. A liquid ß-gal assay was used to quantify the interaction.

GST Pull-Down Assay
Expression and purification of GST fusion proteins were performed as described previously (51). The full-length human TGIF and Sin3A proteins were translated and labeled in vitro using the TNT-coupled reticulocyte lysate system (Promega Corp.). Equal amounts of GST-AR or -TGIF fusion proteins coupled to glutathione Sepharose beads were incubated with radiolabeled TGIF or Sin3A proteins at 4 C for 2 h. The buffer used for protein binding is a modified NETN buffer (0.2% Nonidet P-40 (NP-40), 1 mM EDTA, 20 mM Tris-Cl (pH 8.0), 100 mM NaCl, 5% glycerol, 4 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). Beads were carefully washed four times in NETN buffer and then analyzed by SDS-PAGE followed by autoradiography.

Immunoprecipitation and Western Blotting
The human Sin3A expression vector, pIRES1HisSin3A (33), alone or with a Flag-tagged pcDNA3-TGIF expression plasmid, were transfected into CV-1 cells. After incubation for 24 h, cells were harvested into a buffer containing 20 mM HEPES (pH 8.0), 0.5% NP-40, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 50 mM NaF, 0.3 mM sodium vanadate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5% glycerol). Whole-cell lysates were incubated with mouse normal IgG or Flag monoclonal antibody (Sigma, St Louis, MO) at 4 C for 2 h. Pre-equilibrated protein A-Sepharose beads were then added and, after 1 h of incubation, collected by centrifugation and gently washed three times with the same buffer as described above. Specific protein complexes were eluted with 100 ng/ml of Flag peptide in a buffer containing 10 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 0.1% NP-40. The eluted samples were boiled in SDS sample buffer and resolved on a 10% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and probed with a 1:500 dilution of a polyclonal antibody against the N terminus of mSin3A (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. sc-994). Proteins were detected using an ECL kit (Amersham Pharmacia Biotech, Arlington Heights, IL).

Far Western Blot Analyses
The overexpressed Sin3A and Flag-tagged HDAC1 proteins were precipitated with specific antibodies. The precipitated Sin3A and HDAC1-F were then resolved on an 8% SDS-PAGE and transferred onto nitrocellulose membranes. One set of membranes was analyzed by Western blotting with Sin3A or Flag antibodies. The other was first denatured in 6 M guanidine hydrochloride buffer [50 mM HEPES (pH7.5), 50 mM NaCl, 0.1 mM EDTA, 10 mM MgCl2, 1 mM DTT, 0.1 mM ZnSO4, and 10% glycerol). The membrane was renatured by removing the guanidine hydrochloride gradually (from 6 M to 0.5 mM) in changing the buffer every 2–3 h and was then blocked in renaturation buffer with 5% fat-free milk overnight. The blocked membrane was then incubated with {gamma}-P32-labeled TGIF (amino acids 108–192) for 10–14 h in the renaturation buffer with 0.25% fat-free milk. The membrane was then washed in the same buffer three times at room temperature. Each wash was for 30 min. The membrane was then air-dried and exposed to x-ray film using intensifying screens.


    ACKNOWLEDGMENTS
 
We are especially grateful for the various reagents received from Drs. David Feldman, Nancy Weigel, Albert Brinkmann, Richard Pestell, Myles Brown, Chawnshang Chang, Anthony Hollenberg, David Moore, and Roger Clerc. We thank Drs. Fajun Yang and Olga Petrauskene for invaluable technical assistance and useful discussions and Mr. Homer Abaya for administrative assistance and help in preparing this manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant CA-70297 (to Z.S.) and American Cancer Society Grant RPG98213 (to Z.S.).

Abbreviations: ARE, Androgen responsive element; CBP, cAMP response element-binding protein-binding protein; CMV, cytomegalovirus; CREB, cAMP response element-binding protein; DBD, DNA binding domain; DTT, dithiothreitol; ß-gal, ß-galactosidase; GST, glutathione-S-transferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; HSP, heat-shock protein; MMTV, mouse mammary tumor virus; NCoR, nuclear receptor corepressor; NP-40, Nonidet P-40; PSA, prostate-specific antigen; SMRT, silencing mediator for RAR and TR; SRC, steroid receptor coactivator; SV40, simian virus 40; TGIF, 5'TG3' interacting factor; TSA, trichostatin.

Received for publication May 14, 2001. Accepted for publication August 3, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Kouzarides T 1999 Histone acetylases and deacetylases in cell proliferation. Curr Opin Genet Dev 9:40–48[CrossRef][Medline]
  2. Grunstein M 1997 Histone acetylation in chromatin structure and transcription. Nature 389:349–352[CrossRef][Medline]
  3. Struhl K 1998 Histone acetylation and transcriptional regulatory mechanisms. Genes Dev 12:599–606[Free Full Text]
  4. Xu L, Glass CK, Rosenfeld MG 1999 Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Genet Dev 9:140–147[CrossRef][Medline]
  5. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953–959[Medline]
  6. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  7. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  8. Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM, Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 279:703–707[Abstract/Free Full Text]
  9. Blanco JC, Minucci S, Lu J, Yang XJ, Walker KK, Chen H, Evans RM, Nakatani Y, Ozato K 1998 The histone acetylase PCAF is a nuclear receptor coactivator. Genes Dev 12:1638–1651[Abstract/Free Full Text]
  10. Chen JD and Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  11. Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377:451–454[CrossRef][Medline]
  12. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  13. Chang CS, Kokontis J, Liao ST 1988 Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science 240:324–326[Medline]
  14. Wilson EM, Simental JA, French FS, Sar M 1991 Molecular analysis of the androgen receptor. Ann NY Acad Sci 637:56–63[Medline]
  15. Sanchez ER, Faber LE, Henzel WJ, Pratt WB 1990 The 56–59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins. Biochemistry 29:5145–5152[Medline]
  16. Sullivan WP, Vroman BT, Bauer VJ 1992 Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies. J Steroid Biochem Mol Biol 43:37–41[CrossRef][Medline]
  17. Jenster G 1999 The role of the androgen receptor in the development and progression of prostate cancer. Semin Oncol 26:407–421[Medline]
  18. Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast TH, Trapman J, Brinkmann AO 1991 Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol Endocrinol 5:1396–1404[Abstract]
  19. Fu M, Wang C, Reutens AT, Wang J, Angeletti RH, Siconolfi-Baez L, Ogryzko V, Avantaggiati ML, Pestell RG 2000 p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation. J Biol Chem 275:20853–20860[Abstract/Free Full Text]
  20. Ikonen T, Palvimo JJ, Janne OA 1997 Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J Biol Chem 272:29821–29828[Abstract/Free Full Text]
  21. Lonard DM, Nawaz Z, Smith CL, O’Malley BW 2000 The 26S proteasome is required for estrogen receptor-{alpha} and coactivator turnover and for efficient estrogen receptor-{alpha} transactivation. Mol Cell 5:939–948[Medline]
  22. Bertolino E, Reimund B, Wildt-Perinic D, Clerc RG 1995 A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif. J Biol Chem 270:31178–31188[Abstract/Free Full Text]
  23. Wotton D, Lo RS, Lee S, Massague J 1999 A Smad transcriptional corepressor. Cell 97:29–39[Medline]
  24. Wotton D, Lo RS, Swaby LA, Massague J 1999 Multiple modes of repression by the Smad transcriptional corepressor TGIF. J Biol Chem 274:37105–37110[Abstract/Free Full Text]
  25. Yeh S, Chang C 1996 Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proc Natl Acad Sci USA 93:5517–5521[Abstract/Free Full Text]
  26. Dube JY, Chapdelaine P, Guerin S, Leclerc S, Rennie PS, Matusik RJ, Tremblay RR 1995 Search for androgen response elements in the proximal promoter of the canine prostate arginine esterase gene. J Androl 16:304–311[Abstract/Free Full Text]
  27. Cleutjens KB, van Eekelen CC, van der Korput HA, Brinkman AO, Trapman J 1996 Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specfic antigen promoter. J Biol Chem 271:6379–6388[Abstract/Free Full Text]
  28. Pang S, Dannull J, Kaboo R, Xie Y, Tso CL, Michel K, deKernion JB, Belldegrun AS 1997 Identification of a positive regulatory element responsible for tissue-specific expression of prostate-specific antigen. Cancer Res 57:495–499[Abstract]
  29. Hoeck W, Hofer P, Groner B 1992 Overexpression of the glucocorticoid receptor represses transcription from hormone responsive and non-responsive promoters. J Steroid Biochem Mol Biol 41:283–289[CrossRef][Medline]
  30. Mink S, Ponta H, Cato AC 1990 The long terminal repeat region of the mouse mammary tumour virus contains multiple regulatory elements. Nucleic Acids Res 18:2017–2024[Abstract]
  31. Taunton J, Hassig CA, Schreiber SL 1996 A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272:408–411[Abstract]
  32. Na SY, Choi HS, Kim JW, Na DS, Lee JW 1998 Bcl3, an I{kappa}B protein, as a novel transcription coactivator of the retinoid X receptor. J Biol Chem 273:30933–30938[Abstract/Free Full Text]
  33. Hassig CA, Fleischer TC, Billin AN, Schreiber SL, Ayer DE 1997 Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89:341–347[Medline]
  34. Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold A, Rohrschneider LR 1996 p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity. Genes Dev 10:1084–1095[Abstract]
  35. Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382:319–324[CrossRef][Medline]
  36. Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE, Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89:373–380[Medline]
  37. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2001 Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103:843–852[CrossRef]
  38. Zhang X, Jeyakumar M, Petukhov S, Bagchi MK 1998 A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor. Mol Endocrinol 12:513–524[Abstract/Free Full Text]
  39. Ayer DE, Laherty CD, Lawrence QA, Armstrong AP, Eisenman RN 1996 Mad proteins contain a dominant transcription repression domain. Mol Cell Biol 16:5772–5781[Abstract]
  40. Hurlin PJ, Foley KP, Ayer DE, Eisenman RN, Hanahan D, Arbeit JM 1995 Regulation of Myc and Mad during epidermal differentiation and HPV-associated tumorigenesis. Oncogene 11:2487–2501[Medline]
  41. Hassig CA, Tong JK, Fleischer TC, Owa T, Grable PG, Ayer DE, Schreiber SL 1998 A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proc Natl Acad Sci USA 95:3519–3524[Abstract/Free Full Text]
  42. Grozinger CM, Hassig CA, Schreiber SL 1999 Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc Natl Acad Sci USA 96:4868–4873[Abstract/Free Full Text]
  43. Pazin MJ Kadonaga JT 1997 What’s up and down with histone deacetylation and transcription? Cell 89:325–328[Medline]
  44. Rovera G, Mehta S, Maul G 1974 Ghost monolayers in the study of the modulation of transcription in cultures of CV1 fibroblasts. Exp Cell Res 89:295–305[Medline]
  45. Isaacs JT and Kyprianou N 1987 Development of androgen-independent tumor cells and their implication for the treatment of prostatic cancer. Urol Res 15:133–138[Medline]
  46. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP 1983 LNCaP model of human prostatic carcinoma. Cancer Res 43:1809–1818[Abstract]
  47. Yang F, Li X, Sharma M, Zarnegar M, Lim B, Sun Z 2001 Androgen receptor specifically interacts with a novel p21-activated kinase, PAK6. J Biol Chem 276:15345–15353[Abstract/Free Full Text]
  48. Sun Z, Pan J, Hope WX, Cohen SN, Balk SP 1999 Tumor susceptibility gene 101 protein represses androgen receptor transactivation and interacts with p300. Cancer 86:689–696[CrossRef][Medline]
  49. Sharma M, Zarnegar M, Li X, Lim B, Sun Z 2000 Androgen receptor interacts with a novel MYST protein, HBO1. J Biol Chem 275:35200–35208[Abstract/Free Full Text]
  50. James P, Halladay J, Craig EA 1996 Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436[Abstract/Free Full Text]
  51. Sun Z, Pan J, Balk SP 1997 Androgen receptor-associated protein complex binds upstream of the androgen-responsive elements in the promoters of human prostate-specific antigen and kallikrein 2 gene. Nucleic Acids Res 25:3318–3325[Abstract/Free Full Text]