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Molecular Endocrinology 20 (5): 1048-1060
Copyright © 2006 by The Endocrine Society

The Corepressors Silencing Mediator of Retinoid and Thyroid Hormone Receptor and Nuclear Receptor Corepressor Are Involved in Agonist- and Antagonist-Regulated Transcription by Androgen Receptor

Ho-Geun Yoon and Jiemin Wong

Department of Biochemistry and Molecular Biology (H.-G.Y.), Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul 120-752, South Korea; and Department of Molecular and Cellular Biology (J.W.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Jiemin Wong, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: jwong{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have investigated the role of corepressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) in transcriptional regulation by androgen receptor (AR) in the LNCaP prostate cancer cell line. Using specific small interference RNAs to knock down SMRT and/or N-CoR in LNCaP cells, we found that SMRT and N-CoR not only mediate antagonist-dependent inhibition of AR activation but also have a widespread role in suppressing agonist-dependent activation of several AR target genes we have tested, including PSA (prostate-specific antigen), TSC22 (TSC22 domain family member 1), NKX3–1 (NK3 transcription factor locus 1), and B2M(ß-2-microglobulin). By sequencing analysis followed by analysis of physical association by chromatin immunoprecipitation assay, we mapped the putative androgen response elements in the NKX3–1 and B2M. Consistent with a role in both antagonist- and agonist-regulated transcription by AR, chromatin immunoprecipitation analysis revealed that both SMRT and N-CoR were recruited by AR to these genes in the presence of either flutamide or R1881. Knocking down SMRT and N-CoR enhanced the recruitment of the coactivators steroid receptor coactivator 1 and p300 by agonist-bound AR and led to increased hyperacetylation of histone H3 and H4, suggesting that the corepressors actively compete with coactivators for binding to agonist-bound AR. Taken together, our data indicate that SMRT and N-CoR corepressors are involved in transcriptional regulation by both agonist- and antagonist-bound AR and regulate the magnitude of hormone response, at least in part, by competing with coactivators.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANDROGENS ARE critical in the development and maintenance of the male reproductive system (1, 2). The effects of androgens are mediated through the androgen receptor (AR), a member of a large family of ligand-dependent transcriptional factors that belong to the nuclear receptor (NR) superfamily (3). AR is also involved in the development and progression of prostate cancer, which is one of the most frequently diagnosed cancers in males (4). Androgen antagonists, which inhibit the transcriptional activity of AR, are widely used for treatment of prostate cancer. Although this hormone blockade therapy is effective initially, most patients eventually progress into hormone-refractory or androgen-independent prostate cancer (5, 6). Thus, understanding the underlying mechanisms by which androgen antagonists repress AR activity is critical for the development of more effective drugs or therapeutic approaches in the future.

Over the past few years, it has become clear that the transcriptional activity of AR, as well as other members of the NR superfamily, is modulated, either positively or negatively, by coregulatory proteins known as coactivators and corepressors (7, 8, 9, 10). Coactivators are believed to function either as bridging factors between receptors and basal transcription machinery to enhance recruitment of the basal transcription machinery and/or as factors that have the capacity to actively remodel repressive chromatin (9, 10, 11). A large number of proteins have been reported to function as AR coactivators, including the generic coactivators such as the steroid receptor coactivator (SRC) family members and cAMP response element binding protein (CREB)-binding protein/p300 and the relatively AR-specific coactivators such as ARA54 and ARA70 (7, 12). SRC family members interact with both the activation domain 1 and activation domain 2 of AR (13, 14, 15, 16) and recruit cAMP response element binding protein (CREB)-binding protein/p300, which facilitate transcriptional activation, at least in part, through their intrinsic histone acetyltransferase activity.

The nuclear receptor corepressor (N-CoR) (17) and the related silencing mediator for retinoid and thyroid hormone receptors (SMRT) (18) were originally isolated as retinoic acid receptor- and thyroid hormone receptor-interacting proteins that form complexes with receptors in the absence of ligand. Since then, N-CoR and SMRT have been found to interact with antagonist-bound progesterone receptor (19), glucocorticoid receptor (20), and estrogen receptor (21) to repress transcription, and they also serve as corepressors for several additional members of the NR superfamily, including RevErb (22), peroxisome proliferator-activated receptor-{gamma} (23), and chicken ovalbumin upstream promoter-transcription factor I (24). In addition, SMRT and N-CoR have also been implicated as corepressors for diverse transcription factors including Mad/Mxi, BCL6/LAZ3, ETO, CBF, and REST/NRSF in a wide array of biological processes (25). Studies with N-CoR knockout mice have revealed the essential role for N-CoR in early embryonic development, neural cell differentiation, and development progression of specific erythrocytes and thymocytes (26, 27). Recent biochemical and functional analyses of SMRT and N-CoR demonstrate that both SMRT and N-CoR exist and function as large protein complexes within cells and associate with histone deacetylase 3 (HDAC3), GPS2 (a cell signaling protein), and TBL1 and TBLR1 (two highly related WD-40 repeat proteins) (28, 29, 30, 31). Thus, SMRT and N-CoR can be viewed as class I HDAC-containing corepressor complexes that are distinct from the other HDAC-containing corepressor complexes such as Sin3A and NuRD.

SMRT and N-CoR have also been shown to affect the transcriptional activity of AR. Through transient transfection assays, SMRT was initially shown to interact with AR and suppress its transcriptional activity in the presence of antagonists such as flutamide and cyproterone acetate (32). SMRT and N-CoR were also shown to be recruited to the prostate-specific antigen (PSA) promoter in the presence of antagonist but not agonist (33, 34). Interestingly, several studies have implicated the involvement of SMRT and N-CoR in agonist-dependent transcriptional activation by AR (35, 36, 37, 38). Using ChIP assays, a recent study provided direct evidence that N-CoR is recruited to the PSA promoter by AR in the presence of dihydrotestosterone (DHT) (39). Thus, current data suggest that corepressors SMRT and N-CoR could potentially play important roles in modulating both agonist and antagonist-regulated AR function. However, how SMRT and N-CoR influence the transcriptional regulation of endogenous AR target genes in the presence of agonists or antagonists remains unclear.

In this study, we have investigated the roles of SMRT and N-CoR in modulating both agonist- and antagonist-dependent regulation of several AR target genes in LNCaP cells. We show that SMRT and N-CoR not only mediate antagonist-dependent inhibition of AR activation but also have a widespread role in suppressing agonist-dependent activation of several AR target genes. In support, ChIP analysis revealed that both SMRT and N-CoR were recruited by AR to these genes in the presence of either antagonist flutamide or agonist R1881. Knocking down SMRT and N-CoR enhanced the recruitment of the coactivators SRC-1 and p300 by agonist-bound AR and led to increased hyperacetylation of histone H3 and H4, suggesting that SMRT and N-CoR actively compete with coactivators for binding to agonist-bound AR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Both SMRT and N-CoR Are Targeted to the PSA Enhancer in the Presence of Agonist or Antagonists
To test whether corepressors SMRT and N-CoR interact with agonist- or antagonist-bound AR, we sought to determine whether the N-CoR and SMRT corepressor complexes were recruited to the PSA enhancer and promoter in LNCaP cells treated with or without the agonist R1881 or the antagonists flutamide and bicalutamide. The LNCaP adenocarcinoma cell line is an androgen-sensitive prostate cancer line derived from a lymph node metastasis in a human subject. The AR in LNCaP cells contains a threonine to alanine mutation in codon 877 (T877A) that has been shown to broaden its hormone specificity and render flutamide a partial agonist activity in this cell line (40). In pilot experiments, we found that AR was associated with neither the enhancer nor the promoter in the untreated cells and that R1881 or flutamide treatment led to a drastic increase in the association of AR with the enhancer (data not shown) (see Fig. 1AGo). Consistent with the result in a previous report (41), we found that the association of AR with the promoter region is barely detectable even in the presence of R1881 (data not shown). Furthermore, we found that the association of AR with the enhancer peaked around 30 min and persisted at least up to 4 h after addition of R1881 or flutamide (data not shown), a result also in agreement with the previous report (41). Given these observations, we performed ChIP assays for coactivators and corepressors with LNCaP cells treated with agonist or antagonists for 1 h and focused only on the recruitment to the enhancer. A representative result in Fig. 1Go showed that both SMRT and N-CoR were targeted to the PSA distant enhancer in the presence of R1881 as well as flutamide and bicalutamide. In contrast, the coactivators SRC-1 and p300 were recruited to the enhancer by AR only in the presence of R1881. This result substantiates the idea that corepressor proteins can be targeted to the PSA gene by AR in either an agonist- or antagonist-dependent manner. Interestingly, the flutamide-bound AR seems to be able to partially recruit SRC-3, which may in part explain the partial agonist activity observed for 2-hydroxyflutamide, the active metabolite of flutamide, in LNCaP cells (42).


Figure 1
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  		Fig. 1. Corepressors SMRT and N-CoR Were Recruited to the PSA Enhancer Both in the Presence of Agonist R1881 and Antagonists Flutamide and Bicalutamide

The relative positions of previously identified AREs in the promoter and enhancer are illustrated in the top panel. The ChIP assays were performed with LNCaP cells treated with the indicated reagents for 1 h. Because the binding of AR and cofactors to the promoter region was very weak, only the results for the association with the enhancer region were shown. Note that a similar level of SMRT or N-CoR was recruited to the enhancer region when LNCaP cells were treated with either R1881 or flutamide or bicalutamide. IgG, Rabbit antimouse IgG (negative control for ChIP).

 
Corepressors Are Recruited to Various AR Target Genes by Both Agonist- and Antagonist-Bound AR
To determine whether the association of SMRT/N-CoR with both agonist- and antagonist-bound AR is a general mechanism or a gene-specific event, we wished to analyze their association with additional AR target genes in LNCaP cells. Recently, Nelson et al. (43) identified AR target genes in LNCaP cells using cDNA microarrays. Among 146 genes with transcription alteration more than 3-fold after administration of DHT, we tested a set of genes including prostate-specific antigen (PSA), NK3 transcription factor locus 1 (NKX3–1), ß-2-microglobulin gene (B2M), and TSC22 domain family member 1 (TSC22) in response to R1881 by RT-PCR analysis. We observed a robust induction of all these genes after treatment with R1881 for 12 h (Fig. 2AGo). The induction could also be detected after a 3- or 6-h treatment (see supplemental Fig. 1 on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), indicating all these genes are likely the direct AR targets. Interestingly, different antagonists appear to have different effects on expression of these AR target genes. Flutamide and bicalutamide weakly activated TSC22 but had no effect on NKX3–1 and B2M. In addition, flutamide, but not bicalutamide, slightly enhanced transcription of PSA. The effect of these various ligands on all four AR target genes was further analyzed using real-time quantitative PCR and the results are shown in Fig. 2BGo.


Figure 2
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  		Fig. 2. Confirmation of TSC22, NKX3–1, and B2M as AR Target Genes in LNCaP Cells

LNCaP cells were first treated with or without R1881 (1 nM) for 12 h or 24 h or with flutamide (1 µM) or bicalutamide (1 µM) for 24 h. Total RNA was then prepared from each sample, and the levels of TSC22, NKX3–1, PSA, and B2M were analyzed by semiquantitative PCR (A) and real-time quantitative PCR (B). The level of GAPDH mRNA served as a control in panel A. The real time RT-PCR data in panel B were normalized against 18 S rRNA.

 
To examine the association of SMRT and N-CoR with these AR target genes, we first attempted to identify the AREs in the TSC22, NKX3–1, and B2M genes. By sequencing analysis the putative AREs were identified previously for TSC22, NKX3A, and B2M genes, at the positions –1711, –1272, and –440 bp (relative to their transcription start sites), respectively (41). Unfortunately by ChIP we failed to detect the binding of AR to these putative AREs in the presence of agonist R1881 (data not shown). Using MatInspector software (Genomatrix Com.), a program allowing identification of transcription factor binding sites by sequencing comparison, we searched within the 20-kb region upstream of the transcriptional start site for each gene and identified additional putative AREs for the NKX3A and B2M genes at positions –3,013 and –19,288/12,871/1,902 bp, respectively (Fig. 3Go). To test the binding of AR to each of these putative AREs, we carried out ChIP experiments for AR using samples derived from LNCaP cells treated with or without R1881 or flutamide for 1 h. Specific PCR primers were designed to amplify sequences (100–150 bp) surrounding each of the putative AREs. As shown in Fig. 3Go, the binding of AR could be detected for the ARE of NKX3–1 at the position of –3013 and for B2M at the position of –1902.


Figure 3
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  		Fig. 3. Experimental Verification of the AREs in the NKX3–1 and B2M Genes

The 20-kb region upstream of the transcriptional start site for NKX3–1 or B2M was searched for putative AREs using the MatInspector program. P, Promoter. The putative AREs identified are ttGGAGCTctgAGTTctgc for NKX3–1 and caGGTGCTctcTATTctct (ARE1) tgTGTACCacaTTTTcttc (ARE2), and taGGCAAAcagTGCTcttg (ARE3) for B2M gene. The binding of AR to these sites was directly tested by ChIP assay using LNCaP cells treated with or without R1881 or flutamide for 1 h. Note that AR was shown to bind to ARE1 in the NKX3–1 gene and ARE3 of the B2M gene upon treatment with either R1881 or flutamide. Also note that N-CoR was recruited to the aforementioned AREs under the same condition.

 
We also carried out ChIP assays using a N-CoR-specific antibody under the same experimental conditions. The results in Fig. 3Go show that N-CoR was recruited to the ARE region of the NKX3–1 gene in the presence of either R1881 or flutamide. For the B2M gene, N-CoR was found to be recruited to ARE3 but not ARE1 or ARE2. Again, the recruitment of N-CoR was observed in the presence of either R1881 or flutamide. Thus, the recruitment of N-CoR is strictly correlated with the binding of AR and was observed in the presence of either an agonist or an antagonist. A similar result was observed for SMRT (data not shown and see Fig. 7Go). Taken together, we conclude that corepressors SMRT and N-CoR are recruited to various AR target genes by both agonist- and antagonist-bound AR.


Figure 7
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  		Fig. 7. SMRT and N-CoR Compete with Coactivators for Binding to Agonist-Bound AR

LNCaP cells were treated with siRNA against SMRT and/or N-CoR to knock down their expression individually or in combination. The cells were then treated with R1881 (1 nM) for 1 h, and the recruitment of corepressors and coactivators to three genes, PSA, NKX3–1, and B2M, was determined by ChIP assay using antibodies as indicated. The results were analyzed by real-time PCR and are shown as the percentage of input. The results are shown as mean ± SD calculated from two independent experiments. Note that binding of AR to each gene was not affected by siRNA treatment and that siRNA treatment specifically abrogated the recruitment of the corresponding protein. SiCon, Small interference control.

 
Corepressors SMRT and N-CoR Suppress Agonist-Stimulated Activation of AR Target Genes
To examine the roles of SMRT and N-CoR in transcriptional regulation by AR, we used small interference RNA (siRNA) to knock down SMRT and N-CoR in LNCaP cells. Recent studies indicate that 21- to 23-nucleotide double-stranded RNA (siRNA) can act as a guide sequence within a multicomponent nuclease complex to target complementary mRNA for degradation (44, 45). To test the effect and specificity of the siRNA, LNCaP cells were transfected with different amounts of siRNA against N-CoR and/or SMRT. Three days after transfection, LNCaP cells were collected, and the effect of siRNA was analyzed by Western blot. As shown in Fig. 4Go, siRNA specific for N-CoR was able to reduce the level of N-CoR more than 90% when it was used at a concentration of 80 nM or higher. Small interference SMRT (siSMRT) seemed to be even more effective, as knocking down was obvious even at 40 nM. Moreover, combination of small interference N-CoR (siN-CoR) and siSMRT led to knockdown of both SMRT and N-CoR. As specificity controls, we found that the protein levels of HDAC3 and SRC-3 were not affected (Fig. 4AGo).


Figure 4
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  		Fig. 4. Knocking Down SMRT and/or N-CoR in LNCaP Cells Using Specific siRNA

A, LNCaP cells were transfected with control (scramble) siRNA or siRNA against SMRT or N-CoR at a concentration as indicated. Whole-cell extracts were prepared 3 d after treatment, and the levels of SMRT and N-CoR were determined by Western blot using SMRT and N-CoR antibodies. Western results for HDAC3 and SRC-3 serve as specificity controls. B, The effect of knocking down SMRT or N-CoR or both on the basal level of transcription from PSA gene was analyzed by real-time RT-PCR. siCon, Small interference control.

 
We next tested the effect of the siRNAs on the induction of PSA, NKX3–1, and B2M expression by R1881. For this purpose, LNCaP cells were treated with siRNA against SMRT, N-CoR, or both to knock down their expression individually or in combination. Three days after siRNA treatment, the cells were induced without or with 10 nM R1881 for 6 h, and the levels of PSA, NKX3A, and B2M gene expression were determined by quantitative real-time PCR. As shown in Fig. 4BGo, knocking down SMRT, or N-CoR, or both had little, if any, effect on the transcription of PSA gene in the absence of R1881. Similarly, knocking down SMRT, or N-CoR, or both did not affect the basal transcription from NKX3.1 and B2M genes (see supplemental Fig. 2 on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). However, treatment with siRNA against N-CoR and SMRT increased R1881-stimulated transcription of the PSA gene from about 5-fold to about 7-fold and approximately 9.5-fold, whereas combined siRNA treatment increased the induction to about 10-fold (Fig. 5AGo). A more significant effect was observed for the NKX3–1 (Fig. 5BGo). In this case, knocking down either N-CoR or SMRT augmented R1881-stimulated transcription from about 3-fold to approximately 6-fold and 5.5-fold, whereas knocking down both N-CoR and SMRT increased the activation to about 8.5-fold. In the case of B2M gene, knocking down N-CoR, SMRT, or both increased R1881-stimulated transcription from about 6-fold to about 8-fold, about 8-fold, and about 9-fold, respectively (Fig. 5CGo). As a control, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not significantly affected by any of the siRNA treatments (Fig. 5DGo). Thus, knocking down SMRT and N-CoR in general led to increased levels of R1881-stimulated transcription from all three AR target genes, although the extent of the increase is gene specific.


Figure 5
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  		Fig. 5. SMRT and N-CoR Have a Widespread Role in Suppression of Agonist-Stimulated Transcriptional Activation by AR

LNCaP cells were treated with different concentrations of siRNA against SMRT or N-CoR individually or in combination. The cells were treated 3 d after siRNA treatment with 1 nM R1881 for 6 h, and the effect on transcriptional activation of PSA (A), NKX3–1 (B) and B2M (C) was analyzed by quantitative real-time PCR analyses. The levels of transcription are shown as fold induction in comparison with the level of tran-scription in the absence of R1881, which was set as 1. The transcription from GAPDH gene (D) served as a control. Note the siRNA dose-dependent effect on transcription and a general additive effect of siSMRT + siN-CoR in comparison with siSMRT or siN-CoR alone. The results are shown as mean ± SD calculated from three independent experiments. SiCon, Small interference control.

 
Corepressors SMRT and N-CoR Mediate Suppression of AR Activation by Flutamide
To investigate the roles of corepressors in suppression of AR activation by antagonist, we set up experiments in which activation of AR target genes by R1881 (1 nM) was challenged by addition of flutamide. Although flutamide was reported to act as a partial agonist in LNCaP cells, real-time PCR analysis showed that addition of flutamide could suppress R1881-stimulated transcriptional activation in a dose-dependent manner (see data with control siRNA in Fig. 6Go, A–C). The ability for flutamide to suppress AR activation could be explained by its ability to recruit efficiently corepressors SMRT and N-CoR but less efficiently the coactivators such as SRC-1 and p300 in comparison with R1881 (Fig. 1Go). We then knocked down SMRT or N-CoR individually or in combination by siRNA and tested the ability of flutamide to suppress activation by R1881. The results in Fig. 6AGo show that knocking down SMRT or N-CoR partially relieved the suppression of the PSA gene, especially at the high dose of flutamide. A more dramatic effect was observed for the NKX3–1 gene. In this case, knocking down either N-CoR or SMRT partially relieved the suppression by flutamide, whereas knocking down both SMRT and N-CoR completely abrogated the suppression (Fig. 6BGo). The effect on the B2M gene is less dramatic in comparison with the NKX3–1 gene but is clearly more than that on the PSA gene, and an additive effect was observed when both SMRT and N-CoR were knocked down (Fig. 6CGo). Together, these experiments indicate that corepressors SMRT and N-CoR play important roles in antagonist-mediated repression of AR target gene expression, a key feature of hormone therapy for prostate cancer.


Figure 6
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  		Fig. 6. SMRT and N-CoR Play an Important Role in Mediating the Antagonist Effect of Flutamide

LNCaP cells were treated with siRNA against SMRT or N-CoR individually or in combination for 3 d. The cells were then treated with 1 nM R1881 or 1 nM R1881 plus an increasing amount of flutamide (5 and 10 µM) for 12 h, and the levels of transcription from the PSA (A), NKX3–1 (B), and B2M (C) genes were analyzed by quantitative real-time PCR analyses. The levels of transcription are shown as fold induction as compared with the level of transcription in the absence of R1881. The results are shown as mean ± SD calculated from three independent experiments. SiCon, Small interference control.

 
Corepressors Compete with Coactivators for Binding to Agonist-Bound AR
Given that both coactivators and corepressors are targeted to the PSA enhancer region in the presence of R1881 (Fig. 1Go), we reasoned that coactivators and corepressors might compete with each other for binding to AR. To test this idea, we examined whether knocking down corepressors in LNCaP cells would lead to increased recruitment of coactivators to the three AR target genes described above. For this purpose, LNCaP cells were treated with or without siRNA against SMRT or N-CoR individually or in combination. Three days later the cells were treated with or without R1881 for 1 h and processed for ChIP using antibodies against coactivators SRC-1, SRC-3, and p300 and corepressors SMRT and N-CoR. To more precisely measure the recruitment of coactivators and corepressors, we used real-time quantitative PCR analysis. As shown in Fig. 7AGo, R1881 treatment led to a significant increase in binding of AR to the PSA enhancer. The binding of AR was not affected by the siRNA treatments. Consistent with the semiquantitative result in Fig. 1Go, addition of R1881 resulted in recruitment of N-CoR and SMRT to the PSA enhancer. The recruitment of N-CoR or SMRT was abolished by treatment with siN-CoR or siSMRT, confirming the effectiveness and specificity of siRNA. Knocking down either N-CoR or SMRT led to increased recruitment of SRC-1 and p300, although a greater effect was observed when both corepressors were knocked down. Interestingly, knocking down the corepressors did not appear to affect the recruitment of SRC-3. Whether this reflects a saturated recruitment of SRC-3 is not known.

Like the case of the PSA enhancer, we found that the recruitment of SRC-1 and p300 to the NKX3–1 gene was similarly affected (Fig. 7BGo). Knocking down SMRT and N-CoR affected the recruitment of SRC-1 and p300 but had little effect on SRC-3. Finally, knocking down SMRT and N-CoR also enhanced the recruitment of SRC-1 and p300 to the ARE3 of the B2M gene, although to a lesser extent (Fig. 7CGo). Taken together, these results support the idea that the corepressors SMRT and N-CoR compete with coactivators for binding to agonist-bound AR.

The Balancing between Corepressors and Coactivators Determines the Level of Histone Acetylation
Because SMRT and N-CoR are the HDAC3-containing corepressor complexes and p300 contains intrinsic histone acetyltransferase activity, we also analyzed histone acetylation after knocking down SMRT and/or N-CoR by ChIP assay. Specifically, we analyzed the levels of acetylated H3, acetylated H4, and acetylated H3 at lysine 9 over the PSA enhancer (Fig. 8AGo), the ARE1 region of NKX3–1 (Fig. 8BGo), and the ARE3 region of the B2M gene (Fig. 8CGo). For the PSA enhancer, the results in Fig. 8AGo show that knocking down N-CoR led to an approximately 94% increase in H3 acetylation and an approximately 45% increase in H4 acetylation. Knocking down SMRT also resulted in increased acetylation of H3 and H4, although to a lesser extent (20% and 3%, respectively). Although the effect on histone acetylation differed from one gene to another, the general trend is that knocking down either SMRT or N-CoR led to an increase in histone acetylation and that knocking down both SMRT and N-CoR led to a further increase. The changes in histone modifications are likely the combined result of removal of the corepressor SMRT/N-CoR complexes and the increased recruitment of coactivators SRC-1 and p300 as shown in Fig. 7Go.


Figure 8
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  		Fig. 8. Knocking Down SMRT and/or N-CoR Led to Increased Levels of Histone Acetylation on the AR Target Genes

The experiments were carried out as in Fig. 7Go except antibodies specific for acetylated H3 (AcH3), acetylated H4 (AcH4), and acetylated lysine 9 of H3 (AcK9-H3) were used. The results are shown as mean ± SD calculated from two independent experiments. SiCon, Small interference control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SMRT and N-CoR play important roles in development, differentiation and tumorigenesis (25). These diverse functions are believed to be mediated, at least in part, through their ability to serve as corepressors for nuclear hormone receptors and many other transcription factors. Both SMRT and N-CoR exist in and function as large protein complexes containing HDAC3 (28, 29, 30, 31). In general, SMRT and N-CoR interact with and mediate transcriptional repression by unliganded receptors such as TR and retinoic acid receptor- or antagonist-bound receptors such as progesterone receptor and estrogen receptor (46, 47). Before our study, several groups have investigated the interaction of corepressors SMRT and N-CoR with AR and their roles in transcriptional regulation by AR in the presence of agonists or antagonists (32, 33, 34, 35, 36, 39, 48, 49). In several papers, the interaction was shown to occur in the presence of antagonists but not agonists (32, 33, 36), whereas in others the interaction was shown to also occur in the presence of the agonist DHT (35, 39, 49). Our data are consistent with reports that SMRT and N-CoR also interact with agonist-bound AR. We show that SMRT and N-CoR were recruited not only to the PSA enhancer but also to the AREs in the NKX3–1 and B2M genes in the presence of the agonist R1881. In fact, our data indicate that a similar level of SMRT and N-CoR was recruited to PSA enhancer in the presence of either R1881 or pure antagonist bicalutamide (Fig. 1Go). Thus, our study provides firm evidence to support the idea that corepressors modulate both agonist- and antagonist-regulated AR transcriptional activity.

By transient transfection assays, SMRT and N-CoR have been shown to affect both agonist- and antagonist-regulated transcriptional activity of AR (32, 35, 36, 39, 48, 49). Using specific siRNAs to knock down SMRT and/or N-CoR, we have examined the roles of SMRT and N-CoR in the transcriptional regulation of several endogenous AR target genes in LNCaP cells. Consistent with the results that SMRT and N-CoR interact with R1881-bound AR (Fig. 7Go), knocking down SMRT and/or N-CoR led to increased levels of transcription for all three genes tested, although the extent of increase was gene dependent (Fig. 5Go). Using these siRNAs, we also found that SMRT and N-CoR are required for suppression of AR activation by flutamide (Fig. 6Go). This result is not in contradiction with the previous publications that flutamide could function as AR agonist in LNCaP cells as a result of the T877A mutation (53, 54). Indeed, we observed that flutamide, but not bicalutamide, can induce activation of PSA. However, neither NKX3.1 nor BM2 was induced by flutamide, indicating that flutamide functions as a partial agonist in LNCaP cells. A partial agonist activity for flutamide in LNCaP cells is consistent with our ChIP results in Fig. 1Go showing that flutamide treatment resulted in recruitment of SRC-3, but not SRC-1, to the PSA enhancer. Whether the recruitment of SRC-3 accounts for the partial agonist activity of flutamide in LNCaP cells remains to be determined in the future. Nevertheless, the functional dependence of flutamide on SMRT/N-CoR, as demonstrated in this study, implies that the levels of SMRT and N-CoR in patients may be a determinant for the efficacy of antihormone therapy. In addition, developing drugs with increased capability to promote the AR-SMRT/N-CoR interaction would likely improve the efficacy of current antihormone therapy. In this regard, two recent studies showed that RU486 (mifepristone) is better at inducing interaction between AR and N-CoR in comparison with agonist DHT and other antagonists including bicalutamide (39, 49).

Our ChIP assay data indicate that SMRT and N-CoR compete with coactivators such as SRC-1 and p300 for binding to agonist-bound AR, because knocking down SMRT and N-CoR in general led to increased recruitment of SRC-1 and p300 (Fig. 7Go). Thus, one mechanism by which SMRT and N-CoR inhibit AR activation is likely by sequestering AR from interacting with coactivators. SMRT and N-CoR have been shown to interact with both the ligand-binding domain as well as the N-terminal domain of AR through their nuclear-receptor interaction motifs located in the C terminus (32, 35, 36, 39). Given the evidence that SMRT and N-CoR exist in and function as HDAC3-containing corepressor complexes (28, 29, 30, 31), SMRT and N-CoR are likely to also inhibit transcriptional activation of AR target genes through their associated HDAC3 activity. In this regard, the increased histone acetylation as a consequence of knocking down SMRT and/or N-CoR is likely the combined effect of elimination of deacetylation by SMRT/N-CoR complexes and increased recruitment of coactivators SRC-1 and p300 and possibly other coactivators.

Consistent with the result that both SMRT and N-CoR were recruited to the various AREs by AR (Figs. 1Go and 7Go), SMRT and N-CoR, in general, have an additive effect on inhibition of transcription and histone acetylation. Thus, SMRT and N-CoR seem to have a very similar, if not identical, function in regulating AR activity. This conclusion is in agreement with data in literature showing that both SMRT and N-CoR interact with AR (32, 35, 36, 39). It is noteworthy that knocking down SMRT and/or N-CoR affects the level of transcription, the recruitment of coactivators, and histone acetylation to various degrees for the PSA, NKX3–1, and B2M genes, despite the fact that SMRT and N-CoR are targeted to all these genes by agonist-bound AR. Thus, SMRT and N-CoR are likely to have differential effects on different AR target genes. This differential effect on transcription is likely determined by the differences among different genes in the specific organization of the transcriptional regulatory sequences (enhancer and promoter), the transcription factors and coactivators involved, as well as the local and/or regional chromatin structure.

One discrepancy between our data and that of others concerns the association of SMRT and N-CoR with the PSA enhancer or the promoter region. Among studies in which the recruitment of SMRT and N-CoR was directly analyzed by ChIP, the recruitment was either reported to be detected only in the promoter region (33, 34) or only the association with the promoter region was analyzed (39). In contrast, we found that SMRT and N-CoR were recruited primarily to the enhancer rather than the promoter region. Similarly, the binding of AR was detected mainly in the enhancer rather than in the promoter. Our data are consistent with a previous report showing that the recruitment of coactivators was mainly detected in the enhancer but not the promoter region (41). They are also consistent with the result in another paper showing that AR occupancy in the enhancer region is about 20-fold higher than that in the promoter (34). This discrepancy might be due to the difference in antibodies used and remains to be solved.

In sum, we present evidence that in LNCaP cells AR targets both SMRT and N-CoR to various AR target genes in the presence of either the antagonist flutamide or the agonist R1881. By virtue of their ability to interact with agonist- or antagonist-bound AR, SMRT and N-CoR suppress agonist-stimulated transcriptional activation and mediate inhibition of AR activity by antagonists. Exploring the unique antagonist-promoted interaction between AR and corepressors SMRT and N-CoR or possibly other corepressors is likely to lead the development of new therapeutic drugs for prostate cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and siRNA
LNCaP cells were routinely cultured in RPMI 1640 medium supplemented with 10% (wt/vol) fetal bovine serum. For treatment with agonist or antagonists, LNCaP cells were transferred into phenol red-free RPMI 1640 medium with 10% charcoal-stripped fetal calf serum (Life Technologies, Gaithersburg, MD) for at least 3 d followed by replacement of the media with fresh charcoal-stripped fetal calf serum supplemented with 1 nM of the synthetic androgen R1881 (NEN/Life Science Products, Boston, MA) or 1 µM of antagonists including flutamide or bicalutamide. For the small interference RNA (siRNA) experiments, LNCaP cells were seeded the night before transfection at such a density that cells reach about 30–40% confluence by the time of transfection. The siRNAs against N-CoR and SMRT were previously described (30). A combination of 40–160 nM of each siRNAs was used for transfection using Lipofectamine 2000 (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Transfected cells were continued in culture for 3 d before harvesting for further analyses. The efficiency of the siRNA knockdown was determined by Western analysis using corresponding specific antibodies.

ChIP Assays
For ChIP assays, we first isolated chromatin as described elsewhere (50). In brief, approximately 2 x 109 LNCaP cells in 150-mm dishes were first treated with PBS containing 1% formaldehyde for 10 min, washed twice with PBS, and incubated with 100 mM Tris (pH 9.4)-10 mM dithiothreitol at 30 C for 15 min. The cells were then rinsed twice with PBS and resuspended in 600 µl of solution A buffer (10 mM HEPES (pH 7.9), 0.5% Nonidet P-40, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) by pipetting. After a short spin (5 min at 3000 rpm), the pellets were resuspended in solution B (20 mM HEPES (pH 7.9), 25% glycerol, 0.5% Nonidet P-40, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) containing protease inhibitors by vigorous pipetting to extract nuclear proteins. After centrifugation at 13,000 rpm for 30 min, the nuclear pellets were resuspended in immunoprecipitation buffer (1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0; 150 mM NaCl; and protease inhibitors) and sonicated to break chromatin into fragments with an average length of 0.5–1 kb. The ChIP assays were then performed with indicated antibodies, essentially as described, but omitting sodium dodecyl sulfate in all buffers (51). The antibodies against acetylated H3, H4, H3-K9, and p300 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The antibodies against N-CoR, SMRT, SRC-1, and SRC3 were rabbit antibodies generated against the recombinant N-CoR [amino acids (aa) 1–373], SMRT (aa 1165–1363), SRC-1 (aa 1067–1441), and SRC-3 (aa 582–842) as described previously (29, 52). Primers used for ChIP analysis are as follows. NKX3–1(ARE1): 5'-TTGCATAAATTAGGGGAGAACATACCA-3' and 5'-GAGGGACCCAGCTGCGATTCA-3'; B2M(ARE1): 5'-GATGGCCAGCCTTCCCCTCACCTC-3' and 5'-AACTCTTCCCCTAAAACAACTG-3'; B2M(ARE2): 5'-ATAATAATCACATCAGGGAAAATGGTGTAT-3' and 5'-AGTAAGCCAGGCATAGAAAGACAAAC-TT-3'; B2M(ARE3): 5'-AGACTTCCCAAATTTTGCCATCCTA-3' and 5'-AAAGGCCTGAAATGTTAGT-GTTGAGT-3'.

RNA Extraction and RT-PCR
Total RNA was isolated from prostate tissues using the RNeasy Mini kit (QIAGEN, Chatsworth, CA), according to the manufacturer’s specifications. Total RNA from each sample was reverse transcribed with random primers using a StrataScript reverse transcriptase kit (Stratagene, La Jolla, CA) followed by quantitative PCR. Primers for amplification of the TSC22 transcript were 5'-GACTTGATAATAGCTCCTCTGGT-3' and 5'-ATTTTTCTCTATTAGTTCTTTGATTTG-3'. Primers for the NKX3–1 mRNA were 5'-AGCCGCTCACGTCCTTCCTCATCC-3' and 5'-GGGGCCCGGTGCTCAGCTGGTCGTTCT-3'. Primers for the PSA mRNA were 5'-GCCCACCCAGGAGCCAGCACT-3' and 5'-GGCCCCCAGAATCACCCGAGCAG-3'. Primers for the B2M mRNA were 5'-TCCAGCGTACTCCAAAGATTCAGGTT-3' and 5'-GTTCACACGGCAGGCATACTCATCTT-3'. Primers for the GAPDH mRNA were 5'-CGCGGGGCTCTCCAGAACATCATCC-3' and 5'-CTCCGACGCCTGCTTCACCACCTTCTT-3'. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.

Real-Time PCR Analysis
RT-PCR analysis and quantification were performed with Taqman One-Step RT-PCR Master Mix Reagents or SYBR Green PCR Master mix Reagents [depending on the target RNA (see below)] using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Singularity and specificity of amplification were checked by Dissociation Analysis Software (Applied Biosystems). All samples were normalized to 18S rRNA (primer and probe set for human 18S rRNA purchased from Applied Biosystems). Primer sequences for amplification of the NKX3–1 mRNA used in the quantitative RT-PCR (qRT-PCR) were forward (F) 5'-CTGTCAGCCCCTGAACGG-3', reverse (R) 5'-AACCATATCTTCACTTGGGTCTCC-3', and probe 5'-FAM-CCACCTGGCCAAGCCCCTCAAGCTC-TAMRA-3'. Primer sequences for amplification of the TSC22 mRNA used in the qRT-PCR were forward primer 5'-CAGTGGAGCTGGATCTAGGAGTTTA-3'; reverse primer 5'-TGCACCAGAGGAGCTATTATCAAGTC-3'; and probe 5'-FAM-GTGAGACTTGATAATAGCTCC-TAMRA-3'. Primer sequences for amplification of the PSA RNA used in the qRT-PCR were forward primer 5'-AGTCTGAGGAGGTGTTCTGGTG-3'; reverse primer 5'-GAGGTCGTGGCTGGAGTCATCA-3'; and probe 5'-FAM-GGAACAAAAGCGTGATCTTGC-TAMRA-3'. Primer sequences for amplification of the B2M mRNA used in the qRT-PCR were forward primer 5'-GCTGTGCTCGCGCTACTCTC-3'; reverse primer 5'-CAATGTCGGATGGATGAAACC-3'; and probe 5'-FAM-GGTTTACTCACGTCATCCAGCAG-TAMRA-3'). Primer sequences for ChIP analysis are as follows. NKX3–1(ARE): forward primer 5'-ATGGCCATGGGAGGAGCAG-3', reverse primer 5'-CCATTTTTCGCGGTGAGA-3', and 5'-FAM-ACTCAGAG-CTCCAA-TAMRA-3'; B2M(ARE): forward primer 5'-GCCATCCTAGGCAAACAGTGC-3', reverse primer 5'-TGTATTTTTCTTGGACTTACCATTTATGAC-3', and 5'-FAM-GTCATAAATGGTAAGTC-TAMRA-3'; PSA(ARE): forward primer 5'-GCCTGGATCTGAGAGAGATATCATC-3', reverse primer 5'-ACACCTTTTTTTTTCTGGATTGTTG-3', and 5'-FAM-TGCAAGGATGCCTG-TAMRA-3'. All reactions were performed in triplicate. Relative expression levels and SD values were calculated using the comparative method.


   ACKNOWLEDGMENTS
 
We thank David Stewart for critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants DK065264 and DAMD17-03-1-0165 (to J.W.) and a grant (A05-0125-AA0718-05N1-00010A) from the Korea Health 21 Research and Development Project, Ministry of Health and Welfare, Republic of Korea (to H.G.Y.).

The authors have nothing to declare.

First Published Online December 22, 2005

Abbreviations: aa, Amino acids; AR, androgen receptor; ARE, androgen receptor response element; B2M, ß-2-microglobulin gene; ChIP, chromatin immunoprecipitation; DHT, dihydrotestosterone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; N-CoR, nuclear receptor corepressor; PSA, prostate-specific antigen; NKX3–1, NK3 transcription factor locus 1; NR, nuclear reactor; qRT-PCR, quantitative RT-PCR; siN-CoR, small interference N-CoR; siRNA, small interference RNA; siSMRT, small interference SMRT; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; TSC22, TSC22 domain family member 1.

Received for publication August 5, 2005. Accepted for publication December 14, 2005.


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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Coregulators:   p300  |  SRC-1  |  AIB1  |  NCOR  |  SMRT
Ligands:   Bicalutamide  |  R1881



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[Abstract] [Full Text] [PDF]

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T. J. Peterson, S. Karmakar, M. C. Pace, T. Gao, and C. L. Smith
The Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Corepressor Is Required for Full Estrogen Receptor {alpha} Transcriptional Activity
Mol. Cell. Biol., September 1, 2007; 27(17): 5933 - 5948.
[Abstract] [Full Text] [PDF]