Modulation of the Expression and Transactivation of Androgen Receptor by the Basic Helix-Loop-Helix Transcription Factor Pod-1 through Recruitment of Histone Deacetylase 1
Cheol Yi Hong,
Eun-Yeung Gong,
Kabsun Kim,
Ji Ho Suh,
Hyun-Mi Ko,
Hyun Joo Lee,
Hueng-Sik Choi and
Keesook Lee
Hormone Research Center, School of Biological Sciences and Technology (C.Y.H., E.-Y.G., K.K., J.H.S., H.J.L., H.-S.C., K.L.), and Department of Biological Sciences (H.-M.K.), Chonnam National University, Gwangju 500-757, Republic of Korea
Address all correspondence and requests for reprints to: Keesook Lee, Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: klee{at}chonnam.ac.kr.
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ABSTRACT
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Androgen receptor (AR) is important in male sexual differentiation and testicular function. Here, we demonstrate the regulation of AR expression and its transactivation by the basic helix-loop-helix (bHLH) transcription factor Pod-1, the expression of which in postnatal testis reciprocally coincides with the expression of AR. Pod-1 represses the promoter activity of AR, possibly through its E-box. An AR promoter region of 169 bp, which harbors one canonical E-box, is sufficient for the Pod-1-repression and bound by purified Pod-1 proteins. Pod-1 also suppresses the transactivation of AR. Transient transfection analyses of mammalian cells show that Pod-1 represses AR transactivation in a dose-dependent manner. Furthermore, yeast two-hybrid, glutathione-S-transferase-pull-down, and coimmunoprecipitation analyses reveal that Pod-1 directly associates with AR through its N-terminal region and through the DNA binding-hinge domain of AR. Interestingly, Pod-1 recruits histone deacetylase (HDAC)-1 to inhibit both promoter activity and transactivation of AR. Overexpression of HDAC1 further inhibits the Pod-1-mediated repressions and Pod-1 directly interacts with HDAC1. Furthermore, chromatin immunoprecipitation assay reveals that HDAC1 is recruited with Pod-1 to the endogenous AR promoter and the androgen-regulated Pem promoter. Taken together, these results suggest that Pod-1, which controls AR transcription and function, may play an important role in the development and function of the testis.
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INTRODUCTION
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ANDROGENS ARE IMPORTANT in male sexual differentiation and testicular function (1, 2). Androgens act through androgen receptor (AR), a ligand-dependent transcription factor. Therefore, knowledge of the molecular mechanisms of AR transactivation as well as AR expression will lead to an understanding of the molecular pathway(s) of androgen action. Previous studies have reported that the expression of AR is controlled by single promoters in a number of tissues, and the major elements for AR gene regulation are located within approximately 1000 nucleotides 5' of the translation initiation codon (3, 4, 5). Although sequence analysis of this region has shown neither a TATA nor a CAAT box, mouse AR promoter contains an initiator element preceding the +1 site of transcription and a perfect GC box in a GC-rich region approximately 45 bp upstream (6). The initiator and GC box are both known to be required for transcription from some TATA-less genes (7, 8).
AR is a member of the nuclear receptor superfamily. The structure of AR is similar to those of other nuclear receptors with three separate functional domains: activating domain, DNA binding domain (DBD), and ligand binding domain (LBD) (9). Upon ligand binding, the AR undergoes conformational changes that facilitate the formation of AR dimers in complex with specific DNA sequences, called androgen response elements (AREs), to enhance transcription of target genes (10, 11). The function of AR in gene activation is modulated by other proteins called coactivators and corepressors. Coactivators potentiate ligand-dependent transactivation of the receptor with diverse modes of action, including direct interaction with basal transcription factors and covalent modification of histones and other proteins (12, 13, 14, 15). In contrast, corepressors may recruit histone deacetylase (HDAC) activity to the receptor complex, which keeps the chromatin structure (16, 17) in a state that the general transcriptional machinery and other transcription factors are blocked from functionally interacting with promoters.
Pod-1 is one of the basic helix-loop-helix (bHLH) transcription factors, which are key players in a wide array of development and differentiation of various cell types (18, 19, 20, 21). Members of the bHLH transcription factor family are divided into several classes based upon tissue distribution, dimerization capability, and DNA binding specificity (22, 23). Class II, to which Pod-1 belongs, also includes MyoD, myogenin, Atonal, and Neuro/Beta2, and exhibits a tissue-restricted pattern of expression. Pod-1 is most highly expressed in the mesenchyme of developing organs, including the lung, kidney, and gut. Pod-1, which is also expressed in mouse fetal gonad, shows a stage-dependent pattern of expression in developing testis. Its expression in the testis highly persists during later fetal stages, but gradually declines thereafter during the neonatal stage through 20 d after birth (24). Pod-1 expression in postnatal testis, which mostly occurs in Sertoli cells, reciprocally coincides with progression of the first wave of meiosis and the expression of AR (24, 25). Recently, the Pod-1 knockout mouse has been analyzed to show disrupted gonadogenesis, which indicates that Pod-1 is essential for the normal development of the testis (26). However, the detailed mechanism of Pod-1 action during gonadogenesis and the role of Pod-1 in postnatal testis remain unclear.
The present study demonstrates that Pod-1 represses the activity of AR promoter, as well as its transactivation. Pod-1 binds to the E-box element in the AR promoter to repress its transcription. Meanwhile, Pod-1 suppression of AR transactivation operates through its direct interaction with AR, which is, to the best of our knowledge, the first case that a member of bHLH transcription factor family represses the function of nuclear receptor. Interestingly, Pod-1 recruits HDAC-1 to inhibit both the expression and transactivation of AR, which suggests that a similar mechanism is involved in Pod-1-suppression of its target and of AR target promoters. Theses results suggest that Pod-1 may also play a key role in the development and function of the testis.
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RESULTS
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Pod-1 Represses the Promoter Activity of the AR Gene
Pod-1 expression reciprocally coincides with the expression of AR in the testis (24, 25). Furthermore, sequence analysis shows that some canonical E-boxes, to which Pod-1 potentially binds, reside within 1.5 kb of mouse AR promoter. Thus, we investigated whether Pod-1 regulates AR expression. We performed transient transfection analyses with Pod-1 expression plasmid and a luciferase reporter directed by the 1.5-kb region of mouse AR promoter in 15P-1 Sertoli cells. As shown in Fig. 1A
, Pod-1 inhibited the activity of 1.5-kb AR promoter by approximately 5-fold. Furthermore, various deletion mutants of the AR promoter also showed similar repression, up to 5-fold, due to the expression of Pod-1. Interestingly, 169 bp (133
+36) of the AR promoter (DM2), which harbors one canonical E-box, was sufficient for the Pod-1-mediated repression of the reporter gene expression.

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Fig. 1. Repression of the Promoter Activity of the AR Gene by Pod-1
A, Pod-1 inhibits the activity of AR promoter. Diagram of the deletion mutants of mouse AR promoter is shown. The full length is approximately 1.5 kb (541 +1028, +1 is the transcription initiation site). The reporter plasmid of full-length (mAR-Luc) or serial deletion mutants (DM1 6-Luc) of AR promoter was cotransfected with or without 150 ng of Pod-1 expression vector into 15P-1 Sertoli cells. B, Pod-1 targets the E-box element in DM2 AR promoter. The E-box element in DM2 AR promoter was mutated by PCR-based site-direct mutagenesis. Wild-type (DM2-Luc and DM7-Luc) or mutant (mtDM2-Luc and mtDM7-Luc) (70 ng) was cotransfected with or without 100 ng of Pod-1 expression vector into 15P-1 cells. C, The activity of AR promoter is inhibited by Pod-1 but is not inhibited by E12 or Beta2. 15P-1 Sertoli cells were cotransfected with 70 ng of DM2-Luc, 30 ng of Pod-1, and indicated amounts of E12 (30 or 90 ng) or Beta2 (30 or 90 ng) expression plasmid. All values represent mean ± SEM of at least three separate experiments. RLU, Relative light unit.
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To address the involvement of the E-box in the Pod-1-repression of AR promoter, we tested the DM2 construct with a mutated E-box (mtDM2) (Fig. 1B
). The result revealed that mutation of the E-box abolished the inhibitory effect of Pod-1 on the AR promoter, exhibiting insignificant repression of the reporter expression by Pod-1. We also observed similar insignificant repression with mtDM7 construct which contains 1063 bp (133
+930) of the AR promoter (DM7) with the same mutated E-box in mtDM2 construct. These results suggest that the E-box is involved in the Pod-1-repression of AR promoter and the E-box in DM2 is a major target of Pod-1.
The E-box-mediated repression of AR promoter appeared to be Pod-1 specific because other members of the bHLH transcription factor family, such as E12 and Beta2, did not inhibit the reporter expression from DM2 (Fig. 1C
). Interestingly, coexpression of E12 with Pod-1 derepressed the Pod-1 repression of AR promoter. E12 is a Class I bHLH transcription factor which often heterodimerizes with Class II bHLH transcription factors, such as Pod-1. Meanwhile, coexpression of Beta2, a member of the Class II bHLH transcription factor group, with Pod-1 insignificantly affected the Pod-1-repression of AR promoter. Taken together, these results suggest that Pod-1 may regulate the expression of AR via the E-box element within the AR promoter.
Pod-1 Binds to an E-box within the AR Promoter
To explore whether Pod-1 is recruited to the endogenous AR promoter, MSC-1 cells that express functional AR were transfected with HA-Pod-1 expression plasmid or pcDNA3HA empty vector. Chromatin immunoprecipitation (ChIP) assays were then carried out with anti-HA antibody. As shown in Fig. 2A
, upon its expression, HA-Pod-1 was recruited to the AR promoter region spanning the E-box.

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Fig. 2. Pod-1 Binds to an E-box of AR Promoter
A, Pod-1 is recruited to the AR promoter. ChIP assays were performed using anti-HA antibody in MSC-1 cells transfected with HA-Pod-1 expression plasmid. The immunoprecipitates were analyzed by PCR using a pair of specific primers spanning a DM2 region containing the E-box element in the AR promoter. A control PCR for nonspecific immunoprecipitation was done using primers specific to the ß-actin coding region. B, Pod-1 specifically binds to the E-box element within the DM2 region of AR promoter. GST, GST-Pod-1, and GST-E12 proteins were purified by glutathione Sepharose-4B beads. EMSAs were performed using either the wild-type E-box-containing or the mutant E-box-containing the DM2 region as a probe.
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To examine whether Pod-1 binds to an E-box element within the AR promoter, we performed EMSA using purified glutathione-S-transferase (GST)-Pod-1 protein and the 169-bp DM2 region of AR promoter as a probe (Fig. 2B
). GST-Pod-1 protein bound to the probe (DM2) that contains the wild-type E-box producing two complexes but did not bind to the probe (mtDM2) that contains a mutant E-box (Fig. 1B
). Because either the competition with the cold probe or the addition of anti-Pod-1 antibody eliminated the formation of the DM2/GST-Pod-1 complexes, the GST-Pod-1 binding to DM2 appears to be specific. Additionally, neither GST nor GST-E12 proteins bound to the probe (DM2) that contains the wild-type E-box. These results suggest that Pod-1 protein binds to the E-box element within the AR promoter.
Pod-1 Suppresses the Transactivation of AR
Because AR transactivation is often modulated by transcription factors such as nuclear factor-
B and c-Jun (27, 28), we investigated whether Pod-1 regulates AR transactivation and expression. Transient transfection analyses were performed with Pod-1 and AR expression plasmids together with a reporter plasmid, ARE2-TATA-Luc (29), which contains two copies of androgen response element, in 15P-1 Sertoli cells (Fig. 3A
). Surprisingly, Pod-1 suppressed the transcriptional activity of AR in a dose-dependent manner. Furthermore, this suppression of AR transactivation seemed to be specific to Pod-1 because the expression of other bHLH transcription factors, such as E12 and Beta2, caused no significant suppression of AR transactivation (Fig. 3B
). Interestingly, coexpression of E12, but not Beta2, with Pod-1 derepressed the Pod-1-mediated suppression of AR transactivation.

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Fig. 3. Suppression of AR Transactivation by Pod-1
A, Pod-1 suppresses the transactivation of AR. ARE2-TATA-Luc (30 ng) was cotransfected with 5 ng of AR and increasing amounts of Pod-1 expression plasmid (0, 5, 25, 50, and 150 ng) into 15P-1 cells. B, Pod-1 represses the transactivation of AR, whereas E12 and Beta2 did not. ARE2-TATA-Luc (30 ng) was cotransfected with 5 ng of AR, 25 ng of Pod-1, and indicated amounts of E12 (50 and 100 ng) or Beta2 (50 and 100 ng) expression vector into 15P-1 cells. C, Pod-1 represses the transcriptional activity of AR with a natural AR target promoter. Pem promoter-reporter (Pem-Luc) plasmid (50 ng) was cotransfected with 5 ng of AR, with or without 120 ng of Pod-1, into HeLa cells. Transfected cells were treated with or without 100 nM of testosterone for 24 h. All values represent mean ± SEM of at least three separate experiments.
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The suppression of AR transactivation by Pod-1 was confirmed with a natural AR target promoter, Pem, which is expressed in testicular Sertoli cells under the control of androgen. As expected, the coexpression of Pod-1 repressed the transcriptional activity of AR on the Pem promoter (Fig. 3C
). Together, these results suggest that Pod-1 specifically suppresses the transcriptional activity of AR, whereas E12 and Beta2 do not.
Pod-1 and AR Form a Complex in Vivo and Are Recruited to a Natural AR Target Promoter
To investigate whether the functional interaction between Pod-1 and AR involves their physical association, we performed yeast two-hybrid analyses using Pod-1 fused to the LexA DBD (LexA-Pod-1) and AR domain mutants (Fig. 4A
) fused to the B42 activation domain (B42-AR domain mutants). As shown in Fig. 4C
, LexA-Pod-1 alone showed a basal level of ß- galactosidase activity, as did B42-AR-Tau alone and B42-AR-LBD alone, whereas B42-AR-DBD and hinge region (DBDh) alone showed very weakly induced ß-galactosidase activity. However, the presence of LexA-Pod-1 with B42-AR-DBDh strongly induced ß-galactosidase activity. Regardless of the presence of testosterone, the presence of LexA-Pod-1 with B42-AR-Tau or B42-AR-LBD very weakly induced the expression of the reporter gene. These results indicate that Pod-1 interacts with AR, and that the DBDh region of AR is the major determinant for their interaction.

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Fig. 4. Association of Pod-1 with AR
A, Schematic representation of the full-length AR and different deletion mutants used in yeast two-hybrid and GST-pull-down assays. B, Schematic representation of the full-length Pod-1 and different deletion mutants used in transient transfections and GST-pull down assay. C, Pod-1 efficiently interacts with AR via its DBDh region in yeast two-hybrid protein-binding assays. The interaction between AR and Pod-1 was scored by the activation of ß-galactosidase reporter. All values represent the mean ± SEM of at least three independent colonies. D, The N-terminal region of Pod-1 directly interacts with AR via its DBDh region in GST-pull-down assays. [35S]methionine-labeled Pod-1 and AR were allowed to bind the GST fusion proteins of AR domain mutants and full-length, N-terminal region (Pod-1N) or C-terminal region (Pod-1C) of Pod-1, respectively. Reactions were carried out with the equivalent amount of each protein as determined by Coomassie blue staining (data not shown). Ten percent of the labeled protein used in the binding reaction was loaded as an input. E, The N-terminal region of Pod-1 is sufficient for the repression of AR transactivation. HeLa cells were cotransfected with 30 ng of ARE2-TATA-Luc and 5 ng of AR expression vector, along with 150 ng of Pod-1, Pod-1N, or Pod1 C expression plasmid. Transfected cells were treated with or without 100 nM of testosterone (T) for 24 h. The proper expression of Pod-1, Pod-1N and Pod-1C proteins was checked by Western blot analysis with anti-HA or anti-Flag antibody. All values represent mean ± SEM of at least three separate experiments. F, Pod-1 was coimmunoprecipitated with AR. MSC-1 cells were transfected with HA-Pod-1 expression plasmid and then treated with or without 100 nM of testosterone (T) for 24 h. Coimmunoprecipitations were conducted with anti-HA or anti-AR antibody. Western blot analyses of immunoprecipitated materials were performed with anti-AR or anti-Pod-1 antibody. Control Western blots (WB) are shown for the expression level of each protein. G, Pod-1 is recruited to an AR target promoter with AR. ChIP assays were performed using anti-AR and anti-HA antibody with MSC-1 cells transfected with HA-Pod-1 expression plasmid and then treated with or without 100 nM of testosterone for 24 h.
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Direct physical interaction of Pod-1 with AR was then assessed by GST-pull-down analyses (Fig. 4D
). [35S]methionine-labeled Pod-1 produced by in vitro translation was incubated with the GST fusion protein of AR domain mutants, whereas [35S]methionine- labeled AR was incubated with the GST fusion protein of Pod-1 (full length), its N-terminal mutant (Pod-1N) or C-terminal mutant (Pod-1C) (Fig. 4B
). Pod-1 interacted with GST-AR-DBDh, but with neither GST-AR-Tau nor GST-AR-LBD. On the other hand, AR interacted with GST-Pod-1N as well as GST-Pod-1 full-length, but not with GST-Pod-1C. These results suggest that Pod-1 directly interacts with AR, and the DBDh of AR and N-terminal region of Pod-1 are responsible for their interaction.
Because AR interacts with the N-terminal region of Pod-1 (Pod-1N), we investigated whether Pod-1N is sufficient for the inhibition of AR transactivation. We cotransfected expression plasmids of Pod-1 or its deletion mutants (Fig. 4B
) along with an AR expression plasmid and ARE2-TATA-Luc into HeLa cells (Fig. 4E
). Interestingly, Pod-1N on its own was capable of inhibiting the transactivation of AR, although it was slightly weaker than full-length Pod-1. However, the C-terminal region of Pod-1 (Pod-1C) did not significantly suppress AR transactivation. These results suggest that only the N-terminal region of Pod-1 is involved in the Pod-1-suppression of AR transactivation.
To assess whether Pod-1 and AR form a complex in vivo, we performed coimmunoprecipitation experiments with MSC-1 cells which had been transfected with HA-Pod-1 expression plasmid and treated with 100 nM of testosterone (Fig. 4F
). Immunoprecipitations were performed with anti-HA or anti-AR antibody. Results showed the Pod-1 associated with AR in vivo in an androgen-dependent manner, despite the fact that the association mainly involves the DBDh region of AR. The region of AR for Pod-1 interaction is probably blocked in the absence of ligand as the domain of AR for DNA binding (9, 10).
Additionally, we tested whether AR/Pod-1 protein complex could be recruited to the natural AR target promoter, Pem. ChIP assays were performed with MSC-1 cells transfected with HA-Pod-1 expression plasmid and treated with 100 nM of testosterone (Fig. 4G
). DNA/protein complexes were immunoprecipitated with either anti-AR or anti-HA antibody. As expected, both AR and Pod-1 were recruited to the Pem promoter in an androgen-dependent manner. All together, these results suggest that Pod-1 and AR form a complex in vivo and recruited to the AR target promoter.
HDAC1 Is Involved in the Pod-1-Mediated Repression of AR Transactivation and the Promoter Activity of AR Gene
To explore how Pod-1 suppresses AR function, we first assessed the involvement of histone deacetylases (HDACs) using the HDAC inhibitor trichostatin A (TSA). In 15P-1 cells, the transactivation of AR was inhibited by Pod-1 coexpression, but the repressed AR transactivation was recovered after treatment with TSA (Fig. 5A
). This TSA relief of Pod-1 suppression seemed to be specific because TSA did not significantly affect AR transactivation in the absence of Pod-1 expression. These results suggest the involvement of HDACs in the Pod-1-mediated suppression of AR transactivation.

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Fig. 5. Involvement of HDAC1 in Pod-1 Repression of AR Transactivation and Promoter Activity
A, TSA rescues Pod-1-mediated repression of AR transactivation. 15P-1 cells were cotransfected with 5 ng of AR and 150 ng of Pod-1 expression plasmids. Transfected cells were treated with or without 100 nM of testosterone for 24 h and 100 nM of TSA for 20 h before harvest. B, HDAC1 was involved in the inhibition of AR transactivation by Pod-1. ARE2-TATA-Luc (30 ng) was cotransfected with 5 ng of AR, 25 ng of Pod-1, and 100 ng of HDAC1 or 4 expression plasmid into 15P-1 cells. Transfected cells were treated with or without 100 nM of testosterone for 24 h. C, Pem-Luc (30 ng) was cotransfected with 5 ng of AR, indicated amounts of Pod-1 (25 and 100 ng), and 100 ng of HDAC1 or -4 expression plasmid into HeLa cells. Transfected cells were treated with or without 100 nM of testosterone for 24 h. D, TSA rescues Pod-1-mediated repression of AR promoter activity. HeLa cells were cotransfected with 70 ng of DM2-Luc reporter and 100 ng of Pod-1 expression plasmid, and then treated with or without 100 nM of TSA for 20 h. E, HDAC1 was involved in the inhibition of AR promoter activity by Pod-1. DM2-Luc (70 ng) was cotransfected with 50 ng of Pod-1 and 50 ng of HDAC1 or -4 expression plasmid into HeLa cells. All values represent mean ± SEM of at least three separate experiments.
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To confirm the involvement of HDACs, we tested effects of HDAC1 and -4 coexpression on the Pod-1 repression of AR transactivation. As shown in Fig. 5B
, expression of Pod-1 with AR inhibited the transactivation of AR, and coexpression of HDAC1 with Pod-1 further inhibited AR transactivation, whereas coexpression of HDAC4 caused little effect on the Pod-1-repression of AR transactivation. Coexpression of HDAC1 or -4 with only AR showed little effect on AR transactivation in the presence of testosterone. The same experiment with Pem-Luc containing a natural AR target promoter in HeLa cells showed a similar result (Fig. 5C
), which suggests that HDAC-1 is involved in the Pod-1-mediated suppression of AR transactivation.
The above results then allowed us to investigate whether HDAC is also involved in the Pod-1-repression of AR promoter activity. The DM2-Luc reporter was cotransfected with Pod-1 with or without TSA treatment. As shown in Fig. 5D
, the activity of DM2 AR promoter was inhibited by Pod-1 expression and TSA treatment almost fully relieved the repressed AR promoter activity. Furthermore, the AR promoter activity repressed by Pod-1 was further inhibited by coexpression of HDAC1, but not by coexpression of HDAC4 (Fig. 5E
). Neither HDAC1 nor HDAC4 alone affected the activity of DM2 AR promoter. Together, these results suggest that HDAC1 is involved in the Pod-1-repression of AR promoter activity, as well as its repression of AR transactivation.
Pod-1 Directly Recruits HDAC1 to Inhibit AR Transactivation and the Promoter Activity of the AR Gene
To determine whether Pod-1 directly associates with HDAC1 in the Pod-1-repression of AR transactivation and promoter activity, GST-pull-down assays were performed. [35S]methionine-labeled HDAC1 produced by in vitro translation was allowed to bind the GST fusion protein of Pod-1 or Pod-1N (Fig. 6A
). Pod-1 interacted strongly with HDAC1. Interestingly, the N-terminal region of Pod-1 (Pod-1N) is sufficient for efficient interaction with HDAC1.

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Fig. 6. Association of HDAC1 with Pod-1
A, Pod-1N directly interacts with HDAC1. [35S]methionine-labeled HDAC1 was allowed to bind the GST fusion proteins of full-length or the N-terminal region of Pod-1 (Pod-1N). Reactions were carried out with the equivalent amount of each protein as determined by Coomassie blue staining (data not shown). Ten percent of the labeled protein used in the binding reaction was loaded as an input. B, Pod-1 forms a complex with AR and HDAC1 in vivo. HA-Pod-1 expression plasmid was transfected into MSC-1 cells. Transfected cells were treated with or without 100 nM of testosterone for 24 h. Immunoprecipitations were conducted with anti-HA antibody. Western blot analyses of immunoprecipitated materials were performed with anti-AR, anti-HDAC1, anti-HDAC4, and anti-Pod-1 antibodies. Control Western blots (WB) are shown for the expression level of each protein. C, Pod-1 endogenously forms a complex with AR and HDAC1 in the testis. Testis extracts were prepared from 20-d-old mice. Immunoprecipitations were conducted with rabbit IgG or anti-AR antibody. Western blot analyses of immunoprecipitated materials were performed with anti-AR, anti-HDAC1, and anti-Pod-1 antibodies. D, HDAC1 is recruited by Pod-1 associated with AR to the Pem promoter. ChIP assays were performed with MSC-1 cells transfected with or without HA-Pod-1, and treated with or without 100 nM of testosterone. E, HDAC1 is recruited by Pod-1 bound to the AR promoter. ChIP assays were performed with MSC-1 cells transfected with or without HA-Pod-1 expression plasmid. Cross-linked DNA fragments were immunoprecipitated with anti-AR, anti-HA, anti-HDAC1, and anti-HDAC4 antibodies. The immunoprecipitates were analyzed by PCR using a pair of specific primers spanning the androgen-response elements in Pem promoter or the Pod-1-binding E-box element in AR promoter. A control PCR for nonspecific immunoprecipitation was performed using primers specific to exon 4 of the ß-actin gene. F, Pod-1 down-regulates the androgen-induced Pem expression by decreasing both AR expression and transactivation. MSC-1 cells were transfected with 300 ng of Pod-1 expression vector together with or without 100 ng of AR expression plasmid. Cells were then treated with or without 100 nM of testosterone for 24 h. Top, Pem transcripts were analyzed by real-time RT-PCR of total RNAs. ß-Actin was used for a quantitative control. Bottom, AR protein levels were analyzed by Western blot analysis.
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To explore whether endogenous HDAC1 forms a complex with AR and Pod-1 in vivo, we performed coimmunoprecipitation experiments with MSC-1 cells transfected with HA-Pod-1 expression plasmid and treated with 100 nM of testosterone. Immunoprecipitations were conducted with anti-HA antibody (Fig. 6B
). As expected, Pod-1 was associated with AR and HDAC1 in vivo. HDAC4 was not detected in MSC-1 cells. We also confirmed that the AR/Pod-1/HDAC-1 complex is endogenously formed in the 20-d testis by coimmunoprecipitation of tissue extracts with anti-AR antibody (Fig. 6C
).
Recruitment of the AR/Pod-1/HDAC1 complex to an AR target promoter was then assessed by ChIP assays with MSC-1 cells transfected with HA-Pod-1 expression plasmid and treated with 100 nM of testosterone (Fig. 6D
). Without testosterone treatment, AR, HA-Pod-1, HDAC1, and HDAC4 were not associated with the ARE-containing region of Pem promoter. However, with testosterone treatment, Pod-1 and HDAC1, as well as AR, were recruited to the region of Pem promoter. No signal was detected from the control PCR for nonspecific immunoprecipitation using primers specific to exon 4 of the ß-actin gene.
Recruitment of HDAC-1 to the AR promoter by Pod-1 was also tested by ChIP assays with MSC-1 cells transfected with or without HA-Pod-1 expression plasmid (Fig. 6E
). With HA-Pod-1 expression, HDAC1, as well as Pod-1, was recruited to the region of AR promoter containing the Pod-1-binding E-box, whereas this did not occur in the absence of HA-Pod-1 expression. Together, the results suggest that Pod-1, both bound to the E-box element and associated with DNA-bound AR, recruits HDAC1 in vivo.
Pod-1 can bind the AR promoter as well as AR bound to the androgen-regulated Pem promoter. The effect of Pod-1 expression on AR protein level was tested by transiently transfecting MSC-1 cells, which express endogenous AR, with Pod-1 expression plasmid. The results showed that Pod-1 lowered the endogenous AR protein level as expected (bottom panel, Fig. 6F
). When Pod-1 was expressed in MSC-1 cells, androgen-induced expression of endogenous Pem gene was significantly inhibited (compare lane 2 with 4 in top panel, Fig. 6F
). This suggests that Pod-1 inhibits Pem expression at least by lowering AR protein level. However, when we overexpressed AR along with Pod-1 in the cells, we also detected Pod-1 suppression of androgen-induced Pem expression (compare lane 6 with 8 in top panel, Fig. 6F
). By overexpressing AR, we tried to minimize the change of total AR protein level in transfected cells (bottom panel, Fig. 6F
), which could be caused from the Pod-1-inhibition of endogenous AR expression. Along with others (Figs. 1
, 3
, and 5
), these results strongly suggest that Pod-1 down-regulates an androgen-signal by decreasing both AR expression and transactivation.
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DISCUSSION
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Testosterone is the fundamental hormone responsible for the initiation and maintenance of spermatogenesis. Because male germ cells do not express a functional AR (30, 31), testosterone is thought to indirectly affect spermatogenesis via somatic testicular cells, such as Sertoli cells, which interact directly with developing germ cells and express a functional AR (30). The absolute requirement of AR action in Sertoli cells for proper spermatogenesis has been recently confirmed with the testis lacking AR in Sertoli cells, in which spermatogenesis arrested at the diplotene premeiotic stage with increased germ cell apoptosis (32, 33, 34). Furthermore, terminal differentiation of spermatids and their release from the seminiferous epithelium have also turned out to be AR dependent (34). Thus, the expression and transactivation of AR in the testis should be strictly regulated for proper testicular function, although it is largely unknown how this regulation occurs. Pod-1, which has been demonstrated in the current study to act as a transcriptional repressor of the AR gene and a corepressor of AR, may constitute an additional level of control for the fine-tuning of AR action in the testis.
The present study demonstrates that Pod-1 functions as a negative transcriptional regulator of the AR gene by binding to the E-box within mouse AR promoter. This agrees with the findings of previous studies that have reported that Pod-1 exerts inhibitory effects on gene expression (24, 35, 36). Interestingly, the E-box element on the AR promoter is active for Pod-1 but is not active for E12 or Beta2. Nevertheless, E12 interferes with the Pod-1-repression of AR promoter and the binding of Pod-1 protein to the AR promoter (Figs. 1C
and 2B
). These interferences may be due to the sequestration of Pod-1 protein from the AR promoter by E12, which can form a Pod-1/E12 heterodimer that does not bind to the E-box of the AR promoter. The association of Pod-1 with E12 has been recently reported (36). Pod-1 may act as a homodimer on the E-box element of the AR promoter. However, we cannot rule out the possibility that Pod-1 acts on the AR promoter as a heterodimer with other members of the bHLH transcription factor family.
Here, we also demonstrate that Pod-1 acts as a corepressor of AR by inhibiting its transactivation. Investigation of other nuclear receptors for Pod-1-suppression of their transactivation revealed that Pod-1 could inhibit the transcriptional activity of glucocorticoid receptor, estrogen receptor ß, retinoic acid receptor
, and retinoid X receptor
(data not shown). These results suggest that Pod-1 may act as a general corepressor of nuclear receptors. Although a large number of bHLHs have been identified in various organisms ranging from yeast to human (37), to the best of our knowledge, Pod-1 is the first bHLH transcription factor that has been shown to act as a corepressor of nuclear receptors.
Pod-1 recruits HDAC1 to repress the expression of the AR gene. This is quite similar to Stra 13, a bHLH factor related with Hairy, which also directly recruits HDAC1 to repress its own gene expression (38). Other bHLH transcription factors, including Hairy, Enhancer of Split, Mad, Mxi1, and Tal1, have been also shown to function as repressors. Although the mechanisms by which they work have not been fully elucidated, some progress has been made with several factors. For example, Hairy recruits a corepressor, known as Groucho, which can functionally interact with the histone deacetylase, Rpd3 (39, 40), whereas Mad heterodimerized with Max recruits the Sin3-N-CoR-HDAC complex (41, 42). On the other hand, Tal1 has been shown to repress gene expression by forming a heterodimer with E47, which is incapable of activating transcription, although it binds to the E-box element (43). These reports suggest a divergence among members of the bHLH family regarding how they function as a transcriptional repressor.
In conclusion, our results demonstrate that the bHLH transcription factor, Pod-1, negatively regulates the transcription of the AR gene and AR transactivation. In both cases, Pod-1 recruits HDAC1 to repress gene expression, implicating a similar mechanism for its function as both a transcriptional repressor and an AR corepressor. Further studies with cell type-specific knockout of the Pod-1 in the testis may allow us to analyze its role and its action mechanism in the development and function of the testis.
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MATERIALS AND METHODS
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Plasmids
pGL3-mAR promoter (1.5 kb) was constructed by subcloning the BamHI-XhoI fragment of pBLCAT3-mAR-promoter (a kind gift from Dr. Tindall, Mayo Clinic Foundation, Rochester, MN) (6) into pGL3basic vector. The luciferase reporter plasmid of deletion mutant 1 (DM1) of mouse AR promoter (DM1-Luc) was constructed by self-ligation of pGL3-mAR promoter cut with ApaI-SacI. DM2
7-Luc constructs were generated by subcloning the NheI-BstXI fragment, ApaI-BstXI fragment, ApaI-HindIII fragment, ApaI-MscI fragment, PstI fragment, and NheI partial-Pst fragment of pBLCAT3-mAR promoter into pGL3 basic vector. Pod-1 expression vector was a kind gift from Dr. Nakatsuji (Kyoto University, Kyoto, Japan) (24). CMV4-E12 and pCR3.1-Beta2 expression vectors were previously described (44). Pem promoter-reporter plasmid (Pem-Luc) was created by PCR amplification using a primer pair (forward, 5'-TATGCTCATGGCATAAGGGG-3'; and reverse, 5'-ACCCTGAATAGGATCAATGATG-3'), which amplifies a promoter region spanning 620
+1. Full-length Pod-1 was subcloned in the frame into pcDNA3HA, pGEX4T-1, and LexA202 vector to generate pcDNA3HA-Pod-1, pGEX4T-1-Pod-1, and LexA-Pod-1, respectively. pcDNA3Flag-Pod-1N and HA-Pod-1C, and pGEX4T-1-Pod-1N and Pod-1C were generated after amplification of the corresponding regions by PCR with the following primer sets: (forward primer, which was originated from pGEX vector; 5'-GGGCTGGCAAGCCACGTTTGGTG-3' and reverse; 5'-CTTCCCCTCCTGGCTGACCC-3') for N-terminal region (Pod-1N) of Pod-1, and (forward; 5'-GGGTCAGCCAGGAGGGGAAG-3' and reverse; 5'-TCAGGACGCGGTGGTTCCAC-3') for C-terminal region (Pod-1C) containing a basic helix-loop-helix region of Pod-1. The B42-AR deletion mutants and pGEX4T-1-AR deletion mutants were generated by cloning the regions spanning Tau, DBD/hinge, and LBD of mouse AR into the B42 vector digested with EcoRI-XhoI restriction enzymes. HDAC1 and -4 expression plasmids were previously described (45, 46). A luciferase reporter plasmid containing two AREs of the C3 gene, pARE2-TATA-Luc (29), is a generous gift from Dr. J. J. Palvimo (University of Helsinki, Helsinki, Finland).
Transient Transfection Assays
HeLa cells, and 15P-1 and MSC-1 Sertoli cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS. Cells were plated in 24-well plates, and were transfected with the indicated amount of expression plasmids, a reporter plasmid, and the control lacZ expression plasmid, pCMVß (CLONTECH, Palo Alto, CA), using Effectene reagent (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer. Total amounts of expression vectors were kept constant by adding appropriate amounts of pcDNA3. Twenty-four hours after transfection, cells were treated with 100 nM of testosterone for 24 h. TSA was treated 20 h before harvesting. Luciferase and ß-galactosidase activities were assayed as previously described (45). The levels of luciferase activity were normalized to the lacZ expression.
Yeast Two-Hybrid Assay
Plasmids encoding LexA fusions and B42 fusions were cotransformed into Saccaromyces cerevisiae EGY48 containing the lacZ reporter plasmid. The transformants were grown in the inducing medium and processed for liquid ß-galactosidase assays as previously described (45).
GST-Pull-Down Assay
GST, GST-Pod-1, GST-AR domain mutants, and GST-Pod-1 deletion mutants were expressed in Escherichia coli BL21 cells and isolated with glutathione-Sepharose-4B beads (Pharmacia, Biotech AB, Sweden). Immobilized GST fusion proteins were then incubated with [35S]methionine-labeled Pod-1, AR, HDAC1, and HDAC4 proteins produced by in vitro translation using the TNT-coupled transcription-translation system (Promega, Madison,WI). The binding reactions were carried out in 250 µl of GST binding buffer [20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10% glycerol, 0.05% Nonidet P-40, 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, and 1.5% BSA] for 4 h at 4 C. The beads were washed three times with 1 ml of GST-binding buffer. Bound proteins were eluted by the addition of 20 µl of SDS-PAGE sample buffer, and were analyzed by SDS-PAGE and autoradiography (45).
Coimmunoprecipitation and Western Blot Assays
In vivo coimmunoprecipitation assays were performed with MSC-1 cells transfected with 2 µg of HA-Pod-1 expression plasmids. Transfected cells were treated with or without 100 nM testosterone for 24 h after transfection and harvested with RIPA cell lysis buffer [50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2.5 mM EGTA, 1% Triton X-100, 50 mM NaF, 10 mM Na4P2O7, 10 mM Na3VO4, 1 µg/ml aprotinin, 0.1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol].
Whole cell lysate or testis extract (400 µg) was incubated with 2 µg of anti-HA or anti-AR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 4 h at 4 C and further incubated for another 2 h after adding 20 µl of protein A agarose bead slurry (Invitrogen, Carlsbad, CA). Agarose beads were washed three times each with RIPA and tissue lysis buffer at 4 C. Bound proteins were separated by SDS-PAGE. Proteins on the gels were transferred to nitrocellulose membranes (Sigma, St. Louis, MO), subjected to Western blot analysis with anti-AR (Santa Cruz Biotechnology), anti-Pod-1 (Santa Cruz Biotechnology), anti-HDAC1, and anti-HDAC4 (Zymed Laboratories Inc., San Francisco, CA). Signals were then detected with an ECL kit (Amersham Pharmacia, Piscataway, NJ).
ChIP Assay
MSC-1 cells were transfected with 2 µg of HA-Pod-1 expression plasmids. Transfected cells were then treated with or without 100 nM testosterone for 24 h and cross-linked with 1% formaldehyde for 10 min at room temperature. ChIP assays were performed as previously described (45). Anti-AR (Santa Cruz Biotechnology), anti-HA (Santa Cruz Biotechnology) or anti-HDAC1 (Zymed Laboratories Inc.) was used for immunoprecipitation. Immunoprecipitated and input-sheared DNAs were subjected to PCR using the following primer pairs: 1) for mouse AR promoter (forward, 5'-GCTAGCTTGCGGTGAGGGGAGG-3'; reverse, 5'-TCCTAGGGATCTCCGAGG GGG-3'), which amplifies an approximately 170-bp region spanning the E-box; 2) for mouse Pem promoter (forward, 5'-AGTCAGCTGAGCTGTAACTG-3'; reverse, 5'-ACCCTGAATAGGATCAATGAT G-3'), which amplifies an approximately 319-bp region spanning androgen-response elements within the Pem promoter; 3) for ß-actin (sense, 5'-GAGACCTTCAACACCCCAGCC-3'; antisense, 5'-CCGTCAGG CAGCTCATAGCTC-3'), which amplifies a 362-bp region spanning exon 4 of the ß-actin gene; 4) for androgen-response elements (forward: 5'-CAGGTGCCAGAACATTTCTC-3' and reverse: 5'-GAGTTTTCACTGC ATACGACG-3'), which amplify an approximately 430-bp region spanning the androgen-response elements within ARE2-TATA-Luc reporter plasmid; 5) for Luc (sense, 5'-GAAGGTTGTGGATCTGGATAC-3'; antisense, 5'-TTTCCGTCATCGTCTTTCCG-3'), which amplifies an approximately 370-bp region spanning the C-terminal part of luciferase-coding region of the ARE2-TATA-Luc reporter.
EMSA
GST, GST-Pod-1, and GST-E12 fusion proteins were expressed in E. coli BL21 cells and purified with glutathione-Sepharose-4B beads (Pharmacia, Biotech AB) with 10 mM reduced glutathione (47). Gel mobility shift assay solution (20 µl) contained 20 mM Tris (pH 8.0), 30 mM KCl, 4% glycerol, 5 mM MgCl2, and 0.5 µg of poly(deoxyinosine-deoxycytosine). One microgram of GST fusion proteins was used in each reaction. Competitor probe was added at 100-fold molar excess. After 10 min incubation with antibody on ice, 10,000 cpm of labeled probes was added and the incubation continued for another 20 min at room temperature. DNA-protein complexes were analyzed on 5% polyacrylamide gel in 0.5x TBE (0.5x TBE = 45 mM Tris, 45 mM boric acid, 1 mM EDTA). Gels were dried and analyzed by autoradiography. Probes were gel-isolated after NotI digestion of T-easy-DM2 or T-easy-mtDM2, which contains a PCR-amplified DM2 or mtDM2 region. PCRs were performed using the following primer pairs: for the wild-type E-box region (forward, 5'-GCTAGCTTGCGGTGAGGGGAGG-3'; reverse, 5'-TCCTAGGGATCTCCGAGGGGGCGCTGGGACCACGAAAGCAAATGCAACAG-3') and for the mutant E-box region (forward, 5'-GCTAGCTTGCGGTGAGGGGAG G-3'; reverse, 5'-TCCTAGGGATCTCCGAG GGGGCGCTGGGACCACGAAAGGAATTCCAACAGTTTGTGAGTCGG-3'). Probes were labeled by a fill-in reaction in the presence of [
-32P] deoxy-CTP.
Real-Time RT-PCR
MSC-1 cells were transfected with Pod-1 alone or Pod-1 together with AR expression plasmids, and then treated with or without 100 nM testosterone for 24 h. Quantitative RT-PCR was performed with total RNAs using a real-time PCR machine (Corbett Research, Sydney, Australia) and QuantiTect SYBR Green RT-PCR (QIAGEN) according to the manufacturers procedure. PCR were carried out with mouse Pem primer set (forward primer, 5'-GATAGACTGATGGATGCCTGT-3'; reverse primer, 5'-TCAAAATCTCGGTGTCGCAAA-3') and ß-actin primer set (forward primer, 5'-GAGACCTTCAACACCCCAGCC-3'; reverse primer, 5'-CCGTCAGGCAGCTCATAGCTC-3') as a quantitative control. Signals were analyzed with the Rotor Gene 2000 (RG-2000) software (Corbett Research, Sydney, Australia).
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ACKNOWLEDGMENTS
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We thank Dr. Tindall and Dr. Nakatsuji for pBLCAT3-mAR-promoter and Pod-1 expression vector, respectively. We also thank Dr. Palvimo for the pARE2-TATA-Luc reporter construct.
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FOOTNOTES
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This work was supported by Korea Research Foundation Grant (KRF-2002-070-C0007).
First Published Online May 26, 2005
Abbreviations: AR, Androgen receptor; AREs, androgen response elements; bHLH, basic helix-loop-helix; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; DBDh, DBD and hinge region; GST, glutathione-S-transferase; HDAC, histone deacetylase; LBD, ligand binding domain; TSA, trichostatin A.
Received for publication October 8, 2004.
Accepted for publication May 18, 2005.
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REFERENCES
|
---|
- Hughes IA, Lim HN, Martin H, Mongan NP, Dovey L, Ahmed SF, Hawkins JR 2001 Developmental aspects of androgen action. Mol Cell Endocrinol 185:3341[CrossRef][Medline]
- Gelmann EP 2002 Molecular biology of the androgen receptor. J Clin Oncol 20:30013015[Abstract/Free Full Text]
- Tilley WD, Marcelli M, McPhaul MJ 1990 Expression of the human androgen receptor gene utilizes a common promoter in diverse human tissues and cell lines. J Biol Chem 265:1377613781[Abstract/Free Full Text]
- Faber PW, Van Rooij HC, van der Korput HA, Baarends WM, Brinkman AO, Grootegood JA, Trapman J 1991 Characterization of the human androgen receptor transcription unit. J Biol Chem 266:1074310749[Abstract/Free Full Text]
- Takane KK, Wilson JD, McPhaul MJ 1992 Decreased levels of the androgen receptor in the mature rat phallus are associated with decreased levels of androgen receptor messenger ribonucleic acid. Endocrinology 129:10931100
- Kumar MV, Jones EA, Grossmann ME, Blexrud MD, Tindall DJ 1994 Identification and characterization of a suppressor element in the 5'-flanking region of the mouse androgen receptor gene. Nucleic Acids Res 22:36933698[Abstract]
- Smale ST, Baltimore D 1989 The "initiator" as a transcription control element. Cell 57:103113[CrossRef][Medline]
- Courey AJ, Holtzman DA, Jackson SP, Tjian R 1989 Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell 59:827836[CrossRef][Medline]
- Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
- Wong C-I, Zhou Z-X, Sar M, Wilson EM 1993 Steroid requirement for androgen receptor dimerization and DNA binding modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains. J Biol Chem 268:1900419012[Abstract/Free Full Text]
- Langley E, Zhou Z-X, Wilson EM 1995 Evidence for an anti-parallel orientation of the ligand-activated human androgen receptor dimer. J Biol Chem 270:2998329990[Abstract/Free Full Text]
- Truss M, Bartsch J, Schelbert A, Hache RJ, and Beato M 1995 Hormone induces binding of receptors and transcription factors to a rearranged nucleosome on the MMTV promoter in vivo. EMBO J 14:17371751[Abstract]
- Bannister AJ, Kouzarides T 1996 The CBP co-activator is a histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
- Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953959[CrossRef][Medline]
- Imhof A, Yang X-J, Ogryzko VV, Nakatani Y, Wolffe AP, Ge H 1997 Acetylation of general transcription factors by histone acetyltransferases. Curr Biol 7:689692[CrossRef][Medline]
- Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD, Torchia J, Yang WM, Brard G., Ngo SD, Davie JR, Seto E, Eisenman RN, Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387:4348[CrossRef][Medline]
- 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 89:373380[CrossRef][Medline]
- Hidai H, Bardales R, Goodwin R, Quertermous T, Quertermous EE 1998 Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries. Mech Dev 73:3343[CrossRef][Medline]
- Lu J, Richardson JA, Olson EN 1998 Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs. Mech Dev 73:2332[CrossRef][Medline]
- Robb L, Mifsud L, Hartley L, Biben C, Copeland NG, Gilbert DJ, Jenkins NA, Harvey RP 1998 Epicardin: a novel basic helix-loop-helix transcription factor gene expressed in epicardium, branchial arch myoblasts, and mesenchyme of developing lung, gut, kidney, and gonads. Dev Dyn 213:105113[CrossRef][Medline]
- Quaggin SE, Vanden Heuvel GB, Igarashi P 1998 Pod-1, a mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mech Dev 71:3748[CrossRef][Medline]
- Murre C, Bain G, van Dijk MA, Engel I, Furnari BA, Massari ME, Matthews JR, Quong MW, Rivera RR, Stuiver MH 1994 Structure and function of helix-loop-helix proteins. Biochim Biophys Acta 1218:129135[Medline]
- Massari ME, Murre C 2000 Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol Cell Biol 20:429440[Free Full Text]
- Tamura M, Kanno Y, Chuma S, Saito T, Nakatsuji N 2001 Pod-1/Capsulin shows a sex- and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1. Mech Dev 102:135144[CrossRef][Medline]
- Rey R, Lordereau-Richard I, Carel JC, Barbet P, Cate RL, Roger M, Chaussain JL, Josso N 1993 Anti-mullerian hormone and testosterone serum levels are inversely during normal and precocious pubertal development. J Clin Endocrinol Metab 77:12201226[Abstract]
- Cui S, Ross A, Stallings N, Parker KL, Capel B, Quaggin SE 2004 Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice. Development 131:40954105[Abstract/Free Full Text]
- Palvimo JJ, Reinikainen P, Ikonen T, Kallio PJ, Moilanen A, Janne OA 1996 Mutual transcriptional interference between RelA and androgen receptor. J Biol Chem 271:2415124156[Abstract/Free Full Text]
- Wise SC, Burmeister LA, Zhou XF, Bubulya A, Oberfield JL, Birrer MJ, Shemshedini L 1998 Identification of domains of c-Jun mediating androgen receptor transactivation. Oncogene 16:20012009[CrossRef][Medline]
- Moilanen A-M, Poukka H, Karvonen U, Hakli M, Jänne OA, Palvimo JJ 1998 Identification of a novel RING finger protein as a coregulator in steroid receptor-mediated gene transcription. Mol Cell Biol 18:51285139[Abstract/Free Full Text]
- Sharpe RM 1994 Regulation of Spermatogenesis. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven; 13632434
- McLachlan RI, ODonnell L, Meachem SJ, Stanton PG, de Kretser DM, Pratis K, Robertson DM 2002 Identification of specific sites of hormonal regulation in spermatogenesis in rats, monkeys, and man. Recent Prog Horm Res 57:149179[Abstract/Free Full Text]
- Chang C, Chen YT, Yeh SD, Xu Q, Wang RS, Guillou F, Lardy H, Yeh S 2004 Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells. Proc Natl Acad Sci USA 101:68766881[Abstract/Free Full Text]
- De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G 2004 A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 101:13271332[Abstract/Free Full Text]
- Holdcraft RW, Braun RE 2004 Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 131:459467[Abstract/Free Full Text]
- Miyagishi M, Hatta M, Ohshima T, Ishida J, Fujii R, Nakajima T, Fukamizu A 2000 Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, Pod-1/Capsulin. Mol Cell Biochem 205:141147[CrossRef][Medline]
- Funato N, Ohyama K, Kuroda T, Nakamura M 2003 Basic helix-loop-helix transcription factor epicardin/capsulin/Pod-1 suppresses differentiation by negative regulation of transcription. J Biol Chem 278:74867493[Abstract/Free Full Text]
- Atchley WR, Fitch WM 1997 A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA 94:51725176[Abstract/Free Full Text]
- Sun H, Taneja R 2000 Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Proc Natl Acad Sci USA 97:40584063[Abstract/Free Full Text]
- Fisher AL, Ohsako S, Caudy M 1996 The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain. Mol Cell Biol 16:26702677[Abstract]
- Chen G, Fernandez J, Mische S, Courey AJ 1999 A functional interaction between the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development. Genes Dev 13:22182230[Abstract/Free Full Text]
- Laherty CD, Yang WM, Sun JM, Davie JR, Seto E, Eisenman RN 1997 Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression. Cell 8:349356
- chreiber-Agus N, DePinho RA 1998 Repression by the Mad (Mxi1)-Sin3 complex. Bioessays 20:808818[CrossRef][Medline]
- Park ST, Sun XH 1998 The Tal1 oncoprotein inhibits E47-mediated transcription. Mechanism of inhibition. J Biol Chem 273:70307037[Abstract/Free Full Text]
- Kim H-J, Kim J-Y, Park Y-Y, Choi H-S 2003 Synergistic activation of the human orphan nuclear receptor SHP gene promoter by basic helix-loop-helix protein E2A and orphan nuclear receptor SF-1. Nucleic Acids Res 31:68606871[Abstract/Free Full Text]
- Hong CY, Park JH, Seo KH, Kim J-M, Im SY, Lee JW, Choi HS, Lee K 2003 Expression of MIS in the testis is downregulated by TNF-
through the negative regulation of SF-1 transactivation by NF-
B. Mol Cell Biol 23:60006012[Abstract/Free Full Text]
- Jeong BC, Hong CY, Chattopadhyay S, Park JH, Gong EY, Kim HJ, Chun SY, Lee K 2004 Androgen receptor corepressor-19 kDa (ARR19), a leucine-rich protein that represses the transcriptional activity of androgen receptor through recruitment of histone deacetylase. Mol Endocrinol 18:1325[Abstract/Free Full Text]
- Sanyal S, Kim JY, Kim HJ, Takeda J, Lee YK, Moore DD, Choi HS 2002 Differential regulation of the orphan nuclear receptor small heterodimer partner (SHP) gene promoter by orphan nuclear receptor ERR isoforms. J Biol Chem 277:17391748[Abstract/Free Full Text]