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Molecular Endocrinology, doi:10.1210/me.2004-0495
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Molecular Endocrinology 20 (1): 100-113
Copyright © 2006 by The Endocrine Society

Association of the mSin3A-Histone Deacetylase 1/2 Corepressor Complex with the Mouse Steroidogenic Acute Regulatory Protein Gene

Brian F. Clem and Barbara J. Clark

Department of Biochemistry and Molecular Biology and The Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Dr. Barbara J. Clark, Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292. E-mail: bjclark{at}louisville.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several factors have been identified in the transcriptional repression of the steroidogenic acute regulatory protein (StAR) gene promoter; yet, no associating corepressor complexes have been characterized for the mouse promoter in MA-10 mouse Leydig tumor cells. We now report that Sp3, CAGA element binding proteins, and a corepressor complex consisting of mSin3A, histone deacetylase (HDAC)1, and HDAC2 associates with a transcriptional repressor region within the mouse StAR promoter. 5'-Promoter deletion analysis localized the negative regulatory region between –180 and –150 bp upstream of the transcription start site, and mutations in both the CAGA and Sp binding elements were required to relieve the repression of basal StAR promoter activity. Protein-DNA binding analysis revealed Sp3 and specific CAGA element-binding protein(s) associated with the repressor region. Coimmunoprecipitation analysis identified the presence of the mSin3A, HDAC1, and HDAC2 corepressor complex in MA-10 cells. Furthermore, chromatin immunoprecipitation assays revealed Sp3, mSin3A, and HDAC1/2 association with the proximal region of the StAR promoter in situ. In addition, HDAC inhibition resulted in a dose-dependent activation of a mouse StAR reporter construct, whereas mutations within the repressor region diminished this effect by 44%. In sum, these data support a novel regulatory mechanism for transcriptional repression of the mouse StAR promoter by DNA binding of Sp3 and CAGA element-binding proteins, and association of the Sin3 corepressor complex exhibiting HDAC activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ACUTE PHASE of steroid biosynthesis in the adrenal and gonads is defined as the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is metabolized by the cytochrome P450 side chain cleavage enzyme into pregnenolone (1, 2, 3). Trophic hormone stimulation of steroidogenic tissues results in the de novo synthesis of the steroidogenic acute regulatory protein (StAR), which mediates this rate-limiting step in acute steroidogenesis (3, 4, 5). The significance of StAR’s role in steroid production is evident in the human disorder congenital lipoid adrenal hyperplasia, in which patients present a loss of steroid hormone production due to the lack of a functional StAR protein (6, 7). A similar phenotype is evident in StAR knockout mice, in which cholesterol accumulates within steroidogenic tissues leading to termination of steroid output (8, 9). Therefore, regulation of acute steroidogenesis is dependent on controlling StAR protein function and expression.

Extensive work has been performed in elucidating the molecular mechanisms by which StAR expression is enhanced by trophic hormone stimulation. The cAMP-protein kinase A signaling cascade increases StAR transcription independently of new protein synthesis, suggesting that posttranslational modifications of existing factors are necessary for StAR gene activation (10, 11). Many transcription factors, including steroidogenic factor 1 (SF-1), cAMP response element binding protein, GATA-4, activator protein 1 family members, and CCAAT/enhancer binding protein-ß, have been identified to be positive mediators of StAR transcription at both basal and trophic hormone-stimulated levels (12, 13, 14, 15, 16, 17, 18, 19, 20). However, fewer studies have examined the role of transcriptional repression as another mechanism for regulating StAR expression.

Transcriptional repression of genes can be achieved through direct or indirect mechanisms (21, 22). Indirectly, repressor proteins may interfere with the positive regulatory actions of certain activator proteins on target gene promoters (21). This can be accomplished by either inhibiting the positive factors from binding to their cis-acting element or by masking the activation function of those factors that are DNA bound (21). Directly, repressor proteins can bind to specific DNA elements and recruit repressor complexes that can modify chromatin structure and silence transcription. This repression event blocks the ability of the basal transcriptional machinery to bind to DNA and initiate transcription. Separately, repressor proteins can bind to the transcriptional machinery itself, thereby prohibiting the start of transcription (22).

To date, several factors have been identified to repress StAR transcription by direct and indirect mechanisms including DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X, gene 1), Yin Yang1 (YY1), Forkhead L2 (FOXL2), and c-fos (23, 24, 25, 26, 27, 28, 29, 30). DAX-1 has been proposed to repress StAR gene expression by either inhibiting SF-1 activation or by directly binding to hairpin structures within the human StAR promoter (24, 30). DAX-1 contains a strong repressor domain at its C terminus, and mutations abolishing this activity have been found in patients with adrenal hypoplasia (31). A direct physical interaction of SF-1 and DAX-1 has been reported, as well as an association with a known corepressor, receptor-interacting protein 140, which decreased activity of a human StAR reporter construct (29, 32). Furthermore, DAX-1 expression was found to be high in MA-10 mouse Leydig tumor cells, which do not constitutively express StAR, yet were absent in R2C rat Leydig tumor cells that have high basal StAR expression (33). YY1 was shown to repress human and rat StAR promoter activity by inhibiting the activity of sterol-regulatory element-binding protein (SREBP)-1a and nuclear factor Y. YY1 colocalizes with SREBP-1a within the nucleus, and YY1 expression results in decreased association of SREBP-1a and nuclear factor Y to their cognate binding elements within the human StAR promoter (23). FOXL2, on the other hand, has been shown to bind to specific elements within the proximal region of the human StAR promoter and repress StAR transcription in Chinese hamster ovary cells (26). Overexpression of c-fos was shown to inhibit rat StAR promoter activity, whereas mutation of c-fos binding sites abolished this association and repression (28).

Previously, our laboratory reported the presence of a putative repressor region within the mouse StAR promoter between –254 and –150 bp of the transcription start site. Deletion of this region resulted in a 4-fold increase in StAR promoter activity in MA-10 cells (34). The purpose of this study was to investigate the mechanism of transcriptional repression attributed to this region of the mouse StAR promoter. We now show the repressor region lies between –180 and –150 bp of the transcription start site, and mutations within a CAGA binding site and an Sp cis-acting element are required for full loss of StAR promoter repression. In addition, Sp3, as well as CAGA element binding protein(s), was shown to associate with the 30-bp repressor region. Furthermore, Sp3 was shown to associate with a corepressor complex consisting of mSin3A, HDAC1, and HDAC2, and this corepressor complex interacts with the StAR proximal promoter. Trichostatin A (TSA) treatment of MA-10 cells resulted in an increase in StAR mRNA levels and StAR reporter gene activity that was reduced with mutations within the repressor region. Therefore, we conclude that Sp3, CAGA binding protein(s), and the Sin3A corepressor complex bind to the StAR proximal promoter, indicating another direct repression mechanism for the mouse StAR promoter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
5'-Deletion Analysis Maps the Putative Repressor Region between –180 and –150 bp of the Transcription Start Site in the Mouse StAR Promoter
To localize the putative repressor region, MA-10 cells were transiently transfected with 5'-deletion StAR promoter constructs between –254 and –150 bp of the transcription start site. A 40-bp deletion producing the –215wt StAR reporter construct did not significantly increase promoter activity (Fig. 1Go). A significant 2.9-fold decrease in activity was evident with a deletion to –180 bp of the wild-type StAR reporter constructs. However, removal of an additional 30 bp to generate the –150-bp reporter construct resulted in a 3.7-fold increase in StAR promoter activity. Because StAR transcription is induced by activation of the cAMP-protein kinase A signaling pathway, we tested whether loss of the putative repressor region would enhance the cAMP-dependent activation StAR promoter. MA-10 cells were transiently transfected with the 5'-deletion StAR promoter constructs and treated with N,O'-dibutyryl-cAMP [(Bu)2cAMP] and the fold induction [(Bu)2cAMP /control] was determined. As shown in Fig. 1BGo, there was no effect on the cAMP-dependent increase in StAR reporter gene activity with loss of the region between –254 and –150 bp. These data suggest that the putative repressor region is located between –180 and –150 bp of the transcription start site within the mouse StAR promoter and that function of this region is independent of the cAMP-dependent response.



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  		Fig. 1. Localization of a StAR Repressor Region between –180 and –150 bp of the Transcription Start Site

MA-10 cells were transiently transfected with 5'-StAR promoter deletion constructs and the phRl-null Renilla vector as described in Materials and Methods. Transfected cells were treated in the absence (A) or presence (B) of 1 mM (Bu)2cAMP, and cell lysates were collected and assayed for luciferase and Renilla activities. Transfections and treatments were performed in triplicate, and the luciferase activity was normalized to Renilla activity. A, Shown are the mean RLU/Renilla values ± SEM from three independent experiments. B, The RLU/Renilla values in the presence of (Bu)2cAMP were normalized to the untreated control values and shown are the mean values ± SEM for the (Bu)2cAMP/control ratio from three independent experiments. *, Statistically significant difference from –254wt promoter activity (P < 0.05). RLU, Relative light units.

 
Sp Family cis-Acting Element and CAGA-Binding Site Mutations Abolish Repression Mediated through the –180 bp/–150-bp Region of the StAR Promoter
Site-directed mutagenesis within the 30-bp (–180/–150) repressor region was performed to determine the cis-acting elements necessary for StAR repression. Within this region, a potential Sp family binding site and a CAGA cis-acting element were identified (Fig. 2AGo). Point mutations were generated within these elements in context of the –254-bp StAR reporter construct to investigate their role in StAR transcriptional regulation. Mutation of the potential Sp element resulted in a 1.5-fold increase in StAR reporter activity (Fig. 2BGo). A significant 2.5-fold increase in StAR promoter activity was evident with the reporter construct containing mutations within the CAGA-binding element. However, mutation of both the CAGA and the Sp cis-acting elements resulted in a 4-fold increase in activity, indicating a total loss of transcriptional repression similar to the activity achieved by deletion of the entire region (–150wt). These data indicate that although the individual elements contribute to repression of StAR promoter activity, both elements are required to fully repress StAR promoter activity.



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  		Fig. 2. CAGA and Sp cis-Acting Elements Are Required for Repression of StAR Promoter Activity

A, Shown is the 30-bp repressor region sequence with the point mutations indicated in bold and underlined. B, MA-10 cells were transiently transfected with either wild-type or mutated StAR promoter reporter constructs, as indicated, and phRL Renilla plasmid and luciferase and Renilla activities were determined as described in Materials and Methods. Samples were performed in triplicate, and luciferase activity was normalized to Renilla activity. Shown are the mean relative light units/Renilla values ± SEM from three to four independent experiments. *, Statistically significant difference from –254wt promoter activity (P < 0.05).

 
Protein-DNA Interactions with the Repressor Region
EMSAs were used to examine protein-DNA complexes with the 30-bp repressor region of the StAR promoter. Two protein-DNA complexes, indicated as complexes I and II, were formed using the repressor region and MA-10 nuclear extracts (Fig. 3Go, lane 1). The specificity of these complexes was confirmed by cold competition assays, whereby excess unlabeled wild-type oligonucleotide (50x or 100x) competed for protein binding (Fig. 3Go, lanes 2 and 3). In addition, a consensus Sp oligo effectively competed for protein binding with the StAR repressor region, suggesting that Sp protein family members are involved in the protein-DNA complexes formed with the StAR promoter (Fig. 3Go, lane 4). To distinguish between Sp family members, antibody supershift assays were performed by the addition of either {alpha}-Sp1 or {alpha}-Sp3 to the EMSA binding reactions. The Sp3 antibody abolished protein complex II and decreased complex I, whereas the Sp1 antibody had no effect (Fig. 3Go, lanes 5 and 6 and 7, respectively).



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  		Fig. 3. Sp3 Associates with the Repressor Region within the StAR Promoter

Protein binding to the repressor region was determined by EMSA using control MA-10 nuclear extracts as described in Materials and Methods. Radiolabeled probe (50 fmol) (–180/–150) was incubated with 20 µg nuclear extract in the absence (lane 1) or presence of 50- or 100-fold molar excess of unlabeled wild-type StAR repressor probe (lanes 2 and 3) or 100-fold molar excess of an unlabeled Sp1 consensus oligonucleotide (lane 4). For antibody supershift assays, antibodies for Sp3 (2 or 4 µg) or Sp1 (4 µg) were added to nuclear extracts for 30 min, after which radiolabeled probe was added and reactions were incubated for an additional 30 min (lanes 5–7). Protein-DNA complexes were separated on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography. Two specific complexes, indicated as complex I and complex II, were detected. N.S., Nonspecific; Ab, antibody; Comp., competition; Cons, consensus.

 
To investigate the effect of mutations on the protein-DNA complexes, oligonucleotides containing specific point mutations were used in EMSA reactions. Mutation of the Sp3 cis-acting element (–180/–150Sp3mut) resulted in the appearance of two new protein-DNA complexes indicated by arrowheads (Complex III and IV) and the loss of complexes I and II (Fig. 4Go, lane 2). Addition of Sp3 antibody to the EMSA reaction had no effect on complexes III and IV, indicating Sp3 is not part of these protein-DNA complexes (Fig. 4Go, lanes 2 and 6). Mutation of the CAGA-binding element (–180/–150CAGAmut) within the repressor region did not decrease protein-DNA complexes I and II, and Sp3 was again verified to be a part of those complexes (Fig. 4Go, lanes 3 and 7). However, a probe containing both the Sp3 and CAGA element mutations abolished all specific protein-DNA complexes within the repressor region (Fig. 4Go, lanes 4 and 8). These results suggest that proteins are capable of binding to the –180 bp/–150 bp single CAGA and Sp3 element mutations, but that both binding sites are necessary for wild-type DNA binding.



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  		Fig. 4. Sp3 and the CAGA cis-Acting Elements Are Required for Protein-DNA Complex Formation in the StAR Repressor Region

Protein binding to wild-type or mutant repressor region oligonucleotides was determined by EMSA using control MA-10 nuclear extracts as described in Materials and Methods and Fig. 3Go. Protein-DNA complexes were separated on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography. Indicated probes are –180/–150 wt (lanes 1 and 5), –180/–150 Sp3mut (lanes 2 and 6), –180/–150 CAGA mut (lanes 3 and 7), and –180/–150 CAGA and Sp3mut (lanes 4 and 8). Mutated sequences for the –180/–150 probes are provided in Fig. 2AGo. N.S., Nonspecific; Ab, antibody.

 
Antibody supershift assays were used to determine the identity of the protein-DNA complexes still present using the Sp3 mutant oligonucleotide (complexes III and IV). To confirm specificity, both complexes were competed with cold molar excess of both the Sp3 mutant and wild-type probe (Fig. 5Go, lanes 1–3), suggesting the CAGA element is responsible for protein binding. Because the CAGA binding element resembles a Smad cis-acting element or a nonconsensus E box-binding motif, different antibodies against specific Smad family members or E box-binding proteins were used in conjunction with the Sp3 mutant probe. However, incubation of EMSA reactions with antibodies for Mad1 or Smad4 had no effect on the specific protein-DNA complex, similar to that observed with the control rabbit IgG (Fig. 5Go, lanes 4–6). In addition, cold competition assays using 100-fold molar excess of a myc/max consensus oligonucleotide was unable to compete for protein components of complexes I and II (data not shown). To investigate other Smad family members, an antibody recognizing both Smad2 and Smad3 was used in the EMSA reactions. However, as with Smad4, the Smad2/3 antibody did not affect complexes I and II with the wild-type repressor probe (data not shown). These data indicate that specific protein-DNA interactions are formed with the CAGA element, but these complexes do not appear to contain Smads or general myc/max/mad binding proteins. Together these results confirm that Sp3 and CAGA-binding protein(s) are components of the protein complexes that interact with the repressor region of the StAR promoter, and mutations of both elements are required for full loss of protein-DNA binding.



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  		Fig. 5. Protein Binding to the CAGA-Binding Motif Does Not Include Mad1 or Smad4

Protein binding to the Sp3 mutant repressor region oligonucleotide was determined by EMSA using control MA-10 nuclear extracts as described in Materials and Methods and Figs. 3Go and 4Go. For antibody supershift assays, 4 µg IgG (lane 4), Mad1 (lane 5), or Smad4 (lane 6) antibody was added to nuclear extracts for 30 min, after which radiolabeled probe was added and reactions were incubated for an additional 30 min (lanes 4–6). Protein-DNA complexes were separated on a 4% nondenaturing polyacrylamide gel and visualized by autoradiography. The sequence for the –180/–150 Sp3mut probe is outlined in Fig. 2AGo. N.S., Nonspecific; Comp., competition.

 
Sp3 Interacts with a Corepressor Complex that Associates with the StAR Proximal Promoter
Because Sp3 has been shown to interact with known repressor complexes consisting of mSin3A, HDAC1, and HDAC2 in various cell lines (35, 36), we tested whether this complex is present within MA-10 cells. First, coimmunoprecipitations were performed in reciprocal fashion against the various members of this complex using {alpha}-Sp3, {alpha}-mSin3A, {alpha}-HDAC1, or {alpha}-HDAC2. As shown in Fig. 6Go, all components of the complex are present in nuclear extracts of MA-10 cells. Immunoprecipitation (IP) for Sp3 followed by Western blot (WB) for mSin3A and HDAC2 indicates that Sp3 physically interacts with mSin3A and HDAC2, suggesting that this repressor complex is formed in MA-10 cells. Conversely, IP for mSin3A followed by WB for Sp3 and HDAC2 further confirms the interaction of this protein complex. In addition, the association of the repressor complex was shown by the presence of Sp3 and mSin3A immunoreactive bands in conjunction with the HDAC2 IP sample. In contrast, IP for HDAC1 followed by WB for Sp3, HDAC2, and mSin3A revealed a physical interaction with HDAC2 and mSin3A but not Sp3, suggesting that HDAC1 and Sp3 do not directly associate. Although we were unable to perform WB for HDAC1 on the IP samples due to interference from the IgG heavy chain signal, our data demonstrate that HDAC1 is expressed in MA-10 cells and the reciprocal IPs support a HDAC1-HDAC2-mSin3A association and Sp3 association with HDAC2 and mSin3A. Together, these data demonstrate that a known repressor complex consisting of mSin3A, HDAC1, and HDAC2 is intact within MA-10 cells, and this complex associates with Sp3 most likely via direct interactions with mSin3A and/or HDAC2.



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  		Fig. 6. Coimmunoprecipitation of Sp3-mSin3A-HDAC Corepressor Complex in MA-10 Cells

Control MA-10 nuclear extracts were incubated with antibodies for either rabbit IgG, Sp3, mSin3A, HDAC1, or HDAC2 as indicated (IP), and immunoprecipitated protein complexes were recovered as described in Materials and Methods. The presence of mSin3A, HDAC2, Sp3, and HDAC1 in nuclear extract and mSin3A, HDAC2, or Sp3 in IP complexes was detected by WB as described in Materials and Methods. Arrows indicate specific mSin3A (145 kDa), HDAC2 (54 kDa), Sp3 (80 kDa), and HDAC1 (52 kDa) proteins. NE, Nuclear extract.

 
Chromatin immunoprecipitation (ChIP) assays were performed to investigate the association of Sp3 and the mSin3A repressor complex with the StAR promoter in situ. Sheared chromatin was immunoprecipitated using {alpha}-Sp3, {alpha}-mSin3A, {alpha}-HDAC1, or {alpha}-HDAC2. Primers specific for the proximal region of the StAR promoter that flank the repressor region and a distal region were used to amplify the immunoprecipitated DNA (Fig. 7AGo). A PCR product was evident when chromatin was immunoprecipitated with antibodies for Sp3, mSin3A, HDAC1, and HDAC2 in conjunction with primers specific for the proximal region of the StAR promoter (Fig. 7BGo). However, little product is observed when either IgG or {alpha}-RbAp48 (another known member of the repressor complex) is used in the IP reactions (Fig. 7BGo and data not shown). Specificity for the interaction of the repressor complex with the proximal StAR promoter is provided by the lack of product formed when using primers specific for a distal region of the StAR promoter with the immunoprecipitated samples (Fig. 7BGo). Treatment of the cells with (Bu)2cAMP for 2 h before ChIP analysis did not qualitatively alter protein interactions with the StAR proximal promoter (Fig. 7CGo), indicating the repressor complex remains constitutively associated with the mouse StAR proximal promoter region.



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  		Fig. 7. Sp3, mSin3A, HDAC1, and HDAC2 Associate with the Proximal Region of the Mouse StAR Promoter

ChIP assays were performed on MA-10 cells as described in Materials and Methods. Sheared chromatin was immunoprecipitated with IgG, {alpha}-Sp3, {alpha}-mSin3A, {alpha}-HDAC1, and {alpha}-HDAC2. PCR was used to amplify recovered chromatin using primers specific for proximal (–222 bp/–114 bp) and distal (–3584 bp/ –3428 bp) regions of the StAR promoter. A, Location of primer pairs used in PCRs for the proximal and distal regions of the StAR promoter. B and C, Ethidium bromide gel of PCR products from input chromatin and ChIP samples using proximal (~108-bp product) and distal (~156-bp product) region primers. C, In some experiments, cells were treated either in the absence (0) or presence of 1 mM (Bu)2cAMP for 120 min before ChIP. Shown are representative gels from four (B) or three (C) independent experiments.

 
CAGA and Sp3 cis-Acting Element Mutations Diminish the TSA-Dependent Activation of the StAR Promoter
To investigate whether HDAC activity is required for function of the repressor complex, we tested the effect of a known HDAC inhibitor, TSA, on StAR reporter gene activity in transiently transfected MA-10 cells. As shown in Fig. 8AGo, treating MA-10 cells with increasing concentrations of TSA resulted in a dose-dependent increase in StAR promoter activity with the wild type (–254wt) StAR promoter construct. To test whether the repressor region was contributing to the potential HDAC-dependent repression, MA-10 cells were transiently transfected with the StAR reporter construct containing the CAGA and Sp3 mutations (–254 CAGA and Sp3 mut), and the effect of TSA on reporter gene activity was determined. Compared with the wild-type StAR promoter construct, mutation of the CAGA and Sp3 elements resulted in 40% and 44% decreases in promoter activity at the 100 nM and 250 nM TSA concentrations, respectively (Fig. 8AGo). To confirm that the loss of repression was due to the identified cis-acting elements, we further tested the –180wt, –180 CAGA and Sp3 mutant, and –150wt StAR promoter constructs for TSA responsiveness. Our data demonstrate that the constructs containing the repressor region (–254wt and –180wt) have a similar increase in activity after treatment with 100 nM TSA and that the TSA effect is diminished approximately 50% for the constructs lacking the functional repressor region (–254CAGA and Sp3 mutant, –180 CAGA and Sp3 mutant, and –150wt) (Fig. 8BGo). These results indicate that HDAC activity contributes to StAR reporter gene repression and that the CAGA-binding motif and the Sp3 cis-acting elements are important for this repressive action. However, deletion or mutation of the repressor elements (–254 CAGA and Sp3 mut) did not completely block the increase in StAR promoter activity with TSA treatment; therefore, HDAC may also function within the –150-bp promoter region. Because the cAMP-dependent element(s) are located within the –150-bp region (34), we tested the effect of TSA treatment on the cAMP-dependent induction of the –254wt, –254CAGA and Sp3 mutant, –180wt, –180 CAGA and Sp3 mutant, and –150wt constructs and demonstrated that there was no significant difference in the cAMP-dependent fold induction with TSA treatment (data not shown), consistent with the data presented in Fig. 1BGo.



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  		Fig. 8. Sp3 and CAGA cis-Acting Element Mutations Reduce the TSA-Dependent Activation of the Mouse StAR Promoter

MA-10 cells were transiently transfected with either wild type or mutated StAR promoter reporter constructs as described in Materials and Methods. Cells were treated in triplicate in serum-free Waymouth’s media containing the indicated concentrations of TSA (A) or 100 nM TSA (B) for 24 h. Cell lysates were collected and luciferase activity was normalized to mg protein. The relative light units/mg protein values in the presence of TSA were normalized to the activity in the absence of TSA (TSA+/TSA–). A and B, Shown are the mean values ± SEM from four independent experiments. C, MA-10 cells were treated with 100 nM TSA for the indicated times, and total RNA was isolated and StAR mRNA levels were determined by quantitative real-time RT-PCR as described in Materials and Methods. Relative expression levels were determined for each experiment and shown are the mean values ± SEM from three independent experiments. *, Statistically significant (P < 0.05) decrease in promoter activity between reporter constructs within treatment concentrations (A) or relative to p-254wt (B). + Indicates a statistically significant (P < 0.05) increase in mRNA relative to untreated control (C). RLU, Relative light units.

 
We next measured StAR mRNA levels in TSA-treated MA-10 cells. As shown in Fig. 8CGo, TSA treatment resulted in a transient increase in basal StAR mRNA levels that peaked between 4 and 6 h, returning to 50% of basal levels after 24 h. The basis for the decline in basal steady-state StAR mRNA levels after 24 h is not known but most likely reflects StAR mRNA turnover. Treatment of the cells with (Bu)2cAMP for 2–6 h after a 24-h pretreatment with TSA had no effect on the cAMP-dependent response (data not shown). Together these data support that the Sp3-CAGA repressor region contributes to StAR gene repression via HDAC association and function and that this region acts independently of the cAMP-dependent activation of StAR promoter in MA-10 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Recent reports have revealed transcriptional repression as a mechanism to regulate StAR gene expression by a variety of transcription factors, including DAX-1, YY1, FOXL2, and c-fos. These proteins possess different activities to induce transcriptional repression by either direct or indirect means (23, 24, 26, 28). Various cis-acting elements or secondary structures have been characterized as providing direct repression within StAR promoters of different species (24, 26, 28). However, only DAX-1 has been shown to repress expression of the mouse StAR promoter (33). We have now characterized a repressor region within the mouse StAR promoter, identified the cis-acting elements and trans-acting factors involved, and, for the first time, identified a corepressor complex involved in negatively regulating the basal transcription of StAR in MA-10 cells.

We previously identified a putative repressor region within the mouse promoter between –254 and –150 bp (34). Analysis of this region revealed no potential secondary hairpin structures or SF-1 binding sites for potential DAX-1-mediated repression. Therefore, we initiated studies to identify the specific cis-acting elements and trans-acting factors responsible for the repression effect. We now show that an Sp3 binding site, as well as a CAGA element located within the –180 and –150-bp region, is responsible for the repression of the StAR promoter. Site-directed mutagenesis revealed that individual mutation within these elements had partial effects on increasing StAR promoter activity, but mutation of both elements together was required to achieve full loss of transcriptional repression.

We have demonstrated that Sp3 binds to the StAR proximal promoter and is dependent upon the CCTCCC sequence located at –159 bp. The Sp protein family, consisting of four major members (Sp1, Sp2, Sp3, and Sp4), has been shown to regulate a vast number of genes within different tissues (37, 38, 39, 40). Sp1 and Sp3 are ubiquitously expressed, whereas Sp2 and Sp4 are more localized, i.e. Sp4 within neuronal cells (41). This family has been classically identified as activators of gene transcription (41); however, Sp3 has also been shown to contain a repressor domain localized to 72 amino acids toward the N terminus preceding the zinc finger region (42). Commonly, Sp3 repression of target genes is associated with competition for Sp1 activation function (43). The Sp3/Sp1 ratio within the nucleus has been shown to regulate the expression of genes including the human type II collagen and the human secretin genes (44, 45). In addition, a recent report identified Sp3 as a transcriptional repressor of GH expression in monocytes (40). Furthermore, Sp3 has recently been shown to physically interact with specific repressor complexes (36, 46), providing a direct mechanism of gene repression by Sp3, independent of Sp1 activator function.

Our binding experiments also revealed protein binding to a CAGA element located at –169 bp that was independent of Sp3. As described above, mutation of this CAGA element resulted in a 2.5-fold increase in StAR promoter activity, suggesting that proteins binding to this element have a functional role in regulating StAR gene expression. CAGA binding sites are common cis-acting elements for Smad protein family members involved in regulating TGFß-targeted gene expression (47, 48, 49). Interestingly, TGFß-1 inhibited StAR mRNA production within bovine adrenocortical cells (50). Furthermore, in H295R human adrenocortical cells, Smad3 was shown to be involved in TGFß-1 repression of StAR mRNA, whereas a dominant-negative mutant of Smad3 abolished this repressive effect (51). Our studies indicate that protein binding to the mouse StAR repressor region does not appear to include Smad2, Smad3, or Smad4, suggesting that the negative regulatory region described in this report may not be the site for TGFß-1 regulation. Lack of Smad binding may be attributed to the fact that a nonconsensus CAGA element is found within the repressor region, and it may not associate with classical Smad-binding proteins. Because this element loosely resembles a nonconsensus E box-binding motif, we tested for known factors that have been identified as E box-binding proteins, i.e. heterodimers of Myc/Mad, which can function to repress target gene transcription (52, 53). Antibody supershift EMSA experiments showed no effect of {alpha}-Mad1 on protein binding to the mouse StAR repressor region, and cold competition assays using a consensus myc/max cis-acting element did not diminish the protein-DNA complexes with the StAR repressor region (data not shown). These data suggest myc/max family members are not capable of associating with the StAR repressor region. The identity of the CAGA-dependent binding proteins is currently under investigation with focus on other potential repressor E box-binding proteins such as the upstream stimulating factor protein family members (54).

Inhibitors of HDAC function, such as TSA and butyric acid, are commonly used to assess HDAC activity on a gene promoter. We demonstrate that inhibition of HDAC function by TSA resulted in a transient increase in StAR steady-state mRNA levels and an increase in mouse StAR promoter activity. Mutations within the functional cis-acting CAGA and Sp3 elements in context of either the –254 or –180-bp StAR promoter constructs or deletion of the entire region (–150wt) caused a reduction in TSA-dependent activation. These data support our model that a loss of Sp3 and CAGA protein binding, which is required for HDAC association, contributes to the loss of repression. In general, an increase in target gene mRNA levels with TSA treatment can been attributed to maintaining an open chromatin structure as a result of preventing histone deacetylation. However, more recent data indicate acetylation of transcription factors, cofactors, and chromatin remodeling proteins promotes transcriptional activation independent of histone acetylation (55, 56, 57), providing other targets for HDAC-mediated transcriptional repression. Because nucleosome organization of transiently transfected plasmid DNA in mammalian cells has been shown to be both random (nonchromatinized) and ordered (chromatinized) (58, 59, 60), the precise mechanism for HDAC function in mouse StAR gene repression remains to be determined.

Our HDAC inhibition studies are in contrast to another report examining HDAC function on StAR promoter activity. These investigators treated human theca cells with valproic acid (VPA), which has HDAC-inhibitory action (61). StAR mRNA and human StAR promoter activity was not changed with VPA treatment, whereas the promoter activities of cytochrome P450c17 and P450 side chain cleavage enzymes were increased (61). Although no direct comparison between inhibitors can be made for our two studies, TSA has been shown to be a more potent HDAC inhibitor compared with VPA in other cell systems at the concentrations used and may explain the discrepancy in results (62). Alternatively, the difference between HDAC inhibitors on StAR expression may be due to differences in StAR promoter sequences (human vs. mouse) as well as cell type (human theca vs. mouse Leydig).

Supporting promoter-specific repression, the functional CAGA-Sp3 element that we have identified in the mouse promoter is not conserved in the human, rat, or pig promoters based on sequence alignment, which demonstrated that the greatest sequence similarity between species was within –120 bp of the transcription start site (17). This sequence difference may also explain why previous studies characterizing the cAMP-responsive regions of human, rat, and pig StAR promoter using 5'-deletion analysis did not report an increase in basal activity with loss of the region corresponding to mouse –180 to –150-bp region (16, 63, 64, 65, 66, 67). Data from a previous study suggest mouse StAR promoter activity in transiently transfected rat granulosa cells was not significantly increased by deletion of the –257 to –152-bp region (18). Thus, a current interest is to determine whether cell type (i.e. granulosa vs. Leydig cell) and species (rat vs. mouse) differences in repressor complex expression may also play a role in mouse StAR gene regulation.

The Sin3 repressor complex is one of two major corepressor complexes that have been characterized within mammalian cells (68, 69). This complex consists of several accessory proteins including functional proteins such as HDAC1/2 as well as mSin3A, which mediate protein-protein interactions. Repressor complexes are recruited to chromatin via specific DNA-bound transcription factors or by other corepressor proteins such as nuclear receptor corepressor and silencing mediator of retinoid and thyroid hormone receptor, which interact with unliganded nuclear receptors (68, 69). One specific transcription factor that has been identified to associate with the mSin3A complex is Sp3 (36). Several target promoters, such as those for the p21Waf1/Cip1 and the human telomerase reverse transcriptase genes, are negatively regulated by Sp3 through recruitment of the mSin3A complex and HDAC proteins (70, 71). Using coimmunoprecipitation experiments, a previous report investigating the LH receptor gene concluded that Sp3 interacted with the Sin3 complex and HDAC proteins through an accessory protein RbAp48 in human placental choriocarcinoma JAR cells (35). In MA-10 cells, we detected the mSin3A-HDAC1/2 repressor complex. However, neither coimmunoprecipitation nor ChIP analysis identified RbAp48 as part of this complex (data not shown). Thus, our data indicate that mSin3A and HDAC2 interact with Sp3 and that HDAC1 interacts with mSin3A and HDAC2 to form a repressor complex in MA-10 cells. These results are similar with another report that has shown a direct interaction of Sp3 with HDAC2 via IP and immunodepletion experiments (72).

One mechanism for transcriptional activation of target genes is to promote the loss of active repression on the gene promoter, such as when a ligand activates nuclear receptors allowing dissociation from known corepressors (73, 74). However, our data indicate that loss or mutation of the –180 to –150-bp repressor region of the mouse StAR promoter does not enhance the cAMP-dependent response. The lack of dissociation of Sp3, mSin3A, HDAC1, or HDAC2 from the proximal StAR promoter after cAMP stimulation suggests the complex is constitutively associated with the mouse StAR gene. The significance of this repressor region could be to serve as a mechanism to ensure low basal levels of StAR expression in steroidogenic cells and to a greater extent in nonsteroidogenic tissues. Future experiments focusing on StAR promoter activity in tissues with varying steroidogenic capacities are needed to investigate the biological relevance of this negative regulatory region, specifically the expression of the repressor complex components described here such as Sp3, mSin3A, HDAC1, and HDAC2.

In sum, we now report a negative regulatory region within the mouse StAR promoter located between –180 to –150 bp upstream of the transcription start site that binds to Sp3 and CAGA element-binding factors. Components of the Sin3 corepressor complex, including mSin3A, HDAC1, and HDAC2, associate with the proximal repressor region of the StAR promoter through Sp3 interactions, providing another mechanism for regulation of the mouse StAR gene expression in MA-10 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Waymouth’s MB/752, and N6,2'-O-dibutyryl-cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). DH5{alpha} competent cells, gentamicin reagent, and Dulbecco’s PBS were purchased from Invitrogen (Carlsbad, CA). TSA was purchased from Calbiochem (San Diego, CA). The protein assay kit was supplied by Bio-Rad Laboratories (Hercules, CA). PCR primers for StAR-luciferase construct generation and ChIP analysis were purchased from Synthetic Genetics (San Diego, CA) and Midland Certified Reagent Co. (Midland, TX). T4-polynucleotide kinase, T4 Ligase, pGL2-luciferase reporter vectors, phRL Renilla-luciferase reporter vectors, and restriction enzymes were purchased from Promega Corp. (Madison, WI). [{gamma}-32P]ATP was purchased from NEN Life Science Products (Boston, MA). Anti-Sp3 (sc-644x), anti-Sp1 (sc-14027x), and anti-mSin3A (sc-994x) antibodies were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-HDAC1 (2062) was purchased from Cell Signaling Technology (Beverly, MA). Anti-HDAC2 (34–6400) was purchased from Invitrogen (Carlsbad, CA). The ChIP assay kit was purchased from Upstate USA, Inc. (Charlottesville, VA).

Cell Culture
The MA-10 mouse Leydig tumor cell line was a gift from Dr. M. Ascoli (Department of Pharmacology, University of Iowa, Iowa City, IA). MA-10 cells were cultured in Waymouth’s MB/752 media supplemented with 15% heat-inactivated horse serum, and 40 µg gentamicin sulfate/ml.

Nuclear Extract Preparations
Nuclear extract preparations from untreated MA-10 cells were performed as previously described (34). A protein assay was performed to determine protein concentrations, and all samples were stored at –80 C until further analysis.

StAR-Luciferase Construct Generation
5'-Deletion constructs of the StAR promoter were generated using PCR. The –254/+35 StAR-luciferase construct was used as a template. PCR was performed using MluI –215 and MluI –180 primers in combination with the GL2 primer to amplify the –215 to +35 (–215wt) and –180 to +35 (–180luc) regions, respectively. PCR products were digested with MluI and HindIII and cloned into the MluI and HindIII sites of the pGL2-basic vector. Site-directed mutagenesis was performed by creating a common restriction site into two separate PCR products that contain mutations within the region of interest (–254 Sp3mut, –254 CAGAmut, –254 CAGA and Sp3mut, and –180 CAGA and Sp3mut). For the single mutation constructs (–254 Sp3mut and the –254 CAGA mut), the 5'-product was generated by using the MluI –254 primer for the top strand and the site-specific mutated primer for the bottom strand. To produce the 3'-product, the site-specific mutated primer was used to amplify the top strand, and the GL2 primer was used to generate the bottom strand. Both PCR products were digested with their respective restriction enzymes and cloned into the MluI and HindIII sites of the pGL2 basic vector. The –254 CAGA and Sp3 mut- and –180 CAGA and Sp3mut-luciferase constructs were generated using the –254 CAGA mut-luc and the corresponding –254 or –180 CAGA and Sp3 mutant primers to produce both mutations. The primers used to produce the site-directed mutants are listed in the oligonucleotides used for EMSA and PCR. Sequences were confirmed by DNA sequencing by the Center for Genetics and Molecular Medicine DNA core at the University of Louisville.

Transient Transfections and Reporter Assays
MA-10 cells were plated at 250,000 cells per well in a 24-well plate 36 h before transfection. Transfections were carried out with 2 µg/ml StAR reporter gene plasmid, 0.5 µg/ml of phRL-null, and 15 µg/ml Lipofectamine reagent in Waymouth’s media without antibiotic or serum for 6 h and then the transfection medium was replaced with complete Waymouth’s media. The cells were treated 24 h after transfection with serum-free media in the absence or presence of 1 mM (Bu)2cAMP for 16 h. Cell lysates were collected, and firefly and Renilla luciferase activities were determined using the Dual Luciferase Reporter assay system (Promega) and the Lumat LB9507 luminometer (Wallac, Inc., Gaithersburg, MD). Each transfection was performed in triplicate, and the data were expressed as the mean relative light units/Renilla± SEM.

For the TSA studies, 2 µg/ml StAR reporter gene plasmid was transfected using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN) in Waymouth’s media with serum. Cells were treated 24 h after transfection in the presence or absence of TSA at the indicated concentrations for 24 h in Waymouth’s media without serum. Firefly luciferase was assayed, and total protein per well was calculated by a Bradford Protein Assay. Each treatment was performed in triplicate, and the mean relative light units/mg protein was determined. The data were expressed as TSA+/TSA-fold induction relative to either the wild-type (–254wt) or double-mutant (–254 CAGA and Sp3 mut) constructs, which were set to a value of 1.

EMSA
To ensure only double-stranded probes were present in the EMSA reactions, annealed oligonucleotides were polyacrylamide gel purified, as previously described (12). Briefly, entire annealing reaction containing a radiolabeled portion was separated by 15% nondenaturing PAGE, and the autoradiograph was placed back onto the gel, after which the bands corresponding to the double-stranded oligonucleotides were excised and purified. DNA concentration was determined by A260 reading using an Ultraspec 3000 (Pharmacia Biotech, Piscataway, NJ). Sequences for EMSA probes are listed under oligonucleotides used for EMSA and PCR. EMSA reactions using –180/–150 wild-type or mutant radiolabeled probes and 20 µg of control MA-10 nuclear extracts with either antibody supershift (Sp3 or Sp1) or cold-competition assays were performed as previously described (69). Reactions were separated on a 4% nondenaturing polyacrylamide gel, and protein-DNA complexes were visualized by autoradiography.

Coimmunoprecipitation and Immunoblot Analysis
For coimmunoprecipitation, 250 µg of control MA-10 nuclear extract was incubated with 3 µg of normal rabbit IgG, {alpha}-Sp3, {alpha}-mSin3A, {alpha}-HDAC2, or 10 µl of {alpha}-HDAC1 in 1 ml of IP buffer (50 mM Tris/Cl, pH 7.4; 1% IGEPAL CA630; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethylsulfonylfluoride; 0.04 U/ml aprotinin; and 0.1 µg/ml leupeptin) overnight with rotation at 4 C. Immunocomplexes were recovered using 100 µl of preequilibrated 50% Protein A-agarose slurry and incubated for 2 h with rotation at 4 C. The protein A immunocomplexes were washed four times with IP buffer, and proteins were eluted from Protein A-Agarose beads by addition of 50 µl of 2x sodium dodecyl sulfate-Laemmli buffer and boiling at 95 C for 10 min. Protein elutions and 10 µg of control nuclear extract were separated by 7.5% SDS-PAGE, transferred to PVDF membrane, after which the membranes were blocked in PBS containing 4% nonfat dry milk and 1% Tween (PBS-Tween) overnight. The membrane was incubated with a 1:2000 dilution of {alpha}-Sp3 or {alpha}-mSin3A, 5 µg of {alpha}-HDAC2 or 10 µg {alpha}-HDAC1 in PBS-Tween with 2% nonfat dry milk, and immunoblot analysis was performed using a horseradish peroxidase-conjugated donkey antirabbit secondary antibody. Immunoreactive bands were detected using the Western Lightning Chemiluminescent Kit (Amersham Pharmacia Biotech, Arlington Heights, IL).

ChIP Assay
ChIP assays were performed as previously described (12). Briefly, MA-10 cells were treated in serum-free medium in the absence or presence of 1 mM (Bu)2cAMP for 2 h and then incubated with 1% formaldehyde for 10 min at 37 C. Chromatin was sheared to approximately less than 1000 bp in size by sonication and immunoprecipitated with 8 µg of IgG, {alpha}-Sp3, or {alpha}-mSin3A, or 5 µg of {alpha}-HDAC2, or 10 µl of {alpha}-HDAC1. Formaldehyde cross-links were reversed, and immunoprecipitated DNA was recovered. PCR was performed using 0.5 µl input chromatin sample and 3 µl immunoprecipitated DNA sample with primer pairs specific for the proximal (–222 bp/–114 bp) or distal (–3584 bp/–3428 bp) regions of the StAR promoter. Sequences for PCR primers used are as follows: mStAR –222 forward, 5'-ATATCCTCTGCCCCATCTCC-3'; mStAR –114 reverse, 5'-ATCCTGCAGTGCTGGCCAAG-3', mStAR –3584 forward, 5'-CATA-CGTGCACTGTCTTAGC-3', mStAR –3428 reverse, 5'-ACTCCTCCAGTAACT-CCTTC-3'.

Quantitative Real-time RT-PCR
MA-10 cells were treated with serum-free medium in the absence or presence of 100 nM TSA for the indicated times, and total RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instruction, and 1 µg RNA was used for reverse transcription following standard procedures. Multiplex PCRs were optimized for StAR and 18S rRNA by establishing a concentration range from 0.1–1.5 ng RNA input and determining the limiting primer concentrations for 18S rRNA and StAR. The final multiplex PCR contained 300 nM StAR forward and reverse primers plus 100 nM StAR Taqman probe, and 25 nM 18S rRNA forward and reverse primers plus 50 nM 18S rRNA probe using Taqman Universal PCR master mix. The reaction conditions were 94 C for 30 sec; 60 C for 20 sec; 72 C for 30 sec for a total of 40 cycles using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA). Under these conditions, the CT value for each tube containing 0.15–1.25 ng total RNA input was linear for both StAR and 18S rRNA. For all experiments, aliquots of the reverse transcription reaction to achieve 0.25 and 0.5 ng cDNA were used as input for real-time PCRs. PCR was performed in duplicate for each concentration, and the relative differences in StAR mRNA were determined using the {Delta}{Delta}CT method. StAR forward primer (5'-CGCAGAGGTTCCACCTGTGT-3') corresponds to 523–542 bp, and the reverse primer (5'-AGTGGATGAAGCACCATGCA-3') corresponds to 617–638 bp; the probe (5'-FAM-CAGGCATGGCCACACA-TAMRA-3') corresponds to 548–563 bp. A 115-bp StAR PCR product was verified by sizing on an agarose gel. 18S rRNA primers and probe were purchased from ABI with the probe 5'-labeled with the VIC reporter dye (Applied Biosystems, Inc., Foster City, CA).

Oligonucleotide Sequences Used for EMSA and PCR
Altered base pairs in the mutated oligos are underlined

For PCR Cloning
MluI –254, 5'-ACTAACGCGTCTACATTTACAACTTTAG-3'; MluI –215, 5'-CGTCACGCGTTCCTCTGCCCCATCTCCG-3'; MluI –180, 5'-TCAGACGC-GTTACCTGCAGAGTCTGGTCC-3'; Sp3 mut, 5'-TACCTGCAGAGTCTGGTCAT-GCATTTACAC-3'; CAGA mut, 5'-ACTAGCGGCCGCTCCTCCCTTTACACAGTC-3'; CAGA and Sp3 mut, 5'-ACTAGCGGCCGCTCATGCATTTACACAGTCTGC-3'.

For EMSA Probes
–180/–150 wt, 5'-TACCTGCAGAGTCTGGTCCTCCCTTTACAC-3'; –180/–150 Sp3 mut, 5'-TACCTGCAGAGTCTGGTCATGCATTTACAC-3', –180/–150 CAGA mut, 5'-TACCTGGAGCGGCCGCTCCTCCCTTTACAC-3'; –180/–150 CAGA and Sp3 mut, 5'-TACCTGGAGCGGCCGCTCATGCATTTACAC-3'.

Statistics
Statistical significance between the wild-type and mutant StAR-promoter reporter constructs was determined by one-way ANOVA followed by Tukey’s posttest using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant. For TSA studies, an unpaired Student’s t test was performed to determine statistical significance between wild-type and mutant StAR-reporter constructs within treatment concentrations. P < 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Elizabeth Hudson for her contributions to these studies and Dr. Carolyn Klinge for her critical review of this manuscript.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK-51656 (to B.J.C.) and training grants from the University of Louisville Center for Genetics and Molecular Medicine and Grant T32 ES011564 from The National Institute of Environmental Health Sciences (to B.F.C.).

First Published Online August 18, 2005

Abbreviations: (Bu)2cAMP, N6,2'-O-dibutyryl-cAMP; ChIP, chromatin immunoprecipitation; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X, gene 1; FOXL2, Forkhead L2; HDAC, histone deacetylase; IP, immunoprecipitation; SF-1, steroidogenic factor 1; SREBP, sterol-regulatory element binding protein; StAR, steroidogenic acute regulatory protein; TSA, trichostatin A; VPA, valproic acid; WB, Western blot; YY1, Yin Yang 1.

Received for publication December 8, 2004. Accepted for publication August 9, 2005.


   REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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