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Endocrinology Vol. 141, No. 4 1345-1355
Copyright © 2000 by The Endocrine Society

ARTICLES

Steroidogenic Factor-1 Influences Protein-Deoxyribonucleic Acid Interactions within the Cyclic Adenosine 3',5'-Monophosphate-Responsive Regions of the Murine Steroidogenic Acute Regulatory Protein Gene1

Clavia R. Wooton-Kee and Barbara J. Clark

Department of Biochemistry and Molecular Biology, 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
 Materials and Methods
 Results
 Discussion
 References
 
De novo synthesis of the steroidogenic acute regulatory protein (StAR) in response to trophic hormonal stimulation of steroidogenic cells is required for the delivery of cholesterol from the mitochondrial outer membrane to the mitochondrial inner membrane and the cytochrome P450 side-chain cleavage enzyme. StAR expression is transcriptionally regulated by cAMP-mediated mechanisms, and we have identified a 45-bp region within the mouse promoter that is important for cAMP responsiveness of the gene. This region, located between -105 and -60 of the start site of transcription, contains a SF-1-binding site, a highly conserved C/EBPß -AP-1-nuclear receptor half-site sequences (CAN region), and a GATA-4-binding site. The SF-1 element and CAN region are required for full basal activity, whereas the GATA-4 element may account for 20% of the cAMP response in MA-10 mouse Leydig tumor cells. A cAMP-dependent protein-DNA complex was observed with the CAN region and mutation of a nonconsensus AP-1 site within this region greatly diminished promoter strength. Complex protein-DNA interactions within the cAMP response region (-105/-60) were shown to require the SF-1 element (-95), suggesting that SF-1 is required for protein-DNA interaction at the CAN (-79) region and maximal activity of the promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID HORMONE biosynthesis in the adrenal and gonads is stimulated by trophic hormones via a cAMP-dependent second messenger system. Within minutes of hormonal stimulation, cholesterol is mobilized to the outer mitochondrial membrane and transferred to the mitochondrial inner membrane where it is converted to pregnenolone by the cytochrome P450 side-chain cleavage enzyme (P450scc) (1, 2, 3, 4). This acute response is the rate-limiting step in steroidogenesis and is dependent upon the de novo synthesis of the steroidogenic acute regulatory protein (StAR) (5). Without a functional StAR protein, cholesterol accumulates in the outer mitochondrial membrane and steroid production ceases. Thus, StAR functions in the transfer of cholesterol to the inner mitochondrial membrane (5). This function of StAR is elegantly demonstrated by characterization of StAR knockout mice that lack steroid production (6). These data confirmed that the StAR mutations identified in patients with congenital lipoid adrenal hyperplasia are the underlying cause for the disorder (7, 8).

Hormonal treatment of steroidogenic cells controls StAR gene expression. Our previous studies have shown that treatment of MA-10 mouse Leydig tumor cells with trophic hormones or the cAMP analog, (Bu)2cAMP, induces StAR message within 30 min, and maximal steady state levels are reached within 4 h (9). This induction of StAR gene transcription does not require de novo protein synthesis, suggesting that posttranslational mechanisms are involved in the acute induction of StAR gene in response to increases in intracellular levels of cAMP (10).

Several factors that participate in StAR gene activation have been reported. One factor is the orphan nuclear receptor steroidogenic factor-1 (SF-1). SF-1 has been shown to have a critical role in the regulation of many of the steroid hydroxylase genes, and gene knockout studies in mice have shown that SF-1 is critical for development of the gonad and adrenal glands (11, 12, 13, 14). Regulation of SF-1 may occur through direct phosphorylation by a protein kinase A (PKA)-dependent pathway. This proposal is supported by studies in which SF-1 trans-activation of the steroid hydroxylase genes was lost in PKA-deficient cell lines (15, 16). Furthermore, potential PKA phosphorylation sites are present in SF-1, and in vitro studies have directly demonstrated a PKA-dependent phosphorylation of SF-1 (17, 18, 19). In addition, activation of the mitogen-activated protein kinase pathway has been shown to phosphorylate and active SF-1, supporting a role for phosphorylation of SF-1 for its function (20). Our initial analysis of two SF-1 sites in the mouse promoter, located at -45 and -135 from the transcription start site, indicated that these elements contributed to basal promoter activity but were not essential for the cAMP-dependent induction (10). The involvement of a SF-1 site in StAR gene activation was more recently confirmed with the rat promoter (21). Mutation of the SF-1 elements at -135 and -95, either individually or in combination, decreased SF-1-dependent reporter gene expression in a nonsteroidogenic cell line cotransfected for SF-1 expression (21). In these studies the (Bu)2cAMP-mediated increase in promoter activity was also depressed, suggesting that SF-1 may mediate part of the cAMP response. However, it is not known what effect the -95 SF-1 mutation would have in a steroidogenic cell line, where factors other than SF-1 may be required for basal and cAMP-dependent StAR transcription. On the other hand, the SF-1 element located at -105/-95 in the human promoter was shown to be critical for basal and maximal cAMP responsiveness in human granulosa-luteal cells and in H295R adrenocortical cells (22, 23). Thus, it appears that the role of SF-1 in StAR expression may be promoter and/or tissue specific.

C/EBPß has also been shown to modulate StAR basal promoter activity. C/EBPß is a basic leucine zipper transcription factor that is expressed in liver, intestine, lung, and adipose tissues (24). More recently, C/EBPß has been identified as the only isoform of C/EBP present in unstimulated primary Leydig cell cultures and MA-10 Leydig cells (25). Two putative CCAAT sites located at -113 and -87 in the murine StAR promoter were identified, and C/EBPß in MA-10 nuclear extracts was shown to bind to the -113 element (26). A weak protein-DNA interaction was observed with the -87 region of the promoter, but the factors were not identified. Mutation of the -87, but not the -113, site abolished SF-1-dependent trans-activation of StAR-luciferase reporter gene expression in transfected COS-1 cells (26). However, transient transfection of MA-10 cells with a StAR-luciferase reporter gene construct containing mutations at either the -87 or -113 C/EBPß-binding site did not affect the cAMP-dependent response, but greatly reduced the basal activity of the promoter. A protein-protein interaction between C/EBPß and SF-1 was further demonstrated; thus, these studies suggested a possible C/EBPß-SF-1 interaction that is required for basal promoter activity.

Although SF-1 and C/EBPß participate in basal expression of StAR, the mechanism for cAMP-dependent induction has not been elucidated. Previously, we narrowed the cAMP-responsive region of murine StAR to -254 bp of the start site of transcription (10). Sequence analysis of this region identified potential binding sites for transcription factors that have been shown to mediate cAMP-dependent responses in other genes. Two elements that are part of the focus in the present study are a C/EBPß/nonconsensus activating protein-1/nuclear receptor half-site located between -87/-79 (CAN region) and a putative binding site for members of the GATA family of zinc finger transcription factors located between -68/-40. Activating protein-1 is composed of either Fos-Jun or Jun-Jun dimers that bind to the consensus sequence TGA(G/C)TCA and regulates genes either constitutively or by cAMP- or Ca2+-mediated mechanisms (27, 28, 29). The GATA family of transcription factors regulates gene expression, differentiation, and cell proliferation by binding to the consensus DNA sequence (A/T)GATA(A/G) (30). More recently, three members of the GATA transcription factor family were shown to be expressed in the developing gonads: GATA-1, GATA-4, and GATA-6 (31, 32, 33). GATA-1 message has been isolated in MA-10 cells, and GATA-1 was shown to up-regulate the basal promoter activity of the rat inhibin {alpha}-subunit gene (34). GATA-4 and GATA-6 have also been identified in testicular tissue, and transient expression of GATA-4 was shown to trans-activate an inhibin {alpha}-promoter/reporter construct in mouse Leydig and granulosa tumor cell lines (35). Thus, a new role is emerging for GATA transcription factors in Leydig cell function.

To clarify the role of SF-1 in StAR expression in MA-10 mouse Leydig tumor cells, we extended our previous studies and demonstrated that SF-1 binds to a third element in mouse promoter at -95. We show that this SF-1 site, denoted SF1–3, is required for full basal activity of the StAR promoter. Additionally, SF-1 functions in part to stabilize protein-DNA interactions at the C/EBPß/nonconsensus activating protein-1 (AP-1)/nuclear receptor half-site (CAN) region located at -79. We also demonstrate that GATA-4 binds to the StAR promoter at -68 bp and contributes to 20% of the cAMP-dependent induction. We conclude that the SF1–3, CAN, and GATA-4 DNA-binding sites serve critical roles in maintaining full basal promoter activity, which, in turn, is necessary for maximal cAMP induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
N6,2'-O-(Bu)2cAMP [(Bu)2cAMP)] was purchased from Sigma (St. Louis, MO). Restriction enzymes, Klenow enzyme, T4 DNA ligase, Luciferase Assay System, and pGL2-luciferase reporter vectors were purchased from Promega Corp. (Madison, WI). [{gamma}-32P]ATP was obtained from NEN Life Science Products (Wilmington, DE). Custom oligonucleotides were purchased from Genosys (The Woodlands, TX) and Genemed Synthesis, Inc. (San Francisco, CA). Glutathione-S-transferase (GST)-SF-1 plasmid was donated by Dr. Keith Parker, University of Texas Southwestern Medical Center (Dallas, TX). Antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Upstate Biotechnology, Inc. (Lake Placid, NY). X-ray film was obtained from Eastman Kodak Co. (Rochester, NY) and NEN Life Science Products (Boston, MA).

Cell culture
The MA-10 mouse Leydig tumor cell line was a gift from Dr. M. Ascoli (Department of Pharmacology, University of Iowa of Medicine, Iowa City, IA). The cells were grown in Waymouth’s MB/752 medium containing 15% horse serum and 40 µg gentamicin sulfate/ml.

Transient transfections and reporter assays
MA-10 cells were plated at 300,000 cells/well in a 24-well plate 24 h before transfection. Two micrograms of StAR-luciferase reporter gene plasmid and 2 µg pCMV-ß-galactosidase (CMV, cytomegalovirus) expression vector plasmid were cotransfected into cells using 15 µg/ml Lipofectamine (Life Technologies, Inc., Grand Island, NY) and Waymouth’s medium without antibiotics and serum as described previously (10). Twenty-four hours posttransfection, the cells were treated with 1 mM (Bu)2cAMP for 16–19 h. The Promega Corp. Luciferase Assay System was used to prepare cell lysates and measure luciferase activity (Lumat LB 9507 luminometer, Wallac, Inc., Gaithersburg, MD). ß-Galactosidase activity was assayed using ß-D-galactopyranosidase (Roche Molecular Biochemicals, Indianapolis, IN) as the substrate and measuring the absorbance at 595 nm. Relative light unit (RLU) values were normalized to ß-galactosidase activity for each sample. Each treatment was performed in triplicate, and the mean ± SEM were determined. The data were expressed as fold induction relative to the -254/+35 luc control, which was set at 100. The mean ± SEM for the fold induction in response to (Bu)2cAMP for all independent transfection experiments was calculated, and a pooled Student’s t test was performed on the data. P < 0.05 was considered statistically significant.

StAR-luciferase constructs
PCR was used to generate the 5'-deletion constructs for the StAR promoter. Linearized -254/+35 StAR promoter-pGL2-luciferase (-254 StAR promoter construct) was used as the template. To amplify the region -150 to +35 (-150/+35 luc), MluI -150 and GL2-primers were used in the PCR reaction, and the product was digested with MluI and HindIII and cloned into the MluI and HindIII sites of pGL2-basic. The regions -105 to +35 (-105/+35 luc) and -68 to +35 region (-68/+35 luc) were amplified using SF1–3 top primer and GL2 primers, and -68/-44 top primer and GL2 primers, respectively, and the PCR product for each clone was Klenow treated, digested with HindIII, and cloned into the SmaI-HindIII sites of pGL2-basic. The site-specific mutants generated in the -254/+35 luc plasmid were constructed by generating two overlapping PCR products that share an engineered restriction site to alter the core sequences of interest. To produce the 5'-PCR product, the GL1 primer was used to amplify the top strand, and the bottom strand was amplified with the site-specific primer. The 3'-PCR product was produced with the site-specific mutant primer (top strand) and GL2 (bottom strand). The PCR products were digested with the appropriate restriction enzymes and cloned into the SmaI-KpnI restriction sites of pGL2-basic. The sequences of oligonucleotides used in the reactions are listed under Oligonucleotides used in EMSA and PCR. The SF1–3mut/GATA-4mut-luc, GATAmut/AP-1mut-luc, and SF1–3mut/AP-1mut-luc constructs were created in a similar manner; the mutant SF1–3 site was engineered into GATAmut-luc using the SF1–3mut-luc primer to generate SF1–3mut/GATAmut-luc, the AP-1mut site was engineered into GATAmut-luc using the AP-1mut-luc primer to generate GATAmut/AP-1mut-luc, and the AP-1mut site was engineered into SF1–3mut-luc using the AP-1mut-luc construct to generate SF1–3mut/AP-1mut-luc. One exception for SF1–3mut/AP-1mut-luc is the 5'-PCR product was digested with MluI and BglII, the 3'-PCR product was digested with BglII and XbaI, and the two products were cloned into the MluI-NheI sites of pGL2 basic. All sequences were confirmed by double stranded sequencing using the Sequenase II kit (United States Biochemical Corp., Cleveland, OH).

Electrophoretic mobility shift assay (EMSA)
Double stranded oligonucleotides were generated by mixing equal molar concentrations of top and bottom strand primers in a high salt annealing buffer [10 mM Tris (pH 7.5), 50 mM NaCl, and 1 mM EDTA] and heating for 2 min at 95 C followed by slow equilibration to room temperature. The oligonucleotides were end labeled with T4 DNA kinase and [{gamma}-32P]ATP (NEN Life Science Products). To generate a probe for the -105/-44 region of StAR, linearized -254 StAR promoter construct was used as the DNA template in a PCR reaction with SF1–3 (top) and -68/-44 (bottom). The -105/-44 fragment containing site-specific mutations or double mutations was generated as described above using the corresponding mutated reporter gene constructs as the template and appropriate oligonucleotides. Each PCR fragment was gel purified and radiolabeled as described above. Restriction digestion analysis was used to confirm the PCR-generated probe contained the desired mutations. MA-10 nuclear extracts (2.5–15.0 µg) or 0.25 µg GST-SF-1 were incubated with 50 fmol radiolabeled oligonucleotide for 30 min on ice. The binding buffer contained the following: 50 mM Tris-Cl (pH 8.0), 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol, and 2.5 µg poly(dI:dC). Cold competition assays were performed with a 20- to 100-fold molar excess of oligonucleotides. Antibody supershift assays were performed with 2.5 or 15 µg MA-10 nuclear extract proteins or 0.25 µg GST-SF-1 plus 4 µg monoclonal GATA-1, polyclonal GATA-4, polyclonal C/EBPß, or polyclonal SF-1 antibodies. For the supershift assays, all components of the binding reaction, excluding the radiolabeled probe, were preincubated on ice for 30 min. Radiolabeled probe was then added, and the incubation was continued for 30 min. Binding reactions were resolved on a 4% nondenaturing polyacrylamide gel. The dried gels were exposed to x-ray film, and the radioactive bands were visualized by autoradiography.

The following oligonucleotides were used in the EMSA binding reactions. Only the top strand is listed, and changed base pairs in the mutated probes are underlined.

Sequences of oligonucleotides used for PCR and EMSA probes
The following sequences were used: SF1–3, 5'-CATTCCATCCTTGACCCTCTGCA-3'; SF1–3mut, 5'-CATTCCATCCTCGAGCCTCTGCA-3'; -68/-44, 5'-TTTTTTATCTCAAGTGATGATGCAC-3'; -68/-44 GATAmut, 5'-TTTTTCCGGACAAGTGATGATGCAC-3'; -68/-44 SREBPmut (SREBP, sterol regulatory binding protein), 5'-TTTTTTATCTCTCGAGATGATGCAC-3'; -87/-64, 5'-TGCACAATGACTGATGACTTTTT-3'; -87/-64 AP-1mut, 5'-TGCACAATAGATCTTGACTTTTT-3'; -87/-64 1/2 mut, 5'-TGCACAATGACTGAGATCTTTTT-3'; GL11, 5'-TGTATCTTATGGTACTGTAACTG-3'; GL21, 5'-CTTTATGTTTTTGGCGTCTTCCA-3'; and -150 Mlu, 5'-ACACACGCGTAGTCTGCTCCCTCCCACCTTGGCCA-3' (1 indicates the primers used for pGL2-basic vector).

Nuclear extract preparation
Confluent MA-10 mouse Leydig tumor cell cultures were treated with fresh Waymouth’s medium in the absence (control) or presence of (Bu)2cAMP for 4 h, and nuclear extracts were prepared following previously described protocols (36). The extracts were flash-frozen in liquid nitrogen and stored at -80 C until use.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
5'-Deletion analysis localizes the cAMP-responsive region between -105 and -68 of the mStAR promoter
To more closely map the cAMP-responsive elements within the -254 bp region of the murine StAR gene, a series of StAR promoter deletion constructs was transiently transfected into MA-10 cells and tested for cAMP responsiveness (Fig. 1Go). (Bu)2cAMP treatment of MA-10 cells resulted in an 8.7-fold increase in luciferase reporter gene activity for the -254 StAR promoter construct. Deletion to -150 caused both basal and (Bu)2cAMP-stimulated promoter activity to increase 4- and 6-fold, respectively, which represents a 70% increase in the cAMP-dependent fold induction compared with the -254 StAR construct. Further deletion to -105 caused a 46% decrease in basal activity compared with -254 StAR, but the fold induction in response to the cAMP stimulus was not diminished. However, a 5-fold decrease in the cAMP-dependent induction was observed with the -68 StAR promoter construct. These results suggest that multiple elements may be required for the cAMP response of the mouse StAR promoter: a possible negative regulatory region located between the -150/-105 and a positive regulatory region located between the -105/-68 region of the promoter. The -254/-65 region of the promoter was cloned upstream of a heterologous promoter (-254/-66StAR-pGL3luc), but the single copy of this region was not sufficient to confer cAMP-dependent responsiveness (data not shown). Therefore, elements 5' and 3' of -68 appear to required for the induction of StAR gene by cAMP.



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  		Figure 1. 5'-Deletion analysis of the mouse StAR promoter. MA-10 cells were cotransfected with the indicated StAR-promoter-luciferase reporter gene constructs (denoted by the position of the base pairs 5-prime to the transcriptional start site) and the pCMV-ß-galactosidase expression vector. The transfected cells were treated in triplicate with fresh Waymouth’s medium in either the absence (basal) or presence of 1 mM (Bu)2cAMP for 16–19 h, and the luciferase activity was determined and normalized to ß-galactosidase expression (RLU/b-gal). For each experiment, the data were expressed as a percentage of the -254 StAR-promoter construct (-254/+35 StAR promoter-pGL2luciferase), which was set at 100%, and are graphed as the mean ± SEM values for the relative luciferase activity from three to five experiments. The fold induction values represent the ratio of (Bu)2cAMP/basal activity for each construct. Shown are the mean ± SEM values for three to five experiments. The inset graph shows the RLU/ß-gal activities of the -254, -105, and -68 StAR-promoter constructs. pGL2-basic represents vector control, and NT represents nontransfected MA-10 cells. *, Statistically significant (P < 0.05) increase (-150/+35) or decrease (-68/+35 luc) relative to the fold induction of the -254 StAR promoter-luciferase construct in response to (Bu)2cAMP.

 
SF-1 and GATA-4 bind to elements within -105/-44 region of the mouse StAR promoter
As the 5'-deletion analysis indicated that the cAMP-responsive region was within -105 bp of the transcriptional start site, we were interested in testing for possible cAMP-dependent protein-DNA interactions within this region. We first compared the sequence of murine StAR promoter (-105 to +35) to the transcription factor database and identified several potential transcription factor-binding sites that included SF-1, C/EBP, AP-1, and GATA-1. Shown in Fig. 2AGo is the sequence of the -150 region of murine StAR, with the location of these elements indicated. An alignment with the human and rat sequences is also shown to indicate the high degree of sequence conservation within this region of the StAR promoter. We previously characterized the SF-1–1 (-135), SF-1–2 (-45), and the C/EBPß (-113) elements (10, 26), whereas the SF-1 site located at -95 bp has been shown to bind SF-1 in the human and rat StAR promoters (21, 37). The region between -87/-64 is not as well conserved and unique to the mouse sequence are potential C/EBPß, nonconsensus AP-1, and nuclear-receptor half-site elements that we refer to as the CAN region. Another highly conserved region, located between -68/-44, is a putative binding site for GATA-1 and SREBP. This region is adjacent to the proposed cAMP-responsive region (-105/-68) identified by 5'-deletion analysis and was deleted in the nonresponsive 3'-deletion construct (-254/-66StAR-pGL3luc), indicating that this region also may contribute to the cAMP response.



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  		Figure 2. A, Comparison of mouse (M), human (H), and rat (R) StAR promoter sequences within -150 bp before the transcriptional start site. The location of the DNA elements for SF-1, C/EBPß-, GATA-4-, and putative SREBP-binding sites are indicated above the core sequences by the respective name of the highlighted factors. The putative binding sites for the CAN region (-87/-64) include C/EBP, AP-1, and an imperfect nuclear receptor half-site (1/2). B, Shown are the region of the promoter and the name given to the introduced mutation in the -254/+35 StAR-promoter constructs and the EMSA probes. The point mutations are indicated in bold.

 
Based on the location of these elements, overlapping DNA probes were generated to encompass the SF-1 (-105/-83 SF1–3), the C/EBPß-AP-1-nuclear receptor half-site sequences (-87/-64 CAN), and the GATA/SREBP element (-68/-44 GATA) and used in EMSAs. Mutations that were introduced into the core binding sequences of the oligonucleotides are shown in Fig. 2BGo. The SF1–3 DNA probe (-105/-83) formed a specific protein-DNA complex that was observed using nuclear proteins isolated from either untreated (lane 1) or (Bu)2cAMP-treated (lane 5) MA-10 cells (Fig. 3Go). The specificity of complex formation was shown by competition with unlabeled SF1–3 probe (lanes 2 and 6), and the complex was not formed when an oligomer that contained two point mutations in the CATCCTTG core sequence (SF1–3mut) was used in the binding reaction (lanes 4 and 8). A protein-DNA complex of similar mobility was observed when the probe was incubated with purified GST-SF-1 (lane 9), and addition of an antibody specific for SF-1 greatly diminished or abolished DNA-protein interaction (lanes 3 and 7). Thus, these data verify that SF-1 also binds to this region of the mouse promoter. As the mobility and intensity of the SF-1 complex are the same with nuclear proteins from either control or (Bu)2cAMP-stimulated cells, these data indicate that SF-1 binding is independent of the cAMP stimulus in MA-10 cells. This element is referred to as SF1–3 to indicate the third SF-1-binding site within 150 bp of the mouse promoter (Fig. 2AGo).



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  		Figure 3. EMSA analysis reveals SF-1 binding to the -105/-83 region of mouse StAR. EMSA was used to examine binding of MA-10 nuclear extract to the radiolabeled SF1–3 probe, which represents the -105/-83 region of the StAR promoter. Binding reactions containing 50 fmol radiolabeled probe and 2.5 µg nuclear extracts prepared from unstimulated (lanes 1–4) or (Bu)2cAMP-treated (lanes 5–8) MA-10 cells or 0.25 µg purified GST-SF-1 protein (lane 9) were preincubated on ice for 30 min. For cold competition or antibody interference analysis, a 100-fold molar excess of cold probe (indicated as + in lanes 2 and 6) or 4 µg SF-1 polyclonal antibody (indicated as Ab in lanes 3 and 7) were preincubated with nuclear extract for 30 min; then the radiolabeled probe was added to all reactions, and the incubation was continued for 30 min on ice. The DNA-protein complexes were resolved on a nondenaturing 4% polyacrylamide gel, the gel was dried, and the complexes were visualized by autoradiography. Mutations introduced into SF1–3 (SF1–3 mut) are listed in Fig. 2BGo. Lane 10 shows incubation of SF-1 polyclonal antibody and radiolabeled probe without nuclear extract. Free probe is not shown on the gel.

 
Two protein-DNA complexes were observed with the CAN (-87/-64) DNA probe (Fig. 4Go, A and B). Complex A is a doublet that is present in binding reactions with nuclear proteins from both control and (Bu)2cAMP-treated MA-10 cells, whereas complex B is a slower migrating band that is present only with nuclear proteins from stimulated cells (Fig. 4AGo). As this region contains a CAAT box, and C/EBPß has been proposed or shown to interact with this region (26, 38), we used a C/EBPß antibody to test for the presence of C/EBPß in complex A or B. As shown in Fig. 4BGo, addition of the antibody did not affect protein-DNA interactions with the CAN probe and nuclear proteins from stimulated cells (lane 2). We verified that a protein-DNA complex is formed with a consensus C/EBP oligonucleotide (lane 3) and that this complex is supershifted by antibody (lane 4), confirming that C/EBPß is present in the nuclear extract (25, 26). Cold competition with the consensus C/EBP oligonucleotide also failed to indicate any involvement of C/EBPß with this element (lane 5). The specificity of complex formation was demonstrated by cold competition with the CAN probe (lane 8) and a mutation in the putative nuclear receptor half-site (1/2mut) was also effective as a competitor probe (lane 6). Mutation in the putative AP-1 site (AP-1mut), on the other hand, did not compete with the CAN probe for protein binding (lane 7). We next tested for Fos/Jun binding to this region, but addition of antibodies that recognize all family members did not affect complex formation (data not shown).



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  		Figure 4. A cAMP-dependent factor(s) binds to the -87/-64 CAN region of mouse StAR. EMSA was used to examine binding of MA-10 nuclear extract to radiolabeled CAN probe, which represents the -87/-64 region of the StAR promoter. A, Concentration-dependent protein-DNA complex formation. Binding reactions were preincubated for 30 min on ice with nuclear extracts prepared from unstimulated (control) or (Bu)2cAMP-treated [(Bu)2] MA-10 cells. Shown is a titration of 5, 10, and 15 µg control or (Bu)2cAMP nuclear extracts, respectively. B, Effects of cold competition and C/EBPß antibody on protein-DNA interactions. Either a 50-fold molar excess of the indicated cold probe or C/EBPß polyclonal antibody (4 µg) antibody was included in a preincubation with nuclear extracts for 30 min; then radiolabeled probe was added, and the incubation was continued for 30 min on ice. The protein-DNA complexes were resolved on a nondenaturing 4% polyacrylamide gel, the gel was dried, and the complexes were visualized by autoradiography. The sequences of the oligomer probes for the CAN region containing mutations in the potential AP-1 (AP-1 mut) and imperfect nuclear receptor half-sites (1/2 mut) are listed in Fig. 2BGo. con. C/EBP, Commercially purchased consensus C/EBP oligomer. The arrows indicate the positions of complexes A and B and free probe. SS, The C/EBPß antibody supershift.

 
A prominent protein-DNA complex was apparent with the GATA oligonucleotide (-68/-44 GATA) using nuclear proteins from either untreated or (Bu)2cAMP-stimulated MA-10 cells (Fig. 5Go). Complex formation was abolished by cold competition with the GATA oligonucleotide (lane 2) or the SREBPmut oligonucleotide that contains a mutation in the putative SREBP site (lane 4). Conversely, using the SREBPmut as a probe did not affect complex formation (lane 3), indicating that the SREBP element is not involved in protein-DNA interactions at this region. On the other hand, no complex was formed with a probe that had a mutation within the GATA DNA-binding site (lane 5), and the mutant GATA probe (GATA mut) did not compete for protein binding (lane 6). A complex of similar mobility was observed using a probe with the consensus sequence for GATA binding (lanes 7 and 8) and cold competition experiments using this consensus GATA probe resulted in the lack of complex formation (lane 10). Although this sequence was identified as a potential GATA-1-binding site, GATA-1 antibodies did not affect complex formation (compare lanes 1 and 9). However, binding to the consensus GATA probe appeared to be diminished by GATA-1 antibodies (compare lanes 7 and 8). Therefore, we tested for other members of the GATA family, and GATA-4 antibodies resulted in a supershift of the complex (lanes 11–13). The supershifted complex was apparent using nuclear extracts from either control (lane 11) or (Bu)2cAMP-treated (lanes 12 and 13) MA-10 cells with the StAR GATA probe (lanes 11 and 12) or the consensus GATA probe (lane 13). A less prominent band appears with the -68/-44 GATA probe; however, the significance of this interaction is not known. At present, these results indicate that GATA-4 in MA-10 cells binds to the mouse StAR promoter.



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  		Figure 5. GATA-4 in MA-10 nuclear extracts binds the highly conserved region (-68/-44) of the mouse StAR promoter. EMSA was used to examine binding of MA-10 nuclear extract to radiolabeled GATA probe, which represents the -68/-44 region of the StAR promoter. Binding reactions were incubated for 30 min on ice with 5 µg nuclear extracts prepared from unstimulated (lanes 1–11) or (Bu)2cAMP-treated (lanes 12 and 13) MA-10 cells. For cold competition or antibody interference analysis, a 20-fold molar excess of the indicated cold probe, denoted by cc (lanes 2, 4, 6, and 10), or 4 µg GATA-1 monoclonal antibody (lanes 8 and 9) or GATA-4 polyclonal antibody (lanes 11–13) were included in a preincubation with nuclear extracts; then the radiolabeled probe was added, and the incubation was continued for 30 min on ice. Lanes 14 and 15 represent probe alone or probe and antibody minus nuclear extract, respectively. The protein-DNA complexes were resolved on a nondenaturing 4% polyacrylamide gel, the gel was dried, and the complexes were visualized by autoradiography. The radiolabeled probes are denoted as GATA for the -68/-44 region, GATA mut for the -68/-44 region containing mutations in the GATA core sequence, and SREBP mut for the -68/-44 region containing mutations in the putative SREBP element. The sequences for these probes are all shown in Fig. 2BGo. The con. GATA probe represents the 20-mer consensus oligonucleotide for GATA that was used as a positive control for GATA-binding proteins. The arrows indicate the supershift with GATA-4 Ab (SS) and the free probe; the open arrowhead represents the specific complex.

 
The -87/-68 region of the StAR promoter is required for maximal basal promoter activity of StAR
To test the functional significance of the specific protein-DNA interactions characterized above on StAR promoter activity, the mutations that abolished protein binding were individually introduced into the -254 StAR promoter construct, and luciferase activity was measured in transiently transfected MA-10 cells (Fig. 6AGo). Mutation of the SF1–3 site resulted in a 43% reduction in basal luciferase activity compared with the -254 StAR promoter construct (WT), but the fold induction in response to (Bu)2cAMP stimulation was not significantly different. Mutation of the GATA-4-binding site also reduced basal promoter strength (~36%); however, the cAMP response was also decreased from 8.7-fold (WT) to 6.9-fold (GATA). Lastly, mutation of the putative AP-1 site (AP-1mut) or the nuclear receptor half-site (1/2mut) resulted in decreased basal activity by 63% or 57%, respectively, but, again, fold activation in response to (Bu)2cAMP was unchanged compared with that with the -254 StAR promoter construct (WT). Although the fold induction by (Bu)2cAMP-treatment was not diminished with the SF1–3 and CAN region mutations, overall promoter strength was reduced. This decrease in response appeared to closely parallel the severity of the mutation on basal promoter activity. These data indicate that the SF1–3, CAN, and GATA sites all contribute to basal promoter strength. However, neither the SF1–3 nor the CAN region is individually required for the cAMP-dependent response of the StAR promoter. On the other hand, GATA-4 may account for 20–30% of the cAMP response of the murine StAR promoter.



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  		Figure 6. Mutational analysis reveals regulatory regions of mouse StAR. MA-10 cells were cotransfected with the indicated StAR promoter-luciferase reporter gene constructs and pCMV-ß-galactosidase as detailed in Materials and Methods. The transfected cells were treated in triplicate with fresh Waymouth’s medium in either the absence (basal) or presence of 1 mM (Bu)2cAMP for 16–19 h, and luciferase activity was determined and normalized to ß-galactosidase expression (RLU/b-gal). For each experiment, the data were expressed as a percentage of the -254 StAR promoter basal activity, which was set at 100%. Graphed are the mean ± SEM values from three to six experiments for the relative luciferase activity of StAR promoter containing mutations in either a single element (A) or two elements (B). The fold induction values represent the ratio of (Bu)2cAMP/basal activity for each construct. Shown are the mean ± SEM values for three to five experiments. *, Statistically significant (P < 0.05) difference relative to the fold induction of the -254 StAR promoter construct in response to (Bu)2cAMP. WT, Wild-type -254/+35 StAR promoter-luciferase construct; pGL2-basic, empty vector; NT, nontransfected.

 
Mutation of multiple elements affects basal activity of the StAR promoter
As mutations in the SF-1- and GATA-4-binding site individually affected StAR basal and cAMP-dependent promoter activity, respectively, as well as the close proximity of the two binding sites, we introduced mutations that abolished both SF-1 and GATA-4 binding into -254 StAR promoter construct and tested the effect on promoter activity in transiently transfected MA-10 cells (Fig. 6BGo). The overall basal activity of StAR promoter was dramatically decreased (88%) compared with that of the -254 StAR promoter construct (WT), indicating that mutation of both SF1–3 and GATA had an additive effect on promoter strength. Despite minimal basal activity, the promoter caused a 4.8-fold increase in the cAMP-dependent response. However, this represents only 45% of the cAMP response for the -254 StAR promoter construct. Thus, the double mutation has a greater effect than the single GATA mutation on the cAMP response. Therefore, we examined the possibility that either the SF1–3 or GATA-4 site acts functionally with the AP-1 site to promote the cAMP response (Fig. 6BGo). Double mutations in the SF-1 and AP-1 elements (SF1–3/AP-1) or the GATA-4 and AP-1 (GATA/AP-1) elements did not result in any greater effect on StAR promoter activity compared with an additive effect of the single mutations alone. The basal activities of SF1–3mut/AP-1mut and GATAmut/AP-1mut were 16% (AP-1 plus SF-1) and 23% (SF-1 plus GATA) that of the -254 StAR promoter construct, respectively, whereas the (Bu)2cAMP-dependent increases were 90% (SF1–3 effect) and 64% (GATA-4 effect) that of the -254 StAR. Thus, it appears that mutation of the AP-1 element has the single greatest effect on StAR promoter activity, due mainly to decreased basal expression, whereas the SF1–3 and GATA-4 double mutations had the most severe effect on promoter function. One possibility is that SF1–3 and GATA-4 function to stabilize a common factor(s) in the CAN (-87/-64) region. To test this proposal, we examined the effects of abolishing one DNA-binding site on protein-DNA interactions at other sites within the -105/-44 bp of StAR promoter.

Protein-DNA interactions within the cAMP-responsive regions of the StAR promoter
Four major protein-DNA complexes (I–IV) were detected by EMSA analysis using a DNA probe that spans the -105 to -44 region (-105/-44) of the StAR promoter (Fig. 7AGo). Complex formation appeared to be independent of cAMP treatment in that similar complexes were formed with nuclear extracts from both control and (Bu)2cAMP-treated MA-10 cells. Antibodies to SF-1, GATA-4, and C/EBPß were used to help identify the presence of these proteins in the four complexes (Fig. 7BGo). The SF-1 antibody abolished complex I and diminished complex III (lane 2), whereas GATA-4 antibodies caused the appearance of a supershifted complex concomitant with diminished complex II (lane 3). C/EBPß antibodies had no effect on protein-DNA interactions within this region of the StAR promoter (lane 4). Cold competition experiments with the oligonucleotides corresponding to the SF1–3 (-105/-44), CAN (-87/-64), and GATA (-68/-44) regions of the promoter were performed to verify the contribution of each site to complex formation. First, cold competition with unlabeled -105/-44 probe greatly diminished complex formation (lane 5). The SF1–3 probe eliminated complex I and diminished complexes III and IV without affecting complex II formation (lane 6), whereas the GATA probe competed for complex II formation without affecting complex I, III, or IV (lane 9). The CAN probe eliminated complex IV (lane 7), whereas the consensus C/EBPß oligonucleotide (lane 8) had no effect on the protein-DNA interactions with the StAR promoter.



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  		Figure 7. The SF1–3- and GATA-4-binding sites are essential for the formation of multiple complexes within the -105/-44 region of StAR. EMSA was used to examine the binding of MA-10 nuclear extract to the radiolabeled -105/-44 region of the StAR promoter. A, Concentration-dependent protein-DNA complex formation. Fifty femtomoles of radiolabeled probe (-105/-44) were incubated with 5, 10, or 15 µg nuclear extracts from untreated (control; lanes 1–3) or (Bu)2cAMP-treated [(Bu)2; lanes 4–6] MA-10 cells. The arrows indicate the positions of four protein-DNA complexes, denoted I–IV. Free probe is not shown on this gel. B, Cold competition or antibody interference analysis. SF-1, GATA-4, or C/EBPß antibody (4 µg) or the indicated cold competitor probes were incubated with 10 µg nuclear extracts [(Bu)2cAMP-treated]; then the radiolabeled -105/-44 probe was added, and the incubation was continued for 30 min on ice. The protein-DNA complexes were resolved on a nondenaturing 4% polyacrylamide gel, the gel was dried, and the complexes were visualized by autoradiography. The cold competitors were -105/-44, SF1–3 (-105/-83), CAN (-87/-64 region), GATA (-68/-44 region), and consensus C/EBP. Arrows indicate complexes I–IV, and the open arrowhead indicates the GATA-4 supershifted complex (SS). C and D, Effects of mutation of an individual element (C) or two elements (D) on protein-DNA interactions. The sequences for the mutations that were introduced into the -105/44 probe are given in Table 2B. The radiolabeled probes used in the binding reaction are indicated: -105/-44, SF1–3 mut, GATA mut, and AP-1 mut, or their combination (D). Experiments in the presence of antibodies or cold competitor CAN probe were performed as described in B.

 
To further assess complex composition, a series of complementary experiments was performed in which the same sequence mutations used in the functional studies were introduced into the -105/-44 probe, and the effects on protein-DNA interactions were determined (Fig. 7CGo). Complex I was abolished, and complexes III and IV were diminished by mutation of the SF1–3 site (SF1–3mut, lane 2), which is consistent with the results of the SF-1 antibody and cold competition experiments. Complex II was not affected by the SF1–3 mutation, and addition of the GATA-4 antibody caused a supershift of the remaining complex II (lane 3). Conversely, mutation of the GATA-4 site eliminated complex II formation (GATAmut, lane 4), and the remaining complexes (I, III, and IV) were greatly diminished by SF-1 antibodies (lane 5). The CAN probe was an effective competitor for complexes III and IV that remained with the GATAmut probe (lane 6) probe, but did not further reduce complex formation with the AP-1mut probe (lane 7), suggesting that factors binding to the CAN region contribute to formation of these complexes. Indeed, mutation of the AP-1-like site eliminated complex IV and surprisingly also reduced all other protein-DNA interactions (lane 10). The remaining complexes I and III were eliminated by the addition of SF-1 antibodies (lane 9), whereas GATA-4 antibodies resulted in a supershift of complex II (lanes 8).

As StAR basal promoter activity was greatly reduced by mutations within two of the three SF1–3, CAN, or GATA elements (Fig. 6BGo), we also tested the effects of these mutations on protein-DNA interactions (Fig. 7DGo). A double mutation in SF-1 and GATA (SF1–3/GATA mut) resulted in the loss of all complex formation (Fig. 7DGo, lane 2), whereas complex II was still present with the probe containing a double mutation in SF1–3 and AP-1 (SF1–3/AP-1 mut, lane 3). Double mutations of GATA and AP-1 caused a marked decrease in protein-DNA interactions for complexes III, and IV and eliminated complex II (lane 4). These data are consistent with an additive effect of the single mutations on protein-DNA interactions, with the exception that complex I did not appear affected by the GATAmut/AP-1mut probe. This was not an expected result based on the decrease in complex I with the AP-1 mut probe (Fig. 7CGo, compare lanes 10 and 11). These results indicate that complexes III and IV are dependent upon SF-1 and possibly, to a lesser extent, GATA-4.

In sum, the individual mutations in the SF1–3 or AP-1-like regions and the SF-1 antibodies do not affect GATA-4 binding; therefore, we conclude that GATA-4 binding within the -105/-44 region is independent of other factors. Further, the GATA-4 mutation does not prevent SF-1-dependent complex formation (I, III, and IV), indicating that SF-1 binding is not dependent on GATA-4 binding. CAN factors appear to contribute to complex IV formation, as mutation of the AP-1-like element or cold competition with the CAN region eliminated this complex. However, protein binding within the CAN region in the context of the longer probe appears to be dependent on SF-1 binding to SF1–3 and may be influenced by GATA-4 binding. Conversely, an intact AP-1 element appears to be required for efficient SF-1 binding, indicating that protein binding to SF1–3 and CAN regions may be interdependent. Together, these data clearly demonstrate that complex I is due to SF-1 binding, and complex II is due to GATA-4 binding. The data are consistent with complexes III and IV being dependent upon SF-1, with complex III most likely containing SF-1, whereas CAN factor binding contributes to complex IV.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies have identified two regulatory regions of the StAR promoter: a negative region located between -254/-150, and a cAMP-responsive region located between -105/-60. Putative negative regulatory regions were previously identified for the rat and porcine promoters as well (21, 39). Our promoter deletion analysis has eliminated SF-1 (SF-1–1) and C/EBPß binding at -135 and -113 as essential for the cAMP response of the StAR gene and has pointed to SF1–3 (-95), CAN-binding protein(s) (-87/-68), and GATA-4 (-68) as potential factors.

Identification of the -105 to -95 region as a SF-1-binding site was an expected result, as this element was previously shown to bind SF-1 in the human and rat StAR promoters (21, 37). However, these are the first studies to show a distinction between regulations of the human and mouse StAR promoter by cAMP in steroidogenic cell lines. Mutation of the SF-1 site (-95) in the human promoter demonstrated 90% and 80% decreases in basal and forskolin-stimulated promoter activity in human granulosa-lutein cells (37). Our recent studies in the human H295R adrenocortical cells also have shown that the SF-1 element at -95 is required for cAMP and angiotensin II induction of human StAR promoter-luciferase reporter gene expression (23). Transient transfection of COS-1 cells with the rat StAR promoter confirmed a SF-1-dependent activation of the StAR promoter that required the SF-1 site at -95 and that mutation of this site resulted in a decrease in the cAMP response that was due to lowered basal promoter activity (21). Our results demonstrate that the mouse and rat promoters are functionally more similar to each other than to the human StAR promoter. This apparent promoter-specific difference may be reflective of a divergence within the StAR promoter sequences between the human and the mouse. The human sequence has an additional five bases downstream of the SF-1 site and does not contain the CAAT box, the the AP-1-like site, or the nuclear receptor half-site that have been shown to be important for the murine promoter in MA-10 cells (26, 38). Thus, it is apparent that SF-1 is important in StAR gene regulation but is not sufficient for the cAMP-dependent response in MA-10 cells. Possibly, the sequence divergence reflects greater interactions of SF-1 with other factors binding in this region of the mouse StAR promoter.

We have confirmed the functional importance of the AP-1-like element within the -87/-68 (CAN) region of mouse StAR promoter for basal transcriptional activity (26, 38). In addition, this study provides the first evidence that (Bu)2cAMP treatment of MA-10 cells results in the formation of a unique protein-DNA complex at this region of the StAR promoter (complex B). Identification of the CAN factor(s) should provide an important link to elucidating the mechanism of action for cAMP-dependent regulation of StAR. Presently, our data suggest that multiple elements may contribute to activation of the StAR promoter. Double mutations in either the SF1–3 and AP-1 or GATA and AP-1 elements have a greater effect on basal promoter activity that represents the combined effects of the single mutations. The GATAmut/AP-1 mut also resulted in decreased cAMP response that is attributed to the GATA element. On the other hand, the effect of the SF1–3 and GATA double mutation on basal promoter activity was greater than the combined effects of the single mutations. As mutations that disrupted the AP-1-like site (AP-1mut, SF1–3mut/AP-1mut, and GATAmut/AP-1mut) have similar effects on basal activity as the SF1–3mut/GATAmut, these data suggest that all three elements work together to promote full basal activity of the StAR promoter that, in turn, is required for a maximal cAMP response. What is not clear at the time is why the cAMP-dependent response, although reduced, remains intact despite apparent elimination of protein-DNA binding interactions within the -105/-44 region of StAR promoter with the SF1–3mut/GATAmut. As we did not observe a cAMP-dependent complex with the -105/-44 probe, the cAMP-dependent CAN-binding factor(s) may not have been detected in this analysis. Indeed, the cAMP-dependent complex was observed only with increased concentrations of nuclear extract, suggesting that this factor(s) may be expressed in lesser abundance. Thus, it is possible that the AP-1 region is still functional in the SF1–3mut/GATAmut double mutation.

Previously, we demonstrated that a protein(s) in MA-10 nuclear extracts binds to the CAAT box located in the -93/-71 region, but with lower affinity compared with the C/EBPß site located at -113 (26). Antibody supershift EMSA experiments showed that C/EBPß binds to the -113 site, but binding to the proximal site was not tested. We now have directly tested C/EBPß binding to the CAN region and verify that C/EBPß does not bind to this region of the StAR promoter. It is possible that our previous studies did not detect the factor(s) we now observe because the probe used previously (-93/-71) did not contain the entire CAN region (-87/-64) binding sites, which may have affected protein binding to this region. Indeed, complexes A and B appear to be dependent upon an intact AP-1-like element and the flanking TGATGA sequence. Functionally, mutation of the putative AP-1 site resulted in approximately a 63% decrease in the basal promoter activity of StAR without affecting the cAMP response, a result very similar to the effects seen by mutating the CAAT box at -87 (26). Therefore, mutation of the CAAT box most likely affects a factor(s) binding to the AP-1-like element in the CAN region or vice versa.

Our results in MA-10 cells are distinct from the recent report of murine StAR promoter activity in rat ovarian cells, which demonstrated that C/EBPß is a prominent component in a DNA-protein complex formed with the -87/-64 region (38). Interestingly, the binding site recognized by C/EBPß in ovarian cell extracts is not the CAAT box core, but is the AP-1-like element. One possibility for this apparent tissue-specific difference in C/EBPß binding is that different factors bind to the CAN region. This tissue-specific difference may reflect the cycloheximide sensitivity of the cAMP response for StAR. Previously, StAR gene expression was reported to be mediated by a cycloheximide-sensitive mechanism in human granulosa-lutein cells (40). C/EBPß expression has been shown to be increased by FSH in ovarian granulosa cells and by (Bu)2cAMP in MA-10 cells (25, 38). Subsequent to submission of this paper, it was reported that C/EBPß is induced in human granulosa cells by 8-bromo-cAMP (41). The data supported C/EBPß in StAR basal promoter activity as well as the proposal that the cycloheximide-sensitive, cAMP-dependent response may be linked to the induction of C/EBPß. Thus, it is probable that a SF-1/C/EBPß or a GATA-4/C/EBPß interaction mediates the observed cycloheximide-sensitive response in rat ovarian granulosa cells. Consistent with this proposal, C/EBPß and SF-1 were shown to interact directly in a GST pull-down assay (26). However, StAR gene expression in MA-10 cells and Y1 mouse adrenocortical cells is not dependent upon de novo protein synthesis (10). Therefore, another factor(s) that is insensitive to cycloheximide may be used in MA-10 Leydig cells and Y1 cells to bind to the CAN region and promote maximal basal activation of StAR. A difference in tissue-specific transcription factor expression may thus account for differences in the acute regulation of StAR in granulosa vs. Leydig cell cultures.

We identified GATA-4 as the factor that binds to the highly conserved -68/-44 region of the mouse StAR promoter. Functionally, GATA-4 contributes to 20% of the cAMP response. Independently, GATA-4 was recently reported to bind to the same region and was shown to be required for maximal cAMP response in rat granulosa cells (38). Mutation of both the GATA-4 and AP-1-like elements decreased StAR basal promoter activity 97% and dramatically decreased the FSH-dependent increase in reporter gene expression in luteal-granulosa cells. In contrast, we demonstrate that a similar double mutation (GATAmut/AP1mut) had no greater effect on StAR promoter activity in MA-10 cells than the combined effects of the individual mutations within these elements. Again, this discrepancy between our results and those reported for rat granulosa cells indicates that the murine StAR gene may be regulated in a tissue-specific manner. In MA-10 cells, we propose the GATA-4 transcription factor has a role in stabilizing the factor(s) bound to the putative AP-1 site and that GATA-4 and SF-1 work together to stabilize this common factor(s). However, these putative CAN-binding proteins and SF-1 can function in the absence of GATA-4.

In sum, SF-1 binding is important for the basal activity of the StAR gene, most likely by affecting the interactions among the other proteins within the CAN region of the promoter. GATA-4 binding is independent of SF-1 binding and has an apparently minimal effect on SF-1- and CAN-protein binding, but may influence factor binding. Interaction between SF-1 and GATA-4 has been shown for regulation of the Müllerian-inhibiting substance gene; therefore, one model for SF-1 and GATA-4 is that they interact and function to stabilize binding of the CAN-binding factor(s) (42). Alternatively, it is possible that there is a redundancy of function for SF-1, CAN-binding protein, and GATA-4. This proposal would be consistent with our mutational analysis that shows that elimination of one binding factor does not compromise the cAMP response of the StAR promoter. Thus, each of the DNA-binding factors may work together to stabilize the unknown cAMP-mediated transcription factors and/or coactivators, but each factor alone may be sufficient to mediate part of the response.


   Acknowledgments
 
We thank Dr. Keith Parker, University of Texas Southwestern Medical Center (Dallas, TX), for providing the GST-SF-1 expression plasmid. We also thank Drs. Carolyn M. Klinge and Keith C. Falkner, Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine (Louisville, KY), for their critical review of this manuscript. Finally, we thank Ms. Rebecca Combs for her excellent technical assistance.


   Footnotes
 
1 This work was supported by NIH Grant DK-51656. Back

Received August 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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