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Biology of Reproduction 64, 1191-1199 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Nuclear Receptor Dax-1 Represses the Transcriptional Cooperation Between GATA-4 and SF-1 in Sertoli Cells1

Jacques J. Tremblay a, and Robert S. Viger2,a

a Ontogeny and Reproduction Research Unit, CHUL Research Centre and Centre for Research in Biology of Reproduction, Department of Obstetrics and Gynecology, Laval University, Ste-Foy, Québec, Canada G1V 4G2

ABSTRACT

A crucial step in mammalian sex differentiation is the regression of the Müllerian ducts in males. This is achieved through the action of Müllerian inhibiting substance (MIS), a key hormone produced by fetal Sertoli cells. Proper spatiotemporal expression of the MIS gene requires the concerted action of several transcription factors that include Sox9, SF-1, WT-1, GATA-4, and Dax-1. Indeed, SF-1 contributes to MIS gene expression by transcriptionally cooperating with other factors such as GATA-4 and WT-1. Dax-1 is coexpressed with SF-1 in many tissues, including the gonads, where it acts as a negative modulator of SF-1-dependent transcription. We now report that Dax-1 can repress MIS transcription in Sertoli cells by disrupting transcriptional synergism between GATA-4 and SF-1. Dax-1-mediated repression of GATA-4/SF-1 synergism did not involve direct repression of GATA-dependent transactivation, but rather, it occurred through a direct protein-protein interaction with DNA-bound SF-1. It is interesting that SF-1, Dax-1, and GATA factors are coexpressed in several tissues such as the pituitary, the adrenals, and the gonads. Because we have shown that other GATA family members also have the ability to synergize with SF-1, Dax-1 repression of GATA/SF-1 synergism may represent an important mechanism for fine-tuning the regulation of SF-1-dependent genes in multiple target tissues.

development, gene regulation, Müllerian ducts, Sertoli cells

INTRODUCTION

Mammalian sexual development is characterized by three distinct, sequential events: the establishment of chromosomal sex at fertilization (XY or XX), gonadal development and differentiation (testis or ovary), and finally, the acquisition of the proper sexual phenotype (male or female). Several transcription factors are known to have crucial roles in gonadal development and sex determination and differentiation: the testis determining factor, Sry; the Sry-related factor, Sox9; steroidogenic factor 1, SF-1; Wilms' tumor-1, WT-1; the LIM-homeobox factor, Lhx9; and the nuclear receptor, Dax-1 [1–3]. In addition to these factors, the list of genes proposed to be implicated in these fundamental developmental processes continues to grow. Examples of the latter include the double-sex and mab-3-related transcription factor 1 (Dmrt-1) and the zinc finger factor GATA-4 [4–7].

The X-linked Dax-1 gene encodes an atypical member of the nuclear receptor superfamily in which the characteristic N-terminal zinc finger region required for DNA binding is absent and is replaced by a stretch of 3.5 repeats of a 65–67 amino acid motif [8]. Dax-1 is expressed in several endocrine tissues [9], including the gonads, where it is present in Sertoli and Leydig cells of the testis and granulosa and theca cells of the ovary [9–11]. Dax-1 plays a critical role in adrenal development and gonadal function; it was originally identified as the causative gene for adrenal hypoplasia congenita (AHC), which is associated with hypogonadotropic hypogonadism (HHG), a recessive disorder affecting males [9, 10, 12, 13]. Indeed, several groups have characterized numerous Dax-1 mutations (nucleotide substitutions, insertions, and deletions) in individuals affected with AHC and HHG [14–24]. Females carrying heterozygous mutations in the Dax-1 gene are normal and there has been no report of homozygous females because males needed to transmit the nonfunctional allele are infertile. Because Dax-1 gene expression is rapidly down-regulated in the developing male gonad immediately after overt testis differentiation in the mouse [10], the Dax-1 gene was initially believed to be an important ovarian determinant. However, in Dax-1-deficient mice, all females were characterized as phenotypically normal [25]. It is interesting that all male Dax-1-deficient mice were hypogonadal despite normal gonadotropin levels, suggesting a primary testicular defect [25]. Indeed, male Dax-1-deficient mice exhibited a complete loss of germ cells by 14 wk, resulting in sterility [25]. Thus, Dax-1 is apparently not required for ovarian differentiation, but rather is essential for the maintenance of the integrity of the seminiferous epithelium of the testis [25]. In addition to Dax-1 mutations, another human disorder has been associated with the Dax-1 gene. In this case, duplication of a small region on the X chromosome that encompasses the Dax-1 gene leads to dosage-sensitive sex reversal in XY males [26]. In certain situations, overexpression of Dax-1 in vivo can result in sex reversal by antagonizing the action of Sry [27]. Thus, adequate Dax-1 levels are required for normal testis function, but too much Dax-1 appears to have an "anti-testis" effect.

At a transcriptional level, Dax-1 was shown to block steroidogenesis at multiple levels by acting as a potent repressor. Dax-1 transcriptional repression can occur through two different mechanisms. First, Dax-1 has been shown to bind DNA hairpin structures found in its promoter, as well as in the promoter of other target genes, to repress transcription [28]. Second, Dax-1 directly interacts, through its N-terminal region, with the orphan nuclear receptor, SF-1, leading to a repression of SF-1-dependent transcription [29–31]. In fact, Dax-1 serves as an adaptor molecule because it recruits via its C-terminal domain, the nuclear receptor corepressors, N-CoR and Alien [29, 32]. It is interesting that this Dax-1 C-terminal repression domain is absent or mutated in patients with AHC and, consequently, Dax-1 can no longer recruit corepressors [31, 33]. Thus, transcriptional silencing by Dax-1 appears to be essential for normal steroidogenic function.

GATA-4 is a member of a new class of transcriptional regulators (called the GATA factors), which have recently been shown to be expressed in the gonads [4, 34–36]. Six vertebrate GATA factors (GATA-1 to GATA-6) have been identified [37]. They act as transcriptional activators by binding to consensus GATA motifs [(A/T)GATA(A/G)] found in the promoter region of numerous target genes. Complementary in vitro and in vivo approaches have established that these factors play essential functions in cell differentiation, organ morphogenesis, and tissue-specific gene expression [37–46]. Three GATA factors are expressed in the gonads: GATA-1 [4, 36], GATA-4 [4, 35], and GATA-6 [34]. GATA-1 and GATA-4 are both found in Sertoli cells but at different stages of development; GATA-1 is primarily expressed in postnatal Sertoli cells [4, 36], whereas GATA-4 is abundantly expressed in Sertoli cells from the onset of gonadal development until shortly after birth [4]. The GATA-4 expression pattern strikingly correlates with that of MIS, a crucial Sertoli cell hormone required for Müllerian duct regression, and hence, proper male sex differentiation. Indeed, we have identified the MIS promoter as the first known target for GATA-4 in fetal Sertoli cells [4]. GATA-4 can activate the MIS promoter on its own but also physically interacts, via its zinc finger domain, with the orphan nuclear receptor, SF-1, to synergistically activate MIS transcription [5]. GATA factors are also expressed in the mouse ovary [4, 34]; target genes, however, remain to be identified.

Although GATA-4, SF-1, and Dax-1 are all coexpressed in Sertoli cells from the earliest stages of gonadal development, the combinatorial effect of these factors on the activity of gonadal target promoters has not been assessed.

MATERIALS AND METHODS

Plasmids

The -180 base pair [bp], -107 bp, -83 bp, and -65 bp murine MIS-luciferase promoter constructs were obtained by polymerase chain reaction (PCR) on mouse genomic DNA as previously described [5]. The -180mut construct, which was obtained by site-directed mutagenesis, contains a mutation (GATA->GGTA) in the consensus MIS promoter GATA element at -75 bp. Shorter versions of this construct, -107mut and -83mut, were obtained by PCR using the -180mut plasmid as a template. The 3x(SF-1:GATA)-MIS luciferase reporter contains three copies of the MIS SF-1 and GATA elements in their natural context, cloned upstream of the minimal MIS promoter (MISmin) [5]. The minimal POMC (POMCmin), 3xSF-1-POMCmin, and -142 bp LHß luciferase reporters were kindly provided by Jacques Drouin [47–49]. In the 3xSF-1-POMCmin construct, SF-1 refers to the SF-1 binding element found in the bovine LHß promoter [47]. The 2xGATA:3xSF-1-POMCmin promoter construct was obtained by cloning two consensus GATA elements upstream of the 3xSF-1-POMCmin reporter. The 2xGATA-POMCmin luciferase reporter was generated by cloning two copies of the MIS GATA element (sense oligonucleotide, 5'-GATCCTGGTGTTGATAGGGGCGTA-3'; antisense oligonucleotide, 5'-GATCTACGCCCCTATCAACACCAG-3') upstream of the minimal POMC promoter. Similarly, the 2xGATA-MISmin reporter was obtained by cloning two copies of the above-mentioned double-stranded oligonucleotide in front of the minimal MIS promoter. The 2xSF-1-MISmin reporter, which contains two copies of the MIS SF-1 element cloned upstream of the minimal MIS promoter, has been previously described [5]. The 2xGATA:2xSF-1-MISmin promoter construct was made by cloning two copies of a consensus GATA element upstream of the 2xSF-1-MISmin reporter. An expression plasmid for GATA-4 was obtained by cloning a PCR fragment, corresponding to the GATA-4 open reading frame into the HindIII/XbaI sites of the Cytomegalovirus (CMV)-driven expression vector, pcDNA3 (Invitrogen, Carlsbad, CA). The PCR fragment was amplified using first-strand cDNA prepared from neonate testis RNA as a template for the PCR reaction (forward primer, 5'-TCAAGCTTCGAAGCTCAGAGCTTGGGGCG-3'; reverse primer, 5'-TATCTAGACCGAGCAGGAATTTGAAGAGGGAA-3'). A mouse Dax-1 expression plasmid was generated by PCR (forward oligo, 5'-GATCTAGACGAGGAGCCTCAGGCCATG-3'; reverse oligo, 5'-CAGGTACCTTCACAGCTTTGCACAGAG-3') using first-strand cDNA prepared from {alpha}T3-1 cells. The {alpha}T3-1 cell line was kindly provided by Pamela Mellon [50]. An expression plasmid encoding the truncated Dax-1 protein ({Delta}Dax-1), which was deleted of its N-terminal region ({Delta}aa1-253), was also produced by PCR on the full-length Dax-1 cDNA using the same 3' oligo described above but with a different 5' oligo (5'-CATCTAGACAGGTGGTGTGCGAGGCAGCG-3'). All of our PCR-amplified expression vectors and promoter constructs were verified by sequencing. The SF-1 expression plasmid was kindly provided by Keith Parker [51].

Isolation of Immature Sertoli Cells

Neonate Sertoli cells were prepared from 3- to 5-day-old Sprague-Dawley rats (Charles River Canada, St-Constant, PQ) using established procedures as outlined by Tung and Fritz [52]. Final Sertoli cell aggregates were resuspended in Eagle minimal essential medium (MEM) containing 10% fetal bovine serum (FBS). The cells were subsequently seeded at high density (2 x 105 cells) in 24-well plastic dishes and cultured at 32°C under 5% CO2. The next morning, cells were rinsed with PBS to remove contaminating germ cells and then fresh media was added to the cells.

Cell Culture and Transfections

African green monkey kidney CV-1 cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% newborn calf serum. Human choriocarcinoma JEG-3 cells were maintained in DMEM containing 10% FBS. All transfections were done in 24-well plates using the calcium phosphate precipitation method [53]. The day prior to transfection, CV-1, JEG-3, and neonate primary Sertoli cells were plated at densities of 2.2 x 104, 4 x 104, and 2 x 105, respectively. Cells were transfected 24 h after the initial plating. Culture media was changed 12–16 h after transfection and the cells were finally harvested the following morning (40–44 h after transfection). Cells were lysed by adding 50 µl of lysis buffer (100 mM Tris-HCl pH 7.9, 0.5% Igepal [Sigma-Aldrich Canada, Oakville, ON], and 5 mM dithiothreitol) directly to the wells. An aliquot of the lysate was then assayed for luciferase activity using an EG&G Berthold LB 9507 luminometer and luciferine (BD Pharmingen, San Diego, CA) as substrate. In all experiments, the total amount of DNA was kept constant at 1.5 µg per well using Sp64 (Promega, Madison, WI) as carrier DNA; several DNA preparations were used to ensure reproducibility of the results. Transfection efficiencies were monitored by cotransfection with a control ß-galactosidase expression plasmid. Data reported represent the average of at least three experiments, each done in duplicate.

Production of MBP Fusion Proteins and In Vitro Protein-Protein Interaction Assays

A recombinant maltose-binding protein (MBP)-Dax-1 fusion protein was obtained by cloning the Dax-1 coding region in frame with MBP using the commercially available pMAL-c fusion protein vector (New England Biolabs, Mississauga, ON, Canada). The MBP-LacZ{alpha} fusion protein was produced by the pMAL-c vector without any cloned insert. The fusion proteins were produced and purified as previously described [5]. In vitro protein-protein interaction studies were performed as previously outlined [5].

Statistical Analysis

With the exception of Figure 5A, statistical analyses were done by one-way ANOVA followed by a Newman-Keuls test to detect significant differences between groups. For the SF-1-dependent activation of multiple promoters in JEG-3 cells (Fig. 5A), significant differences between the control and SF-1 activation groups were determined using Student t-test. For all statistical analyses, P < 0.05 was considered significant.



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  		FIG. 5. Dax-1 represses SF-1-dependent transcription in JEG-3 cells. A) SF-1 is a potent transactivator in GATA-expressing JEG-3 cells. An expression vector encoding SF-1 was transfected in JEG-3 cells (left panel) and in non-GATA expressing CV-1 cells (right panel) along with 500 ng of several different SF-1-dependent luciferase reporters. Open bars, control (empty) plasmid; black bars, 50 ng SF-1 alone. *Significantly different from control (no SF-1; P < 0.05) B) Expression plasmids encoding SF-1 and Dax-1 were cotransfected in JEG-3 cells along with either a reporter consisting of three copies of the MIS SF-1 and GATA elements in their natural context, cloned upstream of the minimal MIS promoter (left panel) or another one consisting of two consensus GATA elements and three SF-1 binding sites fused to the minimal POMC promoter (right panel). Open bars, control (empty) plasmid; hatched bars, increasing doses (5, 20, and 100 ng) of Dax-1 alone; black bars, 50 ng SF-1 alone; stippled bars, increasing doses of Dax-1 (5, 20, and 100 ng) in the presence of 50 ng of SF-1. All results are shown as fold activation relative to control (± SEM). The different promoter constructs are described in Materials and Methods. *Significantly different from SF-1 alone (black bar). ***Significantly different from SF-1 alone and SF-1 in the presence of all Dax-1 doses (P < 0.05)

RESULTS

Dax-1 Specifically Represses SF-1- but Not GATA-4-Dependent Transactivation

Dax-1 represses SF-1-mediated transcription of promoters by acting as a bridging molecule between SF-1 and nuclear corepressors [29, 32]. Before determining the role of Dax-1 in modulating the transcriptional cooperation between GATA-4 and SF-1, we first tested the effect of Dax-1 on GATA-4-dependent transcription in heterologous CV-1 cells. As expected, Dax-1 efficiently repressed the transactivation induced by SF-1 on two separate SF-1 target promoters, the LHß promoter in its natural context (Fig. 1A, left panel), and a synthetic reporter composed of two copies of a consensus GATA element and three copies of an SF-1 element fused to the minimal pro-opiomelanocortin (POMC) promoter (Fig. 1A, right panel). The repressive effect of Dax-1 was strictly dependent on the presence of SF-1 because Dax-1 alone had no effect on the SF-1-dependent promoters (hatched bars in Fig. 1A). As shown in Figure 1B, Dax-1 did not affect GATA-4-dependent transactivation of two separate GATA-responsive reporters. This suggested that unlike SF-1, Dax-1 does not physically interact with GATA-4. To test this hypothesis, we performed an in vitro protein-protein interaction analysis between Dax-1 and GATA-4. Consistent with the fact that Dax-1 and SF-1 physically interact [29–31], an in vitro synthesized, radiolabeled SF-1 protein was specifically retained on agarose beads coated with an MBP-Dax-1 fusion protein (Fig. 2). The interaction was specific for the Dax-1 portion of the fusion protein because no labeled SF-1 was retained with a control MBP-LacZ{alpha} fusion protein. Similar experiments performed using a labeled GATA-4 protein revealed that GATA-4 and Dax-1 do not directly interact (Fig. 2, right panel). These results taken together indicate that transcriptional repression by Dax-1 requires a direct interaction between Dax-1 and its interacting partner, SF-1.



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  		FIG. 1. Dax-1 represses SF-1 but not GATA-dependent transcription. A and B) Effect of Dax-1 on SF-1 and GATA-dependent reporters. Expression plasmids encoding GATA-4, SF-1, and Dax-1 were transfected in CV-1 cells along with 500 ng of different SF-1 and GATA-responsive promoter constructs. Open bars, control (empty) expression plasmid; hatched bars, increasing doses (5, 20, and 100 ng) of Dax-1 alone; black bars, 50 ng of GATA-4 or 10 ng of SF-1 alone; stippled bars, effect of increasing doses of Dax-1 (5, 20, and 100 ng) in the presence of either 50 ng of GATA-4 or 10 ng of SF-1. Promoter activities are expressed as fold activation relative to control (± SEM). The different promoter constructs are described in Materials and Methods. *Significantly different from 10 ng SF-1 alone (black bar). **Significantly different from 10 ng SF-1 alone and 10 ng SF-1 in the presence of the first Dax-1 dose. ***Significantly different from 50 ng GATA-4 in the presence of the second (20 ng) Dax-1 dose (P < 0.05)



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  		FIG. 2. Dax-1 does not physically interact with GATA-4. In vitro pull-down assays were performed using immobilized, bacterially produced MBP fusion proteins (MBP-Dax-1 or MBP-LacZ{alpha} as control) and either in vitro translated 35S-labeled SF-1 or GATA-4 proteins. After extensive washes, bound proteins were separated on a 12% SDS-PAGE gel and subsequently visualized by autoradiography

Dax-1 Represses GATA/SF-1 Synergism in Heterologous Cells

We previously reported that GATA factors can enhance SF-1-dependent transcription through a synergistic interaction with SF-1 [5]. Because Dax-1 is coexpressed with GATA-4 and SF-1 in multiple gonadal cell types, Dax-1 may also be an important modulator of GATA/SF-1 synergism. The effect of increasing doses of Dax-1 on GATA-4/SF-1 synergism was first tested in heterologous CV-1 cells (Fig. 3). Dax-1 efficiently repressed GATA-4/SF-1 synergism on multiple SF-1 target promoters. They included the natural MIS promoter, which contains both GATA and SF-1 binding sites (Fig. 3, upper left panel), two synthetic reporters consisting of multimerized GATA and SF-1 elements in different positions and fused to different minimal promoters (Fig. 3, upper right and lower left panels), and a synthetic reporter containing only SF-1 binding sites (Fig. 3, lower right panel). Because Dax-1 represses GATA-4/SF-1 transcriptional synergism, Dax-1 must still interact with SF-1, despite the GATA-4/SF-1 interaction and regardless of whether or not GATA-4 is bound to DNA. Dax-1 repression is mediated either by the corepressor associated with Dax-1 or via a disruption of the GATA-4/SF-1 protein-protein interaction. To assess the importance of a Dax-1/SF-1 interaction for the repression of GATA-4/SF-1 synergism, we generated a Dax-1 mutant ({Delta}Dax-1) that was deleted of its entire N-terminal domain, the region previously shown to interact with SF-1 [29–31]. As shown in Figure 4, the Dax-1 mutant no longer repressed GATA-4/SF-1 synergism, confirming that a direct interaction between Dax-1 and SF-1 is required to blunt the transcriptional cooperation between GATA-4 and SF-1.



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  		FIG. 3. Dax-1 represses GATA-4/SF-1 synergism on different natural and synthetic promoter constructs. Expression plasmids encoding GATA-4, SF-1, and Dax-1 were transfected in CV-1 cells along with 500 ng of different promoter constructs containing both GATA and SF-1 binding sites (upper panels and lower left panel) or only SF-1 binding sites (bottom right panel). Open bars, control (empty) expression plasmid; gray bars, 50 ng GATA-4 alone; hatched bars, 10 ng SF-1 alone; black bars, 50 ng GATA-4 and 10 ng SF-1 used in combination; stippled bars, effect of increasing doses of Dax-1 (5, 20, and 100 ng) in the presence of 50 ng GATA-4 and 10 ng SF-1 (GATA-4/SF-1 synergy). All data are reported as fold activation relative to control (± SEM). The different promoter constructs are described in Materials and Methods. *Significantly different from 50 ng GATA-4 in combination with 10 ng SF-1 (GATA-4/SF-1 synergy, black bar). **Significantly different from GATA-4/SF-1 synergy and GATA-4/SF-1 synergy in the presence of the first (5 ng) Dax-1 dose. ***Significantly different from GATA-4/SF-1 synergy and GATA-4/SF-1 synergy in the presence of all doses of Dax-1 (P < 0.05)



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  		FIG. 4. Dax-1 must interact with SF-1 to repress GATA-4/SF-1 synergism. Effect of a truncated Dax-1 protein ({Delta}Dax-1), which removes the SF-1 interaction domain, on GATA-4/SF-1 synergism. Expression plasmids encoding GATA-4, SF-1, Dax-1, and {Delta}Dax-1 were transfected in CV-1 cells along with 500 ng of an SF-1-dependent luciferase reporter. Open bar, control (empty) expression plasmid; gray bar, 50 ng GATA-4 alone; hatched bar, 10 ng SF-1 alone; black bar, 50 ng GATA-4 and 10 ng SF-1 used in combination; stippled bars, effect of increasing doses of Dax-1 (5, 20, and 100 ng) in the presence of 50 ng GATA-4 and 10 ng SF-1 (GATA-4/SF-1 synergy); heavy stippled bars, effect of increasing doses of {Delta}Dax-1 (5, 20, and 100 ng) in the presence of 50 ng GATA-4 and 10 ng SF-1 (GATA-4/SF-1 synergy). All data are reported as fold activation relative to control (± SEM). *Significantly different from GATA-4/SF-1 synergy (black bar). **Significantly different from GATA-4/SF-1 synergy and GATA-4/SF-1 synergy in the presence of the first (5 ng) Dax-1 dose (P < 0.05)

The effect of Dax-1 was subsequently tested in human choriocarcinoma JEG-3 cells. Because this cell line endogenously expresses GATA factors [54], transfection of SF-1 alone leads to a potent GATA/SF-1 synergistic activation of multiple SF-1 target promoters when compared with similar transfections done in CV-1 cells, which are devoid of GATA activity (Fig. 5A). In these cells, Dax-1 was also found to repress SF-1-dependent transactivation/synergism (Fig. 5B). The Dax-1-mediated repression was specific because Dax-1 alone had no effect on promoter activity (hatched bars in Fig. 5B). Thus these results demonstrate that Dax-1 can also repress synergism between exogenously expressed SF-1 and endogenous GATA factors.

Dax-1 Blunts the Transcriptional Synergism Between Endogenous GATA-4 and SF-1 in Sertoli Cells

Having confirmed that Dax-1 can repress GATA/SF-1 synergism in a heterologous context, we next determined whether Dax-1 had similar actions in cells that normally express GATA-4 and SF-1. For this purpose, we transfected neonate primary Sertoli cell cultures, which endogenously express GATA-4, SF-1, MIS, and low levels of Dax-1 [11, 55, 56] with a target promoter we have previously shown GATA-4 and SF-1 to synergistically activate, the MIS promoter [5]. Therefore in transfected primary Sertoli cells, the wild-type MIS promoter is at a synergistic level of activation due to synergism of endogenous GATA-4 and SF-1. As shown in Figure 6, increasing doses of Dax-1 resulted in a concomitant decrease in the activity of the -180 bp MIS promoter, and hence, endogenous GATA-4/SF-1 synergism. Dax-1 repression was also observed on a similar MIS reporter containing a mutated GATA element (-180mut), indicating that Dax-1 can repress GATA-4/SF-1 synergism irrespective of whether or not GATA-4 is bound to DNA. Deletion of the Sox9 binding site (-107 bp and -107mut), did not abrogate the ability of Dax-1 to repress MIS promoter activity. Dax-1, however, could no longer repress transcription when the SF-1 binding site was deleted (-83 bp and -83mut). This is consistent with our data in heterologous cells (Fig. 1), which showed that an intact SF-1 binding site was required to recruit Dax-1 and its associated corepressor to target promoters. Therefore, Dax-1 repression of MIS transcription in Sertoli cells is mediated, in part, through a disruption of GATA-4/SF-1 synergism.



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  		FIG. 6. Dax-1 represses MIS promoter activity in Sertoli cells. A to F) Primary Sertoli cells were transfected with 500 ng of different wild-type and GATA-mutated MIS promoter constructs along with either increasing doses (5, 20, and 100 ng) of an expression vector encoding Dax-1. Mutation of the GATA binding site in the proximal MIS promoter at -75 bp (GATA to GGTA) is depicted by an X. For each construct used (A to F), promoter activity in the presence of increasing doses of Dax-1 (stippled bars) is expressed as a percentage of its corresponding activity in the absence of Dax-1 (open bar) (± SEM). *Significantly different from control (no Dax-1). **Significantly different from control and the third Dax-1 dose. ***Significantly different from control and all Dax-1 doses (P < 0.05)

DISCUSSION

Tissue-specific gene expression is controlled, in part, through unique combinatorial interactions, which may be synergistic or antagonistic, among different transcriptional regulators. The establishment of a proper sexual phenotype is a paradigm of how multiple transcription factors can come together to regulate a distinct set of genes at the appropriate time during development. The transcriptional mechanisms that regulate the expression of Müllerian inhibiting substance, a key hormone in the sex differentiation pathway, have been the focus of intensive study. The transcriptional regulatory elements required for tissue-specific expression of the MIS gene are contained within a short stretch of its proximal promoter region. They include binding sites for Sox9, SF-1, and GATA-4. We have previously reported that GATA-4, a member of the GATA family of transcription factors, regulates MIS transcription by directly binding to its consensus site on the MIS promoter and by a synergistic interaction with SF-1 [4, 5]. In the present study, we now show that Dax-1, an unusual member of the nuclear receptor superfamily, can modulate MIS transcription by disrupting GATA-4/SF-1 synergism in Sertoli cells.

In vitro and in vivo experiments have revealed that functional cooperation between Sox9, SF-1, WT-1, GATA-4, and Dax-1 regulates MIS transcription [5, 30, 57]. SF-1/WT-1 [30], SF-1/Sox9 [57], and SF-1/GATA-4 interactions [5] all lead to enhanced MIS transcription. Dax-1 represses MIS transcription activity without binding to DNA [30]. Indeed, Dax-1 was shown to negatively modulate MIS promoter activity by disrupting SF-1/WT-1 synergism through a direct protein-protein interaction with DNA-bound SF-1 [30]. The present work also describes Dax-1 as a potent repressor of GATA-4/SF-1 synergism. Because Dax-1 did not directly affect GATA-4-dependent activation, the mechanism of Dax-1 repression was mediated through a direct interaction with SF-1. The fact that our Dax-1 deletion mutant ({Delta}Dax-1), which cannot interact with SF-1 [29–31], had no effect on GATA-4/SF-1 synergism supports this conclusion. Taken together, our results indicate that Dax-1 acts not only as a repressor of SF-1-dependent transcription but also plays an important role as a negative modulator of transcriptional synergism between SF-1 and other transcription factors. The latter may be a key mechanism for fine-tuning the regulation of SF-1 target genes. We have used the MIS promoter as an example of a gonadal target gene; a model for Dax-1 repression is shown in Figure 7. Similar repressive roles for Dax-1 likely exist in other SF-1 target tissues such as the pituitary, where transcriptional synergism involving either SF-1 and the homeoprotein, Pitx1 [48], or SF-1 and the immediate early factor, Egr-1 [47, 58, 59], has been shown to be an important constituent of LHß gene activation.



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  		FIG. 7. Proposed mechanism for the role of Dax-1 in the modulation of GATA-4/SF-1 synergism and the control of MIS transcription in Sertoli cells. A) Species-conserved elements (Sox9, SF-1, and GATA-4) in the proximal MIS promoter are indicated by the circles. B) Sox9 is essential for the initiation of MIS transcription [68]. An additional factor, possibly GATA-4 (G4), is required for the cell-specific initiation of MIS transcription. C) The up-regulation of MIS expression requires the cooperation of several transcription factors, including GATA-4, SF-1, and WT-1. D and E) The down-regulation of MIS transcription involves the recruitment of Dax-1 and associated corepressors through direct interactions with SF-1. This interaction leads to repression of GATA-4/SF-1 and SF-1/WT-1 synergism that may (E), or may not (D) involve a disruption of preexisting interactions between SF-1 with GATA-4 and SF-1 with WT-1

GATA factors are coexpressed with SF-1 and Dax-1 in many tissues, including the testis, ovary, pituitary, and adrenal [4, 9, 11, 34, 35, 60]. We have previously reported that GATA/SF-1 synergism is not limited to the GATA-4 factor, but rather can occur with other GATA family members [5]. Moreover, we have observed that Dax-1 can also repress synergism between SF-1 and these other GATA factors (data not shown). Thus, Dax-1 acts as a negative modulator of GATA/SF-1 synergism. It is interesting that a similar inhibitory GATA cofactor, called FOG-2 (Friend-of-GATA, has also been described [61–63]. In the heart, FOG-2 is coexpressed with GATA-4, where it behaves as a potent repressor of GATA-4-dependent transcription [61–63]. We have recently observed that FOG-2 also colocalizes with GATA-4 in Sertoli cells, where it potently represses GATA-4/SF-1 synergism and MIS promoter activity in Sertoli cells (unpublished observations). In contrast to Dax-1, however, the transcriptional repression induced by FOG-2 is entirely mediated through a direct interaction with GATA-4.

Although the MIS hormone is primarily expressed during fetal life, it is also found in both the postnatal testis and ovary, albeit at much lower levels [64]. In adults, MIS has been proposed to regulate steroid hormone production by modulating the transcription of genes encoding steroidogenic enzymes [65–67]. Therefore, in addition to regulating sex differentiation, MIS appears to have a later role in maintaining steroid hormone production in the gonads. Consequently, the MIS gene must still be under tight transcriptional control at this later developmental time point. Because SF-1, Dax-1, and GATA factors continue to be expressed postnatally, combinatorial interactions between these factors are undoubtedly an integral component of this transcriptional control.

ACKNOWLEDGMENTS

We thank Keith Parker (mouse SF-1 expression plasmid) and Jacques Drouin (minimal POMC, 3xSF-1-POMC, and -142 bp LHß promoters) for generously providing plasmids used in this study. We also thank Pamela Mellon for the {alpha}T3-1 cell line.

FOOTNOTES

First decision: 18 September 2000.

1 Supported by a grant from the Canadian Institutes of Health Research. J.J.T. is the recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. R.S.V. is the holder of a chercheur-boursier of the Fonds de la recherche en santé du Québec. Back

2 Correspondence: Robert S. Viger, Ontogeny and Reproduction Research Unit, T1-49, CHUL Research Centre, 2705 Laurier Blvd., Ste-Foy, PQ, Canada G1V 4G2. FAX: 418 654 2765; robert.viger{at}crchul.ulaval.ca Back

Accepted: November 21, 2000.

Received: August 29, 2000.

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