Transcription Factor GATA-4 Enhances Müllerian Inhibiting Substance Gene Transcription through a Direct Interaction with the Nuclear Receptor SF-1

Jacques J. Tremblay and Robert S. Viger

Unité de Recherche en Ontogénie et Reproduction Centre Hospitalier Universitaire de Québec Pavillon Centre Hospitalier de l’Université Laval Ste-Foy, Québec, Canada G1V 4G2


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Secretion of Müllerian-inhibiting substance (MIS) by Sertoli cells of the fetal testis and subsequent regression of the Müllerian ducts in the male embryo is a crucial event that contributes to proper sex differentiation. The zinc finger transcription factor GATA-4 and nuclear receptor SF-1 are early markers of Sertoli cells that have been shown to regulate MIS transcription. The fact that the GATA and SF-1 binding sites are adjacent to one another in the MIS promoter raised the possibility that both factors might transcriptionally cooperate to regulate MIS expression. Indeed, coexpression of both factors resulted in a strong synergistic activation of the MIS promoter. GATA-4/SF-1 synergism was the result of a direct protein-protein interaction mediated through the zinc finger region of GATA-4. Remarkably, synergy between GATA-4 and SF-1 on a variety of different SF-1 targets did not absolutely require GATA binding to DNA. Moreover, synergy with SF-1 was also observed with other GATA family members. Thus, these data not only provide a clearer understanding of the molecular mechanisms that control the sex-specific expression of the MIS gene but also reveal a potentially novel mechanism for the regulation of SF-1-dependent genes in tissues where SF-1 and GATA factors are coexpressed.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammalian gonadal development and sex differentiation require the coordinated expression of specific genes in a strict spatio-temporal manner. At present, we know of at least two genes, Ftz-F1 (encoding the steroidogenic factor SF-1) and WT-1 (Wilms’ tumor-1), that are essential for these events because in their absence, the gonads fail to develop (1, 2, 3). Although not required for the initiation of gonadal morphogenesis, the testis-determining gene SRY, present on the Y chromosome (4, 5), is essential for normal male sex determination because mutation of this gene in XY individuals leads to sex reversal (6, 7). SRY is believed to induce testis formation by triggering somatic cell precursors, in the bipotential gonad of XY embryos, to differentiate into Sertoli cells and organize into testicular cords (8). A related gene, SOX9, a member of the HMG box-containing family of proteins, has been proposed to be a downstream target of SRY in the testis since in the mouse, Sox9 expression is restricted to the developing male gonad (9) while in humans, mutation of the SOX9 gene is associated with sex reversal (10, 11).

In addition to their critical roles in gonadal development, the SF-1, WT-1, SOX9, and SRY transcription factors also play equally important roles in the control of sex-specific gene expression. The best studied male-specific gene lying downstream of these factors in the male sex differentiation pathway is Müllerian inhibiting substance (MIS), which encodes a hormone produced by Sertoli cells of the fetal testis. MIS regulates male sexual differentiation by triggering the regression of the Müllerian ducts, the anlagen of the female reproductive tract, in genotypic XY males. MIS gene expression is sexually dimorphic. Sertoli cells begin to express MIS on embryonic day 12.5 (E12.5) in the mouse; expression is maintained throughout fetal development and then declines markedly after birth (12, 13). In contrast, MIS is apparently not expressed in the fetal and early postnatal ovary but can be detected in granulosa cells of the adult ovary (12, 13).

The identification of transacting factors involved in the regulation of the MIS gene has progressed rapidly in the past few years. The first gene shown to be crucial for MIS expression is the orphan nuclear receptor SF-1 (1, 14). Indeed, targeted disruption of the Ftz-F1 gene, which encodes SF-1, prevents Müllerian duct regression (1) and, accordingly, in recent transgenic studies Giuili et al. (14) have demonstrated that an intact SF-1-binding site is required for the sex-specific expression of MIS. Since SF-1 is expressed in many tissues where MIS is not, there has been an active search for potential mechanisms that could restrict MIS expression to the gonads. An attractive possibility, which has been frequently observed in other systems, is that gonad-specific MIS expression is the result of a combinatorial interaction between SF-1 and other transcriptional regulators. Indeed, the ability of SF-1 to activate MIS transcription has recently been shown to be enhanced through direct protein interactions with WT-1 and Sox9 (15, 16). In part, these SF-1/WT-1 and SF-1/Sox9 interactions have been helpful in our understanding of the cell-specific expression of the MIS gene. We have recently reported, however, the presence of GATA-4, a member of the GATA family of transcriptional regulators, in the developing gonads that could also play an integral role in the cell-specific and high level of MIS expression in the gonads (17). In the mouse, GATA-4 protein is abundant in the somatic cell population of the indifferent gonad just before the MIS gene is first turned on (17). Like MIS, GATA-4 expression later becomes restricted to the Sertoli cell lineage of the fetal testis and granulosa cells of the adult ovary (17). Moreover, the proximal MIS promoter contains a functional, species-conserved GATA element (17). Although other GATA factors have been shown to be expressed in the gonads, they are expressed after the MIS gene is markedly down-regulated (18).

The GATA factors, which bind the WGATAR consensus in the 5'-regulatory region of target genes, are presently an intensely studied group of transcriptional regulators due to their established roles in cell differentiation, organ development, and cell-specific gene expression in many systems (19, 20, 21, 22, 23, 24). The GATA family comprises six members (GATA-1 to -6) that can be divided into two subgroups: the hematopoietic (GATA-1 to -3) and the cardiac (GATA-4 to -6) groups. Although GATA factors have similar DNA-binding properties, they exhibit distinct spatial and developmental expression patterns and play essential, nonredundant functions (19, 20, 21, 22, 23, 24). Mounting evidence in the literature suggests that the functional specificity of the different GATA factors appears to be achieved, in part, via direct protein-protein interactions with other cell-restricted factors. Indeed, the zinc finger cofactor FOG interacts with GATA-1 to synergistically activate hematopoietic-specific gene expression (25). GATA-1, -2, and -3 have also been shown to interact with several other factors, including RBTN2, NF-E2, EKLF, TAL1, and Sp1, to regulate the activity of erythroid- and lymphoid-specific promoters and enhancers (26, 27, 28, 29, 30). Similarly, GATA-4 has recently been shown to directly interact and transcriptionally cooperate with the cardiac homeodomain protein Nkx2–5 to synergistically activate the atrial natriuretic peptide (ANF) promoter in the heart (31, 32).

The MIS promoter is a downstream target for GATA-4 and SF-1 in Sertoli cells (12, 14, 17). We used this promoter to examine whether both factors functionally cooperate to regulate gene expression in the gonads. We show here that coexpression of GATA-4 and SF-1 markedly enhances the activity of the MIS promoter and a panel of other SF-1-dependent targets. Moreover, we provide evidence that this synergy is the result of a novel interaction, both in vitro and in vivo, between SF-1 and the zinc finger region of GATA-4. Interestingly, synergy between GATA-4 and SF-1 did not absolutely require GATA binding to DNA. Thus, the present data not only provide new insights into the cascade of factors that control the sex-specific expression of the MIS gene but also reveals a potentially new mechanism for modulating SF-1 activity in tissues where both GATA factors and SF-1 are coexpressed.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Proximal MIS Promoter Contains Several Species-Conserved Elements
MIS gene expression appears to be regulated by the concerted action of several transcription factors. As shown in Fig. 1Go, alignment of the proximal MIS promoter from different species reveals the presence of three highly conserved regions: a SRY-like element recently reported to bind the SRY-related protein Sox9 (15), a binding site for the orphan nuclear receptor SF-1 (12), and a consensus GATA motif that we have recently shown to be recognized by GATA-4 in Sertoli cells (17). Interestingly, the GATA and SF-1 binding sites in the MIS promoter are 10 bp apart. This close proximity raises the intriguing possibility that GATA-4 might represent a novel cofactor for SF-1 in the sex-specific regulation of MIS gene expression in the gonads. Although GATA-4 and SF-1 have been shown to regulate MIS promoter activity on their own (12, 14, 17), transcriptional synergism between the two factors has not been reported in the gonads or other tissues where SF-1 and GATA factors are coexpressed.



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Figure 1. Conserved SF-1- and GATA-Binding Sites Are Present in the Proximal MIS Promoter

Conserved regulatory elements of the MIS promoter are boxed, and their locations relative to the transcriptional start site are shown. Alignment of the mouse (12 ), rat (69 ), human (70 ), bovine (71 ), and pig (72 ) proximal MIS promoter sequences reveals consensus SF-1 and GATA-binding sites that are in close proximity to one another.

 
GATA-4 and SF-1 Synergize to Activate MIS Transcription
We have recently shown GATA-4 to be a potent activator of MIS gene transcription in vitro (17). To better define the role of GATA-4 in the cell-specific and developmental regulation of the MIS gene in vivo, we tested whether GATA-4 transcriptionally cooperates with SF-1 to enhance MIS transcription. In our initial experiments we used a -180 bp MIS promoter construct, which contains both GATA and SF-1 binding sites, since it has been shown to be sufficient for cell-specific expression in vitro and in vivo (14, 16). Although crucial for MIS expression in vivo (14), SF-1 by itself is a poor activator of the MIS promoter (Fig. 2AGo and Ref. 12). In contrast, GATA-4 significantly activates the MIS promoter (Fig. 2AGo). Surprisingly, the presence of both GATA-4 and SF-1 resulted in a synergistic activation of the -180 bp MIS promoter (Fig. 2AGo, left panel). Synergy was also observed between GATA-4 and a shorter SF-1 receptor (SF-1{Delta}LBD), which was deleted of its ligand-binding domain (LBD), suggesting that synergistic activation of the MIS promoter by GATA-4 and SF-1 does not require the LBD of SF-1 (Fig. 2AGo, middle panel). As control, no synergy was observed on the minimal (-65 bp) MIS promoter, which does not contain GATA and SF-1 binding sites (Fig. 2AGo, right panel).



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Figure 2. GATA-4 and SF-1 Transcriptionally Cooperate

A, GATA-4 and SF-1 synergize to enhance MIS transcription. The -180 bp (left panel) or the minimal (-65 bp; right panel) murine MIS promoter was cotransfected in CV-1 cells with either a control (empty) expression vector (open bars) or expression vectors for GATA-4 (hatched bars), SF-1 (wild-type or deleted of its ligand-binding domain, {Delta}LBD; gray bars), or both GATA-4 and SF-1 (solid bars). B, SF-1 synergizes with several GATA family members. Different GATA proteins were tested for their ability to synergize with SF-1 on a synthetic reporter containing three copies of an oligonucleotide containing the MIS SF-1- and GATA-binding sites in their natural context (SF-1:GATA)3, fused to the minimal MIS promoter. C, GATA-4 and SF-1 synergize on other promoters that contain putative GATA and SF-1 binding sites. The effects of GATA-4 (hatched bars), SF-1 (gray bars), or both factors (solid bars) were tested on three different reporters: the -142 bp LHß promoter (right panel), a synthetic promoter containing two GATA- and three SF-1-binding sites upstream of the minimal POMC promoter (middle panel), and the minimal POMC promoter (right panel). Results are shown as fold activation (±SEM).

 
We next determined whether synergy between GATA and SF-1 could also be observed on a series of natural and artificial promoters that contain consensus SF-1- and GATA-binding elements (Fig. 2Go, B and C). The first consisted of three copies of an oligonucleotide containing the MIS SF-1 and GATA sites in their normal context fused to the unresponsive minimal MIS promoter [(SF-1:GATA)3, Fig. 2BGo]. As observed for the -180 bp MIS promoter, SF-1 remained a weak activator of this reporter despite the presence of three SF-1 binding sites. Several members of the GATA factor family were then tested for synergy with SF-1 (Fig. 2BGo). Surprisingly, all GATA factors strongly synergized with SF-1, suggesting that the mechanism underlying GATA/SF-1 synergy is likely mediated via a domain common to all GATA factors. Indeed, we have identified this domain to be the zinc finger region (see Fig. 7Go, discussed below). As shown in Fig. 2CGo, synergy between GATA-4 and SF-1 was not restricted to the GATA/SF-1 elements of the MIS promoter. The -142 bp LHß promoter, which contains both SF-1 (33) and putative GATA-binding sites, was also synergistically activated by GATA-4 and SF-1 (Fig. 2CGo, left panel). Similarly, a synthetic reporter containing two GATA elements and three copies of the SF-1-binding site from the LHß promoter upstream of the minimal POMC promoter exhibited strong synergism in the presence of both factors (Fig. 2CGo, middle panel). As observed for the minimal MIS promoter, GATA-4 and SF-1 failed to activate the minimal POMC promoter, which lacks GATA- and SF-1-binding sites (Fig. 2CGo, right panel). Thus, the synergy between GATA-4 and SF-1 appears to be independent of promoter context.



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Figure 7. The First or Second Zinc Finger of GATA-4 Can Interact with SF-1

A, In vitro pull-down assays were used to map the domain of GATA-4 involved in the direct interaction with SF-1. GATA-4 deletion mutants were labeled in vitro and then tested for their ability to interact with either MBP-SF-1 (lanes 2) or MBP-LacZ{alpha} (lanes 3) as control. After extensive washes, bound proteins were separated on 12% SDS-PAGE gels and then visualized by autoradiography. B, The domain of the GATA-4 protein involved in an in vivo interaction with SF-1 was identified using the same reporter system described in Fig. 6BGo. Results are shown as GATA-4-dependent enhancement of SF-1 activity (±SEM). The stippled area indicates no enhancement.

 
The C-Terminal Domain and Zinc Finger Region of GATA-4 Are Required for Synergy with SF-1
To map the domain(s) of the GATA-4 protein required for synergy with SF-1, a series of GATA-4 deletion mutants were used. A schematic representation of the wild-type and mutant GATA proteins is illustrated in Fig. 3AGo. The DNA-binding activity of the GATA proteins was first assessed by gel shift assay. With the exception of the two deletion mutants ({Delta}internal and {Delta}N3, Fig. 3BGo, lanes 9 and 10) that remove the second zinc finger, which has been shown to be essential for DNA binding (34, 35), all GATA-4 proteins were expressed in the nucleus at similar levels, as evidenced by their binding to the MIS GATA element (Fig. 3BGo). The transcriptional properties of the GATA-4 deletion mutants were subsequently tested in cotransfection experiments using a reporter containing two highly responsive GATA motifs upstream of the minimal MIS promoter. As shown in Fig. 3CGo, deletion of the entire N-terminal domain up to the second zinc finger ({Delta}N2) led to a 2-fold increase in transcriptional activity when compared with the wild-type GATA-4 protein. Further N-terminal deletion that removed the second zinc finger ({Delta}N3), or an internal deletion that removed the second zinc finger but kept the first zinc finger and nuclear localization signal intact ({Delta}internal), completely abolished transcriptional activity. A recent study has reported that the second zinc finger of GATA-4, in addition to the nuclear localization signal, is also involved in the nuclear localization of the protein since its deletion results in diffuse protein expression both in the nucleus and cytoplasm (34). However, even when {Delta}internal and {Delta}N3 were used at higher DNA concentrations to ensure sufficient nuclear targeting of the proteins, these mutants still failed to bind DNA and activate a GATA-dependent reporter (Fig. 3Go, B and C). Interestingly, deletion of the C-terminal domain (from aa 333 to 440, {Delta}C1) led to an important decrease in transcriptional activity, suggesting that a potent activation domain (AD) is present within this region of the GATA-4 protein. The remaining activity (~25% of wild-type) represents another AD that has previously been identified in the N-terminal region of GATA proteins (34). Consistent with these observations, deletion of both the N- and C-terminal domains ({Delta}N1{Delta}C1 and {Delta}N2{Delta}C1) completely abrogated GATA responsiveness (Fig. 3CGo). Taken together, these results indicate that the GATA-4 protein contains two ADs that flank the DNA-binding domain, the most potent of which is located in the C-terminal region.



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Figure 3. DNA-Binding and Transcriptional Properties of GATA-4 Deletion Mutants

A, Schematic representation of the different GATA-4 deletion mutants used in this study. The two zinc finger regions (ZnF) contained within the DNA-binding domain (gray box) are shown as two circles; the basic region, representing the nuclear localization signal, is indicated by (++). B, The DNA-binding activity of the different GATA-4 deletion mutants (lanes 3–10) were verified by gel shift assay using the MIS GATA element as probe (17 ). C, Transcriptional properties of the GATA-4 deletion mutants. Expression plasmids encoding the different GATA-4 deletion mutants were cotransfected in CV-1 cells along with a highly responsive GATA reporter containing two GATA motifs upstream of the minimal MIS promoter. Results are shown as fold activation (±SEM).

 
With the GATA-4 deletion mutants in hand, we proceeded to evaluate their potential to synergize with SF-1 on the highly responsive (SF-1:GATA)3-MIS reporter. As shown in Fig. 4Go, two GATA-4 domains proved to be crucial for synergy with SF-1: the C-terminal AD and the second zinc finger. The presence of both domains was required for synergy, since deletion of either domain (as reflected in the {Delta}C1 and {Delta}N3 mutants), abolished the GATA-4-dependent enhancement of SF-1 activity (Fig. 4Go). Consistent with the requirement of these two domains, deletion of the entire N-terminal domain and the first zinc finger ({Delta}N2) did not abrogate synergy with SF-1.



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Figure 4. The C-Terminal Domain and Zinc Finger Region of GATA-4 Are Required for Synergy with SF-1

The ability of the GATA-4 deletion mutants to synergize with SF-1 on the (SF-1:GATA)3-MIS reporter was assessed by cotransfection experiments in CV-1 cells. Results are shown as GATA-4-dependent enhancement of SF-1 activity (±SEM).

 
Synergy between GATA-4 and SF-1 Requires SF-1 Binding to DNA
To assess the binding site requirements for synergy between GATA-4 and SF-1, deletions and mutations of the proximal MIS promoter were generated (Fig. 5Go). Synergy between GATA-4 and SF-1 was still observed on a smaller -107 bp MIS promoter fragment which, like the -180 bp construct, retains both the GATA- and SF-1-binding sites (Fig. 5AGo). A -83 bp MIS promoter fragment, which conserves the GATA but removes the SF-1-binding site, was activated solely by GATA-4 (Fig. 5BGo). No synergy was observed when both factors were used in combination, indicating that a GATA motif by itself is not sufficient for transcriptional synergism with SF-1. However, coexpression of SF-1 with GATA-4 substantially repressed the GATA-4-dependent activation of this promoter construct (Fig. 5BGo). This repression likely represents squelching due to an interaction between GATA-4 and SF-1, as previously reported for other transcription factors (36, 37, 38, 39). Similar results were obtained using other GATA-dependent promoters that lack SF-1 sites: the -114 bp cardiac B-type natriuretic peptide (BNP) promoter, which contains GATA- but no SF-1-binding sites (Fig. 5FGo), or two synthetic reporters containing two GATA motifs upstream of either the minimal MIS (Fig. 5DGo) or POMC (Fig. 5EGo) promoters. In contrast to the need for an SF-1 site, mutation of the GATA motif (GATA -> GGTA) in the MIS promoter, which was previously shown to abolish DNA binding and activation by GATA-4 (17), did not prevent synergy between GATA-4 and SF-1 (Fig. 5CGo). The fact that SF-1 can synergize with GATA-4 without the latter binding to DNA suggests that GATA-4/SF-1 synergism might be the result of a direct protein-protein interaction between the two factors.



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Figure 5. Synergy between GATA-4 and SF-1 on Target Promoters Requires an Intact SF-1-Binding Site

The transactivation potential of GATA-4 (hatched bars), SF-1 (gray bars), or both factors together (solid bars) on a series of natural and synthetic promoters was tested by cotransfection experiments in CV-1 cells. To assess the binding site requirements for synergy between GATA-4 and SF-1, several reporters were used: A, a -107 bp MIS promoter construct that retains both SF-1- and GATA-binding sites; B, a deletion to -83 bp that removes the SF-1 binding site; C, the -180 bp MIS promoter containing a point mutation (GATA -> GGTA) in the MIS GATA element, a single nucleotide substitution previously shown to completely abrogate GATA binding (17 ); D and E, reporters containing two GATA motifs upstream of the minimal MIS and POMC promoters, respectively; F, the -114 bp BNP promoter, which is a downstream target for GATA-4 in the myocardium (65 ). Results are shown as fold activation (±SEM).

 
GATA-4 and SF-1 Interact in Vitro and in Vivo
To test the hypothesis that GATA-4 and SF-1 directly interact, we first used an in vitro pull-down assay. As shown in Fig. 6AGo, in vitro translated 35S-labeled GATA-4 was specifically retained by an immobilized maltose-binding protein (MBP)-SF-1 fusion protein (lane 2 in left panel). No labeled protein was retained, however, when a MBP-LacZ{alpha} fusion was used as control (lane 3 in left panel of Fig. 6AGo). This indicated that the GATA-4/SF-1 interaction was not mediated by the MBP moiety of the MBP-SF-1 fusion protein. Moreover, the direct interaction between GATA-4 and SF-1 was specific since no interaction was observed when an unrelated 35S-labeled luciferase protein was used in the assay (Fig. 6AGo, right panel).



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Figure 6. GATA-4 and SF-1 Interact in Vitro and in Vivo

A, In vitro pull-down assays were performed using immobilized, bacterially produced MBP fusion proteins (MBP-SF-1 or MBP-LacZ{alpha} as control) and either in vitro translated 35S-labeled GATA-4 (left panel) or luciferase (right panel) proteins. After extensive washes, bound proteins were separated on a 12% SDS-PAGE gel and subsequently visualized by autoradiography. B, The ability of GATA-4 to enhance SF-1-dependent transcription through an in vivo interaction with SF-1 was assessed by cotransfection experiments in CV-1 cells. Three different reporter constructs, containing only SF-1-binding sites fused to different minimal promoters, were used: three copies of the SF-1-binding element from the LHß promoter fused to the minimal POMC promoter (left panel) (33 ); two copies of the MIS SF-1 element fused to the minimal MIS promoter (middle panel); five copies of the SF-1 binding element from the steroid 21-hydroxylase promoter fused to the minimal PRL promoter (right panel) (66 ). Hatched bars, GATA-4 alone; gray bars, SF-1 alone; solid bars, GATA-4 and SF-1 in combination. Results are shown as fold activation (±SEM).

 
The significance of a protein-protein interaction between GATA-4 and SF-1 in vivo was ascertained by a series of cotransfection experiments (Fig. 6BGo). In these experiments, three different SF-1-dependent reporters were used: three copies of the SF-1 binding site from the LHß promoter fused to the minimal POMC promoter (left panel), two copies of the MIS SF-1 site upstream of the minimal MIS promoter (middle panel), and five copies of the SF-1 binding site from the steroid 21-hydroxylase promoter fused to the minimal PRL promoter (right panel). As expected, SF-1, but not GATA-4, activated the three reporters. However, when GATA-4 and SF-1 were used in combination, GATA-4 significantly enhanced the SF-1-dependent activation of all three constructs. Since these SF-1-dependent reporters do not contain GATA motifs to permit GATA binding, the synergy observed when both GATA-4 and SF-1 were present indicates an in vivo interaction between the two factors.

SF-1 Interacts with Either Zinc Finger of GATA-4
The domain(s) of the GATA-4 protein involved in the direct interaction with SF-1 was initially mapped using in vitro pull-down assays (Fig. 7AGo). N-terminal deletions right up to the second zinc finger ({Delta}N1, {Delta}N2) did not affect the ability of the mutant GATA-4 protein to interact with SF-1. Similarly, a deletion removing the entire C-terminal domain ({Delta}C1) did not prevent an interaction with SF-1. Deletions that removed both zinc fingers of GATA-4 ({Delta}N3 or {Delta}C2), however, abolished the interaction with SF-1. Taken together, these data narrowed the interaction domain to the zinc finger region of GATA-4, a region we previously defined to be required for transcriptional synergy with SF-1 (Fig. 4Go). Indeed, both zinc fingers of GATA-4 were found to interact with SF-1. The second zinc finger ({Delta}N2{Delta}C1), however, appeared to bind more strongly to SF-1 than the first zinc finger ({Delta}internal).

The mapping of the GATA-4 interaction domain was confirmed in vivo by cotransfection experiments using the SF-1-dependent reporters previously described. As shown in Fig. 7BGo, we found that when either the first (aa 201 to 266) or second (aa 242 to 301) zinc finger region of GATA-4 was fused to the C-terminal AD required for synergy ({Delta}N2 and {Delta}internal), a significant enhancement of SF-1-dependent activity was observed. This enhancement was not observed with the C-terminal domain alone ({Delta}N3), even when used at higher doses of DNA to ensure nuclear localization. Thus, both zinc fingers of the GATA-4 protein can interact in vivo with the nuclear receptor SF-1.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MIS is a crucial hormone required for Müllerian duct regression, and hence male sex differentiation. MIS gene expression is tightly regulated during gonadal development; lack of expression causes persistent Müllerian duct syndrome in humans, a condition in which affected males exhibit female internal reproductive structures (40). The study of the molecular determinants involved in MIS expression has led to the identification of several transcription factors involved in the activation of this gene (14, 15, 16, 17, 41). In the present study, we report the transcriptional cooperation and direct physical interaction between the nuclear receptor SF-1 and GATA-4, a member of the GATA family of transcriptional regulators, in the synergistic activation of the MIS promoter. Moreover, these data allow us to propose a model in which GATA-4, in association with SF-1 and other transcriptional regulators, contributes to the sexually dimorphic expression of the MIS gene in the gonads.

GATA-4/SF-1 Synergism Is Mediated via the Zinc Finger Region of GATA-4
As depicted in Fig. 8AGo, the GATA-4 domain required for the physical interaction with SF-1 was mapped to the DNA-binding region. A more detailed analysis of the GATA-4 interaction domain revealed that both GATA-4 zinc fingers interacted with SF-1, resulting in transcriptional synergism in the presence of the C-terminal AD of GATA-4 (Fig. 7Go). Since the DNA-binding domain is highly conserved among the GATA factors, it was not surprising to observe synergy (Fig. 2BGo) and physical interaction (data not shown) between SF-1 and different GATA family members. Our data are consistent with previous reports that have also identified the GATA zinc finger region as a crucial domain involved in protein-protein interactions with other transcription factors. For example, GATA-1, -2, and -3 have all been shown to interact with the Krüppel family factors, Sp1 and EKLF, through their respective zinc finger regions (26). Similarly, self-association of GATA-1 occurs through its zinc finger domains (42). The C-terminal zinc finger of GATA-4 has also been recently shown to interact with the cardiac homeoprotein Nkx2–5 (31, 32, 43), whereas the interaction between GATA-1 and its cofactor FOG is mediated exclusively through the N-terminal zinc finger of GATA-1 (25). Thus, the zinc finger region of GATA proteins appears to be crucial for protein-protein interaction, as illustrated in the present work involving GATA-4 and SF-1.



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Figure 8. Involvement of GATA-4 in MIS Gene Expression

A, Schematic representation of the GATA-4 protein. The GATA-4 domains required for transcriptional synergism and protein-protein interaction with SF-1 are indicated. DBD, DNA-binding domain; NLS, nuclear localization signal; ZnF, zinc finger. B, Ontogeny of MIS gene expression in the mouse testis and ovary. The relative embryonic and postnatal (adult) stages of MIS expression in the gonads are shown along with the expression of transcription factors previously shown to activate MIS transcription (9 12 17 60 61 ). C, Proposed mechanism for GATA-4 in the control of MIS transcription. Different transcriptional regulators (Sox9, WT-1, and SF-1) proposed to be involved in the regulation of the MIS gene are also shown. GATA-4, bound to its site in the proximal MIS promoter, markedly enhances MIS transcription via a direct interaction with SF-1. As described in this work, GATA-4 can also synergize with SF-1 to activate SF-1-dependent promoters without GATA binding to DNA. Thus, in addition to the MIS promoter shown here, GATA factors also have the potential to stimulate SF-1 target genes in the absence of GATA-binding elements. Protein-protein interactions are indicated by ({equiv}).

 
GATA Factors, New Partners for SF-1 in the Control of SF-1 Target Genes
Several classes of transcription factors that share highly conserved DNA-binding domains exhibit similar DNA-binding properties in vitro but, in contrast, possess highly specialized functions in vivo (19, 20, 44, 45). The GATA family of proteins, which all recognize the WGATAR consensus, represents such a class of transcription factors. The functional specificity of the different GATA proteins appears to be achieved, at least in part, through direct protein-protein interactions with other ubiquitous or cell-restricted factors, a mechanism that is evolutionarily conserved in fungi (46, 47), flies (48), and mammals (28, 29, 30, 31, 32, 49, 50, 51, 52). Similarly, functional interactions between SF-1 and other transcription factors have been recently reported in the literature. For example, SF-1 has been shown to directly interact and synergize with the homeoprotein Ptx1 (33, 53) and the immediate early response factor Egr-1 (33, 54) to regulate gene expression in the pituitary, and with Wilms’ tumor-1, WT-1, in the gonads (16). Furthermore, the Drosophila homolog of SF-1, Ftz-F1, has been shown to physically and transcriptionally cooperate with the homeoprotein Ftz (55, 56). The data presented here represent the first demonstration of a functional interaction between two important regulators of cell differentiation, organogenesis, and cell-specific gene expression: the nuclear receptor SF-1 and GATA-4, a member of the GATA family of proteins.

We initially thought that synergy between GATA-4 and SF-1 would be specific to the MIS promoter, given that the SF-1- and GATA-binding sites are in close proximity to each other (Fig. 1Go). Remarkably, we also observed strong synergistic activation by GATA-4 and SF-1 on a variety of other natural and synthetic SF-1-dependent promoters such as the LHß promoter (Fig. 2Go). The LHß promoter is a well characterized downstream target for SF-1 in pituitary gonadotropes (57, 58). Interestingly, the proximal LHß promoter contains an SF-1 binding site (33) and a potential GATA motif. Moreover, GATA factors, including GATA-4, are coexpressed with SF-1 in pituitary gonadotropes (59), suggesting that transcriptional cooperation between GATA and SF-1 may also be implicated in pituitary-specific gene expression. The fact that synergy with SF-1 was also observed with other GATA family members (Fig. 2BGo) and the fact that GATA/SF-1 synergy did not absolutely require GATA binding to DNA (Figs. 5CGo, 6BGo, and 7BGo) are further indications that transcriptional cooperation between GATA factors and SF-1 may have broader implications. Thus, the functional interaction between GATA-4 and SF-1, described here, appears to represent a more generalized mechanism for the regulation of SF-1 target genes in tissues where both SF-1 and GATA factors are coexpressed.

A Role for GATA-4 in Male Sex Differentiation
One of the critical steps in the establishment of the proper male phenotype is the regression of the Müllerian ducts, which is mediated through the action of MIS. At present, four factors have been proposed to be involved in the sex-specific regulation of the MIS gene: SF-1, WT-1, SOX9, and GATA-4. As shown in Fig. 8BGo, the onset of MIS gene expression occurs around E12.5 in the mouse, shortly after testis differentiation. Based on solid evidence recently reported in the literature (14, 16), MIS gene expression in vivo appears to absolutely require the presence of both SF-1 and WT-1. It is interesting to note, however, that these two factors appear in the developing gonadal primordium of the mouse (E9.5–10.5) somewhat before the MIS gene is actually first turned on (E12.5) (13, 60, 61, 62). This observation invariably suggests that another factor is likely missing at this time. A recent report has proposed Sox9 to be this factor since it was shown to interact with SF-1 (15). Several other lines of evidence, however, appear to contradict this possibility. First, Sox9 expression (15), like SF-1 and WT-1, considerably precedes that of MIS (13). Second, in granulosa cells of the adult ovary, MIS is expressed in the absence of Sox9 (9, 13). Lastly, in the chick, MIS was shown to be expressed before Sox9 (63). Taken together, these observations support the existence of another factor that participates in the cell-specific expression of the MIS gene. Our previous findings on the sexually dimorphic expression pattern of the GATA-4 transcription factor in the developing gonads, combined with the present functional interaction data with SF-1 on the MIS promoter, support the notion that GATA-4 is involved in MIS expression in vivo. Indeed, abundant GATA-4 expression begins in the bipotential gonad of the mouse on E11.5 (17), just before the detection of MIS transcripts (13). Moreover, GATA-4, like MIS, is expressed in granulosa cells of the adult mouse ovary (13, 17). Thus, as presented in Fig. 8CGo, these data are consistent with a model in which GATA-4 is an integral component of the combinatorial code of factors required for MIS gene expression and, consequently, male sex differentiation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The -180, -83, and -65 bp murine MIS-luciferase promoter constructs have been described previously (17). The -180-bp MIS promoter harboring a mutation in the GATA element at -75 bp (GATA -> GGTA) was obtained using the pALTER site-directed mutagenesis system (Promega Corp., Madison, WI); the mutation was confirmed by DNA sequencing. The -107 bp MIS promoter was amplified by PCR using the -180 bp construct as template and then cloned in the BamHI/HindIII site of the luciferase expression vector pXP1, as previously described for the other MIS promoter constructs (17). Since the pXP1 vector normally contains two GATA motifs upstream of its multiple cloning site, all pXP1 luciferase reporter constructs were modified to delete these highly responsive GATA sites. This was achieved by replacing a 600-bp NdeI-BamHI fragment with a similar fragment (minus the GATA sites) that was generated by PCR (forward primer: 5'-TTCACACCGCATATGGTGCACT-3'; reverse primer: 5'-ACGGATCCAAGCTTACATTGATGAGTTTGGACAAAC-3') on the promoterless pXP1 vector. Thus, the (GATA)2-MIS luciferase reporter consists of the minimal (-65 bp) MIS promoter in the unmodified pXP1 vector. The (SF-1:GATA)3-MIS luciferase reporter was obtained by cloning three copies of a double-stranded MIS SF-1:GATA element (sense oligonucleotide: 5'-GATCCAGGCACTGTCCCCCAAGGTCACCTTTGGTGTTGATAGGGGCGA-3'; antisense oligonucleotide: 5'-GATCTCGCCCCTATCAACACCAAAGGTCACCTTGGGGGACAGTGCCTG-3') upstream of the minimal MIS promoter in the modified pXP1 vector. Similarly, the (SF-1)2-MIS luciferase reporter was obtained by cloning two copies of the MIS SF-1 element (sense oligonucleotide: 5'-GATCCCCCAAGGTCACCTTTA-3'; antisense oligonucleotide: 5'-GATCTAAAGGTGACCTTGGGGG-3') in front of the minimal MIS promoter in the modified pXP1 vector. The minimal POMC, (SF-1)3-POMC, (GATA)2-POMC, (GATA)2/(SF-1)3-POMC, and -142 bp LHß luciferase reporters were kind gifts of Jacques Drouin (33, 64). In the POMC constructs, SF-1 refers to the SF-1 element found in the bovine LHß promoter (33). Again, the GATA motifs in the POMC constructs given above come from an unmodified pXP1 luciferase reporter. GATA expression vectors, the -114 bp BNP reporter, and certain GATA-4 deletion mutants ({Delta}N1, {Delta}N2, {Delta}C1, {Delta}C2, {Delta}N1C1, {Delta}N2C1) were kindly provided by Mona Nemer. The remaining GATA-4 deletion mutants ({Delta}N3 and {Delta}internal) were generated by PCR on the wild-type GATA-4 cDNA and then cloned into the XbaI/BamHI site of the pCG expression vector (31, 65). The forward primer for the {Delta}N3 construct was 5'-CTTCTAGAGGGGTTCCCAGGCCTCTTGCA-3, and the reverse primer was 5'-CAGGATCCAAGTCCGAGCAGGAATTT-3'. The {Delta}internal construct was obtained by initially cloning the first zinc finger of GATA-4, obtained by PCR, into pCG (forward primer: 5'-CTTCTAGACAACCCAATCTCGATATG-3'; reverse primer: 5'-ATGGATCCTTAGCTAGCCAGCCGGCGCTGAGGCTTGATGAGGGGC-3'). The latter was digested with NheI/BamHI (the NheI site being provided by the reverse primer given above), and the XbaI-BamHI C-terminal PCR fragment used to produce {Delta}N3 was cloned in frame to yield {Delta}internal. The SF-1 expression plasmid and (SF-1)5-PRL reporter were generously provided by Keith Parker. The (SF-1)5-PRL construct consists five copies of the 21-hydroxylase SF-1 element cloned upstream of the minimal PRL promoter (66).

Cell Culture and Transfections
African green monkey kidney CV-1 and murine L cells were grown in DMEM supplemented with 10% newborn calf serum. Transfections were done in 12-well plates using the calcium phosphate precipitation method (67). CV-1 cells were plated at a density of 60,000 cells per well 24 h before transfection. Cell media were changed 12–16 h later, and cells were harvested the next morning. Cells were lysed by adding 100 µl of lysis buffer (100 mM Tris-HCl, pH 7.9, 0.5% Igepal (Sigma Chemical Co., St. Louis, MO), and 5 mM dithiothreitol) directly to the wells. Luciferase activity (60 µl aliquot of lysate) was then assayed using an EG&G Berthold (Bad Wildbad, Germany) LB 9507 luminometer. In synergy experiments, optimal doses of SF-1 (10 ng/well) and GATA-4 (100 ng/well) expression vector were used. It is important to note that the level of activation obtained when both GATA-4 and SF-1 were present (synergy) could never be achieved by simply increasing the amount of GATA-4 or SF-1 when used alone (independent activation). In fact, the optimal dose of GATA-4 for maximum activation was 100 ng/well, which is the same dose used in the synergy experiments. Thus, the synergy observed when both factors are present indicates a true synergy. In all experiments, the total amount of DNA was kept constant at 6 µg per well using Sp64 (Promega Corp., Madison, WI) as carrier DNA; several DNA preparations of the plasmids were used to ensure reproducibility of the results. Data reported represent the average of 3 to 10 experiments, each done in duplicate.

Production of MBP Fusion Proteins
A recombinant MBP-SF-1 fusion protein was obtained by cloning the entire coding region of murine SF-1 in frame with MBP using the commercially available pMAL-c fusion protein vector (New England Biolabs, Inc., Mississauga, Ontario, Canada). The MBP-LacZ{alpha} fusion protein was provided by the wild-type pMAL-c vector. The two fusion proteins constructs (MBP-SF-1 and MBP-LacZ{alpha}) were introduced into the Escherichia coli strain BL21, and fusion proteins were produced by inducing the respective bacterial cultures with isopropylthiogalactoside. Bacterial cultures were lysed by sonication, and the fusion proteins were purified using an amylose resin (New England Biolabs, Inc.) as outlined by the manufacturer.

Protein-Protein Binding Assays
Protein-protein binding studies were done using 35S-labeled in vitro translated GATA-4 proteins (wild-type and deletion mutants) and the purified MBP-SF-1 and MBP-LacZ{alpha} fusion proteins coupled to amylose resin. The 35S-labeled GATA-4 and luciferase proteins were obtained using the TNT system from Promega Corp.; the amino acid positions of the different GATA-4 proteins used are given in Fig. 3AGo. Protein-protein interaction assays were done using 1 µg of MBP-fusion protein and 10 µl of in vitro translated 35S-labeled protein essentially as described by Durocher et al. (31). Bound complexes were separated by SDS-PAGE, and retained proteins were revealed by autoradiography.

DNA-Binding Assays
Recombinant GATA proteins (wild-type and deletion mutants) were obtained by transfecting L cells (which are devoid of GATA activity) with the different GATA-4 expression plasmids described above. Nuclear extracts were prepared 48 h after transfection by the procedure outlined by Schreiber et al. (68). DNA-binding assays were performed using a 32P-labeled double-stranded oligonucleotide corresponding to the conserved MIS promoter GATA element at -75 bp. Binding reactions and electrophoresis conditions were as previously described (17).


    ACKNOWLEDGMENTS
 
We are grateful to Mona Nemer for providing the GATA expression vectors, several GATA deletion mutants, and the -114 bp BNP promoter; Jacques Drouin for the -142 bp LHß and POMC promoter constructs; and Keith Parker for generously providing the SF-1 expression plasmid and (SF-1)5-PRL reporter. Eric Legault and Veronique Tremblay are also thanked for their help in the cloning and sequencing of some of the MIS promoter constructs.


    FOOTNOTES
 
Address requests for reprints to: Dr. Robert S. Viger, Unité de Recherche en Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Pavillon Centre de Recherche du CHUL, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2.

This work was supported by a grant from the Medical Research Council of Canada to R.S.V.

Received for publication March 23, 1999. Accepted for publication May 13, 1999.


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