Modulation of Endogenous GATA-4 Activity Reveals Its Dual Contribution to Müllerian Inhibiting Substance Gene Transcription in Sertoli Cells

Jacques J. Tremblay, Nicholas M. Robert and Robert S. Viger

Ontogeny and Reproduction Research Unit, Centre Hospitalier de l’Université Laval (CHUL) Research Centre; and Centre for Research in Biology of Reproduction, Department of Obstetrics and Gynecology, Laval University, Ste-Foy, Quebec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Robert S. Viger, Ontogeny and Reproduction Research Unit, T1–49, Centre Hospitalier de l’Université Laval (CHUL) Research Centre, 2705 Laurier Boulevard, Ste-Foy, Quebec, Canada G1V 4G2. E-mail: Robert.Viger{at}crchul.ulaval.ca


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Secretion of Müllerian inhibiting substance by fetal Sertoli cells is essential for normal male sex differentiation since it induces regression of the Müllerian ducts in the developing male embryo. Proper spatiotemporal expression of the MIS gene requires a specific combination of transcription factors, including the zinc finger factor GATA-4 and the nuclear receptor steroidogenic factor-1, which both colocalize with Müllerian inhibiting substance in Sertoli cells. To establish the molecular mechanisms through which GATA-4 contributes to MIS transcription, we have generated and characterized novel GATA-4 dominant negative competitors. The first one, which consisted solely of the GATA-4 zinc finger DNA-binding domain, was an efficient competitor of GATA transcription mediated both by direct GATA binding to DNA and protein-protein interactions involving GATA factors. The second type of competitor consisted of the same GATA-4 zinc finger DNA-binding domain but harboring mutations that prevented DNA binding. This second class of competitors repressed GATA-dependent transactivation by specifically competing for GATA protein-protein interactions without affecting the DNA-binding activity of endogenous GATA factors. These competitors, along with the GATA-4 cofactor FOG-2 (friend of GATA-2), were used to specifically modulate endogenous GATA-4 activity in Sertoli cells. Our results indicate that GATA-4 contributes to MIS promoter activity through two distinct mechanisms. Moreover, the GATA competitors described here should provide invaluable in vitro and in vivo tools for the study of GATA- dependent transcription and the identification of new target genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GATA FACTORS are a group of evolutionarily conserved transcriptional regulators. They share a highly conserved DNA-binding domain that consists of two zinc fingers of the form C-X2-C-X17-C-X2-C. The C-terminal zinc finger is required for site-specific recognition and DNA binding to the core WGATAR motif, whereas the N-terminal zinc finger contributes to the full specificity and stability of the DNA binding (1, 2, 3). The N- and C-terminal fingers are also crucial domains involved in protein-protein interactions with other transcription factors (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The vertebrate family of GATA factors is comprised of six proteins (GATA-1 to GATA-6) that can be separated into two subgroups based on sequence homology and tissue distribution: the hematopoietic group (GATA-1/2/3) and the cardiac group (GATA-4/5/6). Although GATA factors have similar DNA-binding properties, they exhibit within each group, distinct spatial and developmental expression patterns and play essential, nonredundant functions in cell differentiation, organ development, and cell- specific gene expression (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Indeed, targeted disruption of the six murine GATA genes have revealed critical roles for these factors in such diverse processes as hematopoiesis, heart tube formation, and genitourinary tract development (17, 18, 19, 20, 21, 22, 23, 24, 25). Moreover, aberrations in GATA function have now been recently linked with human disease where a mutation of the GATA-1 gene has been associated with dyserythropoietic anemia and thrombocytopenia, and GATA-3 haplo-insufficiency has been associated with human hypoparathyroidism, sensorineural deafness, and renal anomaly (HDR) syndrome (26, 27). In addition to their early developmental roles, GATA factors are likely involved in many other physiological processes because of their strong expression in a variety of other tissues, such as the brain, gut, pituitary, and gonads, where numerous downstream target genes have been identified (7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37).

Since members of the GATA family share a highly homologous DNA-binding domain, they all exhibit similar DNA-binding properties (38, 39), and consequently, have been reported to be functionally interchangeable in some in vitro assays (40, 41). This clearly contrasts, however, with their nonredundant functions in vivo (17, 18, 19, 20, 21, 22, 23, 24, 25, 40, 42). The functional specificity of the different GATA factors appears to be achieved, in part, through combinatorial interactions with other transcription factors. Indeed, GATA-1, -2, and -3 have been shown to interact with several cell-restricted or ubiquitously expressed factors such as rhombotin-2 (RBTN2), nuclear factor erythroid 2 (NF-E2), erythroid Krüppel-like factor (EKLF), activating protein 1 (AP-1), stem cell leukemia (SCL/Tal1), pituitary factor 1 (Pit1), PU.1, and stimulatory protein 1 to control the activity of erythroid-, lymphoid-, and pituitary-specific promoters and enhancers (4, 7, 8, 9, 11, 12, 43, 44). Similarly, GATA-4 has been reported to interact with the homeoprotein Nkx2.5 and the myocyte enhancer factor MEF2 to direct cardiac-specific gene expression (5, 6, 13), and with the orphan nuclear receptor steroidogenic factor 1 (SF-1) to synergistically activate several gonadal promoters (10, 28). In addition to the aforementioned GATA-interacting factors, a novel class of multizinc finger GATA cofactors, named the FOG (friend-of-GATA) proteins, have been identified as specific modulators of GATA-dependent transcriptional activity (45, 46, 47, 48, 49). To date, two FOG proteins have been characterized, FOG-1 and FOG-2, and each is coexpressed with a specific subgroup of GATA factors: FOG-1 with the hematopoietic group, and FOG-2 with the cardiac group (45, 46, 48, 49). At the transcriptional level, the FOG proteins act as either enhancers or repressors of GATA-1 and GATA-4 activity, respectively, depending on the cell and promoter context (46, 47, 48, 49, 50, 51). The FOG-1 and FOG-2 proteins modulate GATA-dependent transcription by interacting with the N-terminal zinc finger of their respective GATA factors (47). Moreover, the in vivo relevance of the FOG proteins in GATA-mediated gene expression has been revealed by gene inactivation experiments: mice lacking FOG-1 die in utero due to arrested erythroid differentiation (52), while genetic ablation of FOG-2 or a mutation of the GATA-4 protein that impairs its ability to interact with FOG-2 leads to defects in heart morphogenesis and coronary vascular development (53, 54, 55).

Several genes are known to have crucial roles in gonadal development and sex determination and differentiation. They include Ftz-F1 (SF-1), WT-1 (Wilms’ tumor-1), Sry, Lhx9, and Sox9 (56, 57, 58, 59, 60, 61, 62). In mammals, the crucial step leading to male sex determination and differentiation is the induction of testis formation. In males, the bipotential gonad is directed away from ovarian development and toward testicular differentiation through the action of Sry (57). Since Sry expression is limited to a discrete period of gonadal differentiation (63), it acts as a switch to turn on a network of downstream molecular factors involved in testicular development and male sex differentiation. The earliest marker of testis formation is Müllerian inhibiting substance (MIS), a hormone produced by fetal Sertoli cells. MIS regulates male sex differentiation by triggering regression of the Müllerian ducts, the anlagen of the female reproductive tract, in XY males. MIS 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 (64, 65). In contrast, MIS expression is absent in fetal ovaries but later appears in granulosa cells of the ovary during postnatal life (64, 65). Through an analysis of the conserved 5'-regulatory elements of the MIS gene in vitro and in vivo, several transcription factors have been proposed to participate in MIS transcription such as SF-1 and Sox9 (64, 66, 67). However, since Sox9 and SF-1 are colocalized in several tissues that do not express MIS, other factors must exist to restrict MIS expression to the gonads. In fact, cooperation between transcription factors has been shown to contribute to MIS promoter activity (10, 68, 69).

We have recently reported the presence of GATA-4 in the developing testis that could also play an integral role in the cell-specific and high level of MIS expression in the gonads (30). Since GATA factors are well established regulators of cell differentiation and organ morphogenesis in other systems, GATA factors are interesting candidates for the cell- and sex-specific regulation of MIS expression. In the mouse, GATA-4 protein is abundant in the somatic cell population of the bipotential gonad just before the MIS gene is first turned on (30). Like MIS, GATA-4 expression becomes highly restricted to Sertoli cells of the fetal testis and is down-regulated in the ovary (30). We have also shown that GATA-4 can activate the MIS promoter on its own but also physically interacts with SF-1, leading to a synergistic activation of the MIS promoter in a heterologous context (10). However, the importance of GATA-4, alone and in association with SF-1, for MIS transcription in a more in vivo context, such as primary Sertoli cells, has not yet been established. In the present study, we have generated and characterized several mutated GATA-4 proteins that were used, along with the GATA-4 cofactor FOG-2, as novel tools to modulate endogenous GATA-4 activity in MIS- expressing primary Sertoli cell cultures. Using these tools, we show that GATA-4 contributes to MIS promoter activity through two distinct mechanisms, which are both essential components of an elaborate regulatory complex that controls the spatiotemporal expression of the MIS gene. Moreover, we also provide new molecular tools that should be invaluable for dissecting the molecular mechanisms through which GATA factors contribute to tissue-specific gene expression in other systems.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An Intact GATA Binding Site Is Required for Full MIS Promoter Activity in Sertoli Cells
We have previously reported that GATA-4 can activate the MIS promoter by itself and through a synergistic interaction with the orphan nuclear receptor SF-1 in heterologous cells (10, 30). However, the importance of GATA-4, alone and in association with SF-1, for MIS transcription in cells that normally express the hormone in vivo has not yet been clearly established. Primary Sertoli cell cultures prepared from neonate (3- to 5-day-old) rat testes provide an excellent model system to study MIS regulatory elements since these cells express the MIS gene as well as transcription factors proposed to play a role in its cell- and sex-specific expression, including SF-1 and GATA-4 (Fig. 1Go). Indeed, GATA-4 protein is detectable in neonate Sertoli cell cultures (30), and the MIS promoter is active in these cells (Fig. 2Go). In addition to GATA-4 and SF-1, primary Sertoli cell cultures also express FOG-2 (Fig. 1Go), a recently described GATA-4 cofactor known to modulate GATA-4 transcriptional activity (46, 48, 49, 50).



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Figure 1. Neonate Rat Primary Sertoli Cell Cultures Express Characteristic Sertoli Cell Markers

The expression of MIS, GATA-4, SF-1, and FOG-2 in postnatal testis as well as in neonate primary Sertoli cell cultures was assessed by RT-PCR. The pairs of oligonucleotide primers used in the PCR reactions are described in Materials and Methods. The integrity and relative loading of the different cDNAs were assessed by amplifying tubulin as a control gene.

 


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Figure 2. Full MIS Promoter Activity in Sertoli Cells Requires an Intact GATA Element

Primary Sertoli cells (left panel) and CV-1 cells (devoid of GATA activity, right panel) were transfected with various MIS promoter constructs: -180 bp, -180 mut, -83 bp, and -83 mut. The -83 bp deletion constructs no longer contain the Sox9 and SF-1 binding sites. Mutation of the GATA binding site (GATA to GGTA) is depicted by an X. Absolute promoter activities of the different MIS constructs are expressed as relative light units (±SEM). *, Significantly different from the -180 bp MIS construct; **, significantly different from the -180 bp and -180 mut MIS constructs.

 
To determine the significance of the conserved GATA binding site in the proximal MIS promoter, a point mutation (GATA to GGTA), known to completely abrogate GATA binding (30), was introduced into the MIS GATA element. In the context of the -180-bp promoter, this mutation led to a 40% decrease in MIS promoter activity in Sertoli cells but not in CV-1 cells that are essentially devoid of GATA activity (Fig. 2Go). Similarly, a deletion of the MIS promoter to -83 bp, which removes both the Sox9 and SF-1 binding sites but keeps the GATA element intact, resulted in a greater than 50% decrease in promoter activity when compared with the activity of the -180-bp MIS promoter (Fig. 2Go, left panel). Finally, the -83-bp reporter harboring a mutation in the GATA binding site (-83 mut) was the least active, representing only 30% of the activity (a 70% decrease) of the intact -180-bp MIS promoter (Fig. 2Go). The effects of these deletions and mutations were specific since they did not significantly affect MIS promoter activity in the heterologous CV-1 fibroblast cell line (Fig. 2Go, right panel). Thus, the integrity of the conserved GATA binding site, along with the presence of other regulatory elements, is essential for maximal MIS promoter activity in Sertoli cells. These results are in agreement with a recent study that characterized the requirement of GATA sites for the activity of the human MIS promoter (70).

A Truncated GATA-4 Protein Acts as a Dominant Negative Competitor of GATA Transcriptional Activity
To elucidate the molecular mechanism through which GATA-4 contributes to MIS promoter activity, we have generated a truncated GATA-4 protein (DF WT for double finger wild type) that consists solely of its zinc finger DNA-binding domain (Fig. 3AGo). The DNA binding and transcriptional properties of the DF WT protein were first characterized in an heterologous system. Since the DF WT protein retains its zinc finger region, it bound to DNA as efficiently as the full-length GATA-4 protein (Fig. 3BGo). Gel shift data also confirmed that the DF WT protein is targeted to the nucleus and is expressed at a level similar to that of the full-length GATA-4 protein. In addition to DNA-binding, the DF WT protein was able to physically interact with SF-1 (Fig. 6DGo). However, since the DF WT protein lacks transactivation domains, it could no longer activate a highly responsive synthetic GATA-dependent reporter (Fig. 3CGo). To test the validity of the DF WT protein as a dominant negative competitor, we first determined its ability to repress the transactivation of a highly responsive GATA-dependent reporter in CV-1 cells overexpressing GATA-4. As shown in Fig. 3CGo, the DF WT protein behaved as a dominant negative competitor since it repressed in a dose-dependent manner the GATA-dependent transactivation of a GATA-responsive synthetic reporter composed of two GATA elements from the MIS promoter fused to the minimal MIS promoter. This effect was specific since the DF WT protein, by itself, had no significant effect on the same GATA-dependent reporter (gray bars in Fig. 3CGo), nor did it repress the SF-1-dependent activation of the LHß promoter, a known SF-1 natural target (stippled bars in Fig. 3DGo).



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Figure 3. Characterization of a Truncated GATA-4 Protein That Acts as Dominant Negative Competitor of GATA Activity

A, Schematic representation of the full-length GATA-4 protein and a truncated GATA-4 protein that consists solely of its zinc finger region (DF WT). 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 (++). A diagram depicting the wild-type amino acid sequence of the second zinc finger is also shown. B, The DNA-binding activity of the DF WT protein was assessed by gel shift assay using the previously defined MIS GATA element as probe (30 ). C and D, Effects of DF WT and the GATA cofactor FOG-2 on GATA-4- and SF-1-dependent transactivation. Expression plasmids encoding full-length GATA-4, DF WT, and FOG-2 were transfected in CV-1 cells along with either a highly responsive GATA reporter consisting of two GATA motifs upstream of the minimal MIS promoter (C) or the previously characterized SF-1-dependent LHß promoter (D). In all transfections, the amount of reporter DNA was kept constant at 500 ng per culture well. Open bar, Control (empty) vector; gray shaded bars, increasing doses of DF WT (50, 100, 150 ng) and FOG-2 (10, 50, 100 ng); black bar, 50 ng GATA-4; stippled bars, 50 ng GATA-4 and increasing doses (50, 100, 150 ng) of DF WT; hatched bars, 50 ng GATA-4 and increasing doses (10, 50, 100 ng) of FOG-2. Data are reported as fold activation (±SEM). *, Significantly different from the activation mediated by GATA-4 (black bar) alone; **, significantly different from the activations mediated by GATA-4 alone and GATA-4 in the presence of the first DF WT or FOG-2 dose.

 


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Figure 6. DNA-Binding and Protein-Protein Interaction Properties of Two Mutated Forms of the Truncated GATA-4 Protein DF WT

A, Schematic representation of two truncated GATA-4 proteins (DF {Delta}T279 and DF C294A) harboring mutations in the second zinc finger. 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 (++). A diagram depicting the amino acid sequence of the second zinc finger is also shown. The threonine at amino acid 279 is deleted in DF {Delta}T279, whereas the cysteine-to-alanine substitution at amino acid 294 prevents the second zinc finger from forming in DF C294A. B, The DNA-binding activity of the DF {Delta}T279 and DF C294A proteins was assessed by gel shift assay using the MIS GATA element as probe. C, The three DF GATA-4 proteins are expressed at similar levels. Forty-microgram aliquots of nuclear extracts prepared from L cell fibroblasts overexpressing the different HA-Tag DF proteins were separated by SDS-PAGE and Western blotted to Hybond-polyvinylidene difluoride membrane as described in Materials and Methods. Immunodetection was achieved using a commercially available antibody directed against the HA tag. D, The wild-type and mutated DF GATA-4 proteins physically interact with SF-1. 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 DF WT, DF {Delta}T279, or DF C294A GATA-4 proteins. After extensive washes, bound proteins were separated on a 12% SDS-PAGE gel and subsequently visualized by autoradiography.

 
FOG-2 is a multizinc finger protein that associates with GATA-4 to modulate GATA-dependent transcription (46, 48, 49, 51). As demonstrated in Fig. 3CGo, FOG-2 is a potent repressor of GATA-4-dependent transactivation in CV-1 cells. Like the DF WT protein, FOG-2 specifically attenuated GATA-mediated transactivation since no effects were observed on SF-1-dependent transactivation or when the cofactor was used alone. Thus, FOG-2 and the DF WT protein can be used to modulate GATA-4 activity through different mechanisms: passive competition (DF WT) and active repression (FOG-2).

The DF WT Competitor and FOG-2 Are Negative Modulators of GATA-4/SF-1 Synergism
Since GATA-4 contributes to MIS promoter through a synergistic interaction with SF-1 (10), we tested whether the DF WT competitor and FOG-2, in addition to abating GATA-dependent transactivation, could abrogate GATA-4/SF-1 synergism. The effects of DF WT and FOG-2 on GATA-4/SF-1 synergism were first tested in heterologous CV-1 cells using a highly responsive synthetic reporter that consists of three copies of an oligonucleotide containing the MIS SF-1 and GATA sites in their normal context (SF-1:GATA)3 fused to the unresponsive minimal MIS promoter. This synthetic reporter was previously shown to exhibit strong synergism in the presence of both factors by comparison with the native -180-bp MIS promoter, which only contains one binding site for each factor (10). As shown in Fig. 4Go, the DF WT protein and FOG-2 dramatically reduced transcriptional synergism between GATA-4 and SF-1.



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Figure 4. The DF WT Competitor and FOG-2 Repress GATA-4/SF-1 Synergism

Expression plasmids encoding full-length GATA-4, SF-1, DF WT, and FOG-2 were transfected in CV-1 cells along with a synthetic reporter containing three copies of an oligonucleotide consisting of the MIS SF-1 and GATA binding sites in their natural context (SF-1: GATA)3, fused to the minimal MIS promoter. In all transfections, the amount of reporter DNA was kept constant at 500 ng per culture well. Open bar, Control (empty) vector; light gray shaded bar, 50 ng GATA-4; dark gray shaded bar, 10 ng SF-1; black bar, 50 ng GATA-4 and 10 ng SF-1 (GATA-4/SF-1 synergy); stippled bars, effect of increasing doses of the GATA dominant negative competitor DF WT (50, 100, 150 ng) on GATA-4/SF-1 synergy; hatched bars, effect of increasing doses of FOG-2 (10, 50, 100 ng) on GATA-4/SF-1 synergy. Data are reported as fold activation (±SEM). *, Significantly different from the synergistic (black bar) activation mediated by GATA-4 and SF-1.

 
Modulation of Endogenous GATA Activity in Sertoli Cells Affects MIS Promoter Activity
Having demonstrated the validity of the DF WT competitor and FOG-2 as useful tools to modulate GATA activity using a heterologous system, they were subsequently used to study the contribution of GATA-4 to MIS promoter activity by modulating endogenous GATA-4 activity in Sertoli cells. When overexpressed in Sertoli cells, the DF WT competitor and FOG-2 markedly decreased, in a dose-dependent manner, the activity of the wild-type -180-bp MIS promoter, indicating that endogenous GATA-4 is required for full promoter activity in these cells (Fig. 5Go, A and E). Similar results were also observed with the -83-bp MIS promoter construct, which retains the GATA element but removes the Sox9 and SF-1 binding sites (Fig. 5Go, C and G). The observed effects were specific since the activity of a MIS reporter lacking an intact GATA element (-83 mut) was not affected by the DF WT competitor (Fig. 5DGo) or by FOG-2 (Fig. 5HGo). Interestingly, the activity of a MIS promoter construct containing a mutated GATA element together with an intact SF-1 site (-180 mut), was still decreased by DF WT and FOG-2 (Fig. 5Go, B and F). This indicates that endogenous GATA-4 also contributes to MIS promoter activity through a direct protein-protein interaction with DNA-bound SF-1. This is consistent with our previous findings in heterologous cells, which showed that physical interaction and synergism between GATA-4 and SF-1 does not necessarily require GATA-4 binding to DNA (10).



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Figure 5. The DF Cells WT Competitor and FOG-2 Repress MIS Promoter Activity in Sertoli Cells

Primary Sertoli cells were transfected with 500 ng of different wild-type (A, C, E, and G) and GATA-mutated (B, D, F, and H) MIS promoter constructs along with either increasing doses (50, 100, 250 ng) of the DF WT competitor (stippled bars, left panel) or the GATA cofactor FOG-2 (hatched bars, right panel). Mutation of the GATA binding site in the proximal MIS promoter at -75 bp (GATA to GGTA) is depicted by an X. Promoter activities are expressed relative to the activity of the reporters cotransfected with the control (empty) vector (± SEM). *, Significantly different from control (no DF WT or FOG-2 present); **, significantly different from control and the first DF WT or FOG-2 dose; ***, significantly different from control and all DF WT or FOG-2 doses.

 
Mutated Forms of the DF WT Protein, Which Are Unable to Bind DNA, Specifically Compete GATA-4/SF-1 Synergism
Our previous data in CV-1 cells and the present data in Sertoli cells suggest that GATA-4 not only contributes to MIS promoter activity by directly binding to its consensus element on the MIS promoter (Fig. 2Go), but also via a direct protein-protein interaction with SF-1, which is independent of GATA binding to DNA. To assess the contribution of a bona fide GATA-4/SF-1 interaction to MIS promoter activity, we generated two mutated forms of the DF WT competitor that lack the ability to bind DNA but retain the capacity to physically interact with SF-1 (Fig. 6Go). Thus, in contrast to the DF WT competitor, the mutated DF WT proteins should only compete for GATA-4/SF-1 interactions and not GATA-4 binding to DNA. As illustrated in Fig. 6AGo, the first mutation (DF {Delta}T279) consisted of a deletion of the amino acid threonine at position 279. This amino acid, although not involved in the structural integrity of the zinc finger, has been shown to be essential for recognition of the GATA motif and hence, DNA-binding (71). The second mutation consisted of a cysteine-to- alanine substitution at amino acid 294. Because this cysteine residue plays an integral part in the formation of the C-terminal zinc finger, the DF C294A protein, like DF {Delta}T279, should be unable to bind DNA. As expected, neither the DF {Delta}T279 nor the DF C294A protein bound to a consensus GATA element when tested in a gel shift assay (Fig. 6BGo). However, both mutated DF proteins were expressed at the same level as DF WT, as demonstrated by Western blot assays using an anti-hemagglutin (HA) antibody and N-terminally HA-tagged DF proteins (Fig. 6CGo), or a commercially available GATA-4 antibody and DF proteins containing the epitope-bearing GATA-4 C-terminal domain (data not shown). Moreover, all three proteins retained the ability to physically interact with SF-1 as assessed by an in vitro pull-down assay (Fig. 6DGo). Since we have previously shown that both the N- and C-terminal zinc fingers of GATA-4 are capable of interacting with SF-1 (10), the mutations produced in the C-terminal finger were not expected to affect the ability of the DF {Delta}T279 and DF C294A proteins to interact with SF-1.

As for the DF WT protein, the transcriptional properties of the DF {Delta}T279 and DF C294A proteins were first assessed in heterologous CV-1 cells (Fig. 7Go). Like their wild-type counterpart, the DF {Delta}T279 and DF C294A proteins did not transactivate GATA-dependent reporters due to the lack of transactivation domains and the absence of DNA binding (data not shown). However, in comparison to DF WT, the DF {Delta}T279 and DF C294A proteins were ineffective at competing the GATA-dependent activation of a simple, but highly GATA-responsive, reporter that consisted of two copies of the GATA element of the MIS promoter fused to the unresponsive minimal MIS promoter (Fig. 7AGo). Since the mutated DF proteins retain their ability to interact with SF-1, they were expected to repress the synergy between GATA-4 and SF-1 as effectively as the DF WT protein. Indeed, this is what was observed using two different synthetic reporters that both contain SF-1 binding sites and which exhibit strong GATA-4/SF-1 synergism as previously reported (10). As shown in Fig. 7Go, B and C, the three DF competitors were able to efficiently repress GATA-4/SF-1 synergism regardless of the presence (Fig. 7BGo) or absence (Fig. 7CGo) of a GATA binding site. The decrease in GATA-4/SF-1 synergism was attributable to competition with full-length GATA-4 for protein-protein interaction with SF-1 rather than by a direct disruption of SF-1-dependent transactivation (Fig. 7DGo).



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Figure 7. Transcriptional Properties of the Mutated DF GATA-4 Proteins, DF {Delta}T279 and DF C294A

A, Effects of DF {Delta}T279 and DF C294A on GATA-4-dependent transactivation. Expression plasmids encoding full-length GATA-4, DF WT, DF {Delta}T279, and DF C294A were transfected in CV-1 cells along with 500 ng of a highly responsive GATA reporter consisting of two GATA motifs upstream of the minimal MIS promoter or (D) 500 ng of a reporter consisting of five copies of the SF-1 binding element from the steroid 21-hydroxylase promoter fused to the minimal PRL promoter (80 ). Open bar, Control (empty) vector; gray shaded bar, 50 ng GATA-4; black bars, 50 ng GATA-4 and increasing doses of DF WT (50, 100, 150 ng); hatched bars, 50 ng GATA-4 and increasing doses (50, 100, 150 ng) of DF {Delta}T279; stippled bars, 50 ng GATA-4 and increasing doses (50, 100, 150 ng) of DF C294A. *, Significantly different from the activation mediated by GATA-4 (gray bar) alone; **, significantly different from the activations mediated by GATA-4 alone and GATA-4 in the presence of the first DF WT, DF {Delta}T279, or DF C294A dose. ***, Significantly different from the activations mediated by GATA-4 alone and GATA-4 in the presence of all DF WT, DF {Delta}T279, or DF C294A doses. B and C, The mutated DF GATA-4 proteins, DF {Delta}T279 and DF C294A, repress GATA-4/SF-1 synergism. Expression plasmids encoding full-length GATA-4, SF-1, DF WT, DF {Delta}T279, and DF C294A were transfected in CV-1 cells along with synthetic reporters containing either three copies of an oligonucleotide consisting of the MIS SF-1 and GATA binding sites in their natural context (SF-1: GATA)3 (B), or two copies of the SF-1 binding site from the MIS promoter (C), in both cases fused to the minimal MIS promoter. *, Significantly different from the activation mediated by GATA-4/SF-1 synergism (gray bars); **, significantly different from the activations mediated by GATA-4/SF-1 synergism and GATA-4/SF-1 synergism in the presence of the first DF WT, DF {Delta}T279, or DF C294A dose. D, The two mutated DF proteins do not affect SF-1-dependent transactivation. CV-1 cells were transfected as described in panel A with a highly SF-1 responsive reporter that consists of five copies of the SF-1 binding site from the steroid 21-hydroxylase promoter fused to the minimal PRL promoter (80 ). All data are reported as fold activation (±SEM).

 
Overexpression of DF {Delta}T279 and DF C294A Competitors in Sertoli Cells Reveals the Dual Contribution of GATA-4 to MIS Promoter Activity
Once the DNA-binding and SF-1 interaction properties of the mutated DF proteins were characterized, they were used to study the contribution of an endogenous GATA-4/SF-1 protein interaction to MIS promoter activity in Sertoli cells. When overexpressed in Sertoli cells, the DF {Delta}T279 and DF C294A proteins repressed MIS promoter activity in a dose-dependent manner (Fig. 8Go). Furthermore, this repression was independent of the presence of a GATA binding site (compare Fig. 8Go, panels A and E, with Fig. 8Go, panels B and F). Since we showed that the mutated DF proteins cannot compete for GATA binding to DNA (Fig. 7AGo), their effect on MIS promoter activity was likely mediated by competing a direct protein-protein interaction between GATA-4 and a DNA-bound transcription factor. Consistent with this hypothesis, the two mutated DF proteins still repressed MIS promoter activity when only the Sox9 binding site was removed (data not shown), but they had no significant effects on MIS promoter constructs (-83 bp and -83 mut) that lacked an SF-1 binding site (Fig. 8Go, C, D, G and H). Taken together, these results indicate that a direct interaction between GATA-4 and SF-1 is an additional and essential component of the molecular mechanism through which GATA-4 contributes to MIS promoter activity in Sertoli cells.



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Figure 8. The Mutated DF GATA-4 Proteins, DF {Delta}T279 and DF C294A, Repress MIS Promoter Activity in Sertoli Cells

Primary Sertoli cells were transfected with 500 ng of different wild-type (A, C, E, and G) and GATA-mutated (B, D, F, and H) MIS promoter constructs along with either increasing doses (50, 100, 250 ng) of the DF {Delta}T279 (hatched bars, left panel) or DF C294A (stippled bars) constructs. Mutation of the GATA binding site in the proximal MIS promoter at -75 bp (GATA to GGTA) is depicted by an X. Promoter activities are expressed relative to the activity of the reporters cotransfected with the empty (no DF {Delta}T279 or DF C294A present) vector (±SEM). *, Significantly different from control (no DF {Delta}T279 or DF C294A present); **, significantly different from control and the first DF {Delta}T279 or DF C294A dose; ***, significantly different from control and all DF {Delta}T279 or DF C294A doses.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sexual dimorphism in mammals is initiated by the action of Sry, a Y-chromosome gene that triggers the gonadal primordium in normal XY males to differentiate into a testis. Male sexual development is then controlled by two testicular hormones: testosterone, which is synthesized and secreted by interstitial Leydig cells, and MIS, which is produced by Sertoli cells in the seminiferous cords. MIS regulates sex differentiation by inducing the regression of the Müllerian duct (the precursor to the female reproductive tract) in males. MIS gene expression is tightly regulated during gonadal development; lack of expression causes persistent Müllerian duct syndrome, a condition in which affected males exhibit both male and female internal reproductive organs (72). Due to its sexually dimorphic expression pattern, the MIS gene is an ideal candidate for elucidating transcription factors involved in sex-specific gene expression. However, the transcriptional control of the MIS gene has been somewhat of a conundrum. Although only a short 180 bp of 5'-flanking region of the MIS gene appears to be required for cell- and sex-specific expression in vivo and in vitro (64, 66), we do not yet fully understand the molecular determinants that control its expression. The proximal MIS promoter contains species-conserved regulatory elements, but alone they cannot account for MIS expression in vivo. The proper spatiotemporal expression of the MIS gene must require the combinatorial interaction of several transcription factors including SF-1, Sox9, WT-1, and GATA-4 (30, 68, 69). In the present study, we report the generation and characterization of novel GATA-4 mutant proteins that behave as dominant negative competitors of GATA activity. These competitors, along with GATA-specific cofactor FOG-2, were used to define the molecular mechanism through which GATA-4 contributes to MIS promoter activity in Sertoli cells.

A Dominant Negative Competitor to Study GATA-Dependent Gene Expression
Over the past decade, GATA factors have emerged as a family of critical regulators of several key processes that are essential for the viability of the early developing embryo. Although gene inactivation experiments have undeniably proven crucial roles for GATA factors in early vertebrate development, they have been less useful for the study of their later recruitment as regulators of tissue-specific gene expression in vivo since five of six GATA-/- mice die in utero (17, 18, 19, 20, 21, 22, 23). To overcome this problem and to address the function and mechanism of action of GATA factors in tissue-specific gene expression, we have generated and characterized a truncated GATA-4 protein (DF WT) that behaves as a dominant negative competitor of GATA-dependent transcription. We then used it to elucidate the mechanism by which GATA-4 regulates MIS promoter activity. Dominant negative competitors consisting solely of a DNA-binding domain are clearly important tools that have been successfully used to study the function of transcription factors other than GATA (73, 74). Interestingly, previous attempts to generate such molecules for GATA-3 have failed since the mouse GATA-3 protein, much like the chicken GATA-1 protein, possesses an activation domain located between the two zinc fingers (2, 75). For GATA-3, mutation of this particular activation domain was required for it to act as a dominant negative in vitro and in vivo (75, 76). Unlike GATA-1 and GATA-3, an interfinger activation domain is not present in the GATA-4 protein since our DF WT protein could not activate transcription of GATA-dependent reporters (Fig. 3CGo), despite the fact that it was expressed at similar levels as the full-length GATA-4 protein, was translocated to the nucleus, and efficiently bound DNA (Fig. 3BGo). Moreover, since the DF WT protein not only repressed GATA-4-mediated transactivation but transactivation induced by other members of the GATA family (data not shown), the DF WT protein can be considered as a generalized competitor of GATA activity. Taken together, our data from experiments first performed in heterologous cells confirm that the DF WT protein acts as a dominant negative competitor of GATA-dependent transactivation, thus providing an invaluable tool to study the contribution of GATA factors to the regulation of putative GATA-dependent genes.

Indeed, when tested in our primary Sertoli cell cultures, the DF WT competitor proved to be an efficient dose-dependent repressor of MIS promoter activity, by competing with endogenous GATA-4 for binding to the MIS GATA element (Fig. 5Go). The DF WT-mediated decrease in MIS promoter activity was consistent with the 40% decrease in MIS promoter activity in Sertoli cells when the MIS GATA itself was mutated (Fig. 2Go). Together, these data demonstrate that endogenous GATA-4 contributes to MIS promoter activity in Sertoli cells by directly binding to its site in the MIS promoter. Interestingly, the activity of MIS reporters that harbored a mutated GATA element was also repressed, albeit to a lesser extent than their wild-type counterparts, by the DF WT competitor (Fig. 5Go). The latter suggests that endogenous GATA-4 also contributes to MIS promoter activity in Sertoli cells by a mechanism that is independent of GATA-4 binding to DNA. This mechanism is not dependent on the presence of the Sox9 binding site but absolutely requires an intact SF-1 binding site (again compare Fig. 5Go, panels B and D). The need for an SF-1 binding site invariably suggests that endogenous GATA-4 and SF-1 physically and functionally cooperate in Sertoli cells to control MIS transcription, a notion that is consistent with our previous data in heterologous cells (10). The requirement of GATA-4 for MIS promoter activity in Sertoli cells was further confirmed by using FOG-2, a well characterized repressor of GATA-4-dependent transcription in vitro (48, 49, 50). Since FOG-2 binds the corepressor CtBP2 (50), it was not surprising that FOG-2 was a more active repressor of MIS promoter activity in Sertoli cells than was the DF WT competitor (Fig. 5Go, E–H). Moreover, since we have shown that FOG-2 is coexpressed with GATA-4 in Sertoli cells (Fig. 1Go), the control of GATA-4 activity in these cells must be tightly linked to the expression level of FOG-2.

Novel Molecular Tools to Dissect the Molecular Mechanisms of GATA-Mediated Gene Expression
As exemplified by the role of GATA-4 in MIS transcription, the cell- and promoter-specific activity of the various GATA factors is most often achieved through direct protein-protein interactions with other cell- restricted cofactors (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 28, 43, 44). As previously mentioned, protein-protein interactions involving GATA proteins are often mediated through their highly conserved zinc finger domain. Therefore, to specifically study the DNA binding-independent contribution of GATA-4 to MIS promoter activity, we generated two additional dominant negative proteins (DF {Delta}T279 and DF C294A) that specifically compete for GATA protein-protein interactions without affecting the DNA-binding activity of endogenous GATA factors.

As expected, the mutated DF proteins could not compete GATA-4-mediated transactivation but were as efficient as the wild-type DF protein (DF WT) in competing GATA-4/SF-1 synergism, indicating that the mutated DF proteins do effectively compete with full-length GATA-4 for interaction with SF-1. The fact that an intact SF-1 binding site was absolutely required for the mutated DF proteins to repress MIS promoter activity in Sertoli cells (compare the -180-bp and -83-bp MIS reporters in Fig. 8Go), demonstrate that the mutated DF proteins are efficient competitors of the endogenous GATA-4/SF-1 interaction. Moreover, these results confirm that this interaction is essential for full MIS promoter activity in Sertoli cells. The dual nature of the wild-type vs. the mutated DF proteins is further supported by the fact that when the three DF constructs (WT, {Delta}T279, C294A) were used at similar doses, the mutated DF proteins were less potent than the DF WT protein at repressing activity of the wild-type MIS promoter in Sertoli cells (compare Fig. 5AGo with Fig. 8Go, A and E). This observation is consistent with the fact that the mutated DF proteins compete only the GATA-4/SF-1 interaction and not GATA-4 binding to DNA, whereas the DF WT protein effectively competes both activities.

Dual Contribution of GATA-4 to MIS Transcription in Sertoli Cells
The GATA-4 transcription factor is abundantly expressed in the developing gonads, and its expression pattern closely parallels that of the MIS hormone. Although many elegant studies have provided critical information regarding the transcriptional control of the MIS gene (64, 66, 67, 68, 69), we do not yet fully comprehend how MIS expression is restricted to Sertoli cells in a developmental and sex-specific fashion. By modulating endogenous GATA-4 activity in Sertoli cells, we have shown that GATA-4 contributes to MIS promoter activity through two complementary mechanisms and therefore, GATA-4 constitutes a novel regulator that helps to explain the cell- and sex-specific expression of the MIS gene in vivo. The model presented in Fig. 9Go depicts the dual contribution of GATA-4 to MIS promoter activity. First, direct GATA-4 binding to its site leads to a modest activation of the MIS promoter (Fig. 9AGo). Clearly, GATA binding alone cannot account for the specificity of MIS expression since the Sox9 binding site in the MIS promoter is absolutely required for MIS transcription to initiate in vivo (67). However, it is tempting to speculate that the initiation and low level of MIS expression, which have been reported in the absence of an intact MIS SF-1 promoter element (66, 67), require GATA-4 binding to the MIS promoter or possibly a Sox9/GATA-4 cooperation. The latter would also help to explain the tissue-specific expression of MIS since Sox9 and SF-1 are present in multiple cell lineages where MIS is not. Second, the high level of MIS expression in vivo, which requires an intact SF-1 binding site (66, 67), is consistent with the data presented here showing that high MIS promoter activity can be achieved, at least in part, when GATA-4 directly interacts with SF-1, either when SF-1 alone (Fig. 9CGo) or both factors are bound to their respective sites on the MIS promoter (Fig. 9BGo). It is also possible that two molecules of GATA-4 can simultaneously interact with SF-1 leading to an even stronger transcriptional activation of MIS (Fig. 9DGo). Finally, we showed that FOG-2 is present in primary Sertoli cells and that it can strongly repress the GATA-4-dependent activation of the MIS promoter (Fig. 9EGo). Therefore, the level of FOG-2 expression in Sertoli cells is also likely to be an important player in the combinatorial code of transcription factors required to control MIS gene expression in vivo, in a similar way that Dax-1 represses SF-1-mediated MIS promoter activity (69, 77).



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Figure 9. Dual Contribution of GATA-4 to MIS Transcription and Proposed Mechanism for the Role of FOG-2 in the Control of MIS Gene Expression in Sertoli Cells

Species-conserved elements (Sox9, SF-1, and GATA) in the proximal MIS promoter are indicated by the stippled, shaded, and hatched boxes, respectively. A, GATA-4 alone, by binding to its consensus element in the proximal MIS promoter, leads to weak transcriptional activation (small arrow). B–D, Protein-protein interactions ({equiv}) between GATA-4 and SF-1 result in a marked enhancement in MIS promoter activity (heavy arrow). Three possible scenarios for a GATA-4/SF-1 synergistic interaction are shown. Thus, direct GATA-4 binding to the MIS promoter and the synergistic interaction with SF-1 reflect the dual contribution of GATA-4 to MIS promoter activity. E, The GATA cofactor FOG-2, which interacts with the repressor CtBP2 (50 ), can also associate with GATA-4, leading to transcriptional silencing of the MIS promoter.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The -180, -180 mut, and -83 murine MIS-luciferase promoter constructs were generated by PCR on mouse genomic DNA as described previously (10). The -180 mut construct, which was obtained by site-directed mutagenesis, contains a mutation (GATA -> GGTA) in the consensus MIS promoter GATA element at -75 bp (10). A shorter version of this construct, -83 mut, was obtained by PCR using the -180 mut plasmid as 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). The 2XGATA-MISmin 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 MIS promoter. The -142 bp LHß luciferase reporter was kindly provided by Jacques Drouin (78, 79). A cytomegalovirus-driven expression plasmid for rat GATA-4 was made as described in Ref. 28 . The truncated GATA-4 protein containing only its two zinc fingers (DF WT) was amplified by PCR on the full-length GATA-4 cDNA (forward primer: 5'-CGAAGCTTATGGCGAGACACCCCAATCTCGATATG-3'; reverse primer: 5'-ATGGATCCTTAACCTGCTGGTGTCTTAGATTTATT-3') and cloned into the HindIII/BamHI sites of pcDNA3. The double zinc finger GATA-4 protein containing a deletion of a threonine at position 279 in its second zinc finger (DF {Delta}T279) was generated by site-directed mutagenesis using the pALTER system on DF WT. A second mutated double-fingered GATA-4 protein (DF C294A) was generated by first transferring a HindIII/BamHI fragment obtained from DF WT into pBluescript SK (Stratagene, La Jolla, CA). The resulting plasmid was digested with HindIII/NcoI, thereby liberating the wild-type GATA-4 zinc finger region (the NcoI site is a naturally occurring site immediately downstream of the second zinc finger). The leftover backbone vector was purified, and the GATA-4 zinc finger region was replaced with a similar fragment generated by PCR (amplified on the full-length GATA-4 cDNA using forward primer: 5'-CGAAGCTTATGGCGAGACACCCCAATCTCGATATG-3' and reverse primer: 5'-AACCCCATGGAGCTTCATGTAGAGGCCGGCGGCATTGCAAACAGGCTCGCC-3') but containing a Cys-to-Ala substitution at amino acid 294. A HindIII/BamHI digest of the resulting plasmid produced a fragment that was transferred into pcDNA3 to finally yield DF C294A. The three GATA-4 double-fingered constructs (DF WT, DF {Delta}T279, DF C294A) were also transferred into pRSET (Invitrogen, San Diego, CA) for in vitro35S protein labeling. Additionally, a double-stranded oligonucleotide (sense: 5'-CTAGCTACCCATACGACGTTCCAGATTACGCTT-3'; antisense: 5'-CTAGAAGCGTAATCTGGAACGTCGTATGGGTAG-3') encoding for an HA epitope was N-terminally cloned in frame with each of the three GATA-4 DF constructs. The integrity of all the above mentioned constructs was verified by sequencing. The SF-1 expression plasmid and (SF-1)5-PRL reporter were generously provided by Keith Parker (80). The FOG-2 expression plasmid was kindly provided by Eric Olson (48).

Isolation of Immature Sertoli Cells
Highly enriched populations of neonate Sertoli cells were prepared from 3- to 5-d-old Sprague Dawley rats (Charles River Laboratories, Inc., St-Constant, Quebec, Canada) as previously described (77).

Cell Culture and Transfections
African green monkey kidney CV-1 and mouse fibroblast L cells were grown in DMEM supplemented with 10% newborn calf serum. Primary Sertoli cells were maintained in Eagle’s MEM containing 10% FCS. All transfections were done in 24-well plates using the calcium phosphate precipitation method as previously described (77). The day before transfection, CV-1 and primary Sertoli cells were plated at densities of 2.2 x 104 and 2.0 x 105 cells per well, respectively. Cells were transfected 24 h after the initial plating. Culture media were changed 12–16 h after transfection. The cells were finally harvested the following morning, and an aliquot of the lysate was then assayed for luciferase activity as described elsewhere (28, 77). Several DNA preparations of the plasmids 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.

RNA Isolation and RT-PCR
Total cellular RNA was prepared from rat testis and neonate Sertoli cells by the single-step acid guanidinium thiocyanate-phenol-chloroform method (81). RT-PCR was used to detect the presence of GATA-4, SF-1, MIS, and FOG-2 mRNAs in the primary neonate Sertoli cell cultures. Different first-strand cDNAs were synthesized from total RNA using AMV reverse transcriptase (Amersham Pharmacia Biotech, Baie-D’Urfé, Quebec, Canada). They were then used as templates in the PCR reactions using VENT DNA polymerase (New England Biolabs, Inc., Mississauga, Ontario, Canada) and oligonucleotide primers specific to GATA-4 (forward primer: 5'-CTTCTAGACAACCCAATCTCGATATG-3'; reverse primer: 5'-CAGGATCCAAGTCCGAGCAGGAATTG-3'), SF-1 (forward primer: ACTCTAGAGCGGGCATGGACTACTCG-3'; reverse primer: 5'-CGGGTACCGCACCTTCGTGCCTAGTCG-3'), MIS (forward primer: 5'-GAACCTTTGTGCCTGGTG-3'; reverse primer: 5'-AGGGTCTCTAGGAAGGGGTC-3'), and FOG-2 (forward primer: 5'-CCCTCGAGGGTGACTGCTTTCTTTAGTAACTC-3', reverse primer: 5'-ATGTGCCTACCTGAGCAGGAACA-3'). To verify the specificity of the amplified bands, PCR products were Southern blotted to nylon membrane and hybridized with their respective 32P-labeled cDNAs.

Production of Maltose-Binding Protein (MBP) Fusion Proteins and in Vitro Protein-Protein Binding Assays
A recombinant MBP-SF-1 fusion protein was obtained by cloning the entire coding region of mouse SF-1 in frame with MBP using the commercially available pMAL-c fusion protein vector (New England Biolabs, Inc.). The MBP-LacZ{alpha} fusion protein was produced by the pMAL-c vector without any cloned insert. The two fusion proteins (MBP-SF-1 and MBP-LacZ{alpha}) were produced and purified as previously described (10). In vitro protein-protein interaction studies were done as previously outlined (10).

DNA-Binding and Western Blot Assays
Recombinant GATA proteins (full-length GATA-4, DF WT, DF {Delta}T279, DF C294A, HA-Tag DF WT, HA-Tag DF {Delta}T279, and HA-Tag DF C294A) 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 following transfection by the procedure outlined by Schreiber et al. (82). DNA-binding assays were performed using a 32P-labeled double-stranded oligonucleotide corresponding to the conserved GATA element in the proximal MIS promoter (30). Binding reactions and electrophoresis conditions were as previously described (30). In Western analyses, 40 µg aliquots of nuclear extract containing the three HA-Tag GATA-4 DF proteins were separated by SDS-PAGE and then transferred to Hybond polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Immunodetection of the HA-Tag GATA proteins was achieved using a HA antibody (CLONTECH Laboratories, Inc. Palo Alto, CA) and a Vectastain-ABC-Amp Western blot detection kit (Vector Laboratories, Inc. Burlingame, CA).

Statistical Analysis
Statistical analyses were done by one-way ANOVA, followed by Tukey’s honestly significant difference tests to detect differences between groups. The analyses were done with the aid of the SPSS, Inc. software package (SPSS, Inc., Chicago, IL). P < 0.05 was considered significant.


    ACKNOWLEDGMENTS
 
We thank Keith Parker (mouse SF-1 expression plasmid and 5x SF-1 reporter), Eric Olson (mouse FOG-2 expression plasmid), and Jacques Drouin (-142 bp LHß promoter) for generously providing plasmids used in this study. We also thank Daniel Cyr for providing some rat testis RNA samples.


    FOOTNOTES
 
This work was supported by a grant from the Canadian Institutes of Health Research to R.S.V. J.J.T. is a recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. R.S.V. is a new investigator of the Canadian Institutes of Health Research and Chercheur-Boursier of the Fonds pour la recherche en santé du Québec.

Abbreviations: DF WT, Double finger wild type; FOG, friend of GATA; HA, hemagglutin; MBP, maltose-binding protein; MIS, Müllerian inhibiting substance; SF-1, steroidogenic factor-1.

Received for publication September 1, 2000. Accepted for publication May 24, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Martin DI, Orkin SH 1990 Transcriptional activation and DNA binding by the erythroid factor GF- 1/NF-E1/Eryf 1. Genes Dev 4:1886–1898[Abstract]
  2. Yang HY, Evans T 1992 Distinct roles for the two cGATA-1 finger domains. Mol Cell Biol 12:4562–4570[Abstract]
  3. Omichinski JG, Trainor C, Evans T, Gronenborn AM, Clore GM, Felsenfeld G 1993 A small single-"finger" peptide from the erythroid transcription factor GATA-1 binds specifically to DNA as a zinc or iron complex. Proc Natl Acad Sci USA 90:1676–1680[Abstract]
  4. Merika M, Orkin SH 1995 Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and EKLF. Mol Cell Biol 15:2437–2447[Abstract]
  5. Durocher D, Charron F, Warren R, Schwartz RJ, Nemer M 1997 The cardiac transcription factors Nkx-2.5 and GATA-4 are mutual cofactors. EMBO J 16:5687–5696[Abstract/Free Full Text]
  6. Lee Y, Shioi T, Kasahara H, et al. 1998 The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression. Mol Cell Biol 18:3120–3129[Abstract/Free Full Text]
  7. Gordon DF, Lewis SR, Haugen BR, et al. 1997 Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin ß-subunit promoter. J Biol Chem 272:24339–24347[Abstract/Free Full Text]
  8. Gregory RC, Taxman DJ, Seshasayee D, Kensinger MH, Bieker JJ, Wojchowski DM 1996 Functional interaction of GATA-1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters. Blood 87:1793–1801[Abstract/Free Full Text]
  9. Ono Y, Fukuhara N, Yoshie O 1998 TAL1 and LIM-only proteins synergistically induce retinaldehyde dehydrogenase 2 expression in T-cell acute lymphoblastic leukemia by acting as cofactors for GATA-3. Mol Cell Biol 18:6939–6950[Abstract/Free Full Text]
  10. Tremblay JJ, Viger RS 1999 Transcription factor GATA-4 enhances Müllerian inhibiting substance gene transcription through a direct interaction with the nuclear receptor SF-1. Mol Endocrinol 13:1388–1401[Abstract/Free Full Text]
  11. Nerlov C, Querfurth E, Kulessa H, Graf T 2000 GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood 95:2543–2551[Abstract/Free Full Text]
  12. Rekhtman N, Radparvar F, Evans T, Skoultchi AI 1999 Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. Genes Dev 13:1398–1411[Abstract/Free Full Text]
  13. Morin S, Charron F, Robitaille L, Nemer M 2000 GATA-dependent recruitment of MEF2 proteins to target promoters. EMBO J 19:2046–2055[Abstract/Free Full Text]
  14. Weiss MJ, Orkin SH 1995 GATA transcription factors: key regulators of hematopoiesis. Exp Hematol 23:99–107[Medline]
  15. Simon MC 1995 Gotta have GATA. Nat Genet 11:9–11[Medline]
  16. Molkentin JD 2000 The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem 275:38949–38952[Free Full Text]
  17. Pevny L, Simon MC, Robertson E, et al. 1991 Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349:257–260[CrossRef][Medline]
  18. Kuo CT, Morrisey EE, Anandappa R, et al. 1997 GATA-4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev 11:1048–1060[Abstract]
  19. Molkentin JD, Lin Q, Duncan SA, Olson EN 1997 Requirement of the transcription factor GATA-4 for heart tube formation and ventral morphogenesis. Genes Dev 11:1061–1072[Abstract]
  20. Tsai FY, Keller G, Kuo FC, et al. 1994 An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371:221–226[CrossRef][Medline]
  21. Pandolfi PP, Roth ME, Karis A, Leonard MW, Dzierzak E, Grosveld FG, Engel JD, Lindenbaum MH 1995 Targeted disruption of the GATA-3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat Genet 11:40–44[Medline]
  22. Morrisey EE, Tang Z, Sigrist K, et al. 1998 GATA-6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev 12:3579–3590[Abstract/Free Full Text]
  23. Koutsourakis M, Langeveld A, Patient R, Beddington R, Grosveld F 1999 The transcription factor GATA-6 is essential for early extraembryonic development. Development 126:723–732[Abstract/Free Full Text]
  24. Molkentin JD, Tymitz KM, Richardson JA, Olson EN 2000 Abnormalities of the genitourinary tract in female mice lacking GATA-5. Mol Cell Biol 20:5256–5260[Abstract/Free Full Text]
  25. Zhou Y, Lim KC, Onodera K, et al. 1998 Rescue of the embryonic lethal hematopoietic defect reveals a critical role for GATA-2 in urogenital development. EMBO J 17:6689–6700[Abstract/Free Full Text]
  26. Nichols KE, Crispino JD, Poncz M, et al. 2000 Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA1. Nat Genet 24:266–270[CrossRef][Medline]
  27. Van Esch H, Groenen P, Nesbit MA, et al. K 2000 GATA-3 haplo-insufficiency causes human HDR syndrome. Nature 406:419–422[CrossRef][Medline]
  28. Tremblay JJ, Viger RS 2001 GATA factors differentially activate multiple gonadal promoters through conserved GATA regulatory elements. Endocrinology 142:977–986[Abstract/Free Full Text]
  29. Tamura S, Wang XH, Maeda M, Futai M 1993 Gastric DNA-binding proteins recognize upstream sequence motifs of parietal cell-specific genes. Proc Natl Acad Sci USA 90:10876–10880[Abstract]
  30. Viger RS, Mertineit C, Trasler JM, Nemer M 1998 Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter. Development 125:2665–2675[Abstract/Free Full Text]
  31. Steger DJ, Hecht JH, Mellon PL 1994 GATA-binding proteins regulate the human gonadotropin {alpha}-subunit gene in the placenta and pituitary gland. Mol Cell Biol 14:5592–5602[Abstract]
  32. Dasen JS, O’Connell SM, Flynn SE, et al. 1999 Reciprocal interactions of Pit1 and GATA-2 mediate signaling gradient-induced determination of pituitary cell types. Cell 97:587–598[Medline]
  33. Bossard P, Zaret KS 1998 GATA transcription factors as potentiators of gut endoderm differentiation. Development 125:4909–4917[Abstract/Free Full Text]
  34. van Doorninck JH, van Der WJ, Karis A, et al. 1999 GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei. J Neurosci (Online) 19:RC12
  35. Nardelli J, Thiesson D, Fujiwara Y, Tsai FY, Orkin SH 1999 Expression and genetic interaction of transcription factors GATA-2 and GATA-3 during development of the mouse central nervous system. Dev Biol 210:305–321[CrossRef][Medline]
  36. Gao X, Sedgwick T, Shi YB, Evans T 1998 Distinct functions are implicated for the GATA-4, -5, and -6 transcription factors in the regulation of intestine epithelial cell differentiation. Mol Cell Biol 18:2901–2911[Abstract/Free Full Text]
  37. Mushiake S, Etani Y, Shimada S, et al. 1994 Genes for members of the GATA-binding protein family (GATA-GT1 and GATA-GT2) together with H+/K(+)-ATPase are specifically transcribed in gastric parietal cells. FEBS Lett 340:117–120[CrossRef][Medline]
  38. Ko LJ, Engel JD 1993 DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol 13:4011–4022[Abstract]
  39. Merika M, Orkin SH 1993 DNA-binding specificity of GATA family transcription factors. Mol Cell Biol 13:3999–4010[Abstract]
  40. Blobel GA, Simon MC, Orkin SH 1995 Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins. Mol Cell Biol 15:626–633[Abstract]
  41. Visvader JE, Crossley M, Hill J, Orkin SH, Adams JM 1995 The C-terminal zinc finger of GATA-1 or GATA-2 is sufficient to induce megakaryocytic differentiation of an early myeloid cell line. Mol Cell Biol 15:634–641[Abstract]
  42. Takahashi S, Shimizu R, Suwabe N, Kuroha T, Yoh K, Ohta J, Nishimura S, Lim KC, Engel JD, Yamamoto M 2000 GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies. Blood 96:910–916[Abstract/Free Full Text]
  43. Osada H, Grutz G, Axelson H, Forster A, Rabbitts TH 1995 Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA-1. Proc Natl Acad Sci USA 92:9585–9589[Abstract]
  44. Kawana M, Lee ME, Quertermous EE, Quertermous T 1995 Cooperative interaction of GATA-2 and AP1 regulates transcription of the endothelin-1 gene. Mol Cell Biol 15:4225–4231[Abstract]
  45. Tsang AP, Visvader JE, Turner CA, et al. 1997 FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90:109–119[CrossRef][Medline]
  46. Tevosian SG, Deconinck AE, Cantor AB, et al. 1999 FOG-2: a novel GATA-family cofactor related to multitype zinc-finger proteins friend of GATA-1 and U-shaped. Proc Natl Acad Sci USA 96:950–955[Abstract/Free Full Text]
  47. Fox AH, Liew C, Holmes M, Kowalski K, Mackay J, Crossley M 1999 Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers. EMBO J 18:2812–2822[Abstract/Free Full Text]
  48. Lu JR, McKinsey A, Xu H, Wang DZ, Richardson JA, Olson EN 1999 FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors. Mol Cell Biol 19:4495–4502[Abstract/Free Full Text]
  49. Svensson EC, Tufts RL, Polk CE, Leiden JM 1999 Molecular cloning of FOG-2: a modulator of transcription factor GATA-4 in cardiomyocytes. Proc Natl Acad Sci USA 96:956–961[Abstract/Free Full Text]
  50. Holmes M, Turner J, Fox A, Chisholm O, Crossley M, Chong B 1999 hFOG-2, a novel zinc finger protein, binds the co-repressor mCtBP2 and modulates GATA-mediated activation. J Biol Chem 274:23491–23498[Abstract/Free Full Text]
  51. Svensson EC, Huggins GS, Dardik FB, Polk CE, Leiden JM 2000 A functionally conserved N-terminal domain of the friend of GATA-2 (FOG-2) protein represses GATA-4dependent transcription. J Biol Chem 275:20762–20769[Abstract/Free Full Text]
  52. Tsang AP, Fujiwara Y, Hom DB, Orkin SH 1998 Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG. Genes Dev 12:1176–1188[Abstract/Free Full Text]
  53. Tevosian SG, Deconinck AE, Tanaka M, et al. 2000 FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium. Cell 101:729–739[Medline]
  54. Svensson EC, Huggins GS, Lin H, et al. 2000 A syndrome of tricuspid atresia in mice with a targeted mutation of the gene encoding FOG-2. Nat Genet 25:353–356[CrossRef][Medline]
  55. Crispino JD, Lodish MB, Thurberg BL, et al. 2001 Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes Dev 15:839–844[Abstract/Free Full Text]
  56. Foster JW, Dominguez-Steglich MA, Guioli S, et al. 1994 Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 372:525–530[Medline]
  57. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R 1991 Male development of chromosomally female mice transgenic for Sry. Nature 351:117–121[CrossRef][Medline]
  58. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[Medline]
  59. Pelletier J, Bruening W, Li FP, Haber DA, Glaser T, Housman DE 1991 WT1 mutations contribute to abnormal genital system development and hereditary Wilms’ tumour. Nature 353:431–434[CrossRef][Medline]
  60. Pritchard-Jones K, Fleming S, Davidson D, et al. 1990 The candidate Wilms’ tumour gene is involved in genitourinary development. Nature 346:194–197[CrossRef][Medline]
  61. Wagner T, Wirth J, Meyer J, et al. 1994 Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79:1111–1120[Medline]
  62. Birk OS, Casiano DE, Wassif CA, et al. 2000 The LIM homeobox gene Lhx9 is essential for mouse gonad formation. Nature 403:909–913[CrossRef][Medline]
  63. Koopman P, Münsterberg A, Capel B, Vivian N, Lovell-Badge R 1990 Expression of a candidate sex-determining gene during mouse testis differentiation. Nature 348:450–452[CrossRef][Medline]
  64. Shen WH, Moore CC, Ikeda Y, Parker KL, Ingraham HA 1994 Nuclear receptor steroidogenic factor 1 regulates the Müllerian inhibiting substance gene: a link to the sex determination cascade. Cell 77:651–661[Medline]
  65. Münsterberg A, Lovell-Badge R 1991 Expression of the mouse anti-Müllerian hormone gene suggests a role in both male and female sexual differentiation. Development 113:613–624[Abstract]
  66. Giuili G, Shen WH, Ingraham HA 1997 The nuclear receptor SF-1 mediates sexually dimorphic expression of Müllerian inhibiting substance, in vivo. Development 124:1799–1807[Abstract/Free Full Text]
  67. Arango NA, Lovell-Badge R, Behringer RR 1999 Targeted mutagenesis of the endogenous mouse MIS gene promoter: in vivo definition of genetic pathways of vertebrate sexual development. Cell 99:409–419[Medline]
  68. De Santa Barbara P, Bonneaud N, Boizet B, et al. 1998 Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene. Mol Cell Biol 18:6653–6665[Abstract/Free Full Text]
  69. Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN, Hammer GD, Ingraham HA 1998 Wilms’ tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 93:445–454[Medline]
  70. Watanabe K, Clarke TR, Lane AH, Wang X, Donahoe PK 2000 Endogenous expression of Müllerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites. Proc Natl Acad Sci USA 97:1624–1629[Abstract/Free Full Text]
  71. Omichinski JG, Clore GM, Schaad O, et al. 1993 NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1. Science 261:438–446[Medline]
  72. Rey R, Picard JY 1998 Embryology and endocrinology of genital development. Baillieres Clin Endocrinol Metab 12:17–33[Medline]
  73. Gauthier-Rouviere C, Cai QQ, Lautredou N, Fernandez A, Blanchard JM, Lamb NJ 1993 Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo. Exp Cell Res 209:208–215[CrossRef][Medline]
  74. Zhou T, Cheng J, Yang P, et al. 1996 Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells. J Exp Med 183:1879–1892[Abstract]
  75. Smith VM, Lee PP, Szychowski S, Winoto A 1995 GATA-3 dominant negative mutant. Functional redundancy of the T cell receptor {alpha} and ß enhancers. J Biol Chem 270:1515–1520[Abstract/Free Full Text]
  76. Zhang DH, Yang L, Cohn L, et al. 1999 Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11:473–482[Medline]
  77. Tremblay JJ, Viger RS 2001 Nuclear receptor Dax1 represses the transcriptional cooperation between GATA-4 and SF-1 in Sertoli cells. Biol Reprod 64:1191–1199[Abstract/Free Full Text]
  78. Tremblay JJ, Drouin J 1999 Egr-1 is a downstream effector of GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance LHß gene transcription. Mol Cell Biol 19:2567–2576[Abstract/Free Full Text]
  79. Tremblay JJ, Marcil A, Gauthier Y, Drouin J 1999 Ptx1 regulates SF-1 activity by an interaction that mimics the role of the ligand-binding domain. EMBO J 18:3431–3441[Abstract/Free Full Text]
  80. Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract]
  81. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]
  82. Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid detection of octamer binding proteins with ’mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17:6419–6419[Medline]