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Journal of Molecular Endocrinology (2009) 42 149-160    DOI: 10.1677/JME-08-0089
© 2009 Society for Endocrinology
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The effect of human GATA4 gene mutations on the activity of target gonadal promoters

 Marie France Bouchard1,2,*, Hiroaki Taniguchi1,2,* and Robert S Viger1,2,3

1 Reproduction, Perinatal and Child Health, Centre de Recherche du Centre Hospitalier Universitaire de Québec (CRCHUQ), Room T1-49, 2705 Laurier Boulevard, Québec City, Québec, Canada G1V 4G22 Centre de Recherche en Biologie de la Reproduction (CRBR), Université Laval, Québec City, Québec, Canada3 Department of Obstetrics and Gynecology, Faculty of Medicine, Université Laval, Québec City, Québec, Canada

(Correspondence should be addressed to R S Viger; Email: robert.viger{at}crchul.ulaval.ca)

* *(M F Bouchard and H Taniguchi contributed equally to this work) Back


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Declaration of interest
 Funding
 References
 
GATA transcription factors are crucial regulators of cell-specific gene expression in many tissues including the gonads. Although clinical cases of reproductive dysfunction have yet to be formally linked to GATA gene mutations, they have begun to be reported in other systems. Heterozygous GATA4 mutations have been associated with cases of congenital heart defects. Little is known, however, about the effect of these mutations on gonadal gene transcription. Since individuals carrying these mutations do not appear to suffer from gross reproductive defects, we hypothesized that this might be due to the differential transcriptional properties of the mutant proteins on heart versus gonadal target genes. Five mutations (S52F, E215D, G295S, V266M, and E359X) were recreated in the rat GATA4 protein. Several parameters were used to analyze the transcriptional properties of the mutants: activation of known gonadal target promoters (Star, Cyp19a1, and Inha), DNA binding, and interaction with GATA4 transcriptional partners. Three mutations (S52F, G295S, and E359X) reduced GATA4 transcriptional activity on the different gonadal promoters. With the exception of the G295S mutant, which showed a significant loss of DNA-binding affinity, the decrease in activity of the other GATA4 mutants was not associated with a change in DNA binding. All GATA4 mutants retained their ability to interact and cooperate with their major gonadal partners (NR5A1 and NR5A2) thereby compensating in part for the loss in intrinsic GATA4 transcriptional activity. Thus, unlike the heart, where the GATA4 mutations have deleterious effects, our data suggest that they would have a lesser impact on gonadal gene transcription and function.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Declaration of interest
 Funding
 References
 
GATA4 is a member of the GATA family of zinc finger DNA-binding proteins that recognizes the consensus DNA motif (A/T) GATA (A/G) in the promoter region of target genes (Molkentin 2000). In the reproductive tract, GATA4 is prominently expressed in somatic cells of both the testis and ovary, where it has been shown and/or proposed to regulate the transcription of numerous target genes (Viger et al. 2008). These targets include genes coding for hormones or components of the steroidogenic pathway such as anti-Müllerian hormone (Amh), inhibin subunit {alpha} (Inha), steroidogenic acute regulatory protein (Star), P450 aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), P450 17{alpha}-hydroxylase/17,20 lyase (Cyp17a1), and 3β-hydroxysteroid dehydrogenase/{Delta}5–{Delta}4 isomerase type 2 (HSD3B2; Viger et al. 2008). In gonadal cells, GATA4 activity has been shown to be modulated via cooperative interactions with other transcriptional regulators and/or cofactors (Viger et al. 2008). These include the nuclear receptors steroidogenic factor 1 (SF-1/NR5A1) and liver receptor homolog 1 (LRH-1/NR5A2), which cooperate with GATA4 to enhance transcription from the promoters of many of the GATA4 targets listed above (Viger et al. 2008). GATA4 transcriptional activity can also be modulated by post-translational modifications such as phosphorylation (Viger et al. 2008). Activation of the cAMP/PKA signaling pathway is a major mechanism for conveying the hormone responsiveness of many gonadal genes (Richards 1994). GATA4 can be directly phosphorylated by PKA resulting in a dramatic enhancement of its transactivation potential on many GATA4-dependent gonadal promoters (Tremblay et al. 2002, Tremblay & Viger 2003a).

While human GATA mutations affecting normal reproductive function have yet to be identified, GATA gene sequence variations have begun to be linked to human diseases in other systems. For example, GATA1 mutations have been reported in inherited anemia and thrombocytopenia (Nichols et al. 2000), as well as in Down's syndrome-related acute leukemia (Wechsler et al. 2002). Although no mutation of the GATA2 gene has yet been demonstrated in hematological diseases, it has been reported that decreased GATA2 expression is observed in CD34 positive cells in patients with aplastic anemia (Fujimaki et al. 2001). GATA3 mutations have been associated with HDR syndrome, a genetic disease characterized by hypoparathyroidism, sensorineural deafness, and renal dysplasia (Van Esch et al. 2000). GATA6 frameshift mutations in the DNA-binding domain and C-terminus have been reported to cause malignant astrocytomas (Kamnasaran et al. 2007).

There have now been reports of several GATA4 gene mutations affecting cardiac development and function (Garg et al. 2003, Okubo et al. 2004, Hirayama-Yamada et al. 2005, Nemer et al. 2006, Tang et al. 2006). Most of these mutations occur in amino acid residues highly conserved across species ranging from zebrafish to human, underlying the relative importance of these residues for proper GATA4 function (the characteristics of some of these GATA4 mutations are summarized in Table 1). A missense mutation, G296S, causes a decrease in GATA4 transcriptional activity by weakening its DNA-binding capacity and by disrupting the cooperative interaction with its transcriptional partner TBX5, ultimately leading to reduced expression of target genes in the developing heart (Garg et al. 2003). Another missense mutation, E216D, also causes a reduction in GATA4 transcriptional activity although the DNA-binding ability of the protein and its physical (protein–protein) and functional (transcriptional) interaction with Friend of GATA 2 (FOG2) on cardiac targets are not apparently affected (Nemer et al. 2006). A third mutant, S52F, has also been reported to reduce GATA4 transactivation potential, although its DNA-binding capacity remains intact (Hirayama-Yamada et al. 2005). Similarly, two frameshift mutations, causing the loss of a large portion of the C-terminal end of GATA4, S358X (Okubo et al. 2004), and E359X (Garg et al. 2003), appear to be transcriptionally inactive although the molecular interactions with DNA and/or other proteins have not been investigated. Additional GATA4 mutations have been described: V267M and V380M amino acid substitutions of GATA4 protein have also been related to heart defects (Tang et al. 2006), but the molecular basis for the cardiac phenotypes have yet to be established.


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  		Table 1 Characteristics of the GATA4 mutations studied

 
To date, human GATA4 mutations have been identified based on their deleterious effects on heart development and function. The impact of these mutations on GATA4 function outside of the cardiac system has not been investigated. This includes the reproductive tract where GATA4 has been shown to play a critical role in early gonadal morphogenesis, at least in the mouse (Tevosian et al. 2002, Bouma et al. 2007, Manuylov et al. 2007). In the present study, we have recreated five human GATA4 mutations (listed in Table 1), known to affect heart function, in the rodent protein and tested their functional properties – DNA-binding capacity, transactivation potential, physical and functional interaction with known regulatory partners – on a variety of GATA4-dependent gonadal promoters. Our results show that GATA4-dependent activation of different gonadal promoters can be significantly affected by some of these mutations. However, the different GATA4 mutants retained their ability to interact and cooperate with their major gonadal partners thereby compensating in part for the loss in intrinsic GATA4 transcriptional activity. Thus, unlike the heart where these GATA4 mutations have major deleterious effects, our data suggest that they would have less dramatic consequences on gonadal gene transcription and function.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Declaration of interest
 Funding
 References
 
Plasmids

The murine –902 bp (Star), –218 bp PII aromatase (Cyp19a1), and –679 bp inhibin {alpha} (Inha) promoter constructs have been described previously (Tremblay & Viger 2001). The wild-type GATA4 expression vector has also been reported previously (Tremblay et al. 2001). The different GATA4 human mutations were recreated using the rat GATA4 coding sequence by site-directed mutagenesis. The mutations were introduced according to the manufacturer's instructions using a QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA, USA) and the oligonucleotide primers indicated in Table 2. Expression plasmids for mouse NR5A1 (Lala et al. 1992), NR5A2 (Galarneau et al. 1996), and PKA catalytic subunit (Mayr & Montminy 2001) were kindly provided by Drs Keith Parker (University of Texas Southwestern Medical Center, Dallas, TX, USA), Luc Bélanger (Centre de recherche en cancérologie, Centre de recherche du CHUQ, Université Laval, Québec City, Canada), and Marc Montminy (The Salk Institute for Biological Studies, La Jolla, CA, USA) respectively.


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  		Table 2 Primers used to generate the different GATA4 mutants

 
Cell culture and transfections

African green monkey kidney CV-1 cells and mouse TM3 Leydig cells were used for all transfection studies. As a heterologous line (not expressing GATA or NR5A factors), CV-1 cells are a very convenient line for comparing the transcriptional properties of the different GATA4 mutants presented herein. TM3 cells express endogenous GATA proteins and are representative of a gonadal somatic cell line (Tremblay & Viger 2001). CV-1 cells were grown in DMEM supplemented with 10% newborn calf serum. TM3 cells were cultured in a 1:1 mixture of Ham's F12 and DMEM containing 5% horse serum and 2.5% FBS. HeLa cells, used to overexpress the different GATA4 mutants for western analysis, were grown in DMEM/F12 supplemented with 10% fetal bovine serum. All transfections were done in 24-well plates using the calcium phosphate precipitation method (Chen & Okayama 1987). Cells were plated at an initial density of 2x104 cells/well and transfected 24 h later. Culture medium was changed 12–16 h after transfection, and the cells were finally harvested the following day by lysing the cells in 50 µl lysis buffer (100 mM Tris–HCl (pH 7.9), 0.5% Igepal (Sigma–Aldrich Oakville, Canada), and 5 mM dithiothreitol). An aliquot of the lysate was then assayed for luciferase activity using a Luminoskan Ascent microplate luminometer (Fisher Scientific Limited, Ottawa, Canada) and luciferine (BD Biosciences, Mississauga, Canada) as substrate. In all experiments, the total amount of DNA was kept constant at 2 µg/well using Sp64 (Promega Madison, WI, USA) as carrier DNA. Data reported represent the average of at least three experiments (range of 3–15), each done in duplicate.

Nuclear extracts and western blot

Nuclear extracts were prepared by the procedure outlined by Schreiber et al. (1989). In western analyses, 30 µg aliquots of nuclear extracts from HeLa cells overexpressing GATA4 (wild-type or mutated) were separated by SDS-PAGE then electrotransferred to Hybond polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Baie d'Urfé, Canada). GATA4 proteins were detected using a commercially available GATA4 polyclonal antibody (# sc-1237X, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a VECTASTAIN-ABC-Amp Western blot detection kit (Vector Laboratories Canada, Burlington, Canada).

Electrophoretic mobility shift assay

Recombinant GATA4 wild-type and mutated proteins were in vitro translated using a QuickCoupled TNT in vitro transcription and translation system (Promega). Electrophoretic mobility shift assays (EMSA; DNA-binding) were performed using a 32P-labeled double-stranded oligonucleotide corresponding to the conserved GATA element of the proximal murine Star promoter (Tremblay & Viger 2001). The oligos used were (GATA site is italicized) – sense: 5'-GATCCACTTTTTTATCTCAAGTGA-3', antisense: 5'- GATCTCACTTGAGATAAAAAAGTG-3'). Binding reactions were done using 2 µl of in vitro translated protein in 20 µl buffer (4 mM Tris–HCl (pH 7.9), 24 mM KCl, 0.4 mM EDTA (pH 8.0), 0.4 mM dithiothreitol, 5 mM MgCl2, 10% glycerol, and 100 ng polydeoxyinosinic-deoxycytidylic acid) for 1 h at 4 °C. GATA4-containing complexes were subsequently analyzed by electrophoresis through a 4% non-denaturing polyacrylamide gel in 0.5xTris–borate–EDTA buffer at 200 V for 1.5 h at 4 °C followed by autoradiography.

Production of histidine fusion proteins

Recombinant histidine (HIS)–NR5A1 and HIS–NR5A2 fusion proteins were obtained by cloning the corresponding coding sequences in frame with HIS using the commercially available pRSETB fusion protein vector (Invitrogen, Burlington, Canada). The resulting constructs were introduced into the Escherichia coli strain BL21, and the fusion proteins were produced by inducing the bacterial cultures with isopropyl-1-thio-D-galactopyranoside. After the induction, the bacterial cultures were lysed by sonication and the fusion proteins were purified using a Talon metal affinity resin (BD Biosciences) according to instructions outlined by the manufacturer.

In vitro pull-down (protein–protein interaction) assay

Protein–protein interaction studies were done using 35S-labeled in vitro translated wild-type or mutated GATA4 proteins, and the purified HIS–NR5A1, HIS–NR5A2, or HIS–β-galactosidase (HIS–LacZ) fusion proteins coupled to a Talon metal affinity resin (BD Biosciences). The 35S-labeled GATA4 proteins were obtained using the TNT system from Promega. The pull-down procedure has been described previously (Martin et al. 2005).

Statistical analysis

Statistical comparisons between multiple groups (Figs 2A, 4A and B, and 5A and B) were analyzed by one-way ANOVA followed by a Student–Newman–Keuls test. Where normality and/or equal variance among groups was not met, data were analyzed with equivalent (Kruskal–Wallis ANOVA/Student–Newman–Keuls) non-parametric tests. P<0.05 was considered significant. All statistical analyses were done with the aid of the SigmaStat 3.5 software package (Systat Software Inc., Point Richmond, CA, USA).


Figure 2
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  		Figure 2 The various GATA4 mutations differentially affect the transcriptional properties of the GATA4 protein on different gonadal promoters. (A) The ability of the wild-type (WT) and mutant GATA4 proteins to transactivate the murine Star, aromatase (Cyp19a1), and inhibin {alpha} (Inha) promoters when overexpressed in CV-1 fibroblasts or TM3 mouse Leydig cells. For each cell line, cells were co-transfected with the indicated luciferase reporters along with either an empty expression vector (serving as control) or expression vectors (50 ng) for the different GATA4 proteins as indicated. All promoter activities are reported as fold activation over control (±S.E.M). Groups with similar letters are not significantly different from one another (P>0.05). (B) HeLa cells were transfected with expression vectors encoding WT GATA4 or the different mutants. Western blot analysis shows that all proteins were similarly expressed in isolated nuclear extracts.

 

Figure 5
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  		Figure 5 The ability of NR5A family members to transcriptionally cooperate and physically interact with the GATA4 mutants. (A) Cooperation with NR5A1. (B) Cooperation with NR5A2. CV-1 and TM3 cells were co-transfected with the Cyp19a1 luciferase reporter (which contains binding sites for both GATA and NR5A proteins) along with either an empty expression vector (control) or different combinations of expression vectors for WT or mutated GATA4 (50 ng), NR5A1 (25 ng), and NR5A2 (100 ng) as indicated. All promoter activities are reported as fold activation over control. Promoter activity stimulated by NR5A1 or NR5A2 alone is indicated by the dashed line. For each GATA4 protein tested, an asterisk (*) denotes a statistically significant difference (P<0.05) between the synergism group (gray bar; GATA4 and NR5A factors both present) and groups where the GATA4 (black bar) and NR5A (open bar) factors were used alone. (C) In vitro pull-down assays were used to demonstrate that a physical protein–protein interaction between NR5A factors and GATA4 is retained with the different GATA4 mutants. Input indicates 10% of the labeled protein used in the pull-down assay.

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Declaration of interest
 Funding
 References
 
The GATA4 mutants have reduced transcriptional activity on gonadal promoters

To investigate, the effect of human GATA4 mutations reported to affect heart development and function on the transcriptional regulation of gonadal target genes; we recreated five mutations in context of the highly conserved rat orthologue (Fig. 1 and Table 1). The different GATA4 mutants were first compared with the wild-type protein for their ability to transactivate three known GATA-responsive gonadal promoters (Star, Cyp19a1, and Inha) in heterologous CV-1 fibroblasts that lack endogenous GATA4 expression and TM3 mouse Leydig cells that express endogenous GATA proteins (Fig. 2A). Much like their action on heart-specific promoters (Garg et al. 2003, Okubo et al. 2004, Hirayama-Yamada et al. 2005, Nemer et al. 2006, Tang et al. 2006), the GATA4 G295S, E359X, and S52F mutations also markedly reduced the ability of the GATA4 protein to transactivate the three gonadal promoters in CV-1 cells (Fig. 2A). This effect was intrinsic to the mutant proteins since the mutations themselves did not affect the expression level or the cellular localization of the overexpressed proteins (Fig. 2B). In contrast to these three mutations however, the E215D and V266M mutants retained significant transcriptional activity (Fig. 2A). For the E215D mutant, its transactivation potential was equivalent to the wild-type GATA4 protein. Similar results were also observed in TM3 cells; transcriptional activities were correspondingly lower, however, due to the presence of existing GATA factors in these cells.


Figure 1
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  		Figure 1 Amino acid positions of five known GATA4 heterozygous point mutations associated with human disease. (A) Structure and amino acid sequence of the human GATA4 protein. The locations of the various mutations recreated in this study (S52, E216, V267, G296, and E359) are indicated by black circles. (B) The mutated GATA4 amino acid residues (black boxes) are conserved between human and rodent species. The E359X mutation produces a frameshift truncation; the modified sequence is indicated in bold italic, above and downstream of the mutation. The rodent equivalents of the different GATA4 human mutations are provided in Table 1.

 
DNA-binding affinity of the GATA4 mutants

The reduced transcriptional activity observed for some of the GATA4 mutants suggested that the amino acid modifications involved might directly affect GATA4 DNA binding. To study this possibility, DNA binding of wild-type and mutant GATA4 proteins on the conserved GATA element of the murine Star promoter was compared by EMSA (Fig. 3). Consistent with the initial study characterizing the binding of the mouse orthologue of the human G296S GATA4 mutant protein on a heart-specific promoter GATA element (Garg et al. 2003), the rodent G295S mutant also showed markedly reduced binding compared with the wild-type GATA4 protein on a typical gonadal promoter GATA element (Fig. 3A, left panel). Weak binding of the G295S mutant was still observed even at a fivefold excess of protein (Fig. 3A, right panel), suggesting that impaired DNA binding contributes to the reduced transcriptional activity of this particular mutant. In contrast to the G295S variant, the other GATA4 mutants (E215D, S52F, V266M, and E359X) showed strong DNA binding reminiscent of the wild-type protein (Fig. 3B).


Figure 3
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  		Figure 3 DNA-binding properties of the GATA4 mutants. Electrophoretic mobility shift assays were used to assess the binding of in vitro produced WT or mutated GATA4 proteins to a double-stranded 32P-labeled oligonucleotide corresponding to the consensus GATA element (nucleotide position –63 bp) of the murine Star promoter. (A) Left panel: the G295S mutation reduces DNA binding. Binding of GATA4 WT and G295S mutant proteins are competed with increasing doses (solid triangle) of unlabeled oligonucleotides (molar excesses of 2x, 5x, and 10x). Right panel: the GATA4 G295S mutant shows decreased DNA binding, even at a concentration that is five times higher than that of the WT GATA4 protein. (B) The other GATA4 mutants (E215D, S52F, V266M, and E359X) exhibit DNA-binding capacities similar to the WT GATA protein. The solid triangles indicate increasing amounts (2x, 5x, and 10x) of unlabeled (self) competitor oligonucleotides.

 
PKA-dependent enhancement of GATA4-mediated transactivation

We have previously reported that the transcriptional activity of GATA4 in gonadal cells is enhanced by cAMP/PKA-mediated phosphorylation and that this enhancement is an important mechanism for up-regulating expression of certain hormone-dependent gonadal target genes (Tremblay et al. 2002, Tremblay & Viger 2003a). We therefore surmised that mutations in GATA4 might impair its ability to respond to PKA stimulation. To test this possibility, PKA catalytic subunit was co-transfected with wild-type GATA4 or the different GATA4 mutants (Fig. 4). PKA enhancement of GATA4 activity was assessed on both the Star (Fig. 4A) and Cyp19a1 (Fig. 4B) promoters. As expected, PKA markedly enhanced wild-type GATA4 activity on both promoters in both CV-1 and TM3 cells. Interestingly, PKA overexpression still resulted in a significant enhancement of the activity of most, if not all, of all the GATA4 mutant proteins (Fig. 4A and B). For some of the mutants, PKA-mediated enhancement was even greater than the wild-type GATA4 protein. In CV-1 cells, this was particularly evident for the S52F, G295S, and E359X proteins where transcriptional activity in the absence of PKA was very low. Moreover, PKA was able to raise the activity of these three mutants to the level of the unstimulated wild-type GATA4 protein.


Figure 4
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  		Figure 4 The ability of PKA to enhance the transcriptional properties of the GATA4 mutants on different gonadal promoters. CV-1 and TM3 cells were co-transfected with either a Star (A) or Cyp19a1 (B) luciferase reporter along with either an empty expression vector (control) or expression vectors (50 ng) for WT or mutated GATA4 in the presence or absence of PKA catalytic subunit (50 ng). All promoter activities are reported as fold activation over control (±S.E.M). Promoter activity stimulated by PKA alone is indicated by the dashed line. For each GATA4 protein tested, an asterisk (*) denotes a statistically significant difference (P<0.05) between the stimulated group (+PKA) and both the unstimulated group (–PKA) and PKA alone.

 
Functional cooperation between GATA4 and its transcriptional partners NR5A1 and NR5A2

The nuclear receptor NR5A1 has been shown to be an important transcriptional partner for GATA factors in enhancing the promoter activities of several genes normally expressed in endocrine cells (Viger et al. 2008). More recent studies have extended these findings to include the related factor NR5A2 (Bouchard et al. 2005, Martin et al. 2005, Robert et al. 2006). We therefore tested whether the different GATA4 mutations could disrupt the functional synergism between GATA4 and NR5A1 (Fig. 5A) or between GATA4 and NR5A2 (Fig. 5B) on the Cyp19a1 promoter. The Cyp19a1 promoter is a well-characterized target for GATA4 and NR5A1 or NR5A2 (Tremblay & Viger 2001, Clyne et al. 2002, 2004, Bouchard et al. 2005). As expected, NR5A1 and NR5A2 strongly synergized with wild-type GATA4 in both CV-1 and TM3 cells (Fig. 5A and B). In CV-1 cells, synergism was still observed with four out of the five GATA4 mutants with NR5A1 and all mutants with NR5A2. A similar pattern was observed in TM3 cells except for the S52F, G295S, and E359X mutants. Interestingly, the hG296S mutant that was originally shown to be unable to interact with its cardiac-specific partner TBX5 (Garg et al. 2003), maintained its ability to functionally cooperate with its partners in the context of gonadal gene transcription, at least in CV-1 cells.

The functional cooperation between GATA4 and NR5A factors requires that the proteins contact each other via a direct protein–protein interaction (Bouchard et al. 2005, Martin et al. 2005). In vitro pull-down assays were used to assess whether the different GATA4 mutants could still physically interact with either NR5A1 or NR5A2 (Fig. 5C). In these experiments, NR5A1 and NR5A2 are expressed as HIS-tagged fusion proteins immobilized to a metal resin. The different GATA4 proteins are then expressed as 35S-labeled proteins. Labeled protein retained on the resin indicates an interaction which is visualized on an acrylamide gel. As shown in Fig. 5C (upper panels), both the wild-type and G295S GATA4 proteins were retained by the NR5A fusion proteins. A HIS–LacZ fusion protein was used as a non-specific control. Similar experiments were carried out with the remaining GATA4 mutants that showed interactions similar to the wild-type protein (Fig. 5C, lower panel).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Declaration of interest
 Funding
 References
 
Initial in vitro work characterizing GATA4 target genes in gonadal cells (LaVoie 2003, Tremblay & Viger 2003b, Viger et al. 2004), along with subsequent genetic studies in the mouse (Tevosian et al. 2002, Bouma et al. 2007, Manuylov et al. 2007), has provided strong evidence that the GATA4 transcription factor plays a central role in reproductive function. Despite the lack of direct genetic evidence, the expectation is that this critical role extends to human reproductive function. The paucity of data in the literature describing GATA4 mutations associated with human disease is not unexpected given the early developmental roles that have been ascribed to this factor (Kuo et al. 1997, Molkentin et al. 1997). This notwithstanding, heterozygous GATA4 mutations have been associated with several cases of congenital heart defects (Garg et al. 2003, Okubo et al. 2004, Hirayama-Yamada et al. 2005, Nemer et al. 2006, Tang et al. 2006). Little is known, however, about the molecular basis for these heart phenotypes and even further whether these mutations could compromise GATA4 function in a gonadal context. In the present study, we report the effect of five different human GATA4 mutations (S52F, E216D, V267, G296S, and E359X) known to affect heart function on the transcriptional properties of GATA4 on target gonadal promoters.

To study the different mutations, GATA4 mutants were recreated in the rat orthologous protein. At the outset, it is unlikely that these mutations have a major detrimental effect in the reproductive organs, as they do in the heart, since individuals carrying these mutations appear to be able to procreate (Garg et al. 2003, Okubo et al. 2004, Hirayama-Yamada et al. 2005, Nemer et al. 2006, Tang et al. 2006). Absence of gross phenotypic abnormalities, however, does not necessarily preclude an effect at the molecular level. Indeed, in our analysis of the mutations, three GATA4 mutants (S52F, G295S, and E359X) exhibited markedly weak transcriptional activity, compared with the wild-type protein, on three separate gonadal promoters (Fig. 2A). This effect is similar to what has been demonstrated on cardiac-specific promoters (Garg et al. 2003, Schluterman et al. 2007). The drop in transactivation potential associated with these mutants was not simply due to the amount of protein present, or to impaired cellular localization, since the GATA4 mutants and wild-type protein were expressed at comparable levels in HeLa cell nuclear extracts (Fig. 2B). Interestingly, the G295S mutant was the only one to show a dramatic reduction in DNA-binding affinity on a gonadal GATA promoter element even when used at concentrations five times that of the wild-type protein (Fig. 3A). These findings are consistent with the previously reported data in the heart (Garg et al. 2003), and suggest that the G295S mutant would poorly activate gonadal promoters due to a decrease in DNA binding. The role of the E359X variant remains to be better defined since it is still unclear whether the protein is properly and stably translated in an in vivo context. Based on our in vitro data, we can assume that the 44 nonsense amino acids added to the C-terminal domain of the prematurely truncated GATA4 protein adversely affects its function. The S52F mutant had decreased transcriptional activity (Fig. 2A), but fully retained its DNA-binding capacity (Fig. 3B). Similarly, Schluterman et al. (2007) recently showed that the S52F mutation had decreased transcriptional activity on the heart-specific ANF and {alpha}-MHC promoters. They suggested that the mutation at position 52 altered a serine residue within the GATA4 N-terminal activation domain thereby disrupting the function of this region by changing its structure. Thus, conformational changes induced by the mutations likely explain the reduced transcriptional activity of some of the mutant proteins on both cardiac and gonadal target promoters.

Interestingly, the GATA4 E215D mutant had the same level of activity as the wild-type protein on gonadal promoters (Fig. 2A). This is different from the heart where the same mutant had reduced transcriptional activity on the ANF promoter without changes in DNA-binding ability or cellular localization of the protein (Nemer et al. 2006). These results are not a complete surprise since an engineered GATA4 mutation (E215K), targeting the same amino acid and leading to the loss of interaction between GATA4 and FOG2, also retained strong transcriptional activity on different gonadal promoters (Robert et al. 2002). The differential activation elicited by the E215D mutant is then most likely due to differences in promoter context. Differential promoter sensitivity was also observed in the present study where the V266M mutant fully activated the Star promoter but had reduced activity on the Cyp19a1 and Inha promoters (Fig. 2A). Therefore, with respect to the E215D mutant, important differences in the way the mutated protein behaves at a molecular (i.e., transcriptional) level might be the reason why the heart, from a phenotypic standpoint, is more sensitive to the E215D mutation.

The E216D, V266M, and G295S mutations are located in close vicinity to the GATA4 zinc finger domains that are essential for DNA binding (Fig. 1A). The zinc finger region of GATA proteins, including GATA4, also mediates important protein–protein interactions with other transcriptional regulators that help define the proper spatiotemporal expression of target genes (Molkentin 2000, Viger et al. 2008). Indeed, in the heart, Garg et al. showed that the probable molecular basis for the pronounced cardiac phenotype associated with the G295S mutation involved a failed protein–protein interaction with TBX5, a transcription factor responsible for normal heart development and function (Garg et al. 2003). In endocrine cells, we and others have shown that the nuclear receptors NR5A1 and NR5A2 are important transcriptional partners for GATA4 (Viger et al. 2008). Although NR5A1 is expressed in the heart, at least at the transcript level (Ramayya et al. 1997, Nishimura et al. 2004), there is currently no evidence for a functional role for this receptor as a transcriptional partner for GATA4 in this tissue. Unlike TBX5 in the heart (Garg et al. 2003), the functional synergism between the G295S mutant and its gonadal partners NR5A1 and NR5A2 was not compromised (Fig. 5A and B). The G295S mutant likely retains its ability to transcriptionally cooperate with NR5A factors because it can still physically interact with the two receptors (Fig. 5C). Similar to the G295S mutant, the E215D variant could also transcriptionally cooperate with NR5A1 and NR5A2 (Fig. 5). Since this amino acid substitution does not impair the interaction of GATA4 with FOG2 (Nemer et al. 2006), the E215 residue is likely important for the recruitment of a yet unidentified transcriptional partner, essential for proper heart tube formation, but not necessarily required for gonadal development and function. Thus, again, the observation that the mutant GATA4 proteins partially retained some of their transcriptional properties on target gonadal promoters provides some molecular basis as to why these mutations do not appear to cause any pronounced phenotypic defects in the reproductive tract but do so in the heart. It also stresses the importance of specific protein–protein interactions for the regulation of GATA4 activity in different cellular contexts. In contrast to the gonads, the heart might simply be more sensitive to disruptions in GATA4 function.

Another possibility for the lack of reproductive phenotypes associated with reported GATA4 mutations might be redundancy. Sertoli cells of the testis, for example, express multiple GATA factors including GATA1, GATA4, and GATA6 (Viger et al. 2004). In this case, loss of GATA4 function might be compensated by either GATA1 or GATA6. The likelihood of compensatory mechanisms for GATA function in the gonads is supported by at least one genetic study in the mouse which showed that disruption of GATA1 function in Sertoli cells produces no major gonadal defect, most likely due to compensation by other GATA family members (Lindeboom et al. 2003). Yet another possibility is the fact that although some of the GATA4 mutants exhibited a significant reduction in transcriptional activity on different gonadal target (Fig. 2A), many of these proteins showed a marked enhancement of activity in the presence of PKA to levels approaching or even surpassing the unstimulated wild-type protein (Fig. 4).

Taken together, our data suggest that the human GATA4 mutations that have been associated with severe congenital heart abnormalities would likely have less dramatic consequences on gonad-specific gene transcription, since the mutant proteins retain many of their transcriptional properties on different GATA4-dependent gonadal target promoters. During the course of this study, more germ line heterozygous GATA4 mutations have since been reported (Rajagopal et al. 2007, Reamon-Buettner et al. 2007, Tomita-Mitchell et al. 2007). Interestingly, some of these occur in the GATA4 intronic sequences or the 3' non-translated region of the GATA4 mRNA. These specific mutations leave the coding sequence unaltered and have been predicted to affect RNA folding, probably leading to its decay (Reamon-Buettner et al. 2007). Thus, far, only fully penetrant germ line GATA4 mutations have been reported as a result of screening large families where congenital heart defects are present in more than one generation. Recently, the screening of large cohorts of individuals presenting cardiac malformations uncovered the first non-fully penetrant GATA4 mutations (Rajagopal et al. 2007, Tomita-Mitchell et al. 2007). Given the identification of these novel GATA4 mutations, a similar screen of individuals presenting reproductive abnormalities, and especially that involving gonadal sex reversal is both warranted and needed.


   Declaration of interest
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Declaration of interest
Funding
 References
 
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Declaration of interest
 Funding
References
 
R S Viger is holder of the Canada Research Chair in Reproduction and Sex Development. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to R S Viger.


   Acknowledgements
 
We thank Drs Keith Parker (SF-1 expression vector), Luc Bélanger (LRH-1 expression vector), and Marc Montminy (PKA catalytic {alpha} subunit expression vector) for generously providing plasmids used in this study.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Declaration of interest
 Funding
 References
 
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Received in final form 30 October 2008
Accepted 13 November 2008
Made available online as an Accepted Preprint 13 November 2008




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