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research-article |
Ontogeny-Reproduction Research Unit,5 Centre de Recherche du Centre Hospitalier Universitaire de Québec, Centre de Recherche en Biologie de la Reproduction, Department of Obstetrics and Gynecology, Laval University, Québec City, Québec, Canada G1K 7P4
Faculty of Veterinary Medicine,6 University of Montreal, St-Hyacinthe, Québec, Canada J2S 7C6
ABSTRACT
The GATA4 transcription factor is an important developmental determinant for many organs, such as the heart, gut, and testis. Despite this pivotal role, our understanding of the transcriptional mechanisms that control the proper spatiotemporal expression of the GATA4 gene remains limited. We have generated transgenic mice expressing a green fluorescent protein (GFP) marker under the control of rat Gata4 5' flanking sequences. Several GATA4-expressing organs displayed GFP fluorescence, including the heart, intestine, and pancreas. In the gonads, while GATA4 is expressed in pregranulosa, granulosa, and theca ovarian cells, and Sertoli, Leydig, and peritubular testicular cells, the first 5 kb of Gata4 regulatory sequences immediately upstream of exon 1 were sufficient to direct GFP reporter expression only in testis and, specifically, in Sertoli cells. Onset of GFP expression occurred after Sertoli cell commitment and was maintained in these cells throughout development to adulthood. In vitro studies revealed that the first 118 bp of the Gata4 promoter is sufficient for full basal activity in several GATA4-expressing cell lines. Promoter mutagenesis and DNA-binding experiments identified two GC-box motifs and, particularly, one E-box element within this –118-bp region that are crucial for its activity. Further analysis revealed that members of the USF family of transcription factors, especially USF2, bind to and activate the Gata4 promoter via this critical E-box motif.
developmental biology, gene regulation, Sertoli cells, testis
The GATA family of transcription factors encodes zinc finger DNA-binding proteins that recognize the consensus DNA sequence WGATAR, which is found in the regulatory region of several genes required for the differentiation and/or morphogenesis of numerous vital organs. Among these factors, GATA4 is a major developmental determinant of several organs, especially the heart [1, 2]. Indeed, Gata4 knockout mice (Gata4tm1Jml/Gata4tm1Jml and Gata4tm1Eno/Gata4tm1Eno) die by Embryonic Days 7.5–9.5 (e7.5–e9.5) from cardiac dysgenesis, whereas transgenic overexpression of GATA4 in the heart (Tg(Gata4)1Jmol) results in cardiac hypertrophy [3]. Besides the heart, GATA4 is expressed in multiple ventrally located organs, such as the liver, intestine, stomach, pancreas, gonads, and adrenals, with the highest levels occurring most often during fetal life [4–11]. Using the zebrafish as model, an in vivo role for GATA4 in some of these other target organs also has recently been demonstrated [12]. The ever-expanding list of GATA4 target genes also illustrates the pivotal role of this factor in the molecular cascade of events leading to organogenesis during fetal development and proper organ function later in life [13–16].
Reproductive tissues such as testis and ovary are prominent sites of GATA4 expression. GATA4 expression in the incipient genital ridges is highly conserved between the mouse [6, 10], rat [17], human [18], pig [19], and chick [20]. Although GATA4 is present early in the genital anlage of both sexes, its role in the male gonad has so far been better defined because of its capacity to regulate certain male-specific genes, such as Amh/Mis and Dmrt1 [10, 21]. Because of the early embryonic lethality of Gata4–/– mice [1, 2], no insights into gonad morphogenesis can be ascertained from these animals. A later study by Tevosian et al., which used a targeted knock-in mutation of the Gata4 gene (Gata4tm1Sho/Gata4tm1Sho) to produce a mutant GATA4 protein that cannot bind to Friend of GATA (FOG) cofactors, has shown that a functional GATA4 protein is indeed required for proper testis morphogenesis [22]. In the testis, GATA4 also has been suggested to play an important role in the regulation of steroidogenesis via its ability to regulate the promoter activities of several genes (STAR, CYP11A1, HSD3B2, and CYP19A1) involved in this process [23–25].
The gene regulation of the different GATA family members appears to be highly complex [26]. Features common to GATA genes include the absence of a TATA box, the presence of alternative untranslated 5' exons, and tissue-specific expression via distant enhancer regulatory modules [26]. To date, although many GATA4 target genes have been identified, and despite the importance of this factor in many developmental processes, surprisingly little is known about the factors and/or mechanisms that regulate the GATA4 gene itself. In zebrafish, 14.8 kb of gata4 upstream region has been shown to be sufficient to drive transgene expression in the heart, pharyngeal endoderm, and liver [27], whereas in the mouse, a short enhancer sequence 40 kb upstream of the Gata4 transcription initiation site recently has been reported to direct expression to the embryonic liver [28]. Apart from this lone mouse study, the promoter sequences responsible for directing Gata4 expression to its other characteristic sites during development remain undefined. We now show that 5 kb of regulatory sequences upstream of its first exon are sufficient to direct reporter gene expression in Sertoli cells of the testis throughout ontogeny.
A genomic fragment containing the Gata4 promoter region was obtained by screening a rat genomic library (Clontech Laboratories, Mountain View, CA) with a probe generated against the 5' untranslated region of the rat Gata4 gene. A 13-kb SalI genomic fragment, located within rat genomic contig NC_005114, was obtained and subsequently characterized by enzymatic digestion and sequencing. A 1000-bp Gata4 promoter fragment (–1000 to +85 bp) first was obtained by PCR using the original genomic clone as a template and the primer pair described in Table 1. The amplified product was subcloned into the BamHI/KpnI sites of a modified pXP1 luciferase reporter vector, as previously described [29]. Promoter deletions to –471 bp and –222 bp were obtained by SacI and RsaI digests of the 1000-bp construct, respectively. The remaining 5' deletions were generated by PCR using the 1000-bp construct as a template and the primers listed in Table 1. The longest 5-kb Gata4 promoter fragment was obtained by subcloning a 4-kb-plus fragment, which was obtained via a SacI digest of the original genomic clone, into the unique SacI site present in the –471-bp construct. Gata4 promoter constructs containing mutations in specific regulatory motifs were obtained by site-directed mutagenesis using a QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) and the primers listed in Table 1. All mutations were confirmed by sequencing. The USF1 and USF2 expression plasmids were kindly provided by Dr. Michèle Sawadogo (University of Texas M.D. Anderson Cancer Center, Houston, TX).
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The 5-kb rat Gata4 promoter fragment was cloned upstream of a green fluorescent protein (GFP) reporter. The GFP transgene was linearized as a 6-kb NotI fragment and was purified at a concentration of 1 ng/ml in TE buffer (5 mM Tris, 0.1 mM EDTA, pH 7.4) in preparation for pronuclear microinjection. Single-cell purebred FVB/N embryos were recovered and injected with the transgene using standard methods [30, 31]. To aid in the visual identification of transgenic animals, a tyrosine minigene construct was co-injected as previously described [32]. In total, 10 independent lines of transgenic mice were generated based on pigmentation in adult animals and on the presence of fluorescence in embryos and neonatal animals. Because fluorescence patterns between lines were qualitatively similar but varied in intensity of fluorescence, two lines that showed stronger expression (lines 2 and 6A) were retained for further studies. Based on cosegregation fluorescence and pigmentation markers between generations, the two transgenes had co-integrated into the genomes of both these lines; the site of integration for line 6A was autosomal, whereas the site of integration for line 2 was on the X chromosome. The work presented in this study was generated from line 6A. Generation of mouse lines and characterization of phenotypes (as described below) were performed in accordance with the University of Montreal institutional guidelines for animal use in research.
Tissue Collection and Processing
For embryo studies, transgenic or wild-type FVB/N mice (purchased from The Jackson Laboratory, Bar Harbor, ME) were mated overnight, and noon of the day that a vaginal plug was observed was designated as e0.5. Dissected organs or embryos were observed immediately under a standard fluorescence stereomicroscope and then either processed for further immunohistochemical analysis or placed in RNAlater solution (Ambion, Austin, TX) and stored at –80°C for subsequent RNA or protein extractions. For the immunohistochemical analyses, embryos or organs were fixed in 4% paraformaldehyde-PBS (pH 7.2) at 4°C for 1 h to overnight. Tissues then were either embedded in paraffin and cut into 7-µm-thick sections or cryoprotected in 18% sucrose in PBS, embedded in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN), cut into 8-µm-thick sections, and mounted onto 3-aminopropyltriethoxysilane (Sigma-Aldrich, Mississauga, ON, Canada)-treated glass slides.
Whole-Mount Gene Expression Analyses
Whole-mount in situ hybridizations were performed as previously described [33]. Digoxygenin-labeled probes were synthesized using a DIG RNA labeling kit from Roche (Roche Diagnostics Canada, Laval, QC, Canada), according to the manufacturer's instructions. The Gata4 probe was synthesized from a PCR product corresponding to the 3' end of the open reading frame and was subcloned into pGEM-T vector (Promega, Madison, WI). The GFP probe was generated from the whole EGFP cDNA (Clontech) subcloned into pBluescript (Stratagene). The Sry probe was generated from the Sry cDNA subcloned into pGEM-T vector and linearized with HindIII, thus excluding the HMG box. Images were taken with a Nikon DXM1200 digital camera mounted on a Leica MZFLIII stereomicroscope.
Sections were treated for antigen retrieval, blocked with 10% horse serum, and finally incubated overnight at 4°C with primary antibody diluted in blocking solution (PBS containing 0.1% BSA). Primary antibodies were directed against GFP (1:2000 dilution; catalog no. RDI GRNFP4abr; RDI division of Fitzgerald Industries, Concord, MA), GATA4 (1:1000; catalog no. sc-1237X; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Müllerian hormone (AMH/MIS; 1:1000; catalog no. sc-6886; Santa Cruz Biotechnology), or PECAM/CD31 (1:200; BD Biosciences, Mississauga, Canada). After washing in PBS, sections were incubated for 45 min with either horseradish peroxidase-conjugated anti-rabbit antibody (Envision+ system; DAKO, Mississauga, ON, Canada), biotinylated anti-goat antibody (1:500; Vector Laboratories, Burlington, ON, Canada), anti-rat antibody (1:200; Vector Laboratories), and finally 30 min with a peroxidase-conjugated streptavidin-horseradish complex (LSAB+ Kit; DAKO). The reaction product was developed using 3,3'-diaminobenzidine tetrahydrochloride (DAKO). Sections were counterstained with hematoxylin and mounted with glycerol-gelatin (Sigma-Aldrich). For negative controls, primary antibody was omitted. For double immunolabeling, frozen sections were incubated overnight with GFP antibody (1:1000), followed by sequential incubations with anti-rabbit Ig-Alexa Fluor 546 secondary antibody (1:500; Invitrogen, Burlington, ON, Canada), anti-GATA4 antibody (1:500), and anti-goat Ig-Alexa Fluor 488 secondary antibody (1:300; Invitrogen). GFP was detected after PECAM immunohistochemistry with the GFP antibody (1:1000), followed by anti-rabbit Ig-Alexa Fluor 546 secondary antibody (1:500). Fluorochrome-labeled sections were mounted in vectashield containing DAPI (Vector Laboratories). Slides were analyzed with a Zeiss Akioskop II epifluorescence microscope (Carl Zeiss Canada, Toronto, ON, Canada) connected to a digital camera (Spot RT Slider; Diagnostic Instruments, Sterling Heights, MI).
Cell Culture and Transfection Assays
Mouse MSC-1 Sertoli cells [34] (provided by Dr. Michael Griswold, Washington State University, Pullman, WA), rat DC3 granulosa cells [35] (provided by Dr. Riaz Farookhi, McGill University, Montreal, ON, Canada), and human HeLa epithelial carcinoma cells (ATCC, Manassas, VA) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Monkey CV-1 fibroblast cells (ATCC) were grown in DMEM containing 10% newborn calf serum. Mouse MA-10 Leydig cells [36] (provided by Dr. Mario Ascoli, University of Iowa, Iowa City, IA) were cultured in Waymouth MB752/1 medium (Sigma-Aldrich) supplemented with 15% horse serum. Cell transfections were performed in 24-well plates using the calcium phosphate precipitation method [37]; phRL-TK Renilla luciferase vector (20 ng per well) was used as internal control, and the Dual Luciferase Reporter Assay System (Promega) was used to measure luciferase activities. For the USF1/2 siRNA knockdown experiment, MSC-1 Sertoli cells were seeded in 24-well plates and then transfected in serum- and antibiotic-free medium using 5 µl Lipofectamine 2000 reagent (Invitrogen) along with 0.5 µg of –222-bp Gata4 promoter-luciferase reporter and 36 nM of either control siRNA (catalog no. sc-44230; Santa Cruz Biotechnology) or siRNAs for mouse USF1 or USF2 (catalog nos. sc-36784 and sc-36785; Santa Cruz Biotechnology). After 3 h of incubation, the medium was changed, and the cells were incubated for an additional 36 h and then finally harvested and analyzed for luciferase activity as described above. For all experiments, the data reported represent the average of at least three experiments, each done in duplicate.
Nuclear Extracts, Western Blot, and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared using the procedure outlined by Schreiber et al. [38]. Recombinant proteins were either in vitro translated using a T7 QuickCoupled TNT system (Promega) or overexpressed via transfection of HeLa cells. In the Western blot analysis, 30 µg adult tissue protein, 20 µg cell nuclear extract, and 2 µl in vitro-produced protein were separated by SDS-PAGE and transferred to Hybond polyvinylidene difluoride membrane (GE Healthcare Bio-Sciences, Baie-d'Urfé, QC, Canada). USF2 proteins were detected using commercial antisera (catalog. nos. sc-8983X and sc-862X, respectively, Santa Cruz Biotechnology) and a VECTASTAIN-ABC-Amp Western blot detection kit (Vector Laboratories). Electrophoretic mobility shift assay (EMSA) assays using 10 µg nuclear extract or 1 µl TNT recombinant protein were performed as previously described [39]. The probes and competitor oligonucleotides are listed in Table 1. Supershift experiments were performed by adding 2 µg of either USF1 or USF2 antiserum to the binding reactions.
The Proximal Gata4 Promoter Exhibits Sexually Dimorphic Activity in the Developing Gonads In Vivo
In order to define the regulatory regions of the Gata4 gene necessary to reproduce its endogenous expression, we generated transgenic mice expressing GFP under the control of a 5-kb genomic fragment upstream of exon 1 of the rat Gata4 gene (Fig. 1). The rat Gata4 5' flanking sequence, which is 81% identical to the mouse, was obtained by screening a rat genomic library using a portion of the 5' untranslated region of the Gata4 cDNA. One 13-kb positive clone was isolated from 5 x 105 independent clones and found to contain more than 5 kb of Gata4 promoter sequence (Fig. 1). This promoter region was inserted upstream of the GFP fluorescent visual marker and used to generate several independent lines of transgenic mice. Several GATA4-expressing organs displayed GFP fluorescence, including heart, intestine, and pancreas (data not shown). As shown in Figure 2, macroscopic views of mouse gonads at different developmental timepoints revealed that the 5-kb Gata4 promoter region is sufficient to direct reporter gene expression in the male gonad from e11.5 to adulthood. The fluorescence appeared to be confined to the growing testicular cords beginning as early as e12.5 and then to the seminiferous tubules of the postnatal testis. After birth, although fluorescence emitted by the whole organ seemed to decrease, closer observation of the tubules by confocal microscopy confirmed the persistence of fluorescence (see P0 in Fig. 2). In immature (3-wk) and adult (4-mo) testes, fluorescence extended from the base of the seminiferous epithelium toward the tubule lumen, reminiscent of Sertoli cell morphology. Interestingly, GFP fluorescence exhibited a striking sexual dimorphism, with no expression apparent in either fetal (Fig. 2) or adult ovaries (data not shown).
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Onset of GATA4 Promoter-Driven GFP Reporter Expression Requires a Male Environment
We precisely timed the onset of transgene expression in embryos staged by tail somite (TS) count (Fig. 3). While endogenous Gata4 expression appeared at the very beginning of urogenital ridge thickening in 33 somite embryos (approximately e9.75) of both sexes (Fig. 3A), expression of the GFP transgene (assessed by whole-mount in situ hybridization) initiated somewhat later, at the TS15 stage (approximately e11.0–e11.25), and only in the developing male gonad (Fig. 3G). This coincided with the onset of testis commitment orchestrated by expression of the testis-determining gene Sry (Fig. 3, B–D). Although GFP mRNA could be detected as early as TS15, GFP protein (visualized by fluorescence) only became apparent by TS17 (Fig. 3L). By TS20 (approximately e12.0), both GFP mRNA and protein were robustly detected. Thus, the activity of the 5-kb Gata4 promoter in the male gonad initiated after pre-Sertoli cells were committed to a Sertoli cell fate.
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Transgene Expression in the Testis Is Specific to Sertoli Cells
To investigate whether the observed GFP fluorescence was expressed in the appropriate GATA4-expressing cell types in the testis, we performed a comparative immunohistochemical analysis of endogenous GATA4 and GFP expression during fetal and postnatal testicular development (Figs. 4 and 5). At e11.5 GATA4 protein was found throughout the male gonad, with the exception of germ cells, whereas GFP staining was present in the core and absent from the surface of the testicular primordium (Fig. 4A). By e13.5, when Sertoli cells had aggregated into testicular cords, as shown by AMH staining, GFP could be detected inside the cords. The GFP staining profile was highly similar to that of AMH, suggesting that the transgene was expressed in Sertoli cells. Double immunolabeling for GFP and GATA4 or with PECAM, which specifically stains germ and endothelial cells, confirmed that GFP expression indeed was limited to Sertoli cells (Fig. 4B). Interestingly, the GFP staining pattern did not entirely overlap with GATA4, since numerous GATA4-positive/GFP-negative cells were present in between the forming cords and at the surface of the fetal testis (see e13.5 and e17.5 in Figs. 4A and 5). At birth, although macroscopic detection of GFP immunofluorescence at the whole-organ level appeared to be decreasing (Fig. 2), testicular cords maintained GFP expression that could be easily detected at the cytologic level (Fig. 5). Indeed, in both the neonate and prepubertal testis, GFP fluorescence and immunoreactivity remained confined to Sertoli cells, closely resembling the AMH staining profile (Fig. 5 and data not shown). This Sertoli cell-specific pattern was maintained in all tubules of the young adult (5-wk-old) testis but contrasted with the endogenous GATA4 protein, which is characteristic of both Sertoli, Leydig, and peritubular cells (Fig. 5). Finally, in the older adult testis (observed at 8 mo), whereas all Sertoli cells continued to express GATA4 protein, GFP staining became progressively restricted to a subpopulation of Sertoli cells but, much like the endogenous GATA4 protein, did not correlate with any specific stage of spermatogenesis (data not shown).
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In the female, further investigations at the immunocytochemical level revealed that whereas GATA4 could be detected in somatic cells from the onset of ovarian primordium organization (Fig. 4A) through to the adult ovary (data not shown), neither GFP fluorescence nor immunoreactivity could be detected at any age.
Thus, taken together, 5 kb of Gata4 5' flanking sequence appears to be sufficient to direct reporter expression to the Sertoli cell lineage throughout development but not to other gonadal cell types (ovary, Leydig, and peritubular) that also normally express GATA4.
Conserved GC- and E-Box Elements Are Crucial for Basal Gata4 Promoter Activity
In order to define the minimal region required for Gata4 promoter activity, we constructed a series of 5' deletions linked to the luciferase reporter gene (Fig. 6). When transfected in either homologous GATA4-expressing gonadal cells lines (MSC-1, MA-10, DC3) or heterologous GATA4-negative fibroblast cells (CV-1), the full-length 5-kb promoter fragment was as active as the shorter –118-bp construct, suggesting that regulatory elements located between 5 kb and 118 bp are dispensable for basal Gata4 promoter activity (Fig. 6). Although certain deletion constructs appeared to be more active than the –118-bp fragment in some of the cell lines tested, these proved not to be significantly different. Therefore, the regulatory elements required for basal Gata4 promoter activity are present in the first 118 bp. Sequence analysis of this region revealed the presence of several species-conserved regulatory motifs tightly clustered between nucleotide positions –106 and –85 bp: two GC-box elements, a binding site for NR5A family nuclear receptors (SF-1 or LRH-1), and an E-box element (Fig. 7).
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The functional relevance of these individual elements then was studied by complementary mutagenesis and EMSA studies (Figs. 8 and 9). As shown in Fig. 8, site-directed mutagenesis of either the distal or proximal GC-box motif significantly decreased Gata4 promoter activity in the homologous MSC-1 Sertoli cell line. Simultaneous mutation of both GC-box motifs had an additive effect similar to the gross deletion of these elements (compare with the –94-bp construct in Fig. 8). In contrast to the individual GC-box mutations, mutation of the conserved E-box motif dramatically decreased basal promoter activity, highlighting the essential nature of this site. Mutation of this crucial E-box element along with the GC-box motifs further decreased promoter activity approaching the level seen when all three sites were deleted (compare with the –73-bp construct in Fig. 8). Finally, mutation of the potential NR5A binding site had no significant effect. Interestingly, the effects of the mutations were similar in other gonadal cell lines (MA-10, DC3) as well as in heterologous CV-1 fibroblasts, suggesting that the factors binding to these sites are ubiquitous in nature.
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To begin to identify the factors that bind to these regulatory elements, EMSA assays were performed (Fig. 9, A and B). Using an oligonucleotide probe encompassing both the distal and proximal GC-box elements (–114 to –90 bp; Fig. 9A), a protein/DNA complex formed with nuclear extracts prepared from MSC-1 cells (lane 2 in Fig. 9A). Binding also was observed with nuclear extracts prepared from the other gonadal cell lines (MA-10, DC3), as well as CV-1 fibroblasts (data not shown). Binding also was specific for the GC-box motifs, since competition with excess unlabeled probe, but not one containing a GC-box mutation, displaced binding (lanes 3–6 in Fig. 9A). Competition with a mutated NR5A element confirmed that the observed binding was not specific for the adjacent NR5A motif (lanes 7 and 8 in Fig. 9A).
A similar EMSA approach was used to assess binding to the E-box site. As shown in Fig. 9B, one major complex in MSC-1 nuclear extracts strongly bound to the E-box-containing probe (–96 to –78 bp) . This binding was specific for the E-box motif, since binding could be competed with unlabeled oligonucleotide (lanes 7 and 8 in Fig. 9B) but not with oligonucleotides containing mutated E-box sequences (lanes 11–14 in Fig. 9B). Once again, binding was not competed with a mutated NR5A sequence (lanes 9 and 10 in Fig. 9B), thereby confirming the inactivity of this site. Similar results were obtained with nuclear extracts from other gonadal cell lines (data not shown). Supershift experiments using antisera against either USF1 or USF2 (lanes 2 and 3 in Fig. 9B) suggest that these factors are the two major proteins present in the E-box binding complex. Western blot confirmed that MSC-1 and other gonadal cells do at least express these factors (Fig. 9C). Mutation of the E-box element in the context of the entire 5-kb Gata4 promoter (Fig. 9D), which resulted in a decrease of more than 75% in promoter activity, confirmed the critical nature of this site. Finally, to confirm that USF1 and USF2 proteins do indeed contribute to Gata4 promoter activity, we used short interfering RNA (siRNA) technology to knock down USF expression in MSC-1 cells (Fig. 9E). Cotransfection of MSC-1 cells with USF2 or both USF1 and USF2 siRNA, but not USF1 alone or the control (scrambled) siRNA, significantly decreased activity of the –222-bp Gata4 promoter construct (Fig. 9E). Thus, these data support a role for at least USF2 in the regulation of the proximal Gata4 promoter via the conserved E-box element.
Despite its important developmental roles, much less is known about the transcriptional mechanisms that regulate the GATA4 gene. We now report a detailed description of the proximal Gata4 5' regulatory sequences and, using transgenic mice, have identified a 5-kb promoter fragment that is sufficient to recapitulate endogenous GATA4 expression in Sertoli cells throughout testicular development.
Sertoli Cell Specificity of the 5-kb Gata4 Promoter
The mammalian gonad is an excellent model for the study of the spatiotemporal expression of the GATA4 gene, since the latter is expressed in multiple cell lineages of both the male (Sertoli, Leydig, peritubular, endothelial) and female (granulosa, theca, endothelial) organs during development [6, 10, 40]. Moreover, in the bipotential male gonad, a functional GATA4 protein, as well as its cofactor Friend of GATA2, are crucial for somatic cell precursors to adopt a Sertoli cell fate and organize into testicular cord structures [22]. Thus, understanding how GATA4 expression is specifically regulated within the Sertoli cell is of paramount importance to better defining the role of this factor in early testis differentiation, and, hence, male sex determination. Interestingly, our results revealed that the Sertoli cell of the testis was the only gonadal cell type of the developing embryo to show GFP reporter expression (Fig. 4). This suggests that cis-regulatory elements present in this promoter region are sufficient to drive GATA4 expression within these cells. However, a detailed analysis of the conserved transcription factor regulatory motifs present in the 5-kb promoter did not reveal any binding sites for Sertoli-specific factors such as SRY or SOX proteins. In this case, Sertoli cell-specific expression of GATA4 might then rely on other unknown transcription factors. On the other hand, expression in Sertoli cells might depend solely on factors binding to regulatory elements present in the most proximal promoter region, including, for example, USF1/USF2 binding to the conserved E-box motif. Although plausible, this simple regulatory mechanism would be unusual for a GATA factor, given the complex regulation already described for some of the other GATA genes [26]. The predominant Sertoli cell-specific activity of the 5-kb Gata4 promoter may represent a fail-safe mechanism to ensure abundant GATA4 expression in Sertoli cells in order to trigger their differentiation during the critical period of testis morphogenesis. This is certainly likely, knowing that development of the other testicular cell lineages depends, at least in part, on the differentiation of Sertoli cells [41].
Surprisingly, the 5-kb promoter fragment did not direct reporter expression in the other known GATA4-expressing gonadal cell lineages, such as steroidogenic Leydig cells and ovarian granulosa cells. This may simply be a reflection of the lower basal GATA4 expression normally found in these other cell types [10]. A more likely possibility is that the 5-kb region might lack critical regulatory elements required for GATA4 expression at these other sites. Indeed, distant regulatory modules are known to enhance the basal levels of GATA expression in diverse cell types during development [26]. This now includes the Gata4 gene, the expression of which in the mouse embryonic liver has been recently reported to depend on a distant hepatic enhancer sequence 40 kb upstream of the Gata4 transcription initiation site [28]. Another interesting possibility is that the GATA4 gene itself might be regulated by alternative tissue- and/or cell-specific promoters. This is highly probable, based on the fact that this type of regulation has been described already for other members of the GATA factor family [26].
Cell-Specific Regulation by Ubiquitous Factors: The Sertoli Paradox
GATA gene promoters are typically TATA-less containing GC-rich sequences in their proximal promoter regions [42, 43]. The proximal Gata4 promoter also is TATA-less and contains several CG boxes (Fig. 7). Consistent with a very recent study published by Ohara et al [44], sequential 5' deletion analysis pinned down the critical regulatory elements of the larger 5-kb Gata4 promoter fragment to within its first 118 bp (Fig. 6). Closer examination of this region revealed the presence of four conserved regulatory motifs: 2 GC-boxes designated as GC-d and GC-p, one E-box, and a motif for the binding of the NR5A family of nuclear receptors. Although the NR5A element proved to be nonfunctional, the GC-boxes and especially the E-box were found to be critical for Gata4 promoter activity (Fig. 8). These motifs are known to be bound by a variety of different transcription factors, which are most often ubiquitously expressed. The ubiquitous nature of these regulatory motifs and their related binding proteins is likely the reason why the short –118-bp Gata4 promoter fragment (Fig. 6) or corresponding mutated constructs (Fig. 8) displayed similar activity when expressed in either GATA4-expressing (MSC-1, MA-10, DC3) or non-GATA4-expressing (CV-1) cell lines. Similar findings also have been recently observed in TM3 Leydig and TM4 Sertoli cells as well as in the I-10 and P19.CL6 cardiac cell lines [44]. Although the GC-box motifs clearly contribute to Gata4 promoter activity, we have yet to identify the nature of the proteins binding to these sites. Supershift experiments using a battery of different antisera (SP1 family members, WT1, EGR1, etc.) have so far been inconclusive, in both gonadal (this study) and cardiac [44] cells alike. Conversely, in gonadal cells, we now demonstrate that the critical GATA4 E-box element can be bound by either USF1 or USF2 (Fig. 9B). Results of our USF1/2 knockdown experiments, however, suggest that on the proximal rat Gata4 promoter, either USF2 homodimers or USF2-containing heterodimers are the more transcriptionally relevant complexes binding to the E-box site. Since the E-box motif is known to be bound by different families of transcription factors containing numerous members, we cannot rule out the possibility that the GATA4 E-box element might be bound by factors other than USF proteins. E-box regulatory motifs also can be the target for negative regulation by inhibitory helix-loop-helix factors. Interestingly, for some Sertoli cell-expressed genes such as the androgen receptor (AR) and steroidogenic factor-1 (NR5A1), transcription mediated through their critical E-box promoter elements has been shown to be repressed by TCF21 (podocyte-expressed 1/capsulin/epicardin) [45–47]. Since the proximal GATA4 promoter, much like the NR5A1 promoter, contains a crucial E-box motif that appears to be regulated by USF factors, it is possible that transcription of the GATA4 gene also might be a target for TCF21-mediated repression in some cellular or physiologic contexts.
The ubiquitous expression of a transcription factor does not necessarily preclude the factor from being involved in tissue- or cell-specific expression. Rather, proper spatiotemporal gene expression is the result of a specific combination of regulatory factors, some of which can be ubiquitously expressed. Indeed, in the testis there is a growing list of Sertoli-expressed genes regulated by ubiquitous transcription factors such as SP1/3: Ctsl [48], Clu [49], Dmrt1 [50], FASL [51]; USF1/2: Prkar2b [52], Fshr [53, 54], or both factors: Nr5a1 [55, 56]. With respect to GATA4, it is therefore conceivable that binding of ubiquitous factors to the GC- and E-box Gata4 promoter elements, which are required for its basal activity in cell lines, also contributes to its tissue specificity in vivo. For Sertoli cells, these elements appear sufficient, whereas as previously mentioned for other gonadal cell types (Leydig, granulosa, etc.), additional upstream regulatory elements and/or alternative promoter usage might be involved.
Recently, different transgenic mouse models have been used as tools to purify gonadal embryonic somatic cells for the purpose of comparing male and female transcriptomes at the time of sex determination or the evolution of the male transcriptome during early testis differentiation [57, 58]. Although useful for preparing enriched cell populations, these models did not permit the isolation of a pure population of Sertoli cells. For the newborn testis, other transgenic mouse models also exist that specifically target the Sertoli cell, but these either display delayed Sertoli cell labeling or spermatogenesis stage-specific expression [59–61]. Another important drawback of some of these systems is the use of reporter genes that are not useful for cell purification, such as beta-galactosidase. Given the specific, constant, and high level of GFP reporter expression driven by the 5-kb Gata4 promoter, our experimental model will likely constitute an invaluable tool for the isolation and study of purified populations of Sertoli cells during testicular ontogeny.
ACKNOWLEDGMENTS
Dr. Michèle Sawadogo (University of Texas M.D. Anderson Cancer Center, Houston, TX) is thanked for generously providing the USF1 and USF2 expression plasmids used in this study. Diana Raiwet, Isabelle Daneau, and Céline Forget are thanked for their technical assistance in generating and maintaining the transgenic mouse lines.
FOOTNOTES
4These authors contributed equally to this work. 
1Supported by grants from the Canadian Institutes of Health Research (CIHR) to D.W.S. and R.S.V. R.S.V. is the titleholder of the Canada Research Chair in Reproduction and Sex Development. S.M.G. holds a postdoctoral fellowship from the CIHR Institute of Gender and Health. A.T. was recipient of a studentship from la Chaire Jeanne et Jean-Louis Lévesque.
Correspondence: 2 Robert S. Viger, Ontogeny-Reproduction Research Unit, CHUL Research Centre (CHUQ), Room T1–49, 2705 Laurier Blvd., Québec City, QC, Canada G1V 4G2. FAX: 418 654 2765; e-mail: robert.viger{at}crchul.ulaval.ca
Correspondence: 3 David W. Silversides, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, CP 5000, St-Hyacinthe, QC, Canada J2S 7C6. FAX: 450 778 8103; e-mail: david.w.silversides{at}umontreal.ca
Received: 27 June 2006.
First decision: 21 July 2006.
Accepted: 18 September 2006.
REFERENCES
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USF, USF1 or USF2 antibody; SS, supershift. D) Site-directed mutagenesis also was used to inactivate the E-box motifs in context of the entire 5-kb promoter fragment. The constructs (0.5 µg per well) were transfected in the MSC-1 Sertoli cell line. Promoter activities are expressed as arbitrary units relative to the wild-type 5-kb construct (±SEM). Asterisks indicate significant differences relative to the wild-type construct (P < 0.001, Student's t-test). E) Knockdown of endogenous USF expression affects Gata4 promoter activity. MSC-1 cells were transfected with the –222-bp Gata4 promoter construct and then received either no treatment (-), control siRNA, or siRNAs specific for USF1 and/or USF-2. Promoter activities are expressed relative to the untreated group (±SEM). Asterisks indicate significant differences relative to the untreated group (P < 0.05, Kruskal-Wallis ANOVA followed by Student-Newman-Keuls test).