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Endocrinology Vol. 142, No. 3 977-986
Copyright © 2001 by The Endocrine Society

ARTICLES

GATA Factors Differentially Activate Multiple Gonadal Promoters through Conserved GATA Regulatory Elements1

Jacques J. Tremblay2 and Robert S. Viger3

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

Address all correspondence and requests for reprints to: Dr. Robert S. Viger, Ontogeny and Reproduction Research Unit, T1–49, CHUL Research Center, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: robert.viger{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The GATA factors are a group of transcriptional regulators that play essential roles in cell differentiation, organ morphogenesis, and tissue-specific gene expression during development. The six vertebrate GATA factors are expressed in a broad spectrum of tissues, including the hemopoietic system, heart, gut, brain, placenta, pituitary, and gonads. Interestingly, GATA-like DNA-binding proteins are found in the gonads of several species, ranging from lower invertebrates to humans, thus supporting an evolutionary conserved and crucial role for these factors in gonadal development and function. Indeed, GATA factors are expressed from the onset of gonadal development and are later found in multiple cell lineages of both the testis and ovary. We now report that GATA-4 differentially activates transcription of several genes expressed in the gonads that encode either steroidogenic enzymes (steroidogenic acute regulatory protein and aromatase), hormones (inhibin {alpha} and Müllerian inhibiting substance) and a transcription factor (SF-1) known to be essential for gonadal development and function. Thus, our results identify GATA-4 as an important regulator of gonadal gene transcription where its specificity of action is mediated through synergistic interactions with other transcription factors such as SF-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TISSUE-SPECIFIC gene expression is controlled in part by trans-acting nuclear regulatory proteins (transcription factors) that bind specific DNA sequences. The consensus DNA sequence WGATAR, named the GATA motif, is an essential cis element present in the promoter region of numerous genes. Over the past decade, a number of investigators have characterized a family of structurally related proteins, known as the GATA factors, that bind this sequence and activate transcription (1, 2). GATA proteins are widely expressed; they are found in organisms ranging from fungi to humans (1, 2, 3). The evolutionary conservation of GATA proteins is a strong indication that these transcriptional regulators play important roles in the development and differentiation of all eukaryotic organisms. Interestingly, the reproductive system is no exception to the widespread expression of GATA factors, as several are present in the mammalian testis and ovary (4, 5, 6, 7). Thus, the GATA family of transcription factors represents a new class of potential regulators of gonadal gene expression.

GATA regulatory elements were originally identified in studies of erythroid-specific gene expression (2, 8). A novel transcription factor that specifically bound to GATA cis-elements was cloned from erythroid cells and named GATA-1 (9). GATA-1 was shown to contain a DNA-binding domain that consisted of two similar zinc fingers with the distinctive form C-X2-C-(X17)-C-X2-C (2, 9). Since the initial cloning of the prototypic GATA-1 factor, five additional vertebrate factors (GATA-2 to GATA-6), with similar DNA-binding domains, have been identified (1, 2). The six vertebrate GATA factors can be separated into two subgroups based on sequence homology and tissue distribution: the hemopoietic group (GATA-1/2/3) and the cardiac group (GATA-4/5/6). Within a single species, the highest identity among GATA factors is observed within their zinc finger DNA-binding domain. This conservation is probably the reason why the different GATA family members exhibit similar DNA-binding properties (10, 11). The C-terminal finger is required for site-specific recognition and DNA binding to the core WGATAR motif, whereas the N-terminal finger contributes to the specificity and stability of the DNA binding (12, 13, 14). Although GATA factors share similar DNA-binding properties, they exhibit distinct spatial and developmental expression patterns and play essential, nonredundant functions (1, 2, 15, 16, 17, 18, 19, 20, 21, 22). The functional specificity of the different GATA factors appears to be achieved in part via protein-protein interactions with other cell-restricted factors. The latter include the FOG-1 and FOG-2 (Friend of GATA) proteins that were cloned as GATA-specific cofactors (23, 24, 25, 26).

GATA-like DNA-binding proteins are found in the gonads of several species, including worms, fruit flies, snakes, birds, rodents, and humans (4, 5, 6, 7, 27, 28, 29, 30). In Drosophila melanogaster, an ovary-specific GATA factor named dGATAb has been shown to activate transcription of the yolk protein genes, Yp1 and Yp2, in ovarian follicles (29). A similar GATA-binding protein has also been reported in the silkworm, Bombyx mori, which regulates follicle-specific expression of the chorion genes (27). Another GATA-like protein, termed the Bkm-binding protein, is also present in the snake ovary and mouse testis (30). Bkm-binding protein was identified by its ability to bind to Bkm, a satellite fraction of the W chromosome in snakes and birds enriched in GATA repeats (30). Of the six vertebrate GATA factors, three are expressed in the mammalian gonads: GATA-1 (6, 7, 31), GATA-4 (4, 5, 6, 32), and GATA-6 (4, 5, 32). In the testis, GATA-1 and GATA-4 both localize to Sertoli cells, the major somatic cells of the seminiferous epithelium of the testis, but at different stages of development (6, 7). GATA-1 is the predominant GATA factor of postnatal Sertoli cells, where its expression may be negatively regulated by one or more paracrine factors produced by germ cells (7). In contrast, we have recently shown that GATA-4 marks Sertoli cells from the onset of gonadal development in the mouse (6). Thus, GATA factors are probably key regulators of Sertoli cell-specific gene expression and function during ontogeny. Indeed, GATA-dependent Sertoli cell promoters have been postulated (5, 6, 33, 34, 35). These include the inhibin {alpha} promoter (5, 33) and the Müllerian inhibiting substance (MIS) promoter, which we identified as the first known downstream target for GATA-4 in fetal Sertoli cells (6, 34). GATA-4 has also been proposed to regulate expression of the gene encoding the steroidogenic acute regulatory protein (StAR) (36, 37). GATA factors are also expressed in the mouse ovary (4, 6, 32); their target genes, however, have yet to be identified.

Although GATA factors are expressed in multiple cell lineages of both the testis and ovary, the identification of target genes has been rather limited. However, the presence of numerous consensus GATA binding motifs in several gonadal promoters invariably suggests that GATA factors regulate a broad spectrum of genes in these tissues. In the present study we have characterized the GATA-dependent activation of five different gonadal promoters. Moreover, we provide evidence that GATA cofactors, such as SF-1, are involved in the GATA-dependent transcription of some, but not all, gonadal target genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
The –180-bp murine MIS-luciferase promoter has been described previously (6, 34). The -218-bp proximal PII aromatase (Cyp19), -588-bp SF-1, -902-bp StAR, and -679-bp inhibin {alpha} promoter sequences were amplified by PCR from mouse genomic DNA using the following sets of oligonucleotide primers: PII aromatase (forward, 5'-CGGGATCCTCTGGAATGAACTTCAGA-3'; reverse, 5'-GGGGTACCGTTTGGCTGTGGCTCCTGTCA-3'), SF-1 (forward, 5'-ATGGATCCACACCCTTAGCCCAGCAGTCT-3'; reverse, 5'-TGGGTACCGGGCTCTCAGAAACTTCTTC-3'), StAR (forward, 5'-ATGGATCCATCTGGCCCAGTACCACCAGGGAT-3'; reverse, 5'-GAGGTACCTGAGTCCTGCAGCTGTGGC-3'), and inhibin {alpha} (forward, 5'-ATGGATCCAGCCC-CCTCCCACAGCCT-3'; reverse, 5'-CGAAGCTTCACGCTGCCCTGTGCCCTTTC-3'). The amplified promoter sequences were cloned into either the BamHI/HindIII or BamHI/KpnI sites of a modified pxP1-luciferase reporter plasmid (34). The -182-bp and -182mut SF-1 constructs were obtained by PCR using the -588-bp SF-1 promoter as template and forward primers (-182 bp, 5'-GCGGATCCATAAAGATAGGGATATTTTTTTTTCTTTTAGAAGAG-3'; -182mut, 5'- GCGGATCCATAAAGGTAGGGATATTTTTTTTTCTTTTAGAAGAG-3') used with the reverse primer described above for the -588-bp SF-1 construct. The remaining SF-1 promoter construct (-210 bp) was obtained by enzymatic digestion of a naturally occurring StyI restriction site in the -588-bp SF-1 promoter. The -71-bp and -71mut StAR promoter deletion constructs were generated by PCR using the -902-bp StAR promoter as template, the reverse StAR primer described above, and forward primers 5'-ACGGATCCTTTTTTATCTCAAGTGATGA-3' (-71 bp) and 5'-ACGGATCCTTTTTTACCTCAAGTGATGATGCACAG-3' (-71mut), respectively. The aromatase promoter construct deleted of its GATA site (-93 bp) was obtained by PCR on the -218-bp aromatase promoter using forward primer 5'-ACGGATCCTTGTTTTGACTTGTAACC-3' and the aromatase reverse primer given above. All SF-1, aromatase, and StAR promoter deletion constructs were cloned into the BamHI/KpnI sites of the same pxP1 luciferase reporter plasmid described above. The -99-bp inhibin {alpha} promoter deleted of its GATA sites was obtained by enzymatic digestion of a naturally occurring NcoI restriction site in the -679-bp inhibin {alpha} promoter. The synthetic 2xGATA luciferase reporters were generated by cloning two copies of a double stranded SF-1 GATA element (sense oligonucleotide, 5'-GATCCCATAAAGATAGGGATATTA-3'; antisense oligonucleotide, 5'-GATCTAATATCCCTATCTTTATGG-3') upstream of the minimal MIS or POMC promoters. An expression plasmid for GATA-6 was obtained by cloning a PCR fragment, corresponding to the GATA-6 open reading frame, into the XbaI/KpnI sites of a Rous sarcoma virus-driven expression vector. The PCR fragment was amplified using first strand complementary DNA (cDNA) prepared from neonate testis RNA as template for the PCR reaction (forward primer, 5'-AGTCTAGAACTGAGCCCCTTCGCGGCCG-3'; reverse primer, 5'- TCGGTACCGCTGCCATCTGGACTGCTGG-3'). The full-length GATA-4 cDNA was cloned by PCR (forward primer, 5'-CGAAGCTTATGGCGAGACACCCCAATCTCGATATG-3'; reverse primer, 5'-ATGGATCCTTAACCTGCTGGTGTCTTAGATTTATT-3') using the neonate testis cDNA as template and inserted into the HindIII/BamHI sites of pcDNA3 (Invitrogen, Carlsbad, CA). A GATA-4 protein ({Delta}N-term) deleted of its N-terminal domain up to the second zinc finger ({Delta}1–254) was obtained by PCR (forward primer, 5'-GATCTAGAAAGCCTCAGCGCCGGCTGTCT-3'; reverse primer, 5'-ATGGATCCTTACGCGGTGATTATGTCCCC-3') using the full-length GATA-4 cDNA as template and cloned into the XbaI/BamHI sites of a cytomegalovirus-driven expression vector. Expression vectors for Sox9, Lhx9, and GATA-1 were also generated by PCR using cDNAs prepared from testis (Sox9 and GATA-1) or embryonic day 9 mouse head (Lhx9) using the following pairs of oligonucleotide primers: Sox9 (forward, 5'-TGAAGCTTCGTATGAATCTCCTGGACCCCTT; reverse, 5'-TGTCTAGACCTCAAGGTCGAGTGAGCTGTG-3'), Lhx9 (forward, 5'-CTGGTACCATGCT-CTTCCACGGAATCTCC-3'; reverse, 5'-CGCTCGAGTTAGAAAAGGTTCGTTAAGGT-3'), and GATA-1 (forward, 5'-CTAAGCTTATGGATTTTCCTGGTCTAGGGG-3'; reverse, 5'-CTGGATCCGTACCTTCAAGAACTGAGTGG-3'). The PCR products were cloned into the HindIII/XbaI (Sox9), KpnI/XhoI (Lhx9), or HindIII/BamHI (GATA-1) sites of the pcDNA3 expression vector (Invitrogen). The validity of all our luciferase promoter constructs and expression plasmids was verified by DNA sequencing. The USF-1 and USF-2 expression plasmids were generously provided by Michèle Sawadogo (38). Expression plasmids for the C/EBP isoforms were provided by Steven McKnight (39). The SF-1 expression plasmid was a gift from Keith Parker (40).

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. Pituitary {alpha}T3–1 (41), MSC-1 Sertoli, and adrenal Y-1 cells were maintained in DMEM containing 10% FBS. The TM3 Leydig cell line was cultured in a 1:1 mixture of Ham’s F-12 and DMEM containing 5% horse serum and 2.5% FBS. All transfections were performed in 24-well plates using the calcium phosphate precipitation method (42). The day before transfection, CV-1, L, TM3, {alpha}T3–1, Y-1, and MSC-1 cells were plated at densities of 2.2 x 104, 5 x 104, 4 x 104, 1.5 x 105, 8 x 104, and 5 x 104 cells/well, respectively. Cells were transfected 24 h after the initial plating. Culture medium was changed 12–16 h after transfection, and the cells were finally harvested the following morning. Cells were lysed by adding 50 µl lysis buffer [100 mM Tris-HCl (pH 7.9), 0.5% Igepal (Sigma-Aldrich Corp., Oakville, Canada), and 5 mM dithiothreitol] directly to the culture wells. An aliquot of the lysate was then assayed for luciferase activity using an E.G.&G Berthold LB 9507 luminometer and luciferine (BD PharMingen, San Diego, CA) as substrate. In all experiments, the total amount of DNA was kept constant at 1.5 µg/well using Sp64 (Promega Corp., Madison, WI) as carrier DNA; several DNA preparations of the plasmids were used to ensure reproducibility of the results. The data reported represent the average of at least three experiments, each performed in duplicate.

DNA binding and Western blot assays
Recombinant GATA-4 protein was obtained by transfecting L cell fibroblasts (which are devoid of GATA-binding activity) with an expression vector encoding the full-length GATA-4 protein. Nuclear extracts were prepared 48 h after transfection by the procedure outlined by Schreiber et al. (43). DNA binding assays were performed using a 32P-labeled double stranded oligonucleotide corresponding to the conserved GATA element in the proximal SF-1 promoter at position -175 bp (sense oligo, 5'-CCCATAAAGATAGGGATATT-3'; antisense oligo, 5'-AATTATCCCTATCTTTATGGG-3'). Binding reactions and electrophoresis conditions were previously described (6). A double stranded mutant (M1) oligonucleotide (sense oligo, 5'-CCCATAAAGGTAGGGATATT-3'; antisense oligo, 5'-AATATCCCTACCTTTATGGG-3') was used to confirm the specificity of GATA-4 binding to the SF-1 GATA element. The sequences of the MIS wild-type and mutated oligonucleotides that were also used as competitors in the gel shift assay have been reported previously (6). In the Western blot analysis, 20-µg aliquots of nuclear extract containing either the full-length or N-terminal-deleted GATA-4 proteins (GATA-4 F.L. and {Delta}N-term) were separated by SDS-PAGE and then transferred to Hybond polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Baie-D’Urfé, Canada). Immunodetection of the GATA proteins was achieved using a GATA-4-specific antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a commercially available Vectastain-ABC-Amp Western blot detection kit (Vector Laboratories, Inc., Burlingame, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GATA factors activate multiple gonadal promoters
The organization of the proximal promoter regions of five gonadal genes harboring species-conserved GATA elements is shown in Fig. 1Go. Consistent with most GATA-dependent promoters described to date, the conserved GATA motifs found in gonadal promoters are located in close proximity to the transcriptional start site (Fig. 1AGo). Although we and others found that these gonadal GATA elements are capable of specifically interacting with exogenously or endogenously expressed GATA factors (Fig. 5Go) and (6, 33, 36), a direct comparison of their ability to respond to GATA factors has not yet been assessed. The potential of GATA-4 to activate gonadal promoters is shown in Fig. 2Go. The GATA-4 transcription factor is a logical choice to evaluate the GATA responsiveness of the selected promoters, as it is the sole GATA family member that is expressed in all cell types (Sertoli, granulosa, thecal, and Leydig) where the promoters are normally active. Interestingly, the gonadal promoters were differentially activated by GATA-4 in heterologous (non-GATA-expressing) CV-1 cells (Fig. 2AGo) despite the fact that the conserved GATA elements found in each respective promoter are not differentially bound by GATA proteins. The MIS and inhibin {alpha} promoters were moderately activated by GATA-4 (3.5- and 7-fold), whereas the PII aromatase (Cyp19) and StAR promoters were strongly activated (11.5- and 22-fold) with an equivalent dose of GATA-4. Interestingly, the SF-1 promoter was very poorly activated by GATA-4. Similar activations were also observed with other GATA factors (GATA-1 and GATA-6) that are known to be expressed in the gonads (data not shown). The observed GATA-4 activations were specific, because the corresponding promoters deleted or mutated of their respective GATA elements were not activated by GATA-4 (Fig. 2BGo). Thus, it appears that promoter context plays an important role in how GATA-containing gonadal promoters respond to exogenously expressed GATA factors.



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  		Figure 1. Consensus GATA regulatory motifs are present in the promoters of several gonad-expressed genes. A, Schematic representation of the 5'-regulatory regions of the MIS, aromatase, SF-1, StAR, and inhibin {alpha} genes showing the relative positions of the conserved GATA binding sites. The -218-bp aromatase 5'-sequence corresponds to the proximal PII aromatase (Cyp19) promoter. B, Nucleotide sequences flanking the different GATA promoter elements. The nucleotide positions correspond to the murine promoter sequences.

 


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  		Figure 5. GATA factors specifically bind the conserved GATA element in the proximal SF-1 promoter. A gel shift mobility assay was used to assess whether recombinant GATA-4 could recognize the SF-1 GATA element at position -175 bp. Nuclear extracts containing recombinant GATA-4 protein (L + GATA-4) were produced by transfecting L cell fibroblasts (which are devoid of GATA activity) with an expression plasmid encoding the full-length GATA-4 protein. Nuclear extract prepared from L cells transfected with the empty expression plasmid (L) was used as a negative control. The nuclear extracts were used with a double stranded 32P-labeled oligonucleotide corresponding to the SF-1 GATA element. GATA-4 binding to the SF-1 GATA element was specifically competed by excess unlabeled oligonucleotides [either self (lanes 4–7) or the previously characterized GATA element from the MIS promoter (lane 9)], but not by mutated oligonucleotides in which the GATA consensus motifs had been changed to GGTA (lane 8 or 10).

 


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  		Figure 2. The promoters of several gonad-expressed genes are differentially activated by GATA factors. A, An expression vector encoding GATA-4 was transfected in heterologous (non-GATA-expressing) CV-1 cells along with a luciferase reporter plasmid containing the murine -180-bp MIS, -218-bp PII aromatase, -588-bp and -182-bp SF-1, -902-bp and -71-bp StAR, or -679-bp inhibin {alpha} promoters. B, Similar experiments were performed using reporters in which the GATA elements were either deleted or mutated (mut): -180mut MIS, -93-bp aromatase, -182mut SF-1, -71mut StAR, and -99-bp inhibin {alpha}. The amount of reporter DNA was kept at 500 ng/culture well, with the exception of the highly active SF-1 promoter, which was used at 50 ng/well. {square}, Control (empty) expression vector; {blacksquare}, promoter trans-activations in the presence of 50 ng GATA-4. Similar activations were also observed for other members of the GATA family of factors. Data are reported as fold activation relative to control (±SEM).

 
Interestingly, the MIS, aromatase, SF-1, StAR, and inhibin {alpha} promoters all contain consensus SF-1 regulatory elements (44, 45, 46, 47, 48). As we previously reported that GATA-4 can cooperate with SF-1 to synergistically activate MIS transcription (34), we tested whether GATA-4/SF-1 synergism also contributes to the transcription of these other gonadal genes. As shown in Fig. 3Go, in addition to MIS, marked GATA-4/SF-1 synergism was observed on the aromatase and inhibin {alpha} promoters, but not on the SF-1 and StAR promoters. Thus, transcriptional synergism constitutes another aspect of the differential activation of gonadal genes by GATA-4.



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  		Figure 3. GATA-4 and SF-1 synergize on multiple gonadal promoters. The -180-bp MIS, -218-bp aromatase, -588-bp SF-1, -902-bp StAR, and -679-bp inhibin {alpha} promoters were cotransfected in CV-1 cells with either a control (empty) expression vector ({square}) or expression vectors for GATA-4 (), SF-1 (), or both GATA-4 and SF-1 ({blacksquare}). Results are shown as fold activation relative to control (±SEM).

 
The proximal SF-1 promoter contains a species-conserved GATA-binding site
Although the importance of SF-1 at all levels of the reproductive axis is well established (49), the transcriptional mechanisms that control its tissue-specific expression are not yet fully understood. As GATA-4 and/or GATA-6 is found in multiple cell lineages where SF-1 is also expressed (4, 5, 6, 50, 51), GATA factors probably contribute to SF-1 transcription. Consistent with this hypothesis, sequence alignment of the mouse, rat, and human proximal SF-1 promoter regions revealed the presence, at -175 bp, of a previously uncharacterized consensus GATA binding motif that is conserved across species (Fig. 4Go). We then tested whether this GATA element could specifically interact with recombinant GATA-4 protein. Gel shift experiments showed that the SF-1 GATA element bound GATA-4 with high affinity (Fig. 5Go). This binding was specific, because it was competed by both the unlabeled SF-1 probe (Fig. 5Go, lanes 4–7) and an oligonucleotide corresponding to the previously defined GATA element from the MIS promoter (Fig. 5Go, lane 9), but not by oligonucleotides containing a mutated GATA motif (Fig. 5Go, lanes 8 and 10).



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  		Figure 4. A conserved GATA-binding site is present in the proximal SF-1 promoter. Conserved regulatory elements of the SF-1 promoter are boxed, and their locations relative to the transcriptional start site are shown. Alignment of the mouse, rat, and human proximal SF-1 promoter sequences reveals a previously uncharacterized consensus GATA binding element 175 bp upstream of the transcription start site.

 
The SF-1 GATA regulatory element is activated in a subset of gonadal cell lines
As the SF-1 promoter contains a conserved GATA motif capable of GATA-4 binding, it was expected to be trans-activated by exogenously expressed GATA-4 in heterologous cells. However, our data in CV-1 cells indicated that GATA-4, regardless of dose, was a poor activator of SF-1 transcription (Fig. 6AGo). Knowing that all GATA family members share similar DNA-binding properties (10, 11), we then tested whether the SF-1 promoter could be activated by other GATA factors known to be expressed in the gonads. As shown in Fig. 6BGo, GATA-1 and GATA-6, much like GATA-4, were unable to augment SF-1 promoter activity in CV-1 cells. To determine whether the lack of GATA-dependent trans-activation in CV-1 cells was not simply due to the absence of some critical factor, similar GATA-4 trans-activation experiments were performed in several other cell lines (Fig. 7Go). They included L cell fibroblasts (another heterologous cell model) and {alpha}T3–1, TM3, Y-1, and MSC-1 cells, which are known to endogenously express SF-1 and GATA factors (4, 44, 52, 53, 54 ; and our unpublished observations). In L, TM3, and Y-1 cells, GATA-4 remained a poor activator of SF-1 transcription (Fig. 7Go). However, in the {alpha}T3–1 and MSC-1 cell lines, the proximal SF-1 promoter was significantly activated by GATA-4 (Fig. 7Go). To confirm that the SF-1 GATA element was indeed functional, we generated two synthetic promoter constructs where the SF-1 GATA element was taken out of its natural promoter context and placed upstream of two heterologous promoters. When tested in trans-activation experiments, these synthetic constructs were markedly activated by GATA-4 (12- to 30-fold), indicating that the SF-1 GATA element is functional when taken out of its natural promoter context (Fig. 8AGo). The GATA-dependent activations were specifically mediated by the SF-1 GATA element and not the minimal promoters, as we have previously shown the latter to be unresponsive to GATA factors (34).



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  		Figure 6. GATA factors are poor activators of SF-1 transcription in heterologous CV-1 cells. A, Increasing doses of an expression vector encoding GATA-4 were transfected in CV-1 cells along with 50 ng of the -210-bp SF-1 promoter. The -210-bp construct contains both the conserved GATA and E box elements. {square}, Control (empty) expression vector; {blacksquare}, effect of increasing of doses (25, 50, 100, 250, and 500 ng) of GATA-4. B, Expression vectors encoding different GATA factors were transfected in CV-1 cells along with 50 ng of the -182-bp promoter. {square}, Control (empty) expression vector; {blacksquare}, effects of two doses (50 and 100 ng) of GATA-1 (G1), GATA-4 (G4), or GATA-6 (G6) on SF-1 promoter activity. All results are shown as fold activation relative to control (±SEM).

 


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  		Figure 7. GATA-4 trans-activation of the SF-1 promoter is cell line dependent. GATA-4-dependent trans-activation of the mouse SF-1 promoter in various gonadal and nongonadal cell lines. Increasing doses of a GATA-4 expression plasmid were transfected in different cell lines along with 50 ng of the -182-bp SF-1 promoter. {square}, Control (empty) expression vector; {blacksquare}, effects of increasing doses of GATA-4 (10, 50, and 100 ng) on SF-1 promoter activity. Data are shown as fold activation relative to control (±SEM). L, Mouse L cell fibroblasts; {alpha}T3–1, mouse pituitary gonadotrope cell line; TM3, mouse Leydig cell line; Y-1, mouse adrenal tumor cell line; MSC-1, mouse Sertoli cell line.

 


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  		Figure 8. The proximal SF-1 promoter contains a functional GATA element. A, The trans-activation potential of GATA-4 was tested on two synthetic promoters consisting of two copies of the conserved SF-1 GATA element placed upstream of either the minimal POMC (left panel) or MIS (right panel) promoters. CV-1 cells were cotransfected with 50 ng of a GATA-4 expression plasmid along with 200 ng of either synthetic promoter. {square}, Control (empty) expression plasmid; {blacksquare}, trans-activation induced by 50 ng GATA-4. B, An N-terminal deletion mutant of GATA-4 ({Delta}N-term) trans-activates the natural SF-1 promoter in heterologous cells. CV-1 cells were cotransfected with increasing doses (10, 50, and 100 ng) of either full-length GATA-4 ({blacksquare}) or the N-terminal deletion mutant () along with 50 ng of the -182-bp SF-1 promoter construct. All promoter activities are shown as fold activation relative to control (±SEM). C, The full-length and {Delta}N-term GATA-4 proteins are expressed at similar levels. Twenty-microgram aliquots of nuclear extracts prepared from L cell fibroblasts overexpressing the full-length and {Delta}N-term GATA-4 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 specific GATA-4 antibody directed against the C-terminal portion of the GATA-4 protein.

 
The fact that GATA-4 trans-activation of the SF-1 promoter can only be achieved in certain cell lines (Fig. 7Go) is an indication that GATA-4 transcriptional activity on the SF-1 promoter is also modulated by cell-specific cofactors and/or a posttranslational modification. This interaction/modification may lead to a conformational change in GATA-4 that is necessary to activate the SF-1 promoter in its natural context. Consistent with this hypothesis, a truncated GATA-4 protein, which was deleted of its entire N-terminal region ({Delta}1–254 amino acids), was able to significantly activate the SF-1 promoter in heterologous CV-1 cells (Fig. 8BGo). The difference in trans-activation potential between the full-length and truncated GATA-4 proteins was not due to differences in expression level, because both recombinant proteins were found to be similarly expressed (Fig. 8CGo). As a first step to address the potential implication of cooperative actions of GATA in SF-1 promoter activation, we performed cotransfection experiments involving GATA-4 and other transcription factors known to be important for gonadal gene expression and possibly SF-1 gene transcription. As shown in Fig. 9AGo, neither the HMG-box factor Sox9 nor the LIM homeobox protein Lhx9, which are both coexpressed with SF-1 at the onset of gonadal development (55, 56, 57), was able to activate the SF-1 promoter on their own or in combination with GATA-4. As a conserved E box for the binding of the bZIP-HLH factors USF-1 and USF-2 and a CCAAT-containing motif for the potential binding of C/EBP proteins are present in the proximal SF-1 promoter, we tested whether these factors could transcriptionally cooperate with GATA-4 to activate the SF-1 promoter (Fig. 9BGo). Although the USF proteins could significantly activate the SF-1 promoter on their own, the addition of GATA-4 did not lead to further activation (Fig. 9BGo, left panel). Interestingly, the C/EBP isoforms were also able to individually activate the SF-1 promoter. Again, transcriptional cooperation was not observed when the C/EBP isoforms were used in combination with GATA-4. However, as the C/EBP isoforms are expressed in Sertoli cells (58), they may be important contributors to SF-1 transcription in the testis.



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  		Figure 9. GATA-4 does not cooperate with a subset of gonad-expressed transcription factors in SF-1 promoter activation. The ability of GATA-4 to transcriptionally cooperate with other gonadal transcription factors was tested by cotransfection experiments in CV-1 cells. A, Effect of GATA-4, alone and in combination with increasing doses of expression plasmids encoding the HMG-box factor Sox9 (left panel) or the LIM-homeobox protein Lhx9 (right panel), on the activity of the -588-bp SF-1 promoter. {square}, Control (empty) expression vector; , 50 ng GATA-4 alone; , increasing doses (50, 100, and 250 ng) of either Sox9 or Lhx9 alone; {blacksquare}, increasing doses (50, 100, and 250 ng) of Sox9 or Lhx9 in the presence of 50 ng GATA-4. B, Effect of GATA-4, alone or in combination, with increasing doses of expression plasmids encoding the bZIP-HLH factors USF-1 and USF-2 (left panel) or the CCAAT-enhancer binding proteins C/EBP{alpha}, C/EBP{beta}, and C/EBP{delta} (right panel) on the activity of the -182-bp SF-1 promoter. {square}, Control (empty) expression vector; , 50 ng GATA-4 alone; , increasing doses (50, 100, and 250 ng) of either the USF proteins or the C/EBP isoforms alone; {blacksquare}, increasing doses (50, 100, and 250 ng) of the USF proteins or the C/EBP isoforms in the presence of 50 ng GATA-4. Data are reported as fold activation relative to control (±SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of the vertebrate family of GATA factors in the developmental control of cell fate, cell differentiation, organ morphogenesis, and tissue-specific gene expression is well established. Studies on the mechanism of action of this group of transcriptional regulators have centered primarily on the hemopoietic and cardiac systems. Interestingly, GATA factors are also abundantly expressed in both the mammalian testis and ovary (4, 5, 6, 7, 32), suggesting that they play equally important roles in regulating gonadal gene expression and function. At present, however, only a handful of gonadal target genes have been proposed.

GATA-4: a key regulator of gonadal gene transcription
The promoters of several genes (MIS, aromatase, SF-1, StAR, and inhibin {alpha}), known to be important for proper gonadal development and steroidogenic function, contain species-conserved GATA regulatory elements in their proximal promoter regions (Fig. 1Go). Moreover, we have shown that these promoters are all activated by GATA-4 (Fig. 2Go), the GATA factor common to granulosa, thecal, Sertoli, and Leydig cells. The requirement for a single factor in the control of multiple target genes appears to be a common regulatory mechanism in many tissues, including the endocrine organs. Indeed, within the pituitary, the homeoprotein Pitx1 was defined as a pan-pituitary regulator of gene expression (59). Similar roles have been ascribed to the homeobox protein STF-1/Pdx1 in the pancreas (60, 61) and the orphan nuclear receptor SF-1 in the hypothalamo-pituitary-gonadal axis (62, 63). Thus, the ability of GATA-4 to activate multiple gonadal promoters shown here supports an analogous role for GATA-4 in gonadal gene expression.

The involvement of pan-regulatory factors in tissue-specific transcription, however, most often requires precise combinatorial interactions with other cell-restricted factors. For example, in the pituitary, the Pitx1 homeobox protein has been reported to interact with different factors present only in a subset of pituitary cell types, thereby creating a cell-specific code for lineage-specific gene expression (64). Similar combinatorial interactions have also been described for the orphan nuclear receptor SF-1 in the control of SF-1 target genes in the gonads (34, 65, 66). In heterologous cells, both Pitx1 and SF-1 differentially activate target promoters despite no real differences in Pitx1 and SF-1 binding affinities to their respective promoter elements (59, 63, 64). Interestingly, this is what was observed for GATA-4 on the different gonadal promoters. Thus, it is likely that GATA-4 contributes to tissue-specific expression in the gonads through cooperative interactions with other transcription factors. Indeed, we have shown that GATA-4 cooperates with SF-1 to enhance MIS transcription (34), and we now extend this to include the aromatase and inhibin {alpha} promoters. Similarly, binding sites for both GATA-4 and C/EBP{beta} were shown to be required for StAR promoter activity (36, 37).

The SF-1 promoter is a potential downstream target for GATA-4
The orphan nuclear receptor SF-1 is a critical regulator of gene expression at all levels of the reproductive axis; it is indispensable for adrenal and gonadal development and is later recruited as an essential factor required for cell-specific gene expression in the pituitary, hypothalamus, adrenals, and gonads (62, 63, 67). Although SF-1 was identified nearly a decade ago (40), surprisingly little is known about the transcriptional mechanisms that control its expression. Recent analyses of the SF-1 promoter have all identified an essential E box motif that binds members of the USF family of bZIP-HLH factors (46, 68, 69, 70, 71). Mutation or deletion of this conserved element leads to a marked decrease in SF-1 promoter activity in multiple cell types, including fibroblasts, which do not express SF-1 (46, 68, 69, 70, 71). Taken together, these data suggest that the E box element alone cannot account for the specificity of SF-1 expression in vivo. Therefore, additional regulatory elements must exist.

Interestingly, GATA factors are found in most, if not all, SF-1 target tissues. Indeed, GATA factors are present in pituitary gonadotropes, Sertoli and Leydig cells of the testis, ovarian granulosa and thecal cells, and cells of the fetal and postnatal adrenal cortex (4, 5, 6, 7, 32, 51, 52). This observation raises the intriguing possibility that GATA factors might be involved in the initiation and/or up-regulation of SF-1 expression in certain tissues, such as the gonads, where both factors are expressed from an early developmental stage (6, 57). Indeed, we have shown that the mouse, rat, and human SF-1 promoters all contain a consensus GATA-binding element. Moreover, different groups have shown that deletion of the region containing the conserved GATA element leads to a drop in SF-1 promoter activity in different gonadal cell lines (69, 70). Although GATA-4 specifically bound this element, GATA-4 was a poor activator of the SF-1 promoter in heterologous cells. Interestingly, these data are reminiscent of the well characterized SF-1 element that is present in the proximal MIS promoter (34, 44). Although this element is essential for MIS promoter activity in vivo (72, 73), it is nonetheless weakly activated by SF-1 alone in in vitro transfection assays (34, 44). With respect to the MIS promoter, SF-1 transcriptional activity was shown to be mediated through synergistic interactions with other transcription factors (34, 65, 66). A similar mechanism may also be required to modulate GATA activity in context of the SF-1 promoter. Indeed, GATA-4-mediated trans-activation of the natural SF-1 promoter was strictly dependent on cell type, and in heterologous cells, the N-terminal region of the GATA-4 protein had to be deleted for activation to occur. Taken together, our data suggest that the GATA-dependent regulation of SF-1 promoter requires the participation of a cofactor, the disruption of an interaction with a repressor, or a specific posttranslational modification of the GATA-4 protein, such as phosphorylation or acetylation. Alternatively, regulatory elements for the binding of GATA cooperative factors might be located outside of the promoter region used in this study. An in vivo analysis of the 5'-regulatory elements of the SF-1 gene would be required to examine this possibility.

Besides the conserved GATA motif in the SF-1 promoter, we recently identified an SF-1-like element in the proximal rat GATA-4 gene promoter (Legault, E., J. J. Tremblay, and R. S. Viger, unpublished observations). Thus, in addition to regulating downstream target genes in the gonads, cross-talk between GATA-4 and SF-1 may be an important mechanism for ensuring the proper spatiotemporal expression of these crucial factors during development.


   Acknowledgments
 
Michèle Sawadogo (USF-1 and USF-2 expression plasmids), Steven McKnight (C/EBP isoform expression plasmids), Keith Parker (SF-1 expression plasmid), Jacques Drouin (minimal POMC promoter), Pamela Mellon ({alpha}T3–1 cell line), and Michael Griswold (MSC-1 cell line) are thanked for generously providing plasmids and cell lines used in this study.


   Footnotes
 
1 This work was supported by a grant from the Canadian Institutes of Health Research (to R.S.V.). Back

2 Recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. Back

3 Chercheur-Boursier of the Fonds de la Recherche en Santé du Québec. Back

Received August 22, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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