Endocrinology Vol. 142, No. 3 977-986
Copyright © 2001 by The Endocrine Society
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, T149, CHUL Research Center, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: robert.viger{at}crchul.ulaval.ca
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Abstract
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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
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.
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Introduction
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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
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.
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Materials and Methods
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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
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
(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
promoter deleted of its GATA sites was obtained by enzymatic digestion
of a naturally occurring NcoI restriction site in the
-679-bp inhibin
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 (
N-term)
deleted of its N-terminal domain up to the second zinc finger
(
1254) 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
T31 (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 Hams 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,
T31,
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 1216 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
N-term) were separated by
SDS-PAGE and then transferred to Hybond polyvinylidene difluoride
membranes (Amersham Pharmacia Biotech,
Baie-DUrfé, 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).
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Results
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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. 1
. 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. 1A
). Although we and others found that these gonadal GATA
elements are capable of specifically interacting with exogenously or
endogenously expressed GATA factors (Fig. 5
) 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. 2
. 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. 2A
) despite the
fact that the conserved GATA elements found in each respective promoter
are not differentially bound by GATA proteins. The MIS and inhibin
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. 2B
). 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 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 47) 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 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 . 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. , Control (empty) expression vector; , 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).
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Interestingly, the MIS, aromatase, SF-1, StAR, and inhibin
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. 3
, in addition to MIS, marked
GATA-4/SF-1 synergism was observed on the aromatase and inhibin
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.
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. 4
). 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. 5
). This binding was specific, because it
was competed by both the unlabeled SF-1 probe (Fig. 5
, lanes 47) and
an oligonucleotide corresponding to the previously defined GATA element
from the MIS promoter (Fig. 5
, lane 9), but not by oligonucleotides
containing a mutated GATA motif (Fig. 5
, 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.
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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. 6A
). 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. 6B
, 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. 7
). They included L cell
fibroblasts (another heterologous cell model) and
T31, 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. 7
). However, in the
T31 and
MSC-1 cell lines, the proximal SF-1 promoter was significantly
activated by GATA-4 (Fig. 7
). 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. 8A
). 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. , Control
(empty) expression vector; , 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. , Control (empty) expression vector; ,
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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The fact that GATA-4 trans-activation of the SF-1
promoter can only be achieved in certain cell lines (Fig. 7
) 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 (
1254 amino acids), was able to significantly activate the
SF-1 promoter in heterologous CV-1 cells (Fig. 8B
). 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. 8C
). 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. 9A
, 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. 9B
). 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. 9B
, 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. , Control (empty) expression vector; , 50 ng GATA-4
alone; , increasing doses (50, 100, and 250 ng) of either Sox9 or
Lhx9 alone; , 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 , C/EBP , and C/EBP
(right panel) on the activity of the -182-bp SF-1
promoter. , 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; , 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
|
|---|
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
), known to be important for proper gonadal development and
steroidogenic function, contain species-conserved GATA regulatory
elements in their proximal promoter regions (Fig. 1
). Moreover, we have
shown that these promoters are all activated by GATA-4 (Fig. 2
), 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
promoters. Similarly, binding sites for both GATA-4 and
C/EBP
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 (
T31 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.). 
2 Recipient of a postdoctoral fellowship from the Natural Sciences
and Engineering Research Council of Canada. 
3 Chercheur-Boursier of the Fonds de la Recherche en Santé du
Québec. 
Received August 22, 2000.
 |
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