Modulation of Endogenous GATA-4 Activity Reveals Its Dual Contribution to Müllerian Inhibiting Substance Gene Transcription in Sertoli Cells
Jacques J. Tremblay,
Nicholas M. Robert and
Robert S. Viger
Ontogeny and Reproduction Research Unit, Centre Hospitalier de
lUniversité Laval (CHUL) Research Centre; and Centre for
Research in Biology of Reproduction, Department of Obstetrics and
Gynecology, Laval University, Ste-Foy, Quebec, Canada G1V
4G2
Address all correspondence and requests for reprints to: Dr. Robert S. Viger, Ontogeny and Reproduction Research Unit, T149, Centre Hospitalier de lUniversité Laval (CHUL) Research Centre, 2705 Laurier Boulevard, Ste-Foy, Quebec, Canada G1V 4G2. E-mail:
Robert.Viger{at}crchul.ulaval.ca
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ABSTRACT
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Secretion of Müllerian inhibiting substance by fetal Sertoli
cells is essential for normal male sex differentiation since it induces
regression of the Müllerian ducts in the developing male embryo.
Proper spatiotemporal expression of the MIS gene
requires a specific combination of transcription factors, including the
zinc finger factor GATA-4 and the nuclear receptor steroidogenic
factor-1, which both colocalize with Müllerian inhibiting
substance in Sertoli cells. To establish the molecular mechanisms
through which GATA-4 contributes to MIS transcription,
we have generated and characterized novel GATA-4 dominant negative
competitors. The first one, which consisted solely of the GATA-4 zinc
finger DNA-binding domain, was an efficient competitor of GATA
transcription mediated both by direct GATA binding to DNA and
protein-protein interactions involving GATA factors. The second type of
competitor consisted of the same GATA-4 zinc finger DNA-binding domain
but harboring mutations that prevented DNA binding. This second class
of competitors repressed GATA-dependent transactivation by specifically
competing for GATA protein-protein interactions without affecting the
DNA-binding activity of endogenous GATA factors. These competitors,
along with the GATA-4 cofactor FOG-2 (friend of GATA-2), were used to
specifically modulate endogenous GATA-4 activity in Sertoli cells. Our
results indicate that GATA-4 contributes to MIS promoter
activity through two distinct mechanisms. Moreover, the GATA
competitors described here should provide invaluable in
vitro and in vivo tools for the study of GATA-
dependent transcription and the identification of new target
genes.
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INTRODUCTION
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THE GATA FACTORS are a group of
evolutionarily conserved transcriptional regulators. They share a
highly conserved DNA-binding domain that consists of two zinc fingers
of the form
C-X2-C-X17-C-X2-C.
The C-terminal zinc finger is required for site-specific recognition
and DNA binding to the core WGATAR motif, whereas the N-terminal zinc
finger contributes to the full specificity and stability of the DNA
binding (1, 2, 3). The N- and C-terminal fingers are also
crucial domains involved in protein-protein interactions with other
transcription factors (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The vertebrate family of
GATA factors is comprised of six proteins (GATA-1 to GATA-6) that can
be separated into two subgroups based on sequence homology and tissue
distribution: the hematopoietic group (GATA-1/2/3) and the cardiac
group (GATA-4/5/6). Although GATA factors have similar DNA-binding
properties, they exhibit within each group, distinct spatial and
developmental expression patterns and play essential, nonredundant
functions in cell differentiation, organ development, and cell-
specific gene expression (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Indeed, targeted
disruption of the six murine GATA genes have revealed critical roles
for these factors in such diverse processes as hematopoiesis, heart
tube formation, and genitourinary tract development
(17, 18, 19, 20, 21, 22, 23, 24, 25). Moreover, aberrations in GATA function have now
been recently linked with human disease where a mutation of the
GATA-1 gene has been associated with dyserythropoietic
anemia and thrombocytopenia, and GATA-3 haplo-insufficiency
has been associated with human hypoparathyroidism, sensorineural
deafness, and renal anomaly (HDR) syndrome (26, 27). In
addition to their early developmental roles, GATA factors are likely
involved in many other physiological processes because of their strong
expression in a variety of other tissues, such as the brain, gut,
pituitary, and gonads, where numerous downstream target genes have been
identified (7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37).
Since members of the GATA family share a highly homologous DNA-binding
domain, they all exhibit similar DNA-binding properties (38, 39), and consequently, have been reported to be functionally
interchangeable in some in vitro assays (40, 41). This clearly contrasts, however, with their nonredundant
functions in vivo (17, 18, 19, 20, 21, 22, 23, 24, 25, 40, 42). The
functional specificity of the different GATA factors appears to be
achieved, in part, through combinatorial interactions with other
transcription factors. Indeed, GATA-1, -2, and -3 have been shown to
interact with several cell-restricted or ubiquitously expressed factors
such as rhombotin-2 (RBTN2), nuclear factor erythroid 2 (NF-E2),
erythroid Krüppel-like factor (EKLF), activating protein 1
(AP-1), stem cell leukemia (SCL/Tal1), pituitary factor 1 (Pit1), PU.1,
and stimulatory protein 1 to control the activity of erythroid-,
lymphoid-, and pituitary-specific promoters and enhancers (4, 7, 8, 9, 11, 12, 43, 44). Similarly, GATA-4 has been reported to
interact with the homeoprotein Nkx2.5 and the myocyte enhancer factor
MEF2 to direct cardiac-specific gene expression (5, 6, 13), and with the orphan nuclear receptor steroidogenic factor 1
(SF-1) to synergistically activate several gonadal promoters (10, 28). In addition to the aforementioned GATA-interacting factors,
a novel class of multizinc finger GATA cofactors, named the FOG
(friend-of-GATA) proteins, have been identified as specific modulators
of GATA-dependent transcriptional activity (45, 46, 47, 48, 49). To
date, two FOG proteins have been characterized, FOG-1 and FOG-2, and
each is coexpressed with a specific subgroup of GATA factors: FOG-1
with the hematopoietic group, and FOG-2 with the cardiac group
(45, 46, 48, 49). At the transcriptional level, the FOG
proteins act as either enhancers or repressors of GATA-1 and GATA-4
activity, respectively, depending on the cell and promoter context
(46, 47, 48, 49, 50, 51). The FOG-1 and FOG-2 proteins modulate
GATA-dependent transcription by interacting with the N-terminal zinc
finger of their respective GATA factors (47). Moreover,
the in vivo relevance of the FOG proteins in GATA-mediated
gene expression has been revealed by gene inactivation experiments:
mice lacking FOG-1 die in utero due to arrested erythroid
differentiation (52), while genetic ablation of FOG-2 or a
mutation of the GATA-4 protein that impairs its ability to interact
with FOG-2 leads to defects in heart morphogenesis and coronary
vascular development (53, 54, 55).
Several genes are known to have crucial roles in gonadal development
and sex determination and differentiation. They include
Ftz-F1 (SF-1), WT-1 (Wilms tumor-1),
Sry, Lhx9, and Sox9
(56, 57, 58, 59, 60, 61, 62). In mammals, the crucial step leading to male sex
determination and differentiation is the induction of testis formation.
In males, the bipotential gonad is directed away from ovarian
development and toward testicular differentiation through the action of
Sry (57). Since Sry expression is limited to a
discrete period of gonadal differentiation (63), it acts
as a switch to turn on a network of downstream molecular factors
involved in testicular development and male sex differentiation. The
earliest marker of testis formation is Müllerian inhibiting
substance (MIS), a hormone produced by fetal Sertoli cells. MIS
regulates male sex differentiation by triggering regression of the
Müllerian ducts, the anlagen of the female reproductive tract, in
XY males. MIS expression is sexually dimorphic. Sertoli
cells begin to express MIS on embryonic day 12.5 (E12.5) in
the mouse; expression is maintained throughout fetal development and
then declines markedly after birth (64, 65). In contrast,
MIS expression is absent in fetal ovaries but later appears
in granulosa cells of the ovary during postnatal life (64, 65). Through an analysis of the conserved 5'-regulatory elements
of the MIS gene in vitro and in vivo,
several transcription factors have been proposed to participate in
MIS transcription such as SF-1 and Sox9 (64, 66, 67). However, since Sox9 and SF-1 are colocalized in several
tissues that do not express MIS, other factors must exist to restrict
MIS expression to the gonads. In fact, cooperation between
transcription factors has been shown to contribute to MIS
promoter activity (10, 68, 69).
We have recently reported the presence of GATA-4 in the developing
testis that could also play an integral role in the cell-specific and
high level of MIS expression in the gonads
(30). Since GATA factors are well established regulators
of cell differentiation and organ morphogenesis in other systems, GATA
factors are interesting candidates for the cell- and sex-specific
regulation of MIS expression. In the mouse, GATA-4 protein
is abundant in the somatic cell population of the bipotential gonad
just before the MIS gene is first turned on
(30). Like MIS, GATA-4 expression
becomes highly restricted to Sertoli cells of the fetal testis and is
down-regulated in the ovary (30). We have also shown that
GATA-4 can activate the MIS promoter on its own but also
physically interacts with SF-1, leading to a synergistic activation of
the MIS promoter in a heterologous context
(10). However, the importance of GATA-4, alone and in
association with SF-1, for MIS transcription in a more
in vivo context, such as primary Sertoli cells, has not yet
been established. In the present study, we have generated and
characterized several mutated GATA-4 proteins that were used, along
with the GATA-4 cofactor FOG-2, as novel tools to modulate endogenous
GATA-4 activity in MIS- expressing primary Sertoli cell cultures.
Using these tools, we show that GATA-4 contributes to MIS
promoter activity through two distinct mechanisms, which are both
essential components of an elaborate regulatory complex that controls
the spatiotemporal expression of the MIS gene. Moreover, we
also provide new molecular tools that should be invaluable for
dissecting the molecular mechanisms through which GATA factors
contribute to tissue-specific gene expression in other systems.
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RESULTS
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An Intact GATA Binding Site Is Required for Full MIS
Promoter Activity in Sertoli Cells
We have previously reported that GATA-4 can activate the
MIS promoter by itself and through a synergistic interaction
with the orphan nuclear receptor SF-1 in heterologous cells (10, 30). However, the importance of GATA-4, alone and in association
with SF-1, for MIS transcription in cells that normally
express the hormone in vivo has not yet been clearly
established. Primary Sertoli cell cultures prepared from neonate (3- to
5-day-old) rat testes provide an excellent model system to study
MIS regulatory elements since these cells express the
MIS gene as well as transcription factors proposed to play a
role in its cell- and sex-specific expression, including SF-1 and
GATA-4 (Fig. 1
). Indeed, GATA-4 protein
is detectable in neonate Sertoli cell cultures (30), and
the MIS promoter is active in these cells (Fig. 2
). In addition to GATA-4 and SF-1,
primary Sertoli cell cultures also express FOG-2 (Fig. 1
), a recently
described GATA-4 cofactor known to modulate GATA-4 transcriptional
activity (46, 48, 49, 50).

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Figure 1. Neonate Rat Primary Sertoli Cell Cultures Express
Characteristic Sertoli Cell Markers
The expression of MIS, GATA-4,
SF-1, and FOG-2 in postnatal testis as
well as in neonate primary Sertoli cell cultures was assessed by
RT-PCR. The pairs of oligonucleotide primers used in the PCR reactions
are described in Materials and Methods. The integrity
and relative loading of the different cDNAs were assessed by amplifying
tubulin as a control gene.
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Figure 2. Full MIS Promoter Activity in
Sertoli Cells Requires an Intact GATA Element
Primary Sertoli cells (left panel) and CV-1 cells
(devoid of GATA activity, right panel) were transfected
with various MIS promoter constructs: -180 bp, -180
mut, -83 bp, and -83 mut. The -83 bp deletion constructs no longer
contain the Sox9 and SF-1 binding sites. Mutation of the GATA binding
site (GATA to GGTA) is depicted by an X. Absolute
promoter activities of the different MIS constructs are
expressed as relative light units (±SEM). *,
Significantly different from the -180 bp MIS construct; **,
significantly different from the -180 bp and -180 mut MIS
constructs.
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To determine the significance of the conserved GATA binding site in the
proximal MIS promoter, a point mutation (GATA to
GGTA), known to completely abrogate GATA binding
(30), was introduced into the MIS GATA element.
In the context of the -180-bp promoter, this mutation led to a 40%
decrease in MIS promoter activity in Sertoli cells but not
in CV-1 cells that are essentially devoid of GATA activity (Fig. 2
).
Similarly, a deletion of the MIS promoter to -83 bp, which
removes both the Sox9 and SF-1 binding sites but keeps the GATA element
intact, resulted in a greater than 50% decrease in promoter activity
when compared with the activity of the -180-bp MIS promoter
(Fig. 2
, left panel). Finally, the -83-bp reporter
harboring a mutation in the GATA binding site (-83 mut) was the least
active, representing only 30% of the activity (a 70% decrease) of the
intact -180-bp MIS promoter (Fig. 2
). The effects of these
deletions and mutations were specific since they did not significantly
affect MIS promoter activity in the heterologous CV-1
fibroblast cell line (Fig. 2
, right panel). Thus, the
integrity of the conserved GATA binding site, along with the presence
of other regulatory elements, is essential for maximal MIS
promoter activity in Sertoli cells. These results are in agreement with
a recent study that characterized the requirement of GATA sites for the
activity of the human MIS promoter (70).
A Truncated GATA-4 Protein Acts as a Dominant Negative Competitor
of GATA Transcriptional Activity
To elucidate the molecular mechanism through which GATA-4
contributes to MIS promoter activity, we have generated a
truncated GATA-4 protein (DF WT for double finger wild type) that
consists solely of its zinc finger DNA-binding domain (Fig. 3A
). The DNA binding and transcriptional
properties of the DF WT protein were first characterized in an
heterologous system. Since the DF WT protein retains its zinc finger
region, it bound to DNA as efficiently as the full-length GATA-4
protein (Fig. 3B
). Gel shift data also confirmed that the DF WT protein
is targeted to the nucleus and is expressed at a level similar to that
of the full-length GATA-4 protein. In addition to DNA-binding, the DF
WT protein was able to physically interact with SF-1 (Fig. 6D
).
However, since the DF WT protein lacks transactivation domains, it
could no longer activate a highly responsive synthetic GATA-dependent
reporter (Fig. 3C
). To test the validity of the DF WT protein as a
dominant negative competitor, we first determined its ability to
repress the transactivation of a highly responsive GATA-dependent
reporter in CV-1 cells overexpressing GATA-4. As shown in Fig. 3C
, the
DF WT protein behaved as a dominant negative competitor since it
repressed in a dose-dependent manner the GATA-dependent transactivation
of a GATA-responsive synthetic reporter composed of two GATA elements
from the MIS promoter fused to the minimal MIS
promoter. This effect was specific since the DF WT protein, by itself,
had no significant effect on the same GATA-dependent reporter
(gray bars in Fig. 3C
), nor did it repress the
SF-1-dependent activation of the LHß promoter, a known
SF-1 natural target (stippled bars in Fig. 3D
).

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Figure 3. Characterization of a Truncated GATA-4 Protein That
Acts as Dominant Negative Competitor of GATA Activity
A, Schematic representation of the full-length GATA-4 protein and a
truncated GATA-4 protein that consists solely of its zinc finger region
(DF WT). The two zinc finger regions (ZnF) contained within the
DNA-binding domain (gray box) are shown as two
circles; the basic region, representing the nuclear
localization signal, is indicated by (++). A diagram depicting the
wild-type amino acid sequence of the second zinc finger is also shown.
B, The DNA-binding activity of the DF WT protein was assessed by gel
shift assay using the previously defined MIS GATA
element as probe (30 ). C and D, Effects of DF WT and the
GATA cofactor FOG-2 on GATA-4- and SF-1-dependent transactivation.
Expression plasmids encoding full-length GATA-4, DF WT, and FOG-2 were
transfected in CV-1 cells along with either a highly responsive GATA
reporter consisting of two GATA motifs upstream of the minimal
MIS promoter (C) or the previously characterized
SF-1-dependent LHß promoter (D). In all transfections,
the amount of reporter DNA was kept constant at 500 ng per culture
well. Open bar, Control (empty) vector; gray
shaded bars, increasing doses of DF WT (50, 100, 150 ng) and
FOG-2 (10, 50, 100 ng); black bar, 50 ng GATA-4;
stippled bars, 50 ng GATA-4 and increasing doses (50,
100, 150 ng) of DF WT; hatched bars, 50 ng GATA-4 and
increasing doses (10, 50, 100 ng) of FOG-2. Data are reported as fold
activation (±SEM). *, Significantly different from the
activation mediated by GATA-4 (black bar) alone; **,
significantly different from the activations mediated by GATA-4 alone
and GATA-4 in the presence of the first DF WT or FOG-2 dose.
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Figure 6. DNA-Binding and Protein-Protein Interaction
Properties of Two Mutated Forms of the Truncated GATA-4 Protein DF WT
A, Schematic representation of two truncated GATA-4 proteins (DF
T279 and DF C294A) harboring mutations in the second zinc finger.
The two zinc finger regions (ZnF) contained within the DNA-binding
domain (gray box) are shown as two
circles; the basic region, representing the nuclear
localization signal, is indicated by (++). A diagram depicting the
amino acid sequence of the second zinc finger is also shown. The
threonine at amino acid 279 is deleted in DF T279, whereas the
cysteine-to-alanine substitution at amino acid 294 prevents the second
zinc finger from forming in DF C294A. B, The DNA-binding activity of
the DF T279 and DF C294A proteins was assessed by gel shift assay
using the MIS GATA element as probe. C, The three DF
GATA-4 proteins are expressed at similar levels. Forty-microgram
aliquots of nuclear extracts prepared from L cell fibroblasts
overexpressing the different HA-Tag DF proteins were separated by
SDS-PAGE and Western blotted to Hybond-polyvinylidene difluoride
membrane as described in Materials and Methods.
Immunodetection was achieved using a commercially available antibody
directed against the HA tag. D, The wild-type and mutated DF GATA-4
proteins physically interact with SF-1. In vitro
pull-down assays were performed using immobilized, bacterially produced
MBP fusion proteins (MBP-SF-1 or MBP-LacZ as control) and either
in vitro translated 35S-labeled DF WT, DF
T279, or DF C294A GATA-4 proteins. After extensive washes, bound
proteins were separated on a 12% SDS-PAGE gel and subsequently
visualized by autoradiography.
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FOG-2 is a multizinc finger protein that associates with GATA-4 to
modulate GATA-dependent transcription (46, 48, 49, 51). As
demonstrated in Fig. 3C
, FOG-2 is a potent repressor of
GATA-4-dependent transactivation in CV-1 cells. Like the DF WT protein,
FOG-2 specifically attenuated GATA-mediated transactivation since no
effects were observed on SF-1-dependent transactivation or when the
cofactor was used alone. Thus, FOG-2 and the DF WT protein can be used
to modulate GATA-4 activity through different mechanisms: passive
competition (DF WT) and active repression (FOG-2).
The DF WT Competitor and FOG-2 Are Negative Modulators of
GATA-4/SF-1 Synergism
Since GATA-4 contributes to MIS promoter through a
synergistic interaction with SF-1 (10), we tested whether
the DF WT competitor and FOG-2, in addition to abating GATA-dependent
transactivation, could abrogate GATA-4/SF-1 synergism. The effects of
DF WT and FOG-2 on GATA-4/SF-1 synergism were first tested in
heterologous CV-1 cells using a highly responsive synthetic reporter
that consists of three copies of an oligonucleotide containing the
MIS SF-1 and GATA sites in their normal context
(SF-1:GATA)3 fused to the unresponsive minimal
MIS promoter. This synthetic reporter was previously shown
to exhibit strong synergism in the presence of both factors by
comparison with the native -180-bp MIS promoter, which only
contains one binding site for each factor (10). As shown
in Fig. 4
, the DF WT protein and FOG-2
dramatically reduced transcriptional synergism between GATA-4 and
SF-1.

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Figure 4. The DF WT Competitor and FOG-2 Repress
GATA-4/SF-1 Synergism
Expression plasmids encoding full-length GATA-4, SF-1, DF WT, and
FOG-2 were transfected in CV-1 cells along with a synthetic reporter
containing three copies of an oligonucleotide consisting of the
MIS SF-1 and GATA binding sites in their natural context
(SF-1: GATA)3, fused to the minimal MIS
promoter. In all transfections, the amount of reporter DNA was kept
constant at 500 ng per culture well. Open bar, Control
(empty) vector; light gray shaded bar, 50 ng GATA-4;
dark gray shaded bar, 10 ng SF-1; black
bar, 50 ng GATA-4 and 10 ng SF-1 (GATA-4/SF-1 synergy);
stippled bars, effect of increasing doses of the GATA
dominant negative competitor DF WT (50, 100, 150 ng) on GATA-4/SF-1
synergy; hatched bars, effect of increasing doses of
FOG-2 (10, 50, 100 ng) on GATA-4/SF-1 synergy. Data are reported as
fold activation (±SEM). *, Significantly different from
the synergistic (black bar) activation mediated by
GATA-4 and SF-1.
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Modulation of Endogenous GATA Activity in Sertoli Cells Affects
MIS Promoter Activity
Having demonstrated the validity of the DF WT competitor and FOG-2
as useful tools to modulate GATA activity using a heterologous system,
they were subsequently used to study the contribution of GATA-4 to
MIS promoter activity by modulating endogenous GATA-4
activity in Sertoli cells. When overexpressed in Sertoli cells, the DF
WT competitor and FOG-2 markedly decreased, in a dose-dependent manner,
the activity of the wild-type -180-bp MIS promoter,
indicating that endogenous GATA-4 is required for full promoter
activity in these cells (Fig. 5
, A and
E). Similar results were also observed with the -83-bp MIS
promoter construct, which retains the GATA element but removes the Sox9
and SF-1 binding sites (Fig. 5
, C and G). The observed effects were
specific since the activity of a MIS reporter lacking an
intact GATA element (-83 mut) was not affected by the DF WT competitor
(Fig. 5D
) or by FOG-2 (Fig. 5H
). Interestingly, the activity of a
MIS promoter construct containing a mutated GATA element
together with an intact SF-1 site (-180 mut), was still decreased by
DF WT and FOG-2 (Fig. 5
, B and F). This indicates that endogenous
GATA-4 also contributes to MIS promoter activity through a
direct protein-protein interaction with DNA-bound SF-1. This is
consistent with our previous findings in heterologous cells, which
showed that physical interaction and synergism between GATA-4 and SF-1
does not necessarily require GATA-4 binding to DNA
(10).

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Figure 5. The DF Cells WT Competitor and FOG-2 Repress
MIS Promoter Activity in Sertoli Cells
Primary Sertoli cells were transfected with 500 ng of different
wild-type (A, C, E, and G) and GATA-mutated (B, D, F, and H)
MIS promoter constructs along with either increasing
doses (50, 100, 250 ng) of the DF WT competitor (stippled bars,
left panel) or the GATA cofactor FOG-2 (hatched bars,
right panel). Mutation of the GATA binding site in the proximal
MIS promoter at -75 bp (GATA to
GGTA) is depicted by an X. Promoter activities are expressed
relative to the activity of the reporters cotransfected with the
control (empty) vector (± SEM). *, Significantly different
from control (no DF WT or FOG-2 present); **, significantly different
from control and the first DF WT or FOG-2 dose; ***, significantly
different from control and all DF WT or FOG-2 doses.
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Mutated Forms of the DF WT Protein, Which Are Unable to Bind DNA,
Specifically Compete GATA-4/SF-1 Synergism
Our previous data in CV-1 cells and the present data in Sertoli
cells suggest that GATA-4 not only contributes to MIS
promoter activity by directly binding to its consensus element on the
MIS promoter (Fig. 2
), but also via a direct protein-protein
interaction with SF-1, which is independent of GATA binding to DNA. To
assess the contribution of a bona fide GATA-4/SF-1 interaction to
MIS promoter activity, we generated two mutated forms of the
DF WT competitor that lack the ability to bind DNA but retain the
capacity to physically interact with SF-1 (Fig. 6
). Thus, in contrast to the DF WT
competitor, the mutated DF WT proteins should only compete for
GATA-4/SF-1 interactions and not GATA-4 binding to DNA. As illustrated
in Fig. 6A
, the first mutation (DF
T279) consisted of a deletion of
the amino acid threonine at position 279. This amino acid, although not
involved in the structural integrity of the zinc finger, has been shown
to be essential for recognition of the GATA motif and hence,
DNA-binding (71). The second mutation consisted of a
cysteine-to- alanine substitution at amino acid 294. Because this
cysteine residue plays an integral part in the formation of the
C-terminal zinc finger, the DF C294A protein, like DF
T279, should
be unable to bind DNA. As expected, neither the DF
T279 nor the DF
C294A protein bound to a consensus GATA element when tested in a gel
shift assay (Fig. 6B
). However, both mutated DF proteins were expressed
at the same level as DF WT, as demonstrated by Western blot assays
using an anti-hemagglutin (HA) antibody and N-terminally HA-tagged DF
proteins (Fig. 6C
), or a commercially available GATA-4 antibody and DF
proteins containing the epitope-bearing GATA-4 C-terminal domain (data
not shown). Moreover, all three proteins retained the ability to
physically interact with SF-1 as assessed by an in vitro
pull-down assay (Fig. 6D
). Since we have previously shown that both the
N- and C-terminal zinc fingers of GATA-4 are capable of interacting
with SF-1 (10), the mutations produced in the C-terminal
finger were not expected to affect the ability of the DF
T279 and DF
C294A proteins to interact with SF-1.
As for the DF WT protein, the transcriptional properties of the
DF
T279 and DF C294A proteins were first assessed in heterologous
CV-1 cells (Fig. 7
). Like their wild-type
counterpart, the DF
T279 and DF C294A proteins did not transactivate
GATA-dependent reporters due to the lack of transactivation domains and
the absence of DNA binding (data not shown). However, in comparison to
DF WT, the DF
T279 and DF C294A proteins were ineffective at
competing the GATA-dependent activation of a simple, but highly
GATA-responsive, reporter that consisted of two copies of the GATA
element of the MIS promoter fused to the unresponsive
minimal MIS promoter (Fig. 7A
). Since the mutated DF
proteins retain their ability to interact with SF-1, they were expected
to repress the synergy between GATA-4 and SF-1 as effectively as the DF
WT protein. Indeed, this is what was observed using two different
synthetic reporters that both contain SF-1 binding sites and which
exhibit strong GATA-4/SF-1 synergism as previously reported
(10). As shown in Fig. 7
, B and C, the three DF
competitors were able to efficiently repress GATA-4/SF-1 synergism
regardless of the presence (Fig. 7B
) or absence (Fig. 7C
) of a GATA
binding site. The decrease in GATA-4/SF-1 synergism was attributable to
competition with full-length GATA-4 for protein-protein interaction
with SF-1 rather than by a direct disruption of SF-1-dependent
transactivation (Fig. 7D
).

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Figure 7. Transcriptional Properties of the Mutated DF GATA-4
Proteins, DF T279 and DF C294A
A, Effects of DF T279 and DF C294A on GATA-4-dependent
transactivation. Expression plasmids encoding full-length GATA-4, DF
WT, DF T279, and DF C294A were transfected in CV-1 cells along with
500 ng of a highly responsive GATA reporter consisting of two GATA
motifs upstream of the minimal MIS promoter or (D) 500
ng of a reporter consisting of five copies of the SF-1 binding element
from the steroid 21-hydroxylase promoter fused to the minimal PRL
promoter (80 ). Open bar, Control (empty)
vector; gray shaded bar, 50 ng GATA-4; black
bars, 50 ng GATA-4 and increasing doses of DF WT (50, 100, 150
ng); hatched bars, 50 ng GATA-4 and increasing doses
(50, 100, 150 ng) of DF T279; stippled bars, 50 ng
GATA-4 and increasing doses (50, 100, 150 ng) of DF C294A. *,
Significantly different from the activation mediated by GATA-4
(gray bar) alone; **, significantly different from the
activations mediated by GATA-4 alone and GATA-4 in the presence of the
first DF WT, DF T279, or DF C294A dose. ***, Significantly different
from the activations mediated by GATA-4 alone and GATA-4 in the
presence of all DF WT, DF T279, or DF C294A doses. B and C, The
mutated DF GATA-4 proteins, DF T279 and DF C294A, repress
GATA-4/SF-1 synergism. Expression plasmids encoding full-length GATA-4,
SF-1, DF WT, DF T279, and DF C294A were transfected in CV-1 cells
along with synthetic reporters containing either three copies of an
oligonucleotide consisting of the MIS SF-1 and GATA
binding sites in their natural context (SF-1: GATA)3 (B),
or two copies of the SF-1 binding site from the MIS
promoter (C), in both cases fused to the minimal MIS
promoter. *, Significantly different from the activation mediated by
GATA-4/SF-1 synergism (gray bars); **, significantly
different from the activations mediated by GATA-4/SF-1 synergism and
GATA-4/SF-1 synergism in the presence of the first DF WT, DF T279,
or DF C294A dose. D, The two mutated DF proteins do not affect
SF-1-dependent transactivation. CV-1 cells were transfected as
described in panel A with a highly SF-1 responsive reporter that
consists of five copies of the SF-1 binding site from the steroid
21-hydroxylase promoter fused to the minimal PRL promoter
(80 ). All data are reported as fold activation
(±SEM).
|
|
Overexpression of DF
T279 and DF C294A Competitors in Sertoli
Cells Reveals the Dual Contribution of GATA-4 to MIS
Promoter Activity
Once the DNA-binding and SF-1 interaction properties of the
mutated DF proteins were characterized, they were used to study the
contribution of an endogenous GATA-4/SF-1 protein interaction to
MIS promoter activity in Sertoli cells. When overexpressed
in Sertoli cells, the DF
T279 and DF C294A proteins repressed
MIS promoter activity in a dose-dependent manner (Fig. 8
). Furthermore, this repression was
independent of the presence of a GATA binding site (compare Fig. 8
, panels A and E, with Fig. 8
, panels B and F). Since we showed that the
mutated DF proteins cannot compete for GATA binding to DNA (Fig. 7A
),
their effect on MIS promoter activity was likely mediated by
competing a direct protein-protein interaction between GATA-4 and a
DNA-bound transcription factor. Consistent with this hypothesis, the
two mutated DF proteins still repressed MIS promoter
activity when only the Sox9 binding site was removed (data not shown),
but they had no significant effects on MIS promoter
constructs (-83 bp and -83 mut) that lacked an SF-1 binding site
(Fig. 8
, C, D, G and H). Taken together, these results indicate that a
direct interaction between GATA-4 and SF-1 is an additional and
essential component of the molecular mechanism through which GATA-4
contributes to MIS promoter activity in Sertoli cells.

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|
Figure 8. The Mutated DF GATA-4 Proteins, DF T279 and DF
C294A, Repress MIS Promoter Activity in Sertoli Cells
Primary Sertoli cells were transfected with 500 ng of different
wild-type (A, C, E, and G) and GATA-mutated (B, D, F, and H)
MIS promoter constructs along with either increasing
doses (50, 100, 250 ng) of the DF T279 (hatched bars, left
panel) or DF C294A (stippled bars) constructs.
Mutation of the GATA binding site in the proximal MIS
promoter at -75 bp (GATA to GGTA) is depicted by
an X. Promoter activities are expressed relative to the activity of the
reporters cotransfected with the empty (no DF T279 or DF C294A
present) vector (±SEM). *, Significantly different from
control (no DF T279 or DF C294A present); **, significantly
different from control and the first DF T279 or DF C294A dose; ***,
significantly different from control and all DF T279 or DF C294A
doses.
|
|
 |
DISCUSSION
|
---|
Sexual dimorphism in mammals is initiated by the action of Sry, a
Y-chromosome gene that triggers the gonadal primordium in normal XY
males to differentiate into a testis. Male sexual development is then
controlled by two testicular hormones: testosterone, which is
synthesized and secreted by interstitial Leydig cells, and MIS, which
is produced by Sertoli cells in the seminiferous cords. MIS regulates
sex differentiation by inducing the regression of the Müllerian
duct (the precursor to the female reproductive tract) in males.
MIS gene expression is tightly regulated during gonadal
development; lack of expression causes persistent Müllerian duct
syndrome, a condition in which affected males exhibit both male and
female internal reproductive organs (72). Due to its
sexually dimorphic expression pattern, the MIS gene is an
ideal candidate for elucidating transcription factors involved in
sex-specific gene expression. However, the transcriptional
control of the MIS gene has been somewhat of a
conundrum. Although only a short 180 bp of 5'-flanking region of the
MIS gene appears to be required for cell- and sex-specific
expression in vivo and in vitro (64, 66), we do not yet fully understand the molecular determinants
that control its expression. The proximal MIS promoter
contains species-conserved regulatory elements, but alone they cannot
account for MIS expression in vivo. The proper
spatiotemporal expression of the MIS gene must require the
combinatorial interaction of several transcription factors including
SF-1, Sox9, WT-1, and GATA-4 (30, 68, 69). In the present
study, we report the generation and characterization of novel GATA-4
mutant proteins that behave as dominant negative competitors of GATA
activity. These competitors, along with GATA-specific cofactor FOG-2,
were used to define the molecular mechanism through which GATA-4
contributes to MIS promoter activity in Sertoli cells.
A Dominant Negative Competitor to Study GATA-Dependent Gene
Expression
Over the past decade, GATA factors have emerged as a family of
critical regulators of several key processes that are essential for the
viability of the early developing embryo. Although gene inactivation
experiments have undeniably proven crucial roles for GATA factors in
early vertebrate development, they have been less useful for the study
of their later recruitment as regulators of tissue-specific gene
expression in vivo since five of six
GATA-/- mice die in utero
(17, 18, 19, 20, 21, 22, 23). To overcome this problem and to address the
function and mechanism of action of GATA factors in tissue-specific
gene expression, we have generated and characterized a truncated GATA-4
protein (DF WT) that behaves as a dominant negative competitor of
GATA-dependent transcription. We then used it to elucidate the
mechanism by which GATA-4 regulates MIS promoter activity.
Dominant negative competitors consisting solely of a DNA-binding domain
are clearly important tools that have been successfully used to study
the function of transcription factors other than GATA (73, 74). Interestingly, previous attempts to generate such molecules
for GATA-3 have failed since the mouse GATA-3 protein, much like the
chicken GATA-1 protein, possesses an activation domain located between
the two zinc fingers (2, 75). For GATA-3, mutation of this
particular activation domain was required for it to act as a dominant
negative in vitro and in vivo (75, 76). Unlike GATA-1 and GATA-3, an interfinger activation
domain is not present in the GATA-4 protein since our DF WT protein
could not activate transcription of GATA-dependent reporters (Fig. 3C
),
despite the fact that it was expressed at similar levels as the
full-length GATA-4 protein, was translocated to the nucleus, and
efficiently bound DNA (Fig. 3B
). Moreover, since the DF WT protein not
only repressed GATA-4-mediated transactivation but transactivation
induced by other members of the GATA family (data not shown), the DF WT
protein can be considered as a generalized competitor of GATA activity.
Taken together, our data from experiments first performed in
heterologous cells confirm that the DF WT protein acts as a dominant
negative competitor of GATA-dependent transactivation, thus providing
an invaluable tool to study the contribution of GATA factors to the
regulation of putative GATA-dependent genes.
Indeed, when tested in our primary Sertoli cell cultures, the DF
WT competitor proved to be an efficient dose-dependent repressor of
MIS promoter activity, by competing with endogenous GATA-4
for binding to the MIS GATA element (Fig. 5
). The DF
WT-mediated decrease in MIS promoter activity was consistent
with the 40% decrease in MIS promoter activity in Sertoli
cells when the MIS GATA itself was mutated (Fig. 2
).
Together, these data demonstrate that endogenous GATA-4 contributes to
MIS promoter activity in Sertoli cells by directly binding
to its site in the MIS promoter. Interestingly, the activity
of MIS reporters that harbored a mutated GATA element was
also repressed, albeit to a lesser extent than their wild-type
counterparts, by the DF WT competitor (Fig. 5
). The latter suggests
that endogenous GATA-4 also contributes to MIS promoter
activity in Sertoli cells by a mechanism that is independent of GATA-4
binding to DNA. This mechanism is not dependent on the presence of the
Sox9 binding site but absolutely requires an intact SF-1 binding site
(again compare Fig. 5
, panels B and D). The need for an SF-1 binding
site invariably suggests that endogenous GATA-4 and SF-1 physically and
functionally cooperate in Sertoli cells to control MIS
transcription, a notion that is consistent with our previous data in
heterologous cells (10). The requirement of GATA-4 for
MIS promoter activity in Sertoli cells was further confirmed
by using FOG-2, a well characterized repressor of GATA-4-dependent
transcription in vitro (48, 49, 50). Since FOG-2
binds the corepressor CtBP2 (50), it was not surprising
that FOG-2 was a more active repressor of MIS promoter
activity in Sertoli cells than was the DF WT competitor (Fig. 5
, EH).
Moreover, since we have shown that FOG-2 is coexpressed with GATA-4 in
Sertoli cells (Fig. 1
), the control of GATA-4 activity in these cells
must be tightly linked to the expression level of FOG-2.
Novel Molecular Tools to Dissect the Molecular Mechanisms of
GATA-Mediated Gene Expression
As exemplified by the role of GATA-4 in MIS
transcription, the cell- and promoter-specific activity of the various
GATA factors is most often achieved through direct protein-protein
interactions with other cell- restricted cofactors (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 28, 43, 44). As previously mentioned, protein-protein
interactions involving GATA proteins are often mediated through their
highly conserved zinc finger domain. Therefore, to specifically study
the DNA binding-independent contribution of GATA-4 to MIS
promoter activity, we generated two additional dominant negative
proteins (DF
T279 and DF C294A) that specifically compete for GATA
protein-protein interactions without affecting the DNA-binding activity
of endogenous GATA factors.
As expected, the mutated DF proteins could not compete
GATA-4-mediated transactivation but were as efficient as the wild-type
DF protein (DF WT) in competing GATA-4/SF-1 synergism, indicating that
the mutated DF proteins do effectively compete with full-length GATA-4
for interaction with SF-1. The fact that an intact SF-1 binding site
was absolutely required for the mutated DF proteins to repress MIS
promoter activity in Sertoli cells (compare the -180-bp and -83-bp
MIS reporters in Fig. 8
), demonstrate that the mutated DF proteins are
efficient competitors of the endogenous GATA-4/SF-1 interaction.
Moreover, these results confirm that this interaction is essential for
full MIS promoter activity in Sertoli cells. The dual nature
of the wild-type vs. the mutated DF proteins is further
supported by the fact that when the three DF constructs (WT,
T279,
C294A) were used at similar doses, the mutated DF proteins were less
potent than the DF WT protein at repressing activity of the wild-type
MIS promoter in Sertoli cells (compare Fig. 5A
with Fig. 8
, A and E). This observation is consistent with the fact that the mutated
DF proteins compete only the GATA-4/SF-1 interaction and not GATA-4
binding to DNA, whereas the DF WT protein effectively competes both
activities.
Dual Contribution of GATA-4 to MIS Transcription in
Sertoli Cells
The GATA-4 transcription factor is abundantly expressed in
the developing gonads, and its expression pattern closely parallels
that of the MIS hormone. Although many elegant studies have provided
critical information regarding the transcriptional control of the
MIS gene (64, 66, 67, 68, 69), we do not yet fully
comprehend how MIS expression is restricted to Sertoli cells
in a developmental and sex-specific fashion. By modulating endogenous
GATA-4 activity in Sertoli cells, we have shown that GATA-4 contributes
to MIS promoter activity through two complementary
mechanisms and therefore, GATA-4 constitutes a novel regulator that
helps to explain the cell- and sex-specific expression of the
MIS gene in vivo. The model presented in Fig. 9
depicts the dual contribution of GATA-4
to MIS promoter activity. First, direct GATA-4 binding to
its site leads to a modest activation of the MIS promoter
(Fig. 9A
). Clearly, GATA binding alone cannot account for the
specificity of MIS expression since the Sox9 binding site in
the MIS promoter is absolutely required for MIS
transcription to initiate in vivo (67).
However, it is tempting to speculate that the initiation and low level
of MIS expression, which have been reported in the absence
of an intact MIS SF-1 promoter element (66, 67), require GATA-4 binding to the MIS promoter or
possibly a Sox9/GATA-4 cooperation. The latter would also help to
explain the tissue-specific expression of MIS since Sox9 and
SF-1 are present in multiple cell lineages where MIS is not.
Second, the high level of MIS expression in vivo,
which requires an intact SF-1 binding site (66, 67), is
consistent with the data presented here showing that high
MIS promoter activity can be achieved, at least in part,
when GATA-4 directly interacts with SF-1, either when SF-1 alone (Fig. 9C
) or both factors are bound to their respective sites on the
MIS promoter (Fig. 9B
). It is also possible that two
molecules of GATA-4 can simultaneously interact with SF-1 leading to an
even stronger transcriptional activation of MIS (Fig. 9D
).
Finally, we showed that FOG-2 is present in primary Sertoli cells and
that it can strongly repress the GATA-4-dependent activation of the
MIS promoter (Fig. 9E
). Therefore, the level of FOG-2
expression in Sertoli cells is also likely to be an important player in
the combinatorial code of transcription factors required to control
MIS gene expression in vivo, in a similar way
that Dax-1 represses SF-1-mediated MIS promoter activity
(69, 77).

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Figure 9. Dual Contribution of GATA-4 to MIS
Transcription and Proposed Mechanism for the Role of FOG-2 in the
Control of MIS Gene Expression in Sertoli Cells
Species-conserved elements (Sox9, SF-1, and GATA) in the proximal
MIS promoter are indicated by the stippled,
shaded, and hatched boxes, respectively. A,
GATA-4 alone, by binding to its consensus element in the proximal
MIS promoter, leads to weak transcriptional activation
(small arrow). BD, Protein-protein interactions ( )
between GATA-4 and SF-1 result in a marked enhancement in
MIS promoter activity (heavy arrow).
Three possible scenarios for a GATA-4/SF-1 synergistic interaction
are shown. Thus, direct GATA-4 binding to the MIS
promoter and the synergistic interaction with SF-1 reflect the dual
contribution of GATA-4 to MIS promoter activity. E, The
GATA cofactor FOG-2, which interacts with the repressor CtBP2
(50 ), can also associate with GATA-4, leading to
transcriptional silencing of the MIS promoter.
|
|
 |
MATERIALS AND METHODS
|
---|
Plasmids
The -180, -180 mut, and -83 murine MIS-luciferase
promoter constructs were generated by PCR on mouse genomic DNA as
described previously (10). The -180 mut construct, which
was obtained by site-directed mutagenesis, contains a mutation
(GATA
GGTA) in the consensus MIS
promoter GATA element at -75 bp (10). A shorter version
of this construct, -83 mut, was obtained by PCR using the -180 mut
plasmid as template. The 3X(SF-1:GATA)-MIS luciferase
reporter contains three copies of the MIS SF-1 and GATA
elements in their natural context, cloned upstream of the minimal
MIS promoter (MISmin). The
2XGATA-MISmin luciferase reporter was
generated by cloning two copies of the MIS GATA element
(sense oligonucleotide: 5'-GATCCTGGTGTTGATAGGGGCGTA-3', antisense
oligonucleotide: 5'-GATCTACGCCCCTATCAACACCAG-3' upstream of the minimal
MIS promoter. The -142 bp LHß luciferase reporter was
kindly provided by Jacques Drouin (78, 79). A
cytomegalovirus-driven expression plasmid for rat GATA-4 was made
as described in Ref. 28 . The truncated GATA-4 protein
containing only its two zinc fingers (DF WT) was amplified by PCR on
the full-length GATA-4 cDNA (forward primer:
5'-CGAAGCTTATGGCGAGACACCCCAATCTCGATATG-3'; reverse primer:
5'-ATGGATCCTTAACCTGCTGGTGTCTTAGATTTATT-3') and cloned into the
HindIII/BamHI sites of pcDNA3. The double zinc
finger GATA-4 protein containing a deletion of a threonine at position
279 in its second zinc finger (DF
T279) was generated by
site-directed mutagenesis using the pALTER system on DF WT. A second
mutated double-fingered GATA-4 protein (DF C294A) was generated by
first transferring a HindIII/BamHI fragment
obtained from DF WT into pBluescript SK (Stratagene, La
Jolla, CA). The resulting plasmid was digested with
HindIII/NcoI, thereby liberating the wild-type
GATA-4 zinc finger region (the NcoI site is a naturally
occurring site immediately downstream of the second zinc finger). The
leftover backbone vector was purified, and the GATA-4 zinc finger
region was replaced with a similar fragment generated by PCR (amplified
on the full-length GATA-4 cDNA using forward primer:
5'-CGAAGCTTATGGCGAGACACCCCAATCTCGATATG-3' and reverse primer:
5'-AACCCCATGGAGCTTCATGTAGAGGCCGGCGGCATTGCAAACAGGCTCGCC-3')
but containing a Cys-to-Ala substitution at amino acid 294. A
HindIII/BamHI digest of the resulting plasmid
produced a fragment that was transferred into pcDNA3 to finally yield
DF C294A. The three GATA-4 double-fingered constructs (DF WT, DF
T279, DF C294A) were also transferred into pRSET
(Invitrogen, San Diego, CA) for in vitro35S protein labeling. Additionally, a
double-stranded oligonucleotide (sense:
5'-CTAGCTACCCATACGACGTTCCAGATTACGCTT-3'; antisense:
5'-CTAGAAGCGTAATCTGGAACGTCGTATGGGTAG-3') encoding for an HA epitope was
N-terminally cloned in frame with each of the three GATA-4 DF
constructs. The integrity of all the above mentioned constructs was
verified by sequencing. The SF-1 expression plasmid and
(SF-1)5-PRL reporter were generously provided by
Keith Parker (80). The FOG-2 expression plasmid was kindly
provided by Eric Olson (48).
Isolation of Immature Sertoli Cells
Highly enriched populations of neonate Sertoli cells were
prepared from 3- to 5-d-old Sprague Dawley rats (Charles River Laboratories, Inc., St-Constant, Quebec, Canada) as previously
described (77).
Cell Culture and Transfections
African green monkey kidney CV-1 and mouse fibroblast L cells
were grown in DMEM supplemented with 10% newborn calf serum. Primary
Sertoli cells were maintained in Eagles MEM containing 10% FCS. All
transfections were done in 24-well plates using the calcium phosphate
precipitation method as previously described (77). The day
before transfection, CV-1 and primary Sertoli cells were plated at
densities of 2.2 x 104 and 2.0 x
105 cells per well, respectively. Cells were
transfected 24 h after the initial plating. Culture media were
changed 1216 h after transfection. The cells were finally harvested
the following morning, and an aliquot of the lysate was then assayed
for luciferase activity as described elsewhere (28, 77).
Several DNA preparations of the plasmids were used to ensure
reproducibility of the results. Transfection efficiencies were
monitored by cotransfection with a control ß-galactosidase expression
plasmid. Data reported represent the average of at least three
experiments, each done in duplicate.
RNA Isolation and RT-PCR
Total cellular RNA was prepared from rat testis and neonate
Sertoli cells by the single-step acid guanidinium
thiocyanate-phenol-chloroform method (81). RT-PCR was used
to detect the presence of GATA-4, SF-1, MIS, and FOG-2 mRNAs in the
primary neonate Sertoli cell cultures. Different first-strand cDNAs
were synthesized from total RNA using AMV reverse transcriptase
(Amersham Pharmacia Biotech, Baie-DUrfé, Quebec,
Canada). They were then used as templates in the PCR reactions using
VENT DNA polymerase (New England Biolabs, Inc.,
Mississauga, Ontario, Canada) and oligonucleotide primers specific to
GATA-4 (forward primer: 5'-CTTCTAGACAACCCAATCTCGATATG-3'; reverse
primer: 5'-CAGGATCCAAGTCCGAGCAGGAATTG-3'), SF-1 (forward primer:
ACTCTAGAGCGGGCATGGACTACTCG-3'; reverse primer:
5'-CGGGTACCGCACCTTCGTGCCTAGTCG-3'), MIS (forward primer:
5'-GAACCTTTGTGCCTGGTG-3'; reverse primer: 5'-AGGGTCTCTAGGAAGGGGTC-3'),
and FOG-2 (forward primer: 5'-CCCTCGAGGGTGACTGCTTTCTTTAGTAACTC-3',
reverse primer: 5'-ATGTGCCTACCTGAGCAGGAACA-3'). To verify the
specificity of the amplified bands, PCR products were Southern blotted
to nylon membrane and hybridized with their respective
32P-labeled cDNAs.
Production of Maltose-Binding Protein (MBP) Fusion Proteins and
in Vitro Protein-Protein Binding Assays
A recombinant MBP-SF-1 fusion protein was obtained by cloning
the entire coding region of mouse SF-1 in frame with MBP using the
commercially available pMAL-c fusion protein vector (New England Biolabs, Inc.). The MBP-LacZ
fusion protein was produced by
the pMAL-c vector without any cloned insert. The two fusion proteins
(MBP-SF-1 and MBP-LacZ
) were produced and purified as previously
described (10). In vitro protein-protein
interaction studies were done as previously outlined
(10).
DNA-Binding and Western Blot Assays
Recombinant GATA proteins (full-length GATA-4, DF WT, DF
T279, DF C294A, HA-Tag DF WT, HA-Tag DF
T279, and HA-Tag DF
C294A) were obtained by transfecting L cells (which are devoid of GATA
activity) with the different GATA-4 expression plasmids described
above. Nuclear extracts were prepared 48 h following transfection
by the procedure outlined by Schreiber et al.
(82). DNA-binding assays were performed using a
32P-labeled double-stranded oligonucleotide
corresponding to the conserved GATA element in the proximal
MIS promoter (30). Binding reactions and
electrophoresis conditions were as previously described
(30). In Western analyses, 40 µg aliquots of nuclear
extract containing the three HA-Tag GATA-4 DF proteins were separated
by SDS-PAGE and then transferred to Hybond polyvinylidene
difluoride membranes (Amersham Pharmacia Biotech).
Immunodetection of the HA-Tag GATA proteins was achieved using a HA
antibody (CLONTECH Laboratories, Inc. Palo Alto, CA) and a
Vectastain-ABC-Amp Western blot detection kit
(Vector Laboratories, Inc. Burlingame, CA).
Statistical Analysis
Statistical analyses were done by one-way ANOVA, followed by
Tukeys honestly significant difference tests to detect differences
between groups. The analyses were done with the aid of the SPSS, Inc. software package (SPSS, Inc., Chicago, IL).
P < 0.05 was considered significant.
 |
ACKNOWLEDGMENTS
|
---|
We thank Keith Parker (mouse SF-1 expression plasmid and 5x SF-1
reporter), Eric Olson (mouse FOG-2 expression plasmid), and Jacques
Drouin (-142 bp LHß promoter) for generously providing plasmids used
in this study. We also thank Daniel Cyr for providing some rat testis
RNA samples.
 |
FOOTNOTES
|
---|
This work was supported by a grant from the Canadian Institutes of
Health Research to R.S.V. J.J.T. is a recipient of a postdoctoral
fellowship from the Natural Sciences and Engineering Research Council
of Canada. R.S.V. is a new investigator of the Canadian Institutes of
Health Research and Chercheur-Boursier of the Fonds pour la recherche
en santé du Québec.
Abbreviations: DF WT, Double finger wild type; FOG, friend of
GATA; HA, hemagglutin; MBP, maltose-binding protein; MIS,
Müllerian inhibiting substance; SF-1, steroidogenic
factor-1.
Received for publication September 1, 2000.
Accepted for publication May 24, 2001.
 |
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