Transcription Factor GATA-4 Enhances Müllerian Inhibiting Substance Gene Transcription through a Direct Interaction with the Nuclear Receptor SF-1
Jacques J. Tremblay and
Robert S. Viger
Unité de Recherche en Ontogénie et Reproduction
Centre Hospitalier Universitaire de Québec Pavillon Centre
Hospitalier de lUniversité Laval Ste-Foy, Québec,
Canada G1V 4G2
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ABSTRACT
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Secretion of Müllerian-inhibiting substance
(MIS) by Sertoli cells of the fetal testis and subsequent regression of
the Müllerian ducts in the male embryo is a crucial event that
contributes to proper sex differentiation. The zinc finger
transcription factor GATA-4 and nuclear receptor SF-1 are early markers
of Sertoli cells that have been shown to regulate MIS
transcription. The fact that the GATA and SF-1 binding sites are
adjacent to one another in the MIS promoter raised the
possibility that both factors might transcriptionally cooperate to
regulate MIS expression. Indeed, coexpression of both
factors resulted in a strong synergistic activation of the MIS
promoter. GATA-4/SF-1 synergism was the result of a direct
protein-protein interaction mediated through the zinc finger region of
GATA-4. Remarkably, synergy between GATA-4 and SF-1 on a variety of
different SF-1 targets did not absolutely require GATA binding to DNA.
Moreover, synergy with SF-1 was also observed with other GATA family
members. Thus, these data not only provide a clearer understanding of
the molecular mechanisms that control the sex-specific expression of
the MIS gene but also reveal a potentially novel mechanism
for the regulation of SF-1-dependent genes in tissues where SF-1 and
GATA factors are coexpressed.
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INTRODUCTION
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Mammalian gonadal development and sex differentiation require the
coordinated expression of specific genes in a strict spatio-temporal
manner. At present, we know of at least two genes, Ftz-F1
(encoding the steroidogenic factor SF-1) and WT-1 (Wilms
tumor-1), that are essential for these events because in their absence,
the gonads fail to develop (1, 2, 3). Although not required for the
initiation of gonadal morphogenesis, the testis-determining gene
SRY, present on the Y chromosome (4, 5), is essential for
normal male sex determination because mutation of this gene in XY
individuals leads to sex reversal (6, 7). SRY is believed to induce
testis formation by triggering somatic cell precursors, in the
bipotential gonad of XY embryos, to differentiate into Sertoli cells
and organize into testicular cords (8). A related gene,
SOX9, a member of the HMG box-containing family of proteins,
has been proposed to be a downstream target of SRY in the testis since
in the mouse, Sox9 expression is restricted to the developing male
gonad (9) while in humans, mutation of the SOX9 gene is
associated with sex reversal (10, 11).
In addition to their critical roles in gonadal development, the SF-1,
WT-1, SOX9, and SRY transcription factors also play equally important
roles in the control of sex-specific gene expression. The best studied
male-specific gene lying downstream of these factors in the male sex
differentiation pathway is Müllerian inhibiting substance
(MIS), which encodes a hormone produced by Sertoli cells of
the fetal testis. MIS regulates male sexual differentiation by
triggering the regression of the Müllerian ducts, the anlagen of
the female reproductive tract, in genotypic XY males. MIS
gene 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 (12, 13). In contrast, MIS is apparently not expressed
in the fetal and early postnatal ovary but can be detected in granulosa
cells of the adult ovary (12, 13).
The identification of transacting factors involved in the regulation of
the MIS gene has progressed rapidly in the past few years.
The first gene shown to be crucial for MIS expression is the
orphan nuclear receptor SF-1 (1, 14). Indeed, targeted disruption of
the Ftz-F1 gene, which encodes SF-1, prevents
Müllerian duct regression (1) and, accordingly, in recent
transgenic studies Giuili et al. (14) have demonstrated that
an intact SF-1-binding site is required for the sex-specific expression
of MIS. Since SF-1 is expressed in many tissues where MIS is
not, there has been an active search for potential mechanisms that
could restrict MIS expression to the gonads. An attractive
possibility, which has been frequently observed in other systems, is
that gonad-specific MIS expression is the result of a
combinatorial interaction between SF-1 and other transcriptional
regulators. Indeed, the ability of SF-1 to activate MIS
transcription has recently been shown to be enhanced through direct
protein interactions with WT-1 and Sox9 (15, 16). In part, these
SF-1/WT-1 and SF-1/Sox9 interactions have been helpful in our
understanding of the cell-specific expression of the MIS
gene. We have recently reported, however, the presence of GATA-4, a
member of the GATA family of transcriptional regulators, in the
developing gonads that could also play an integral role in the
cell-specific and high level of MIS expression in the gonads
(17). In the mouse, GATA-4 protein is abundant in the somatic cell
population of the indifferent gonad just before the MIS gene
is first turned on (17). Like MIS, GATA-4
expression later becomes restricted to the Sertoli cell lineage of the
fetal testis and granulosa cells of the adult ovary (17). Moreover, the
proximal MIS promoter contains a functional, species-conserved GATA
element (17). Although other GATA factors have been shown to be
expressed in the gonads, they are expressed after the MIS
gene is markedly down-regulated (18).
The GATA factors, which bind the WGATAR consensus in the 5'-regulatory
region of target genes, are presently an intensely studied group of
transcriptional regulators due to their established roles in cell
differentiation, organ development, and cell-specific gene expression
in many systems (19, 20, 21, 22, 23, 24). The GATA family comprises six members (GATA-1
to -6) that can be divided into two subgroups: the hematopoietic
(GATA-1 to -3) and the cardiac (GATA-4 to -6) groups. Although GATA
factors have similar DNA-binding properties, they exhibit distinct
spatial and developmental expression patterns and play essential,
nonredundant functions (19, 20, 21, 22, 23, 24). Mounting evidence in the literature
suggests that the functional specificity of the different GATA factors
appears to be achieved, in part, via direct protein-protein
interactions with other cell-restricted factors. Indeed, the zinc
finger cofactor FOG interacts with GATA-1 to synergistically activate
hematopoietic-specific gene expression (25). GATA-1, -2, and -3 have
also been shown to interact with several other factors, including
RBTN2, NF-E2, EKLF, TAL1, and Sp1, to regulate the activity of
erythroid- and lymphoid-specific promoters and enhancers (26, 27, 28, 29, 30).
Similarly, GATA-4 has recently been shown to directly interact and
transcriptionally cooperate with the cardiac homeodomain protein
Nkx25 to synergistically activate the atrial natriuretic peptide
(ANF) promoter in the heart (31, 32).
The MIS promoter is a downstream target for GATA-4 and SF-1 in Sertoli
cells (12, 14, 17). We used this promoter to examine whether both
factors functionally cooperate to regulate gene expression in the
gonads. We show here that coexpression of GATA-4 and SF-1 markedly
enhances the activity of the MIS promoter and a panel of other
SF-1-dependent targets. Moreover, we provide evidence that this synergy
is the result of a novel interaction, both in vitro and
in vivo, between SF-1 and the zinc finger region of GATA-4.
Interestingly, synergy between GATA-4 and SF-1 did not absolutely
require GATA binding to DNA. Thus, the present data not only provide
new insights into the cascade of factors that control the sex-specific
expression of the MIS gene but also reveals a potentially
new mechanism for modulating SF-1 activity in tissues where both GATA
factors and SF-1 are coexpressed.
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RESULTS
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The Proximal MIS Promoter Contains Several Species-Conserved
Elements
MIS gene expression appears to be regulated by the
concerted action of several transcription factors. As shown in Fig. 1
, alignment of the proximal MIS promoter
from different species reveals the presence of three highly conserved
regions: a SRY-like element recently reported to bind the SRY-related
protein Sox9 (15), a binding site for the orphan nuclear receptor SF-1
(12), and a consensus GATA motif that we have recently shown to be
recognized by GATA-4 in Sertoli cells (17). Interestingly, the GATA and
SF-1 binding sites in the MIS promoter are 10 bp apart. This close
proximity raises the intriguing possibility that GATA-4 might represent
a novel cofactor for SF-1 in the sex-specific regulation of
MIS gene expression in the gonads. Although GATA-4 and SF-1
have been shown to regulate MIS promoter activity on their own (12, 14, 17), transcriptional synergism between the two factors has not been
reported in the gonads or other tissues where SF-1 and GATA factors are
coexpressed.

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Figure 1. Conserved SF-1- and GATA-Binding Sites Are Present
in the Proximal MIS Promoter
Conserved regulatory elements of the MIS promoter are
boxed, and their locations relative to the
transcriptional start site are shown. Alignment of the mouse (12 ), rat
(69 ), human (70 ), bovine (71 ), and pig (72 ) proximal MIS promoter
sequences reveals consensus SF-1 and GATA-binding sites that are in
close proximity to one another.
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GATA-4 and SF-1 Synergize to Activate MIS
Transcription
We have recently shown GATA-4 to be a potent activator of
MIS gene transcription in vitro (17). To better
define the role of GATA-4 in the cell-specific and developmental
regulation of the MIS gene in vivo, we tested
whether GATA-4 transcriptionally cooperates with SF-1 to enhance
MIS transcription. In our initial experiments we used a
-180 bp MIS promoter construct, which contains both GATA and SF-1
binding sites, since it has been shown to be sufficient for
cell-specific expression in vitro and in vivo
(14, 16). Although crucial for MIS expression in
vivo (14), SF-1 by itself is a poor activator of the MIS promoter
(Fig. 2A
and Ref. 12). In contrast,
GATA-4 significantly activates the MIS promoter (Fig. 2A
).
Surprisingly, the presence of both GATA-4 and SF-1 resulted in a
synergistic activation of the -180 bp MIS promoter (Fig. 2A
, left panel). Synergy was also observed between GATA-4 and a
shorter SF-1 receptor (SF-1
LBD), which was deleted of its
ligand-binding domain (LBD), suggesting that synergistic activation of
the MIS promoter by GATA-4 and SF-1 does not require the LBD of SF-1
(Fig. 2A
, middle panel). As control, no synergy was observed
on the minimal (-65 bp) MIS promoter, which does not contain GATA and
SF-1 binding sites (Fig. 2A
, right panel).

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Figure 2. GATA-4 and SF-1 Transcriptionally Cooperate
A, GATA-4 and SF-1 synergize to enhance MIS transcription.
The -180 bp (left panel) or the minimal (-65 bp;
right panel) murine MIS promoter was cotransfected in
CV-1 cells with either a control (empty) expression vector (open
bars) or expression vectors for GATA-4 (hatched
bars), SF-1 (wild-type or deleted of its ligand-binding domain,
LBD; gray bars), or both GATA-4 and SF-1
(solid bars). B, SF-1 synergizes with several GATA
family members. Different GATA proteins were tested for their ability
to synergize with SF-1 on a synthetic reporter containing three copies
of an oligonucleotide containing the MIS SF-1- and GATA-binding sites
in their natural context (SF-1:GATA)3, fused to the minimal
MIS promoter. C, GATA-4 and SF-1 synergize on other promoters that
contain putative GATA and SF-1 binding sites. The effects of GATA-4
(hatched bars), SF-1 (gray bars), or both
factors (solid bars) were tested on three different
reporters: the -142 bp LHß promoter (right panel), a
synthetic promoter containing two GATA- and three SF-1-binding sites
upstream of the minimal POMC promoter (middle panel),
and the minimal POMC promoter (right panel). Results are
shown as fold activation (±SEM).
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We next determined whether synergy between GATA and SF-1 could also be
observed on a series of natural and artificial promoters that contain
consensus SF-1- and GATA-binding elements (Fig. 2
, B and C). The first
consisted of three copies of an oligonucleotide containing the MIS SF-1
and GATA sites in their normal context fused to the unresponsive
minimal MIS promoter [(SF-1:GATA)3, Fig. 2B
]. As observed
for the -180 bp MIS promoter, SF-1 remained a weak activator of this
reporter despite the presence of three SF-1 binding sites. Several
members of the GATA factor family were then tested for synergy with
SF-1 (Fig. 2B
). Surprisingly, all GATA factors strongly synergized with
SF-1, suggesting that the mechanism underlying GATA/SF-1 synergy is
likely mediated via a domain common to all GATA factors. Indeed, we
have identified this domain to be the zinc finger region (see Fig. 7
, discussed below). As shown in Fig. 2C
, synergy between GATA-4 and SF-1
was not restricted to the GATA/SF-1 elements of the MIS promoter. The
-142 bp LHß promoter, which contains both SF-1 (33) and putative
GATA-binding sites, was also synergistically activated by GATA-4 and
SF-1 (Fig. 2C
, left panel). Similarly, a synthetic reporter
containing two GATA elements and three copies of the SF-1-binding site
from the LHß promoter upstream of the minimal POMC promoter exhibited
strong synergism in the presence of both factors (Fig. 2C
, middle
panel). As observed for the minimal MIS promoter, GATA-4 and SF-1
failed to activate the minimal POMC promoter, which lacks GATA- and
SF-1-binding sites (Fig. 2C
, right panel). Thus, the synergy
between GATA-4 and SF-1 appears to be independent of promoter
context.

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Figure 7. The First or Second Zinc Finger of GATA-4 Can
Interact with SF-1
A, In vitro pull-down assays were used to map the domain
of GATA-4 involved in the direct interaction with SF-1. GATA-4 deletion
mutants were labeled in vitro and then tested for their
ability to interact with either MBP-SF-1 (lanes 2) or MBP-LacZ
(lanes 3) as control. After extensive washes, bound proteins were
separated on 12% SDS-PAGE gels and then visualized by autoradiography.
B, The domain of the GATA-4 protein involved in an in
vivo interaction with SF-1 was identified using the same
reporter system described in Fig. 6B . Results are shown as
GATA-4-dependent enhancement of SF-1 activity (±SEM). The
stippled area indicates no enhancement.
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The C-Terminal Domain and Zinc Finger Region of GATA-4 Are Required
for Synergy with SF-1
To map the domain(s) of the GATA-4 protein required for synergy
with SF-1, a series of GATA-4 deletion mutants were used. A schematic
representation of the wild-type and mutant GATA proteins is illustrated
in Fig. 3A
. The DNA-binding activity of
the GATA proteins was first assessed by gel shift assay. With the
exception of the two deletion mutants (
internal and
N3, Fig. 3B
, lanes 9 and 10) that remove the second zinc
finger, which has been shown to be essential for DNA binding (34, 35),
all GATA-4 proteins were expressed in the nucleus at similar levels, as
evidenced by their binding to the MIS GATA element (Fig. 3B
). The
transcriptional properties of the GATA-4 deletion mutants were
subsequently tested in cotransfection experiments using a reporter
containing two highly responsive GATA motifs upstream of the minimal
MIS promoter. As shown in Fig. 3C
, deletion of the entire N-terminal
domain up to the second zinc finger (
N2) led to a 2-fold
increase in transcriptional activity when compared with the
wild-type GATA-4 protein. Further N-terminal deletion that
removed the second zinc finger (
N3), or an internal
deletion that removed the second zinc finger but kept the first zinc
finger and nuclear localization signal intact
(
internal), completely abolished transcriptional
activity. A recent study has reported that the second zinc finger of
GATA-4, in addition to the nuclear localization signal, is also
involved in the nuclear localization of the protein since its deletion
results in diffuse protein expression both in the nucleus and
cytoplasm (34). However, even when
internal and
N3 were used at higher DNA concentrations to ensure
sufficient nuclear targeting of the proteins, these mutants still
failed to bind DNA and activate a GATA-dependent reporter (Fig. 3
, B
and C). Interestingly, deletion of the C-terminal domain (from aa 333
to 440,
C1) led to an important decrease in
transcriptional activity, suggesting that a potent activation domain
(AD) is present within this region of the GATA-4 protein. The remaining
activity (
25% of wild-type) represents another AD that has
previously been identified in the N-terminal region of GATA proteins
(34). Consistent with these observations, deletion of both the N- and
C-terminal domains (
N1
C1 and
N2
C1) completely abrogated GATA
responsiveness (Fig. 3C
). Taken together, these results indicate that
the GATA-4 protein contains two ADs that flank the DNA-binding domain,
the most potent of which is located in the C-terminal region.

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Figure 3. DNA-Binding and Transcriptional Properties of
GATA-4 Deletion Mutants
A, Schematic representation of the different GATA-4 deletion mutants
used in this study. 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 (++). B, The DNA-binding activity
of the different GATA-4 deletion mutants (lanes 310) were verified by
gel shift assay using the MIS GATA element as probe (17 ). C,
Transcriptional properties of the GATA-4 deletion mutants. Expression
plasmids encoding the different GATA-4 deletion mutants were
cotransfected in CV-1 cells along with a highly responsive GATA
reporter containing two GATA motifs upstream of the minimal MIS
promoter. Results are shown as fold activation (±SEM).
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With the GATA-4 deletion mutants in hand, we proceeded to evaluate
their potential to synergize with SF-1 on the highly responsive
(SF-1:GATA)3-MIS reporter. As shown in Fig. 4
, two GATA-4 domains proved to be
crucial for synergy with SF-1: the C-terminal AD and the second zinc
finger. The presence of both domains was required for synergy, since
deletion of either domain (as reflected in the
C1 and
N3 mutants), abolished the GATA-4-dependent enhancement
of SF-1 activity (Fig. 4
). Consistent with the requirement of these two
domains, deletion of the entire N-terminal domain and the first zinc
finger (
N2) did not abrogate synergy with SF-1.

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Figure 4. The C-Terminal Domain and Zinc Finger Region of
GATA-4 Are Required for Synergy with SF-1
The ability of the GATA-4 deletion mutants to synergize with SF-1 on
the (SF-1:GATA)3-MIS reporter was assessed by
cotransfection experiments in CV-1 cells. Results are shown as
GATA-4-dependent enhancement of SF-1 activity (±SEM).
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Synergy between GATA-4 and SF-1 Requires SF-1 Binding
to DNA
To assess the binding site requirements for synergy between GATA-4
and SF-1, deletions and mutations of the proximal MIS promoter were
generated (Fig. 5
). Synergy between
GATA-4 and SF-1 was still observed on a smaller -107 bp MIS promoter
fragment which, like the -180 bp construct, retains both the GATA- and
SF-1-binding sites (Fig. 5A
). A -83 bp MIS promoter fragment, which
conserves the GATA but removes the SF-1-binding site, was activated
solely by GATA-4 (Fig. 5B
). No synergy was observed when both factors
were used in combination, indicating that a GATA motif by itself is not
sufficient for transcriptional synergism with SF-1. However,
coexpression of SF-1 with GATA-4 substantially repressed the
GATA-4-dependent activation of this promoter construct (Fig. 5B
). This
repression likely represents squelching due to an interaction between
GATA-4 and SF-1, as previously reported for other transcription factors
(36, 37, 38, 39). Similar results were obtained using other GATA-dependent
promoters that lack SF-1 sites: the -114 bp cardiac B-type natriuretic
peptide (BNP) promoter, which contains GATA- but no SF-1-binding sites
(Fig. 5F
), or two synthetic reporters containing two GATA motifs
upstream of either the minimal MIS (Fig. 5D
) or POMC (Fig. 5E
)
promoters. In contrast to the need for an SF-1 site, mutation of the
GATA motif (GATA
GGTA) in the MIS promoter, which was previously
shown to abolish DNA binding and activation by GATA-4 (17), did not
prevent synergy between GATA-4 and SF-1 (Fig. 5C
). The fact that SF-1
can synergize with GATA-4 without the latter binding to DNA suggests
that GATA-4/SF-1 synergism might be the result of a direct
protein-protein interaction between the two factors.

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Figure 5. Synergy between GATA-4 and SF-1 on Target Promoters
Requires an Intact SF-1-Binding Site
The transactivation potential of GATA-4 (hatched bars),
SF-1 (gray bars), or both factors together (solid
bars) on a series of natural and synthetic promoters was tested
by cotransfection experiments in CV-1 cells. To assess the binding site
requirements for synergy between GATA-4 and SF-1, several reporters
were used: A, a -107 bp MIS promoter construct that retains both SF-1-
and GATA-binding sites; B, a deletion to -83 bp that removes the SF-1
binding site; C, the -180 bp MIS promoter containing a point mutation
(GATA GGTA) in the MIS GATA element, a single nucleotide
substitution previously shown to completely abrogate GATA binding (17 );
D and E, reporters containing two GATA motifs upstream of the minimal
MIS and POMC promoters, respectively; F, the -114 bp BNP promoter,
which is a downstream target for GATA-4 in the myocardium (65 ). Results
are shown as fold activation (±SEM).
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GATA-4 and SF-1 Interact in Vitro and in
Vivo
To test the hypothesis that GATA-4 and SF-1 directly interact, we
first used an in vitro pull-down assay. As shown in Fig. 6A
, in vitro translated
35S-labeled GATA-4 was specifically retained by an
immobilized maltose-binding protein (MBP)-SF-1 fusion protein (lane 2
in left panel). No labeled protein was retained, however,
when a MBP-LacZ
fusion was used as control (lane 3 in left
panel of Fig. 6A
). This indicated that the GATA-4/SF-1 interaction
was not mediated by the MBP moiety of the MBP-SF-1 fusion protein.
Moreover, the direct interaction between GATA-4 and SF-1 was specific
since no interaction was observed when an unrelated
35S-labeled luciferase protein was used in the assay (Fig. 6A
, right panel).

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Figure 6. GATA-4 and SF-1 Interact in Vitro
and in Vivo
A, 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 GATA-4 (left panel) or
luciferase (right panel) proteins. After extensive
washes, bound proteins were separated on a 12% SDS-PAGE gel and
subsequently visualized by autoradiography. B, The ability of GATA-4 to
enhance SF-1-dependent transcription through an in vivo
interaction with SF-1 was assessed by cotransfection experiments in
CV-1 cells. Three different reporter constructs, containing only
SF-1-binding sites fused to different minimal promoters, were used:
three copies of the SF-1-binding element from the LHß promoter fused
to the minimal POMC promoter (left panel) (33 ); two
copies of the MIS SF-1 element fused to the minimal MIS promoter
(middle panel); five copies of the SF-1 binding element
from the steroid 21-hydroxylase promoter fused to the minimal PRL
promoter (right panel) (66 ). Hatched
bars, GATA-4 alone; gray bars, SF-1 alone;
solid bars, GATA-4 and SF-1 in combination. Results are
shown as fold activation (±SEM).
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The significance of a protein-protein interaction between GATA-4 and
SF-1 in vivo was ascertained by a series of cotransfection
experiments (Fig. 6B
). In these experiments, three different
SF-1-dependent reporters were used: three copies of the SF-1 binding
site from the LHß promoter fused to the minimal POMC promoter
(left panel), two copies of the MIS SF-1 site upstream of
the minimal MIS promoter (middle panel), and five copies of
the SF-1 binding site from the steroid 21-hydroxylase promoter fused to
the minimal PRL promoter (right panel). As expected, SF-1,
but not GATA-4, activated the three reporters. However, when GATA-4 and
SF-1 were used in combination, GATA-4 significantly enhanced the
SF-1-dependent activation of all three constructs. Since these
SF-1-dependent reporters do not contain GATA motifs to permit GATA
binding, the synergy observed when both GATA-4 and SF-1 were present
indicates an in vivo interaction between the two
factors.
SF-1 Interacts with Either Zinc Finger of GATA-4
The domain(s) of the GATA-4 protein involved in the direct
interaction with SF-1 was initially mapped using in vitro
pull-down assays (Fig. 7A
). N-terminal
deletions right up to the second zinc finger (
N1,
N2) did not affect the ability of the mutant GATA-4
protein to interact with SF-1. Similarly, a deletion removing the
entire C-terminal domain (
C1) did not prevent an
interaction with SF-1. Deletions that removed both zinc fingers of
GATA-4 (
N3 or
C2), however, abolished the
interaction with SF-1. Taken together, these data narrowed the
interaction domain to the zinc finger region of GATA-4, a region we
previously defined to be required for transcriptional synergy with SF-1
(Fig. 4
). Indeed, both zinc fingers of GATA-4 were found to interact
with SF-1. The second zinc finger (
N2
C1),
however, appeared to bind more strongly to SF-1 than the first zinc
finger (
internal).
The mapping of the GATA-4 interaction domain was confirmed in
vivo by cotransfection experiments using the SF-1-dependent
reporters previously described. As shown in Fig. 7B
, we found that when
either the first (aa 201 to 266) or second (aa 242 to 301) zinc finger
region of GATA-4 was fused to the C-terminal AD required for synergy
(
N2 and
internal), a significant
enhancement of SF-1-dependent activity was observed. This enhancement
was not observed with the C-terminal domain alone (
N3),
even when used at higher doses of DNA to ensure nuclear localization.
Thus, both zinc fingers of the GATA-4 protein can interact in
vivo with the nuclear receptor SF-1.
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DISCUSSION
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MIS is a crucial hormone required for Müllerian duct
regression, and hence male sex differentiation. MIS gene
expression is tightly regulated during gonadal development; lack of
expression causes persistent Müllerian duct syndrome in humans, a
condition in which affected males exhibit female internal reproductive
structures (40). The study of the molecular determinants involved in
MIS expression has led to the identification of several
transcription factors involved in the activation of this gene (14, 15, 16, 17, 41). In the present study, we report the transcriptional cooperation
and direct physical interaction between the nuclear receptor SF-1 and
GATA-4, a member of the GATA family of transcriptional regulators, in
the synergistic activation of the MIS promoter. Moreover, these
data allow us to propose a model in which GATA-4, in association with
SF-1 and other transcriptional regulators, contributes to the sexually
dimorphic expression of the MIS gene in the gonads.
GATA-4/SF-1 Synergism Is Mediated via the Zinc Finger Region of
GATA-4
As depicted in Fig. 8A
, the GATA-4
domain required for the physical interaction with SF-1 was mapped to
the DNA-binding region. A more detailed analysis of the GATA-4
interaction domain revealed that both GATA-4 zinc fingers interacted
with SF-1, resulting in transcriptional synergism in the presence of
the C-terminal AD of GATA-4 (Fig. 7
). Since the DNA-binding domain is
highly conserved among the GATA factors, it was not surprising to
observe synergy (Fig. 2B
) and physical interaction (data not shown)
between SF-1 and different GATA family members. Our data are consistent
with previous reports that have also identified the GATA zinc finger
region as a crucial domain involved in protein-protein interactions
with other transcription factors. For example, GATA-1, -2, and -3 have
all been shown to interact with the Krüppel family factors, Sp1
and EKLF, through their respective zinc finger regions (26). Similarly,
self-association of GATA-1 occurs through its zinc finger domains (42).
The C-terminal zinc finger of GATA-4 has also been recently shown to
interact with the cardiac homeoprotein Nkx25 (31, 32, 43), whereas
the interaction between GATA-1 and its cofactor FOG is mediated
exclusively through the N-terminal zinc finger of GATA-1 (25). Thus,
the zinc finger region of GATA proteins appears to be crucial for
protein-protein interaction, as illustrated in the present work
involving GATA-4 and SF-1.

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Figure 8. Involvement of GATA-4 in MIS Gene
Expression
A, Schematic representation of the GATA-4 protein. The GATA-4 domains
required for transcriptional synergism and protein-protein interaction
with SF-1 are indicated. DBD, DNA-binding domain; NLS, nuclear
localization signal; ZnF, zinc finger. B, Ontogeny of
MIS gene expression in the mouse testis and ovary. The
relative embryonic and postnatal (adult) stages of MIS
expression in the gonads are shown along with the expression of
transcription factors previously shown to activate MIS transcription
(9 12 17 60 61 ). C, Proposed mechanism for GATA-4 in the control of
MIS transcription. Different transcriptional regulators (Sox9, WT-1,
and SF-1) proposed to be involved in the regulation of the
MIS gene are also shown. GATA-4, bound to its site in
the proximal MIS promoter, markedly enhances MIS transcription via a
direct interaction with SF-1. As described in this work, GATA-4 can
also synergize with SF-1 to activate SF-1-dependent promoters without
GATA binding to DNA. Thus, in addition to the MIS promoter shown here,
GATA factors also have the potential to stimulate SF-1 target genes in
the absence of GATA-binding elements. Protein-protein interactions are
indicated by ( ).
|
|
GATA Factors, New Partners for SF-1 in the Control of SF-1 Target
Genes
Several classes of transcription factors that share highly
conserved DNA-binding domains exhibit similar DNA-binding properties
in vitro but, in contrast, possess highly specialized
functions in vivo (19, 20, 44, 45). The GATA family of
proteins, which all recognize the WGATAR consensus, represents such
a class of transcription factors. The functional specificity of the
different GATA proteins appears to be achieved, at least in part,
through direct protein-protein interactions with other ubiquitous or
cell-restricted factors, a mechanism that is evolutionarily conserved
in fungi (46, 47), flies (48), and mammals (28, 29, 30, 31, 32, 49, 50, 51, 52). Similarly,
functional interactions between SF-1 and other transcription factors
have been recently reported in the literature. For example, SF-1 has
been shown to directly interact and synergize with the homeoprotein
Ptx1 (33, 53) and the immediate early response factor Egr-1 (33, 54) to
regulate gene expression in the pituitary, and with Wilms tumor-1,
WT-1, in the gonads (16). Furthermore, the Drosophila
homolog of SF-1, Ftz-F1, has been shown to physically and
transcriptionally cooperate with the homeoprotein Ftz (55, 56). The
data presented here represent the first demonstration of a functional
interaction between two important regulators of cell differentiation,
organogenesis, and cell-specific gene expression: the nuclear receptor
SF-1 and GATA-4, a member of the GATA family of proteins.
We initially thought that synergy between GATA-4 and SF-1 would be
specific to the MIS promoter, given that the SF-1- and GATA-binding
sites are in close proximity to each other (Fig. 1
). Remarkably, we
also observed strong synergistic activation by GATA-4 and SF-1 on a
variety of other natural and synthetic SF-1-dependent promoters such as
the LHß promoter (Fig. 2
). The LHß promoter is a well characterized
downstream target for SF-1 in pituitary gonadotropes (57, 58).
Interestingly, the proximal LHß promoter contains an SF-1 binding
site (33) and a potential GATA motif. Moreover, GATA factors, including
GATA-4, are coexpressed with SF-1 in pituitary gonadotropes (59),
suggesting that transcriptional cooperation between GATA and SF-1 may
also be implicated in pituitary-specific gene expression. The fact that
synergy with SF-1 was also observed with other GATA family members
(Fig. 2B
) and the fact that GATA/SF-1 synergy did not absolutely
require GATA binding to DNA (Figs. 5C
, 6B
, and 7B
) are further
indications that transcriptional cooperation between GATA factors and
SF-1 may have broader implications. Thus, the functional interaction
between GATA-4 and SF-1, described here, appears to represent a more
generalized mechanism for the regulation of SF-1 target genes in
tissues where both SF-1 and GATA factors are coexpressed.
A Role for GATA-4 in Male Sex Differentiation
One of the critical steps in the establishment of the proper male
phenotype is the regression of the Müllerian ducts, which is
mediated through the action of MIS. At present, four factors have been
proposed to be involved in the sex-specific regulation of the
MIS gene: SF-1, WT-1, SOX9, and GATA-4. As shown in Fig. 8B
, the onset of MIS gene expression occurs around E12.5 in the
mouse, shortly after testis differentiation. Based on solid evidence
recently reported in the literature (14, 16), MIS gene
expression in vivo appears to absolutely require the
presence of both SF-1 and WT-1. It is interesting to note, however,
that these two factors appear in the developing gonadal primordium of
the mouse (E9.510.5) somewhat before the MIS gene is
actually first turned on (E12.5) (13, 60, 61, 62). This observation
invariably suggests that another factor is likely missing at this time.
A recent report has proposed Sox9 to be this factor since it was shown
to interact with SF-1 (15). Several other lines of evidence, however,
appear to contradict this possibility. First, Sox9
expression (15), like SF-1 and WT-1, considerably
precedes that of MIS (13). Second, in granulosa cells of the
adult ovary, MIS is expressed in the absence of Sox9 (9, 13). Lastly, in the chick, MIS was shown to be expressed
before Sox9 (63). Taken together, these observations support
the existence of another factor that participates in the cell-specific
expression of the MIS gene. Our previous findings on the
sexually dimorphic expression pattern of the GATA-4 transcription
factor in the developing gonads, combined with the present functional
interaction data with SF-1 on the MIS promoter, support the notion
that GATA-4 is involved in MIS expression in
vivo. Indeed, abundant GATA-4 expression begins in the bipotential
gonad of the mouse on E11.5 (17), just before the detection of MIS
transcripts (13). Moreover, GATA-4, like MIS, is
expressed in granulosa cells of the adult mouse ovary (13, 17). Thus,
as presented in Fig. 8C
, these data are consistent with a model in
which GATA-4 is an integral component of the combinatorial code of
factors required for MIS gene expression and, consequently,
male sex differentiation.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The -180, -83, and -65 bp murine MIS-luciferase promoter
constructs have been described previously (17). The -180-bp MIS
promoter harboring a mutation in the GATA element at -75 bp
(GATA
GGTA) was obtained using the pALTER
site-directed mutagenesis system (Promega Corp., Madison,
WI); the mutation was confirmed by DNA sequencing. The -107 bp MIS
promoter was amplified by PCR using the -180 bp construct as template
and then cloned in the BamHI/HindIII site of the
luciferase expression vector pXP1, as previously described for the
other MIS promoter constructs (17). Since the pXP1 vector normally
contains two GATA motifs upstream of its multiple cloning site, all
pXP1 luciferase reporter constructs were modified to delete these
highly responsive GATA sites. This was achieved by replacing a
600-bp NdeI-BamHI fragment with a similar
fragment (minus the GATA sites) that was generated by PCR (forward
primer: 5'-TTCACACCGCATATGGTGCACT-3'; reverse primer:
5'-ACGGATCCAAGCTTACATTGATGAGTTTGGACAAAC-3') on
the promoterless pXP1 vector. Thus, the (GATA)2-MIS
luciferase reporter consists of the minimal (-65 bp) MIS promoter in
the unmodified pXP1 vector. The (SF-1:GATA)3-MIS luciferase
reporter was obtained by cloning three copies of a double-stranded MIS
SF-1:GATA element (sense oligonucleotide:
5'-GATCCAGGCACTGTCCCCCAAGGTCACCTTTGGTGTTGATAGGGGCGA-3'; antisense
oligonucleotide:
5'-GATCTCGCCCCTATCAACACCAAAGGTCACCTTGGGGGACAGTGCCTG-3') upstream of the
minimal MIS promoter in the modified pXP1 vector. Similarly, the
(SF-1)2-MIS luciferase reporter was obtained by cloning two
copies of the MIS SF-1 element (sense oligonucleotide:
5'-GATCCCCCAAGGTCACCTTTA-3'; antisense oligonucleotide:
5'-GATCTAAAGGTGACCTTGGGGG-3') in front of the minimal MIS promoter in
the modified pXP1 vector. The minimal POMC, (SF-1)3-POMC,
(GATA)2-POMC, (GATA)2/(SF-1)3-POMC,
and -142 bp LHß luciferase reporters were kind gifts of Jacques
Drouin (33, 64). In the POMC constructs, SF-1 refers to the SF-1
element found in the bovine LHß promoter (33). Again, the GATA motifs
in the POMC constructs given above come from an unmodified pXP1
luciferase reporter. GATA expression vectors, the -114 bp BNP
reporter, and certain GATA-4 deletion mutants (
N1,
N2,
C1,
C2,
N1C1,
N2C1) were
kindly provided by Mona Nemer. The remaining GATA-4 deletion mutants
(
N3 and
internal) were generated by PCR
on the wild-type GATA-4 cDNA and then cloned into the
XbaI/BamHI site of the pCG expression vector (31, 65). The forward primer for the
N3 construct was
5'-CTTCTAGAGGGGTTCCCAGGCCTCTTGCA-3, and the reverse primer was
5'-CAGGATCCAAGTCCGAGCAGGAATTT-3'. The
internal construct
was obtained by initially cloning the first zinc finger of GATA-4,
obtained by PCR, into pCG (forward primer:
5'-CTTCTAGACAACCCAATCTCGATATG-3'; reverse primer:
5'-ATGGATCCTTAGCTAGCCAGCCGGCGCTGAGGCTTGATGAGGGGC-3').
The latter was digested with NheI/BamHI (the
NheI site being provided by the reverse primer given above),
and the XbaI-BamHI C-terminal PCR fragment used
to produce
N3 was cloned in frame to yield
internal. The SF-1 expression plasmid and
(SF-1)5-PRL reporter were generously provided by Keith
Parker. The (SF-1)5-PRL construct consists five copies of
the 21-hydroxylase SF-1 element cloned upstream of the minimal PRL
promoter (66).
Cell Culture and Transfections
African green monkey kidney CV-1 and murine L cells were grown
in DMEM supplemented with 10% newborn calf serum. Transfections were
done in 12-well plates using the calcium phosphate precipitation method
(67). CV-1 cells were plated at a density of 60,000 cells per well
24 h before transfection. Cell media were changed 1216 h later,
and cells were harvested the next morning. Cells were lysed by adding
100 µl of lysis buffer (100 mM Tris-HCl, pH 7.9, 0.5%
Igepal (Sigma Chemical Co., St. Louis, MO), and 5
mM dithiothreitol) directly to the wells. Luciferase
activity (60 µl aliquot of lysate) was then assayed using an EG&G
Berthold (Bad Wildbad, Germany) LB 9507 luminometer. In synergy
experiments, optimal doses of SF-1 (10 ng/well) and GATA-4 (100
ng/well) expression vector were used. It is important to note that the
level of activation obtained when both GATA-4 and SF-1 were present
(synergy) could never be achieved by simply increasing the amount of
GATA-4 or SF-1 when used alone (independent activation). In fact, the
optimal dose of GATA-4 for maximum activation was 100 ng/well, which is
the same dose used in the synergy experiments. Thus, the synergy
observed when both factors are present indicates a true synergy. In all
experiments, the total amount of DNA was kept constant at 6 µg per
well using Sp64 (Promega Corp., Madison, WI) as carrier
DNA; several DNA preparations of the plasmids were used to ensure
reproducibility of the results. Data reported represent the average of
3 to 10 experiments, each done in duplicate.
Production of MBP Fusion Proteins
A recombinant MBP-SF-1 fusion protein was obtained by cloning
the entire coding region of murine SF-1 in frame with MBP using the
commercially available pMAL-c fusion protein vector (New England Biolabs, Inc., Mississauga, Ontario, Canada). The MBP-LacZ
fusion protein was provided by the wild-type pMAL-c vector. The two
fusion proteins constructs (MBP-SF-1 and MBP-LacZ
) were introduced
into the Escherichia coli strain BL21, and fusion proteins
were produced by inducing the respective bacterial cultures with
isopropylthiogalactoside. Bacterial cultures were lysed by sonication,
and the fusion proteins were purified using an amylose resin (New England Biolabs, Inc.) as outlined by the manufacturer.
Protein-Protein Binding Assays
Protein-protein binding studies were done using
35S-labeled in vitro translated GATA-4 proteins
(wild-type and deletion mutants) and the purified MBP-SF-1 and
MBP-LacZ
fusion proteins coupled to amylose resin. The
35S-labeled GATA-4 and luciferase proteins were obtained
using the TNT system from Promega Corp.; the amino acid
positions of the different GATA-4 proteins used are given in Fig. 3A
.
Protein-protein interaction assays were done using 1 µg of MBP-fusion
protein and 10 µl of in vitro translated
35S-labeled protein essentially as described by Durocher
et al. (31). Bound complexes were separated by SDS-PAGE, and
retained proteins were revealed by autoradiography.
DNA-Binding Assays
Recombinant GATA proteins (wild-type and deletion mutants) 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 after transfection by the procedure
outlined by Schreiber et al. (68). DNA-binding assays were
performed using a 32P-labeled double-stranded
oligonucleotide corresponding to the conserved MIS promoter GATA
element at -75 bp. Binding reactions and electrophoresis conditions
were as previously described (17).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Mona Nemer for providing the GATA expression
vectors, several GATA deletion mutants, and the -114 bp BNP promoter;
Jacques Drouin for the -142 bp LHß and POMC promoter constructs; and
Keith Parker for generously providing the SF-1 expression plasmid
and (SF-1)5-PRL reporter. Eric Legault and Veronique
Tremblay are also thanked for their help in the cloning and sequencing
of some of the MIS promoter constructs.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Robert S. Viger, Unité de Recherche en Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Pavillon Centre de Recherche du CHUL, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2.
This work was supported by a grant from the Medical Research Council of
Canada to R.S.V.
Received for publication March 23, 1999.
Accepted for publication May 13, 1999.
 |
REFERENCES
|
---|
-
Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear
receptor is essential for adrenal and gonadal development and sexual
differentiation. Cell 77:481490[Medline]
-
Pritchard-Jones K, Fleming S, Davidson D, Bickmore W,
Porteous D, Gosden C, Bard J, Buckler A, Pelletier J, Housman D, van
Heyningen V, Hastie N 1990 The candidate Wilms tumour gene is
involved in genitourinary development. Nature 346:194197[CrossRef][Medline]
-
Pelletier J, Bruening W, Li FP, Haber DA, Glaser T, Housman
DE 1991 WT1 mutations contribute to abnormal genital system development
and hereditary Wilms tumour. Nature 353:431434[CrossRef][Medline]
-
Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL,
Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN 1990 A
gene from the human sex-determining region encodes a protein with
homology to a conserved DNA-binding motif. Nature 346:240244[CrossRef][Medline]
-
Gubbay J, Collignon J, Koopman P, Capel B, Economou A,
Münsterberg A, Vivian N, Goodfellow P, Lovell-Badge R 1990 A gene
mapping to the sex-determining region of the mouse Y chromosome is a
member of a novel family of embryonically expressed genes. Nature 346:245250[CrossRef][Medline]
-
Hawkins JR, Taylor A, Berta P, Levilliers J, Van der Auwera
B, Goodfellow PN 1992 Mutational analysis of SRY: nonsense and missense
mutations in XY sex reversal. Hum Genet 88:471474[Medline]
-
Hawkins JR, Taylor A, Goodfellow PN, Migeon CJ, Smith KD,
Berkovitz GD 1992 Evidence for increased prevalence of SRY mutations in
XY females with complete rather than partial gonadal dysgenesis.
Am J Hum Genet 51:979984[Medline]
-
Palmer SJ, Burgoyne PS 1991 In situ analysis of fetal,
prepuberal and adult XXXY chimaeric mouse testes: Sertoli cells are
predominantly, but not exclusively, XY. Development 112:265268[Abstract]
-
Morais da Silva S, Hacker A, Harley V, Goodfellow P, Swain A,
Lovell-Badge R 1996 Sox9 expression during gonadal development implies
a conserved role for the gene in testis differentiation in mammals and
birds. Nat Genet 14:6268[Medline]
-
Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J,
Pasantes J, Bricarelli FD, Keutel J, Hustert E 1994 Autosomal sex
reversal and campomelic dysplasia are caused by mutations in and around
the SRY-related gene SOX9. Cell 79:11111120[Medline]
-
Foster JW, Dominguez-Steglich MA, Guioli S, Kowk G, Weller PA,
Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN 1994 Campomelic dysplasia and autosomal sex reversal caused by mutations in
an SRY-related gene. Nature 372:525530[Medline]
-
Shen WH, Moore CC, Ikeda Y, Parker KL, Ingraham HA 1994 Nuclear receptor steroidogenic factor 1 regulates the Müllerian
inhibiting substance gene: a link to the sex determination cascade.
Cell 77:651661[Medline]
-
Münsterberg A, Lovell-Badge R 1991 Expression of the
mouse anti-Müllerian hormone gene suggests a role in both male
and female sexual differentiation. Development 113:613624[Abstract]
-
Giuili G, Shen WH, Ingraham HA 1997 The nuclear receptor SF-1
mediates sexually dimorphic expression of Müllerian inhibiting
substance, in vivo. Development 124:17991807[Abstract/Free Full Text]
-
De Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M,
Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P 1998 Direct
interaction of SRY-related protein SOX9 and steroidogenic factor 1
regulates transcription of the human anti-Müllerian hormone gene.
Mol Cell Biol 18:66536665[Abstract/Free Full Text]
-
Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN,
Hammer GD, Ingraham HA 1998 Wilms tumor 1 and Dax-1 modulate the
orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 93:445454[Medline]
-
Viger RS, Mertineit C, Trasler JM, Nemer M 1998 Transcription
factor GATA-4 is expressed in a sexually dimorphic pattern during mouse
gonadal development and is a potent activator of the Müllerian
inhibiting substance promoter. Development 125:26652675[Abstract/Free Full Text]
-
Yomogida K, Ohtani H, Harigae H, Ito E, Nishimune Y, Engel JD,
Yamamoto M 1994 Developmental stage- and spermatogenic cycle-specific
expression of transcription factor GATA-1 in mouse Sertoli cells.
Development 120:17591766[Abstract/Free Full Text]
-
Weiss MJ, Orkin SH 1995 GATA transcription factors: key
regulators of hematopoiesis. Exp Hematol 23:99107[Medline]
-
Simon MC 1995 Gotta have GATA. Nat Genet 11:911[Medline]
-
Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek
MS, Soudais C, Leiden JM 1997 GATA-4 transcription factor is required
for ventral morphogenesis and heart tube formation. Genes Dev 11:10481060[Abstract]
-
Molkentin JD, Lin Q, Duncan SA, Olson EN 1997 Requirement of
the transcription factor GATA-4 for heart tube formation and ventral
morphogenesis. Genes Dev 11:10611072[Abstract]
-
Morrisey EE, Tang Z, Sigrist K, Lu MM, Jiang F, Ip HS,
Parmacek MS 98 A.D. GATA-6 regulates HNF4 and is required for
differentiation of visceral endoderm in the mouse embryo. Genes Dev 12:35793590
-
Koutsourakis M, Langeveld A, Patient R, Beddington R, Grosveld
F 1999 The transcription factor GATA-6 is essential for early
extraembryonic development. Development 126:723732[Abstract/Free Full Text]
-
Tsang AP, Visvader JE, Turner CA, Fujiwara Y, Yu C, Weiss MJ,
Crossley M, Orkin SH 1997 FOG, a multitype zinc finger protein, acts as
a cofactor for transcription factor GATA-1 in erythroid and
megakaryocytic differentiation. Cell 90:109119[CrossRef][Medline]
-
Merika M, Orkin SH 1995 Functional synergy and physical
interactions of the erythroid transcription factor GATA-1 with the
Krüppel family proteins Sp1 and EKLF. Mol Cell Biol 15:24372447[Abstract]
-
Gregory RC, Taxman DJ, Seshasayee D, Kensinger MH, Bieker JJ,
Wojchowski DM 1996 Functional interaction of GATA-1 with erythroid
Krüppel-like factor and Sp1 at defined erythroid promoters. Blood 87:17931801[Abstract/Free Full Text]
-
Osada H, Grutz G, Axelson H, Forster A, Rabbitts TH 1995 Association of erythroid transcription factors: complexes involving the
LIM protein RBTN2 and the zinc-finger protein GATA-1. Proc Natl Acad
Sci USA 92:95859589[Abstract]
-
Gong Q, Dean A 1993 Enhancer-dependent transcription of the
epsilon-globin promoter requires promoter-bound GATA-1 and
enhancer-bound AP-1/NF-E2. Mol Cell Biol 13:911917[Abstract]
-
Walters M, Martin DI 1992 Functional erythroid promoters
created by interaction of the transcription factor GATA-1 with CACCC
and AP-1/NFE-2 elements. Proc Natl Acad Sci USA 89:1044410448[Abstract]
-
Durocher D, Charron F, Warren R, Schwartz RJ, Nemer M 1997 The
cardiac transcription factors Nkx-2.5 and GATA-4 are mutual cofactors.
EMBO J 16:56875696[Abstract/Free Full Text]
-
Lee Y, Shioi T, Kasahara H, Jobe SM, Wiese RJ, Markham BE,
Izumo S 1998 The cardiac tissue-restricted homeobox protein Csx/Nkx2.5
physically associates with the zinc finger protein GATA-4 and
cooperatively activates atrial natriuretic factor gene expression. Mol
Cell Biol 18:31203129[Abstract/Free Full Text]
-
Tremblay JJ, Drouin J 1999 Egr-1 is a downstream effector of
GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance
LHß gene transcription. Mol Cell Biol 19:25672576[Abstract/Free Full Text]
-
Morrisey EE, Ip HS, Tang Z, Parmacek MS 1997 GATA-4 activates
transcription via two novel domains that are conserved within the
GATA-4/5/6 subfamily. J Biol Chem 272:85158524[Abstract/Free Full Text]
-
Visvader JE, Crossley M, Hill J, Orkin SH, Adams JM 1995 The
C-terminal zinc finger of GATA-1 or GATA-2 is sufficient to induce
megakaryocytic differentiation of an early myeloid cell line. Mol Cell
Biol 15:634641[Abstract]
-
Cahill MA, Ernst WH, Janknecht R, Nordheim A 1994 Regulatory
squelching. FEBS Lett 344:105108[CrossRef][Medline]
-
Yang-Yen HS, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin
J, Karin M 1990 Transcription interference between c-jun and
glucocorticoid receptor due to mutual inhibition of DNA-binding
activity. Cell 62:12051215[Medline]
-
Schüle R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J,
Yang N, Verma IM, Evans RM 1990 Functional antagonism between
oncoprotein c-jun and the glucocorticoid receptor. Cell 62:12171226[Medline]
-
Philips A, Maira M, Mullick A, Chamberland M, Lesage S, Hugo
P, Drouin J 1997 Antagonism between Nur77 and glucocorticoid receptor
for control of transcription. Mol Cell Biol 17:59525959[Abstract]
-
Rey R, Picard JY 1998 Embryology and endocrinology of genital
development. Baillieres Clin Endocrinol Metab 12:1733[Medline]
-
Haqq CM, King CY, Donahoe PK, Weiss MA 1993 SRY recognizes
conserved DNA sites in sex-specific promoters. Proc Natl Acad Sci USA 90:10971101[Abstract]
-
Crossley M, Merika M, Orkin SH 1995 Self-association of the
erythroid transcription factor GATA-1 mediated by its zinc finger
domains. Mol Cell Biol 15:24482456[Abstract]
-
Sepulveda JL, Belaguli N, Nigam V, Chen CY, Nemer M, Schwartz
RJ 1998 GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role
for regulating early cardiac gene expression. Mol Cell Biol 18:34053415[Abstract/Free Full Text]
-
Mann RS, Chan SK 1996 Extra specificity from extradenticle:
the partnership between HOX and PBX/EXD homeodomain proteins. Trends
Genet 12:258262[CrossRef][Medline]
-
Drouin J, Lamolet B, Lamonerie T, Lanctôt C, Tremblay JJ 1998 The PTX family of homeodomain transcription factors during
pituitary development. Mol Cell Endocrinol 140:3136[CrossRef][Medline]
-
Svetlov VV, Cooper TG 1998 The Saccharomyces cerevisiae
GATA factors Dal80p and Deh1p can form homo- and heterodimeric
complexes. J Bacteriol 180:56825688[Abstract/Free Full Text]
-
Feng B, Marzluf GA 1998 Interaction between major nitrogen
regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is
required for their synergistic activation of gene expression in
Neurospora crassa. Mol Cell Biol 18:39833990[Abstract/Free Full Text]
-
Haenlin M, Cubadda Y, Blondeau F, Heitzler P, Lutz Y, Simpson
P, Ramain P 1997 Transcriptional activity of pannier is regulated
negatively by heterodimerization of the GATA DNA-binding domain with a
cofactor encoded by the u-shaped gene of Drosophila. Genes
Dev 11:30963108[Abstract/Free Full Text]
-
Blobel GA, Nakajima T, Eckner R, Montminy M, Orkin SH 1998 CREB-binding protein cooperates with transcription factor GATA-1 and is
required for erythroid differentiation. Proc Natl Acad Sci USA 95:20612066[Abstract/Free Full Text]
-
Gordon DF, Lewis SR, Haugen BR, James RA, McDermott MT, Wood
WM, Ridgway EC 1997 Pit-1 and GATA-2 interact and functionally
cooperate to activate the thyrotropin ß-subunit promoter. J Biol
Chem 272:2433924347[Abstract/Free Full Text]
-
Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal H, Forster
A, Rabbitts TH 1997 The LIM-only protein Lmo2 is a bridging molecule
assembling an erythroid, DNA-binding complex which includes the TAL1,
E47, GATA-1 and Ldb1/NLI proteins. EMBO J 16:31453157[Abstract/Free Full Text]
-
Ono Y, Fukuhara N, Yoshie O 1998 TAL1 and LIM-only proteins
synergistically induce retinaldehyde dehydrogenase 2 expression in
T-cell acute lymphoblastic leukemia by acting as cofactors for GATA-3.
Mol Cell Biol 18:69396950[Abstract/Free Full Text]
-
Tremblay JJ, Lanctôt C, Drouin J 1998 The pan-pituitary
activator of transcription, Ptx1 (pituitary homeobox 1), acts in
synergy with SF-1 and Pit1 and is an upstream regulator of the
lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 12:428441[Abstract/Free Full Text]
-
Halvorson LM, Ito M, Jameson JL, Chin WW 1998 Steroidogenic
factor-1 and early growth response protein 1 act through two composite
DNA binding sites to regulate luteinizing hormone ß-subunit gene
expression. J Biol Chem 273:1471214720[Abstract/Free Full Text]
-
Yu Y, Li W, Su K, Yussa M, Han W, Perrimon N, Pick L 1997 The
nuclear hormone receptor Ftz-F1 is a cofactor for the
Drosophila homeodomain protein Ftz. Nature 385:552555[CrossRef][Medline]
-
Guichet A, Copeland JW, Erdelyi M, Hlousek D, Zavorszky P, Ho
J, Brown S, Percival-Smith A, Krause HM, Ephrussi A 1997 The nuclear
receptor homologue Ftz-F1 and the homeodomain protein Ftz are mutually
dependent cofactors. Nature 385:548552[CrossRef][Medline]
-
Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of
luteinizing hormone beta gene promoter activity by the orphan nuclear
receptor, steroidogenic factor-1. J Biol Chem 271:66456650[Abstract/Free Full Text]
-
Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site
is required for activity of the luteinizing hormone ß-subunit
promoter in gonadotropes of transgenic mice. J Biol Chem 271:1078210785[Abstract/Free Full Text]
-
Steger D, Hecht JH, Mellon PL 1994 GATA-binding proteins
regulate the human gonadotropin
-subunit gene in the placenta and
pituitary gland. Mol Cell Biol 14:55925602[Abstract]
-
Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Developmental
expression of mouse steroidogenic factor-1, an essential regulator of
the steroid hydroxylases. Mol Endocrinol 8:654662[Abstract]
-
Pelletier J, Schalling M, Buckler AJ, Rogers A, Haber DA,
Housman D 1991 Expression of the Wilms tumor gene WT1 in the murine
urogenital system. Genes Dev 5:13451356[Abstract]
-
Rackley RR, Flenniken AM, Kuriyan NP, Kessler PM, Stoler MH,
Williams BR 1993 Expression of the Wilms tumor suppressor gene WT1
during mouse embryogenesis. Cell Growth Differ 4:10231031[Abstract]
-
Oreal E, Pieau C, Mattei MG, Josso N, Picard JY, Carre-Eusebe
D, Magre S 1998 Early expression of AMH in chicken embryonic gonads
precedes testicular SOX9 expression. Dev Dyn 214:522532[CrossRef]
-
Jeannotte L, Trifiro MA, Plante RK, Chamberland M, Drouin J 1987 Tissue-specific activity of the pro-opiomelanocortin gene
promoter. Mol Cell Biol 7:40584064[Medline]
-
Grépin C, Dagnino L, Robitaille L, Haberstroh L, Antakly
T, Nemer M 1994 A hormone-encoding gene identifies a pathway for
cardiac but not skeletal muscle gene transcription. Mol Cell Biol 14:31153129[Abstract]
-
Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key
regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852860[Abstract]
-
Chen C, Okayama H 1987 High efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid
detection of octamer binding proteins with mini-extracts, prepared
from a small number of cells. Nucleic Acids Res 17:64196419[Medline]
-
Haqq C, Lee MM, Tizard R, Wysk M, DeMarinis J, Donahoe PK,
Cate RL 1992 Isolation of the rat gene for Müllerian inhibiting
substance. Genomics 12:665669[Medline]
-
Guerrier D, Boussin L, Mader S, Josso N, Kahn A, Picard JY 1990 Expression of the gene for anti-Müllerian hormone. J Reprod
Fertil 88:695706[Abstract]
-
Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung
A, Ninfa EG, Frey AZ, Gash DJ, Chow EP 1986 Isolation of the bovine and
human genes for Müllerian inhibiting substance and expression of
the human gene in animal cells. Cell 45:685698[Medline]
-
Pilon N, Behdjani R, Daneau I, Lussier JG, Silversides DW 1998 Porcine steroidogenic factor-1 gene (pSF-1) expression and analysis of
embryonic pig gonads during sexual differentiation. Endocrinology 139:38033812[Abstract/Free Full Text]