The Murine Dax-1 Promoter Is Stimulated by SF-1 (Steroidogenic Factor-1) and Inhibited by COUP-TF (Chicken Ovalbumin Upstream Promoter-Transcription Factor) via a Composite Nuclear Receptor-Regulatory Element
Richard N. Yu,
Masafumi Ito and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine
Northwestern University Medical School Chicago, Illinois 60611
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ABSTRACT
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The Dax-1 gene encodes a protein that
is structurally related to members of the orphan nuclear receptor
superfamily. Dax-1 is coexpressed with another orphan
nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal,
gonads, hypothalamus, and pituitary gland. Mutations in
Dax-1 cause adrenal hypoplasia congenita, a disorder that
is characterized by adrenal insufficiency and hypogonadotropic
hypogonadism. These developmental and endocrine abnormalities are
similar to those caused by disruption of the murine Ftz-F1
gene (which encodes SF-1), suggesting that these nuclear receptors act
along the same developmental cascade. Cloning of the murine
Dax-1 gene revealed a candidate SF-1-binding site in the
Dax-1 promoter. In transient expression assays in
SF-1-deficient JEG-3 cells, SF-1 stimulated expression of the
Dax-1 promoter. However, deletion or mutation of the
consensus SF-1-binding site did not eliminate SF-1 stimulation. Further
analyses revealed the presence of a cryptic SF-1 site that creates an
imperfect direct repeat of the SF-1 element. When linked to the minimal
thymidine kinase promoter, each of the isolated SF-1 sites was
sufficient to mediate transcriptional regulation by SF-1. Mutation of
both SF-1 sites eliminated SF-1 binding and stimulation of the
Dax-1 promoter. Unexpectedly, mutation of either half of
the composite SF-1 sites increased basal activity in JEG-3 cells,
suggesting interaction of a repressor protein. Gel shift analyses of
the composite response element revealed an additional complex that was
not supershifted by SF-1 antibodies. This complex was eliminated by
mutation of either half-site, and it was supershifted by antibodies
against chicken ovalbumin upstream promoter-transcription factor
(COUP-TF). We propose that Dax-1 is stimulated by SF-1, and
that SF-1 and COUP-TF provide antagonistic pathways that converge upon
a common regulatory site.
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INTRODUCTION
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The Dax-1 gene was identified based upon its
association with adrenal hypoplasia congenita (AHC), an X-linked
disorder that causes adrenal insufficiency and hypogonadotropic
hypogonadism (1, 2). The Dax-1 locus on Xp21 has also been
associated with dosage-sensitive sex reversal in males in which this
region of the X-chromosome is duplicated (3). These features are
reflected in its name, DAX-1 (dosage-sensitive sex-reversal-AHC
critical region on the X-chromosome) (1).
The cloning of Dax-1 revealed that it encodes a variant
member of the orphan nuclear receptor superfamily (1). The highest
degree of homology with other nuclear receptors resides in the
C-terminal portion of the DAX-1 protein. This putative ligand-binding
domain is most homologous with that of chicken ovalbumin upstream
promoter-transcription factor (COUP-TF) and steroidogenic factor-1
(SF-1) (1, 4, 5). However, DAX-1 is unique by virtue of the
amino-terminal domain that lacks the zinc-finger DNA-binding domain
that is characteristic of other nuclear receptors. Instead, DAX-1
contains a unique N-terminal domain composed of a repeating 66- to
67-amino acid motif (1, 6). Recently, DAX-1 has been shown to bind to
palindromic stem-loop sequences that are present in the promoters of
some of its target genes (7), and it acts as a potent repressor of
transcription (1, 8, 9).
DAX-1 is expressed in a tissue-specific manner that reflects sites of
endocrine dysfunction in patients with AHC, including the adrenal
cortex, testis, ovary, anterior pituitary gonadotropes, and ventral
medial hypothalamus (10, 11, 12, 13). These sites of expression are also
characteristic of tissues that express SF-1 (13, 14, 15, 16, 17). Moreover,
disruption of the Ftz-f1 locus that encodes SF-1 results in
a phenotype that partially resembles AHC (18, 19, 20, 21). Mice that are
homozygous for the SF-1 gene knockout exhibit absent adrenal glands and
gonads, as well as hypogonadotropic hypogonadism (15, 16, 20, 22). The
male mice are sex-reversed, apparently reflecting impaired development
of the primordial cells that give rise to the differentiated gonad
(18). There may also be a defect in SF-1 regulation of the
Müllerian-inhibiting substance (MIS) gene (15) and the
steroidogenic enzyme genes (23, 24). The absence of male
sex-differentiation in the SF-1 knockout mice is distinct from the
effects of Dax-1 mutations, which allow normal development
of the male phenotype in affected humans (25).
The similar phenotypic features caused by Ftz-f1 and
Dax-1 mutations, and their similar spatial and developmental
patterns of expression, have led to the suggestion that there is a
functional relationship between these two factors (8, 11, 13, 26).
Although little is known about the function of DAX-1, SF-1 is known to
regulate an array of steroidogenic enzyme genes (24), as well as genes
involved in sex differentiation (15) and gonadotropin regulation (15, 16, 27, 28, 29, 30). It has been suggested that DAX-1 might interact with SF-1
to either stimulate or antagonize its transcriptional properties (7, 8, 13, 24). Alternatively, it is possible that DAX-1 and SF-1 participate
in a developmental cascade in which the product of one gene regulates
expression of the other gene (13, 24, 26). An SF-1-binding site has
been reported in the promoter of the human Dax-1 gene (31)
and the mouse Dax-1 gene (13). In this report, we examined
whether SF-1 regulates the murine Dax-1 gene. Unexpectedly,
we found redundant SF-1-binding sites in the Dax-1 promoter
and demonstrate that this duplicated region also creates a binding site
for COUP-TF, which acts as a repressor.
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RESULTS
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The Murine Dax-1 Promoter Contains a Duplicated Binding
Site for SF-1
Previous analyses of the human Dax-1 promoter indicated
the presence of an SF-1-binding site (13, 31). The murine
Dax-1 gene was isolated by screening a genomic library with
the human Dax-1 cDNA. The 5'-flanking region (2.9 kb) of the
murine Dax-1 promoter was sequenced, and the proximal region
near the transcriptional start site is shown in Fig. 1
. The indicated transcriptional start
site was confirmed using ribonuclease (RNase) protection assays of mRNA
from murine adrenocortical Y1 and murine anterior pituitary gonadotrope
T3 cells (data not shown), and it corresponds to the site reported
by Ikeda et al. (13). Analysis of the Dax-1
promoter sequence revealed that a putative SF-1-binding site (-129 to
-121, TCGAGGTCA) is also present in the murine gene.

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Figure 1. Structure of the Murine DAX-1 Promoter
The DNA sequence of the proximal region of the murine DAX-1 promoter is
shown, and the nucleotides are numbered relative to the transcriptional
start site (arrow), identified by RNase protection
assays. The TATA-box is underlined and the translation
initiation codon (ATG) is boxed with the encoded amino
acids shown below the codons. Two overlapping elements
resembling the SF-1 consensus sequence are indicated by
horizontal arrows as site A (-129 to -121) and site B
(-123 to -115).
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Electrophoretic mobility shift assays (EMSAs) were performed to examine
SF-1 binding to the murine Dax-1 promoter (Fig. 2
). Using in vitro translated
murine SF-1, a major complex was formed that was eliminated by the
addition of an antibody to SF-1 (see below). A mutation (GG
TT) was
introduced into the putative SF-1 site (-129 to -121) to confirm that
it binds SF-1 (Fig. 2A
). This two-nucleotide mutation eliminates SF-1
binding to a consensus SF-1 site (data not shown). Unexpectedly, this
mutation (134 m1a) had no apparent effect on SF-1 binding (Fig. 2B
).
This finding led us to consider whether an additional SF-1-binding site
might reside within this region of the Dax-1 promoter.
Inspection of the sequence suggested a possible cryptic SF-1 site (site
B) adjacent to the consensus sequence (site A). Mutations were
therefore also introduced into site B, or sites A and B. Like the
mutation in site A (134 m1a), the mutation in site B alone (134 m1b)
was not sufficient to eliminate SF-1 binding. However, a double
mutation in sites A and B (134 m1ab) prevented SF-1 binding. An
additional mutation (134 m3), which was predicted not to change
nucleotides critical for SF-1 binding, had no effect on the formation
of the SF-1 complex. These results are consistent with the ability of
SF-1 to bind to either sites A or B in the composite element.

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Figure 2. SF-1 Binds Specifically to the Murine DAX-1
Promoter
A, Oligonucleotide DNA sequences (sense strands) used in the EMSAs.
Mutations introduced into the sequence are indicated
below the wild-type oligonucleotide (134wt). B, EMSAs
were performed using in vitro translated murine SF-1 (3
µl lysate). 32P-labeled probe (20 fmol) was incubated
with 2 µl reticulocyte lysate and resolved on 4% polyacrylamide
gels. The SF-1 band is indicated by an arrow. NP
indicates the use of nonprogrammed reticulocyte lysate.
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The binding characteristics of sites A and B were also examined using
nuclear extracts prepared from
T3 cells and Y1 cells (Fig. 3
). Both of these cell lines have been
shown previously to contain abundant SF-1 protein (22, 32). Using a
25-bp sequence that contains both of the putative SF-1 binding sites
(134wt; -134 to -110 bp), nuclear extracts from
T3 and Y1 cells
resulted in three major protein/DNA complexes, A, B, and C (Fig. 3
, A
and B). An antibody directed against the DNA-binding domain of SF-1
specifically prevented the formation of complex A, indicating that this
band corresponds to bound SF-1. The anti-SF-1 antibody did not alter
complexes B or C, suggesting that they do not contain SF-1.

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Figure 3. Competition for SF-1 Binding to Sites A and B in
the Dax-1 Promoter
EMSAs were performed using nuclear extracts (5 µg) prepared from a
murine T3 pituitary gonadotrope cell line (panel A) and from a
murine Y-1 adrenocortical cell line (panel B). The indicated
32P-labeled probes (20 fmol) were incubated with nuclear
extracts and resolved on 4% polyacrylamide gels. Three distinct
protein/DNA complexes were formed (A, B, and C) with the 134wt probe.
The identity of SF-1 was determined by using a polyclonal rabbit
antimurine SF-1 antibody (complex A).
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Because site B might be configured as either a direct repeat, or as a
palindrome, two sets of mutations were introduced into this site (134
m1b and 134 m1c, respectively) (see Fig. 2A
). As found with in
vitro translated SF-1, mutations of site A (134 m1a) or site B
(134 m1b, 134 m1c) alone were not sufficient to eliminate SF-1 binding
(complex A). The site A mutation (134 m1a) eliminated complexes B and
C, whereas the site B mutations only partially decreased binding to the
slower mobility complexes (complexes B and C). The double mutation of
both sites A and B (134 m1ab) eliminated the binding of all three major
complexes, including SF-1 (complex A). These results confirm that the
murine Dax-1 promoter contains duplicated SF-1 sites and
indicate that additional proteins also bind to this composite
element.
Competition studies were performed to define further the
characteristics of the complexes binding to the composite SF-1 sites
(Fig. 4
). Using extracts from Y1 cells,
competition with a canonical SF-1 sequence (gatcTCAAGGTCAgatc)
inhibited the binding of all three major complexes, although complex C
was affected less than complexes A and B (Fig. 4A
and data not shown).
These competition studies also revealed the presence of minor bands at
the positions of complexes A and B that are not inhibited by the
canonical SF-1 site. As before, an antibody directed against SF-1
impaired the binding of complex A, but did not alter complexes B or C.
An irrelevant control antibody directed against retinoid X receptor-
did not affect the SF-1 complex.

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Figure 4. Competition for Binding to the Murine DAX-1
Composite Element
EMSAs were performed using nuclear extracts (5 µg) prepared from Y-1
cells and the wild-type -134 radiolabeled probe. In panel A,
competition was performed using 100-fold or 500-fold excess of an
unlabeled canonical SF-1 sequence (gatcTCAAGGTCAgatc). Antibodies
include anti-retinoid X receptor- and anti-SF-1. In panels B and C,
the indicated competitor (100-fold excess) oligonucleotides were
incubated in the presence of nuclear extract, and EMSAs were performed
in the absence (panel B) or presence (panel C) of the anti-SF-1
antibody.
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Competition studies were also performed using various mutants of the
Dax-1 composite element (Fig. 4B
). The site A mutation (134
m1a) retained competition for SF-1 (complex A), but failed to compete
for complexes B and C, consistent with the ability of site B to bind
SF-1 but not the other complexes. The single mutations of site B (134
m1b, 134 m1c) retained competition for each of the complexes,
indicating that, like the canonical SF-1 sequence, site A is sufficient
to bind all three complexes. Mutations in both sites A and B (134 m1ab)
did not compete for any of the complexes. Similar competition studies
were also performed in the presence of the anti-SF-1 antibody (Fig. 4C
). The antibody did not affect complexes B or C, even in the presence
of a selective competitor for SF-1 (134 m1a). Taken together, these
results are consistent with SF-1 binding to both sites A and B. In
addition, mutations in site A, but not site B, impair the binding of
complexes B and C.
SF-1 Stimulates the Murine Dax-1 Promoter in
SF-1-Deficient JEG-3 Cells
JEG-3 cells are a placental choriocarcinoma cell line that is
deficient in SF-1 as assessed by RT-PCR and by Western blot analysis
(8). The effect of SF-1 on promoter activity was examined in this cell
line by cotransfecting various Dax-1 promoter deletion
mutants in the absence or presence of an SF-1 expression vector
(cytomegalovirus-driven SF-1). In the absence of cotransfected SF-1,
there was little change in basal Dax-1 promoter activity
until deletion from -114 to -84 bp, which resulted in about 2-fold
stimulation (Fig. 5A
). Further deletion
from -84 to -50 bp markedly decreased promoter activity, suggesting
the presence of a basal element in this region.

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Figure 5. Transcriptional Activity of the Murine DAX-1
Promoter in the Absence or Presence of an SF-1 Expression Construct
A and B, Deletion series of murine DAX-1 promoter-luciferase reporter
constructs transfected into JEG-3 cells with an empty expression
construct (panel A) or with a CMV-driven, SF-1 expression construct
(panel B). C and D, Mutant series of murine DAX-1 promoter-luciferase
reporter constructs transfected into JEG-3 cells with an empty
expression construct (panel C) or with a CMV-driven, SF-1 expression
construct (panel D). Each reporter plasmid (0.5 µg) was transfected
into JEG-3 cells with 100 ng of the empty or SF-1 expression constructs
and cultured for 48 h. Basal activity is shown in ALU.
Fold-stimulation by SF-1 is calculated for each construct as the ratio
of promoter activity in the presence and absence of transfected SF-1.
The error bars represent mean ± SD.
Control refers to a promoterless luciferase plasmid.
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Because the deletion mutations alter the level of basal activity,
the effect of cotransfected SF-1 is presented as fold-stimulation
relative to the basal activity of each construct (Fig. 5B
). The
full-length Dax-1 promoter (2938 bp) was stimulated 17-fold
by cotransfection of SF-1. SF-1 had no effect on the promoterless
plasmid or control viral promoters (data not shown). There was a
progressive decline of SF-1 stimulation between -2938 and -1376 bp
before returning to the maximal level with further deletions between
-861 and -134 bp. Deletion of the SF-1-binding sites (sites A and B)
between -134 and -114 bp resulted in an abrupt loss of SF-1-mediated
transcription.
Point mutations of site A or site B, or in sites A and B, were
used to correlate the functional regulation by SF-1 with its binding
properties. These mutations in the native -134 Dax-1
promoter are identical to those introduced into the EMSA probes (Fig. 2A
). Unexpectedly, the 134 m1a point mutation in site A caused a 4- to
5-fold increase in basal promoter activity relative to the 134wt
reporter gene (Fig. 5C
). This result raised the possibility that a
repressor may bind to site A (this mutation eliminates complexes B and
C). A less pronounced increase in basal activity was also observed with
the 134 m1b mutation in site B, and the double mutant of sites A and B
(134 m1ab) increased basal activity to a level similar to that of the
site A mutant alone. SF-1 stimulation of the 134wt promoter was reduced
from 17-fold to less than 5-fold by each of the individual SF-1-binding
site mutations. These data indicate that each of the SF-1-binding sites
are required for maximal induction of the Dax-1 promoter by
SF-1.
A series of heterologous promoter constructs was also created to
determine whether the isolated composite regulatory element is
sufficient for SF-1-mediated transcriptional regulation (Fig. 6
). The -134 to -110 sequence from the
murine Dax-1 promoter was inserted upstream of a minimal
thymidine kinase (TK) promoter (TK81-luc). Specific point mutations of
site A or site B, or both sites A and B, were examined with the TK
reporter constructs. The results with the TK promoter largely parallel
the findings with the native Dax-1 promoter. Relative to the
wild-type element, the 134 m1a (site A) mutation increased basal
promoter activity, but there was less effect with the 134 m1b and the
double mutant, 134 m1ab (Fig. 6A
). As with the native promoter,
mutation of either site A or site B, or sites A and B, reduced or
eliminated stimulation by SF-1.

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Figure 6. SF-1-Regulatory Elements from the DAX-1 Promoter
Are Functional When Linked to a Heterologous Promoter
Regulatory activity of the isolated composite response element of the
murine DAX-1 promoter. Oligonucleotides containing the wild-type and
mutant SF-1 response elements described in Fig. 2 were fused to a TK
minimal promoter luciferase reporter construct. These constructs were
transfected into JEG-3 cells with an empty expression construct (panel
A) or with a CMV-driven, SF-1 expression construct (panel B). Each
reporter plasmid (1.0 µg) was transfected into JEG-3 cells with 100
ng of the empty or SF-1 expression construct. Luciferase activity
(mean ± SD) was determined after 48 h. Basal
activity is shown in ALU. Fold-stimulation by SF-1 is calculated for
each construct as the ratio of promoter activity in the presence and
absence of transfected SF-1.
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Mutation of the Dax-1 Promoter SF-1 Sites Reduces Basal
Activity in SF-1-Containing
T3 Cells and Y1 Cells
The functional role of the SF-1-binding sites was also analyzed in
cell lines (pituitary gonadotrope
T3 cells and murine adrenocortical
Y1 cells) that express SF-1 endogenously (22, 33). Both cell lines
exhibited a high basal level of expression of the full-length 2.9-kb
Dax-1 promoter construct, consistent with the presence of
SF-1 in these cell lines (Fig. 7
, A and
C). Sequential deletion of the 5'-flanking region did not alter
promoter activity substantially until deletion between -134 and -114
bp (includes sites A and B), which caused
50% loss of activity in
both
T3 and Y1 cells. Further deletion from -114 to -84 bp had
little effect, but deletion to -50 bp reduced activity to background
levels.

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Figure 7. Native Murine DAX-1 Promoter Transcriptional
Activity in SF-1-Containing Cell Lines
Luciferase activity of the deletion series (panels A and C) and mutant
series (panels B and D) of murine DAX-1 promoter luciferase reporter
constructs. Each reporter plasmid (0.5 µg) was transfected into T3
anterior pituitary cells (panels A and B) or into Y1 adrenal cells
(panels C and D). Luciferase activity (mean ± SD) was
measured after 48 h. Basal activity is shown in ALU.
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When -134 bp promoter constructs containing specific point mutations
of the composite regulatory element were transfected, a reduction of
basal luciferase activity was observed upon mutation of either site A
or site B, or both sites A and B (Fig. 7
, B and D). The deletion and
point mutation studies indicate that the SF-1 sites confer 3050% of
the basal activity of the Dax-1 promoter in cell lines that
express high levels of SF-1.
The isolated SF-1 elements linked to the TK promoter confirmed the
results of the native promoter studies in both
T3 and Y1 cells (Fig. 8
). The site A -134/-110 m1a-TK
construct caused 3050% reduction of activity. The site B mutation
(-134/-110 m1b), and the combined site A and site B mutations
(-134/-110 m1ab), reduced activity close to that of the TK promoter
alone.
The Composite SF-1-Binding Site Binds COUP-TF Family Members
As shown above, in addition to SF-1, the 134wt probe binds a
complex (complex B) that has slower mobility and appears to contain
several distinct bands. A series of antibodies were used in an attempt
to identify additional proteins that might bind to SF-1-like sequences.
Using
T3 nuclear extracts, antibodies directed against COUP-TF
caused a supershift of most of the proteins in complex B (Fig. 9A
). As shown before, anti-SF-1 antibody
eliminated the binding of complex A. Antibodies against DAX-1,
cAMP-response element binding protein (CREB), and phospho-CREB did not
alter the binding pattern of the complexes. Inspection of the DNA
sequence of the composite SF-1 site revealed the presence of an
imperfect palindrome (-129/-115) that overlaps the SF-1 direct repeat
(Fig. 9B
). The palindromic element exhibits similarity to several
sequences previously reported to bind and mediate COUP-TF activity (34, 35).

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Figure 9. COUP-TF Binds to the Composite Regulatory Element
A, Demonstration of COUP-TF binding to the SF-1 regulatory composite
element. EMSAs were performed using nuclear extracts (5 µg) prepared
from a murine pituitary gonadotrope cell line, T3.
32P-labeled 134wt probe (20 fmol) was incubated with the
nuclear extracts as described in Fig. 2 . Supershift analyses were
performed using antibodies against DAX-1, SF-1, COUP-TF, CREB, and
phospho-CREB proteins. B, Comparison of the composite element DNA
sequence to known COUP-TF-binding sites. PAL, Palindromic version of
the GGTCA sequence; mPOMC, mouse POMC element. C and D, EMSAs were
performed using in vitro translated Ear3/COUP-TF1 (panel
C) or ARP1/COUP-TF2 (panel D) proteins. Wild-type or mutant
32P-labeled probe (20 fmol) was incubated with in
vitro translated products as described in Fig. 2 . NP indicates
the use of nonprogrammed reticulocyte lysate.
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Murine COUP-TF1 and COUP-TF2 were in vitro translated and
tested in EMSAs for binding to the composite element (Fig. 9
, C and D).
Using the 134wt probe, both COUP-TF1 and COUP-TF2 bound to the
composite site, although greater binding was seen with COUP-TF2. As
seen with the native extracts, mutation of site A eliminated COUP-TF
binding, whereas mutations of sites B or C reduced COUP-TF binding. The
double mutation of sites A and B also eliminated the binding of
COUP-TF. These data indicate that site A is necessary for the binding
of COUP-TF, but sequences in site B also serve to provide maximal
binding of COUP-TF.
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DISCUSSION
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The murine Dax-1 promoter contains a duplicated binding
site for SF-1. Both sites are capable of binding SF-1 and can mediate
transcriptional activation. The combined sites (-134 to -114 region)
function as a composite element that is capable of interacting with a
number of different proteins, resulting in the formation of three major
complexes. Supershift analyses have identified two of the these
complexes as SF-1 (complex A) and COUP-TF (complex B). Complex C has
not yet been characterized. Both half-sites are required for full
SF-1-mediated activation. Disruption of these sites lowered the basal
activity of the native Dax-1 promoter in SF-1-containing
T3 and Y1 cells, demonstrating the requirement for the intact
-134/-114 element in maintaining nominal transcriptional
activity.
Deletion of the -134/-114 region, or specific mutation of half-site
A, increases basal promoter activity in JEG-3 cells, suggesting a
relief of repression that requires the presence of site A. This site
corresponds to the binding site for complex B, which contains COUP-TF.
The inhibitory activity of COUP-TF may be mediated through a direct
interaction with a novel class of ubiquitous proteins (36). These
factors, N-CoR (nuclear receptor corepressor) and SMRT (silencing
mediator for retinoic acid receptor and thyroid hormone receptor) (37, 38), possess strong silencing activity. Thus, inhibition by COUP-TF may
involve the recruitment of corepressors (36), as well as its ability to
block SF-1 interactions with its target site. In the mouse
Dax-1 promoter, site A, but not site B, is required for the
formation of complex B. Based upon the mobility of complex B, it is
likely that COUP-TF binds as a dimer, but further studies will be
required to define the nature of the COUP-TF complexes.
In general, COUP-TF serves as a negative regulator of a wide array of
genes (39). The two major forms of COUP-TF, I and II, are widely
expressed, and their levels are high particularly during organogenesis
and neurogenesis when they are believed to exert a repressive function
on target genes (39, 40, 41). The spatial expression of COUP-TF overlaps
that of DAX-1, although detailed comparisons of their developmental
patterns of expression have not been performed. Targeted disruption of
COUP-TFI and COUP-TFII result in perinatal (39, 42) and embryonic (39)
lethality, respectively. In view of our studies suggesting a role for
COUP-TF in the control of the Dax-1 promoter, it will be of
interest to determine whether the expression of DAX-1 is altered in
these animals.
Inhibition by COUP-TF can be effected through several mechanisms:
direct repression, trans-repression, competition for binding to a
shared DNA element, or competition for cofactors or heterodimeric
partners (39). In the case of the Dax-1 promoter, COUP-TF
appears to bind to the composite element with an affinity equal to or
better than SF-1. Transfection studies to examine the direct effect of
COUP-TF competition on SF-1-mediated transactivation of the
Dax-1 promoter have been limited by the fact that
transfected COUP-TF activates the proximal Dax-1 promoter
and most other TATA-containing promoters tested under the experimental
conditions employed in this study (data not shown). Thus, it is
difficult to distinguish the repressive and activating effects of
COUP-TF that occur through distinct promoter regions. Competition
assays demonstrate that the binding of COUP-TF is specific and that it
is not composed of a heterodimeric complex with SF-1. These results
suggest that the relative levels of SF-1 and COUP-TF may determine
which factors occupy the composite element, thereby determining the
level of promoter activity.
SF-1 and DAX-1 colocalize to identical tissues (cells) within the
hypothalamic-pituitary-gonadal and -adrenal axes (13). In terms of
temporal expression, SF-1 usually precedes or is coexpressed with DAX-1
(13). In light of the phenotypes caused by the mouse Ftz-f1
gene disruption and in the human condition of X-linked AHC with
hypogonadotropic hypogonadism, a relationship between these two factors
seems highly likely. An interaction along this shared developmental
cascade may occur through direct protein-protein interactions, through
positive transcriptional regulation of one factor by the other, or
through intermediary factors. SF-1 and DAX-1 have been shown to
interact directly using in vitro protein interaction
studies, but it is unclear whether such interactions occur in
transfected cells or in vivo (8). DAX-1 has been shown to
inhibit SF-1-mediated transcription (8), and it has also been shown to
bind to hairpin loops that are present in the StAR and
Dax-1 promoters and to inhibit transcription of these genes
(7). A corepressor for DAX-1 has not been identified, but represents an
alternative mechanism by which DAX-1 might alter SF-1-mediated effects
since DAX-1 has been shown to contain a potent repressor domain (8, 9).
The possibility that Dax-1 might be transcriptionally
regulated by SF-1 or vice versa has not been thoroughly
examined. The finding that SF-1 expression precedes or coincides with
expression of DAX-1 (11, 14) corresponds well with the identification
of SF-1-binding sites in the mouse and human Dax-1 promoters
and is consistent with the hypothesis that SF-1 may regulate
Dax-1 promoter activity. On the other hand, DAX-1 expression
was not eliminated in the SF-1 knockout mouse (13), indicating that
SF-1 is not obligatory for the expression of Dax-1. This
study also found that removal of the SF-1 site had little effect on the
activity of the human Dax-1 promoter (13). In contrast to
these results, we find that SF-1 clearly contributes to
Dax-1 promoter activity. The SF-1 sites contributed about
50% to basal Dax-1 promoter activity in cell lines (
T3
and Y-1) that express SF-1 endogenously. However, the role of SF-1 was
revealed more clearly in SF-1-deficient JEG-3 cells in which it caused
17-fold induction. In addition to the presence of endogenous SF-1,
differences between cell lines may also reflect the amounts of COUP-TF,
coactivators, and perhaps endogenous ligands for SF-1. Recently, it has
been reported that SF-1 is activated by oxysterols, which are generated
by the action of P450c27 (e.g. 25-hydroxycholesterol) (43).
These, or other ligands, may be present in JEG-3 cells, which are
highly steroidogenic (44). Mutation of both SF-1 sites clearly does not
eliminate Dax-1 promoter activity, consistent with the idea
that multiple factors are involved in the control of its expression. In
view of this finding, it is possible that SF-1 modulates
Dax-1 expression at certain times during development. This
issue may be complicated further by the fact that DAX-1 may
autoregulate its expression by acting to inhibit SF-1 (7, 8).
The duplicated SF-1 elements in the murine Dax-1 promoter
are not completely conserved in the human gene. We have found that the
human sequence also contains two SF-1-binding sites (data not shown).
In the murine Dax-1 promoter, these sites act additively to
potentiate SF-1-mediated transcriptional activation. It is unlikely
that two SF-1 receptors bind to this region simultaneously because only
monomeric complexes were observed in EMSA assays, and the duplicated
SF-1 sites overlap partially. However, the availability of two sites
for SF-1 binding may increase the likelihood that SF-1 can bind to and
activate the murine promoter. Perhaps of greater significance, the
presence of the second site creates a composite site that may
facilitate the binding of COUP-TF. Further studies will be required to
assess whether this pathway for regulation of the Dax-1
promoter has been evolutionarily conserved. There is reason to suspect
that the interaction between SF-1 and COUP-TF may also occur for other
genes that are targets for SF-1. In the case of the bovine
Cyp17 gene (encoding P450 steroid 17
-hydroxylase), SF-1
and COUP-TF have been shown to bind to repeated sequences (AAGTCA and
AGGTCA) that are spaced by six nucleotides in the repCRS2 element (34, 45). As with the Dax-1 promoter, SF-1 stimulates this
element, and COUP-TF acts as an inhibitor. Thus, it is possible that
SF-1 and COUP-TF represent antagonistic pathways at least for a subset
of target genes.
 |
MATERIALS AND METHODS
|
---|
Cloning of Murine Dax-1 Gene
Dax-1 genomic DNA clones were isolated from a 129Sv/J
murine genomic
DNA library (Stratagene, La Jolla, CA). The library
was screened using full-length human Dax-1 cDNA (8) that was
radiolabeled using a random nonamer-labeling kit (Stratagene). After
purification of DNA from positive clones using a
DNA purification
kit (Promega, Madison, WI), genomic fragments were excised using
NotI and subcloned into the NotI site of pGEM
5Zf(+) (Promega). DNA sequencing was performed using an ABI Prism 377
DNA Sequencer (Perkin-Elmer, Foster City, CA).
Reporter Plasmid Construction
A 2938-bp KpnINcoI mDAX promoter DNA
fragment was isolated from pGEM 5Z-mDX21 and subcloned into the
KpnINcoI polylinker site of the luciferase
reporter construct pGL3 Basic (Promega). This construct,
pGL3B-mDX(-2938), was used to generate sequential 5'-deletions of the
DAX-1 promoter by exonuclease III/mung bean nuclease digestion
(Stratagene). The DNA sequences of the resulting deletion constructs
were confirmed by DNA sequencing. Site-directed mutagenesis and
deletion of more proximal DAX-1 promoter fragments were performed by
PCR amplification using Deep Vent polymerase (New England Biolabs,
Beverly, MA) and synthetic oligonucleotide primers (GIBCO/BRL,
Bethesda, MD). Primer pairs consisted of a 5'-sense DAX-1 promoter
primer and a 3'-antisense luciferase gene primer (LUCseq,
5'-GAATGGCGCCGGGCCTTTCTT-3'). The following DAX-1 promoter primers were
used for site-directed mutagenesis (sense sequence, XhoI
restriction enzyme site in lowercase; DAX promoter in
uppercase):
mDX134wt, 5'-gatcctcgagAGCTTTCGAGGTCATGGCCA-3';
mDX134m1a, 5'-gatcctcgagAGCTTTCGATTTCATGGCCA-3';
mDX134m1b, 5'-gatcctcgagAGCTTTCGAGGTCATTTCCACA-3';
mDX134m1ab, 5'-gatcctcgagAGCTTTCGATTTCATTTCCACA-3';
mDX134m3, 5'-gatcctcgagAGCTTTCGAGGCCATTTCCACA-3'
mDX114wt, 5'-gatcctcgagCACACATTCAAGCACAAAGG-3';
mDX84wt, 5'-gatcctcgagTCTGCGCCCTTGTCCAAGAG-3';
mDX50wt, 5'-gatcctcgagGCTTGCGTGCGCATTCAGTA-3'.
Amplification products were digested with XhoI and
NcoI and subcloned into the XhoINcoI
polylinker site of pGL3 Basic. All DAX-1 promoter reporter constructs
were confirmed by DNA sequencing. The pTK81-mDAX heterologous
constructs were prepared using the following double-stranded
oligonucleotides that correspond to the DAX-1 composite element primers
(sense strand:
81/mDX134wt, 5'-tcgagAGCTTTCGAGGTCATGGCCACACACactagta-3';
81/mDX134m1a, 5'-tcgagAGCTTTCGATTTCATGGCCACACACactagta-3';
81/mDX134m1b, 5'-tcgagAGCTTTCGAGGTCATTTCCACACACactagta-3';
81/mDX134m1ab, 5'-tcgagAGCTTTCGATTTCATTTCCACACACactagta-3'.
Each annealed primer pair was subcloned into the polylinker site of
pTK81 (46, 47), immediately upstream of the thymidine kinase minimal
promoter.
Cell Culture, Transfections, and Luciferase Assays
Murine pituitary gonadotrope
T3 cells (48) and human
placental JEG-3 cells (American Type Culture Collection, HTB-36) were
grown in DMEM supplemented with 10% FBS in a 5% CO2
atmosphere at 37 C. Murine adrenocortical Y1 cells (American Type
Culture Collection, CCL-79) were grown in Hams F10 medium
supplemented with 15% horse serum and 2.5% FBS. Cells were
transfected by the calcium phosphate method as previously described
(49). Luciferase assays (50) were performed 48 h after
transfection and are reported in arbitrary light units (ALU). Basal
activity is expressed in ALU, and fold-stimulation by SF-1 is expressed
for each construct as the ratio of promoter activity in the presence
and absence of transfected SF-1 expression vector. Results are the
mean ± SD of triplicate transfections.
EMSAs
Nuclear extracts were isolated from the indicated cell lines as
previously described (51). Protein concentrations were determined using
the Bradford assay system (Bio-Rad, Hercules, CA). The following
oligonucleotides (sense strand) were used for EMSAs:
mDX134wt, 5'-AGCTTTCGAGGTCATGGCCAC-3';
mDX134m1a, 5'-AGCTTTCGATTTCATGGCCAC-3';
mDX134m1b, 5'-AGCTTTCGAGGTCATTTCCAC-3';
mDX134m1ab, 5'-AGCTTTCGATTTCATTTCCAC-3'.
The oligonucleotide pairs were annealed and labeled with
[
-32P]dCTP using Klenow DNA polymerase. Nuclear
extracts (5 µg) were incubated with 20 fmol radiolabeled,
double-stranded oligonucleotides for 30 min at room temperature in a
volume of 20 µl. Protein-DNA complexes were resolved on 4%
nondenaturing, polyacrylamide gels using 0.5x Tris-borate-EDTA (TBE)
buffer.
In vitro transcription and translation were performed with
the TnT reticulocyte lysate system (Promega) as recommended by the
manufacturer. T3 or T7 RNA polymerase was used for the transcription of
SF-1 (8), COUP-TF1 (52), and COUP-TF2 (53, 54). In vitro
translated products (2 µl) were incubated with 20 fmol radiolabeled,
double-stranded oligonucleotides for 30 min at room temperature in a
final volume of 20 µl. The DNA and protein complexes were resolved as
described above. Antibodies against COUP-TF were provided by M. J.
Tsai (Baylor College of Medicine, Houston, TX). DAX-1 antibodies were
raised in rabbits using a peptide fragment (LTEHIRMMQREYQIR; Research
Genetics, Huntsville, AL). SF-1, CREB, and phospho-CREB antibodies were
obtained from Upstate Biotechnology (Lake Placid, NY).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to M. J. Tsai for kindly providing COUP-TF
antibodies and expression vectors.
This work was performed as part of the National Cooperative Program for
Infertility Research (NIH Grant U54-HD-29164); R.N.Y. was the
receipient of NIH training grant (T32 DK-07169).
 |
FOOTNOTES
|
---|
Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Tarry Building 15709, Chicago, Illinois 60611. E-mail: ljameson{at}nwu.edu
Received for publication July 3, 1997.
Revision received January 21, 1998.
Accepted for publication March 9, 1998.
 |
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