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Division of Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, Illinois 60611
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. 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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). 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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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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). 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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). 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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). 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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T3 Cells and Y1 CellsT3 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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, B and D). The deletion and
point mutation studies indicate that the SF-1 sites confer 30–50% 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 30–50% 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.
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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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, 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 KpnI–NcoI mDAX promoter DNA
fragment was isolated from pGEM 5Z-mDX2–1 and subcloned into the
KpnI–NcoI 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 XhoI–NcoI 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 Ham’s 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).
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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Received for publication July 3, 1997. Revision received January 21, 1998. Accepted for publication March 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 Y.-Y. Park, S.-W. Ahn, H.-J. Kim, J.-M. Kim, I.-K. Lee, H. Kang, and H.-S. Choi An autoregulatory loop controlling orphan nuclear receptor DAX-1 gene expression by orphan nuclear receptor ERR{gamma} Nucleic Acids Res., November 28, 2005; 33(21): 6756 - 6768. [Abstract] [Full Text] [PDF]
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 S. Y. Park, J. J. Meeks, G. Raverot, L. E. Pfaff, J. Weiss, G. D. Hammer, and J. L. Jameson Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development Development, May 15, 2005; 132(10): 2415 - 2423. [Abstract] [Full Text] [PDF]
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 O. Zwermann, F. Beuschlein, E. Lalli, A. Klink, P. Sassone-Corsi, and M. Reincke Clinical and molecular evidence for DAX-1 inhibition of steroidogenic factor-1-dependent ACTH receptor gene expression Eur. J. Endocrinol., May 1, 2005; 152(5): 769 - 776. [Abstract] [Full Text] [PDF]
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G. D. Hammer, K. L. Parker, and B. P. Schimmer Minireview: Transcriptional Regulation of Adrenocortical Development Endocrinology, March 1, 2005; 146(3): 1018 - 1024. [Abstract] [Full Text] [PDF]
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 W. Zheng and C. R. Jefcoate Steroidogenic Factor-1 Interacts with cAMP Response Element-Binding Protein to Mediate cAMP Stimulation of CYP1B1 via a Far Upstream Enhancer Mol. Pharmacol., February 1, 2005; 67(2): 499 - 512. [Abstract] [Full Text] [PDF]
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C. F. Buholzer, J.-F. Arrighi, S. Abraham, V. Piguet, A. M. Capponi, and A. J. Casal Chicken Ovalbumin Upstream Promoter-Transcription Factor Is a Negative Regulator of Steroidogenesis in Bovine Adrenal Glomerulosa Cells Mol. Endocrinol., January 1, 2005; 19(1): 65 - 75. [Abstract] [Full Text] [PDF]
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K. L. Britt, P. G. Stanton, M. Misso, E. R. Simpson, and J. K. Findlay The Effects of Estrogen on the Expression of Genes Underlying the Differentiation of Somatic Cells in the Murine Gonad Endocrinology, August 1, 2004; 145(8): 3950 - 3960. [Abstract] [Full Text] [PDF]
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 L. M. Salvador, M. P. Flynn, J. Avila, S. Reierstad, E. T. Maizels, H. Alam, Y. Park, J. D. Scott, D. W. Carr, and M. Hunzicker-Dunn Neuronal Microtubule-associated Protein 2D Is a Dual A-kinase Anchoring Protein Expressed in Rat Ovarian Granulosa Cells J. Biol. Chem., June 25, 2004; 279(26): 27621 - 27632. [Abstract] [Full Text] [PDF]
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E. Lalli and P. Sassone-Corsi DAX-1, an Unusual Orphan Receptor at the Crossroads of Steroidogenic Function and Sexual Differentiation Mol. Endocrinol., August 1, 2003; 17(8): 1445 - 1453. [Abstract] [Full Text] [PDF]
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H. Mizusaki, K. Kawabe, T. Mukai, E. Ariyoshi, M. Kasahara, H. Yoshioka, A. Swain, and K.-i. Morohashi Dax-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1) Gene Transcription Is Regulated by Wnt4 in the Female Developing Gonad Mol. Endocrinol., April 1, 2003; 17(4): 507 - 519. [Abstract] [Full Text] [PDF]
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 M. Maira, C. Martens, E. Batsche, Y. Gauthier, and J. Drouin Dimer-Specific Potentiation of NGFI-B (Nur77) Transcriptional Activity by the Protein Kinase A Pathway and AF-1-Dependent Coactivator Recruitment Mol. Cell. Biol., February 1, 2003; 23(3): 763 - 776. [Abstract] [Full Text]
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B. Gurates, S. Sebastian, S. Yang, J. Zhou, M. Tamura, Z. Fang, T. Suzuki, H. Sasano, and S. E. Bulun WT1 and DAX-1 Inhibit Aromatase P450 Expression in Human Endometrial and Endometriotic Stromal Cells J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4369 - 4377. [Abstract] [Full Text] [PDF]
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S. Kiiveri, J. Liu, M. Westerholm-Ormio, N. Narita, D. B. Wilson, R. Voutilainen, and M. Heikinheimo Differential Expression of GATA-4 and GATA-6 in Fetal and Adult Mouse and Human Adrenal Tissue Endocrinology, August 1, 2002; 143(8): 3136 - 3143. [Abstract] [Full Text] [PDF]
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 W. Xing, N. Danilovich, and M. R. Sairam Orphan Receptor Chicken Ovalbumin Upstream Promoter Transcription Factors Inhibit Steroid Factor-1, Upstream Stimulatory Factor, and Activator Protein-1 Activation of Ovine Follicle-Stimulating Hormone Receptor Expression via Composite cis-Elements Biol Reprod, June 1, 2002; 66(6): 1656 - 1666. [Abstract] [Full Text] [PDF]
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C. Hoyle, V. Narvaez, G. Alldus, R. Lovell-Badge, and A. Swain Dax1 Expression Is Dependent on Steroidogenic Factor 1 in the Developing Gonad Mol. Endocrinol., April 1, 2002; 16(4): 747 - 756. [Abstract] [Full Text] [PDF]
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P. Koskimies, J. Levallet, P. Sipila, I. Huhtaniemi, and M. Poutanen Murine Relaxin-Like Factor Promoter: Functional Characterization and Regulation by Transcription Factors Steroidogenic Factor 1 and DAX-1 Endocrinology, March 1, 2002; 143(3): 909 - 919. [Abstract] [Full Text] [PDF]
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W. R. Duan, M. Ito, Y. Park, E. T. Maizels, M. Hunzicker-Dunn, and J. L. Jameson GnRH Regulates Early Growth Response Protein 1 Transcription Through Multiple Promoter Elements Mol. Endocrinol., February 1, 2002; 16(2): 221 - 233. [Abstract] [Full Text] [PDF]
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P. S. Babu, D. L. Bavers, F. Beuschlein, S. Shah, B. Jeffs, J. L. Jameson, and G. D. Hammer Interaction Between Dax-1 and Steroidogenic Factor-1 in Vivo: Increased Adrenal Responsiveness to ACTH in the Absence of Dax-1 Endocrinology, February 1, 2002; 143(2): 665 - 673. [Abstract] [Full Text] [PDF]
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 K. L. Parker, D. A. Rice, D. S. Lala, Y. Ikeda, X. Luo, M. Wong, M. Bakke, L. Zhao, C. Frigeri, N. A. Hanley, et al. Steroidogenic Factor 1: an Essential Mediator of Endocrine Development Recent Prog. Horm. Res., January 1, 2002; 57(1): 19 - 36. [Abstract] [Full Text] [PDF]
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 A. Lacroix, N. N'Diaye, J. Tremblay, and P. Hamet Ectopic and Abnormal Hormone Receptors in Adrenal Cushing's Syndrome Endocr. Rev., February 1, 2001; 22(1): 75 - 110. [Abstract] [Full Text]
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C. Aigueperse, P. Val, C. Pacot, C. Darne, E. Lalli, P. Sassone-Corsi, G. Veyssiere, C. Jean, and A. Martinez SF-1 (Steroidogenic Factor-1), C/EBP{beta} (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells Mol. Endocrinol., January 1, 2001; 15(1): 93 - 111. [Abstract] [Full Text]
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 M. L. Bland, C. A. M. Jamieson, S. F. Akana, S. R. Bornstein, G. Eisenhofer, M. F. Dallman, and H. A. Ingraham Haploinsufficiency of steroidogenic factor-1 in mice disrupts adrenal development leading to an impaired stress response PNAS, December 19, 2000; 97(26): 14488 - 14493. [Abstract] [Full Text] [PDF]
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D. Boerboom, N. Pilon, R. Behdjani, D. W. Silversides, and J. Sirois Expression and Regulation of Transcripts Encoding Two Members of the NR5A Nuclear Receptor Subfamily of Orphan Nuclear Receptors, Steroidogenic Factor-1 and NR5A2, in Equine Ovarian Cells during the Ovulatory Process Endocrinology, December 1, 2000; 141(12): 4647 - 4656. [Abstract] [Full Text] [PDF]
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E. Barnea and Y. Bergman Synergy of SF1 and RAR in Activation of Oct-3/4 Promoter J. Biol. Chem., February 25, 2000; 275(9): 6608 - 6619. [Abstract] [Full Text] [PDF]
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M. Ito, Y. Park, J. Weck, K. E. Mayo, and J. L. Jameson Synergistic Activation of the Inhibin {alpha}-Promoter by Steroidogenic Factor-1 and Cyclic Adenosine 3',5'-Monophosphate Mol. Endocrinol., January 1, 2000; 14(1): 66 - 81. [Abstract] [Full Text]
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V. Giguère Orphan Nuclear Receptors: From Gene to Function Endocr. Rev., October 1, 1999; 20(5): 689 - 725. [Abstract] [Full Text]
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K. Kawabe, T. Shikayama, H. Tsuboi, S. Oka, K. Oba, T. Yanase, H. Nawata, and K.-i. Morohashi Dax-1 as One of the Target Genes of Ad4BP/SF-1 Mol. Endocrinol., August 1, 1999; 13(8): 1267 - 1284. [Abstract] [Full Text]
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K. Zeitoun, K. Takayama, M. D. Michael, and S. E. Bulun Stimulation of Aromatase P450 Promoter (II) Activity in Endometriosis and Its Inhibition in Endometrium Are Regulated by Competitive Binding of Steroidogenic Factor-1 and Chicken Ovalbumin Upstream Promoter Transcription Factor to the Same cis-Acting Element Mol. Endocrinol., February 1, 1999; 13(2): 239 - 253. [Abstract] [Full Text]
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M. Ito, J. C. Achermann, and J. L. Jameson A Naturally Occurring Steroidogenic Factor-1 Mutation Exhibits Differential Binding and Activation of Target Genes J. Biol. Chem., October 6, 2000; 275(41): 31708 - 31714. [Abstract] [Full Text] [PDF]
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