From the
Departments of Molecular and Integrative
Physiology and
Internal Medicine, Division of
Endocrinology and Metabolism, University of Michigan, Ann Arbor, Michigan
48109
Received for publication, December 12, 2002 , and in revised form, April 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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The Wnt genes encode a family of highly conserved glycoproteins that have
been implicated in a variety of embryologic programs including gonadal and
adrenal development
(2933).
As such, they are attractive candidates for modulators of SF-1-dependent
transcriptional programs. Wnt ligands bind to cell-surface Frizzled receptors,
initiating a cytoplasmic signaling cascade that results in -catenin
stabilization, cytosolic accumulation, and subsequent translocation to the
nucleus. Within the nucleus,
-catenin classically complexes with a
member of the T-cell factor (TCF) family of transcription factors to activate
target-gene transcription
(34). However, recent studies
indicate that
-catenin can interact directly with a variety of
coregulator proteins and transcription factors, including CRE-binding protein
(CREB)-binding protein (CBP), the SMAD family of proteins, and the nuclear
androgen receptor (AR) to enhance both TCF-dependent
(35,
36) and TCF-independent
transcription
(3739).
In situ hybridization has demonstrated that Wnt-4, Wnt-11,
and Wnt-2b are all expressed in the adrenal cortex. Wnt 11
localizes to the definitive zone, while Wnt-2b localizes exquisitely
to the subcapsular cells beneath the adrenal capsule
(32,
33). Wnt-4 knockout
mice die within 24 h postpartum due to severe urogenital abnormalities
including kidney failure secondary to a failure of nephrogenesis
(40). Female Wnt-4
knockout mice demonstrated surprising masculinization of urogenital ridge
structures due to the aberrant up-regulation of the SF-1 target genes
Müllerian inhibitory substance and P450c17, and the resultant excess
testosterone biosynthesis
(29). In addition, ectopic
expression of the SF-1 target gene, P450c21 (critical for corticosterone
biosynthesis) was observed in the gonads of Wnt-4-null mice. An
increase in plasma corticosterone was also observed in Wnt-4-null
mice. On the contrary, P450-aldosterone synthase and P450c17 (females only)
expression were decreased in the embryonic adrenal of Wnt-4 knockout
mice, supporting a role of Wnt-4 in the development of the organs arising from
the urogenital ridge (31).
Recently, a patient with adrenal hypoplasia and male to female sex-reversal
was shown to have a duplication of chromosomal locus 1p32
[PDB]
-36, which contains
the Wnt-4 gene. Up-regulation of the SF-1 target gene Dax-1
in cells from this patient suggests an interaction between SF-1 and Wnt
signaling on the Dax-1 promoter
(41), as recently confirmed by
the demonstration of synergy between SF-1 and
-catenin on activation of
Dax-1 transcription in the developing female gonad
(42). Because knockout of the
SF-1 target gene,
-inhibin results in a syndrome of gonadal and adrenal
sex steroid excess similar to the Wnt-4-null mice, and transcription
of
-inhibin, like Dax-1
(43), is absolutely dependent
upon SF-1, we hypothesized that components of the Wnt signaling cascade
directly interact with SF-1 to regulate the
-inhibin promoter in the
adrenal cortex
(4446).
In this article we have specifically explored the activation of SF-1-dependent
transcription of the
-inhibin gene by
-catenin in the adrenal
cortex.
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MATERIALS AND METHODS |
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Plasmid ConstructsThe following plasmids have been
previously described: -inhibin-luciferase (and deletion constructs),
pci-neo-HA-SF-1, pcDNA3-S33Y-
-cat, CMV-mTCF-4B-FLAG, Lex-A-Luc,
CMV-LexA-SF-1, CMV-LexA-DBD, TOPFLASH, p-65-luc, and myc-LacZ
(6,
4652).
Inhibin-Luc-
SF-1-RE was generated by introducing a point mutation in to
the SF-1 binding site using QuikChange (Stratagene, La Jolla, CA) according to
the manufacturer's instructions. The oligonucleotides used to generate the
SF-1-RE mutant were:
5'-GTGGGAGATAAGGCTCAGTTCCACAGACATCTGCG-3' and
5'-CAGGGCCACAGACATCTGTATCAGAGATAGGAGGTC-3'
(46).
Luciferase AssaysJEG3 cells were plated at a density of 1
x 105 cells per well into 12-well plates. Twenty-four hours
after plating, cells were transiently transfected using calcium phosphate
coprecipitation (Specialty Media, Phillipsburg, NJ). Cells were lysed 48 h
post-transfection, and a luciferase assay (Promega, Madison, WI) was performed
in triplicate on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA).
Luciferase assays were normalized for transfection efficiency by
co-transfecting CMV-myc-LacZ and subsequent determination of
-galactosidase activity (Applied Biosystems, Foster City, CA).
Co-immunoprecipitation AssayNontransgenic virgin female
mice were injected intraperitoneally with either 150 mM NaCl or 150
mM LiCl with an injection volume of 20 ml/kg
(53). At either two or three
hours post-injection, adrenals were harvested and either snap-frozen in liquid
nitrogen (for subsequent RNA isolation) or stored in ice-cold
phosphate-buffered saline. Adrenals from six to nine mice were pooled and
minced by hand. The minced adrenals were collected into 1 ml of
phosphate-buffered saline and pelleted by spinning at 1000 rpm for 5 min. Cell
membranes were disrupted for 30 min at 4 °C using 400 µl of a buffer
containing 25 mM HEPES, 5 mM KCl, 0.5 mM
MgCl2, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40 at pH 7.6. Nuclei were
isolated from cytoplasmic protein by spinning at 2500 rpm for 1 min. Nuclear
proteins were extracted for 60 min at 4 °C using 500 µl of a buffer
containing 25 mM HEPES, 10% sucrose, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.01% Nonidet
P-40, and 350 mM NaCl. Insoluble proteins were removed by spinning
at maximum speed for 10 min. Protein concentrations were determined by the
method of Bradford (Bio-Rad, Hercules, CA). Two hundred micrograms of total
nuclear lysate for each condition were precleared with 10 µl (bed volume)
of protein A/G-agarose for 30 min at 4 °C. Precleared nuclear extract was
incubated with either rabbit polyclonal -HA antibodies (Santa Cruz
Biotechnology) or rabbit polyclonal
-SF-1 antibodies (Upstate
Biotechnology, Lake Placid, NY) for 16 h at 4 °C with rotation.
Subsequently, 10 µl of protein A/G-agarose was added to samples and
incubated at 4 °C with rotation for 60 min. The samples were then washed
four times with radioimmune precipitation assay buffer (Tris-HCl, 50
mM, pH 7.4; Nonidet P-40, 1%; sodium deoxycholate, 0.25%; NaCl, 150
mM; EDTA, 1 mM; phenylmethylsulfonyl fluoride, 1
mM; leupeptin and pepstatin, 1 µg/ml each; NaF, 1
mM). Immunoprecipates were resolved on 10% denaturing
polyacrylamide gels, transferred to polyvinylidene difluoride membrane
followed by immunoblot analysis using polyclonal
-
-catenin
antibodies (Santa Cruz Biotechnology).
Chromatin ImmunoprecipitationChromatin immunoprecipitation
(ChIP) assays were performed using a chromatin immunoprecipitation assay kit
(Upstate Biotechnology) according to the manufacturer's instructions with the
minor modification that isolated nuclei were used as starting material instead
of whole cells. Briefly, cells were cross-linked with 1% formaldehyde at 37
°C for 10 min. Cells were rinsed three times with ice-cold
phosphate-buffered saline and collected into phosphate-buffered saline and
centrifuged for 5 min at 1000 rpm. Crude nuclei were isolated using the
protocol described for co-immunoprecipitation assays. Nuclei were prepared in
the SDS lysis buffer provided in the kit. Lysates were sonicated at 30% power,
5 x 10 s using a Sonic Dismembrator model 300 (Fisher Scientific,
Pittsburgh, PA) to shear genomic DNA. The following antibodies were used to
perform immunoprecipitations, polyclonal -SF-1 (Upstate Biotechnology),
polyclonal
-
-catenin (Santa Cruz Biotechnology), and polyclonal
rabbit IgG antibodies (Pierce). PCR amplification of the proximal
-inhibin promoter region spanning the SF-1 response element was
performed using the following primers: 5'-GGGGTGGTGCATTCTGTCCT-3'
and 5'-GCTGCCCTGTGCCCTTTCTGT-3'. 5 µl of extracted DNA and 35
rounds of amplification were used. PCR amplification of the distal
-inhibin promoter (7.5 to 7 kb) was performed using the
following primers: 5'-GGACCCCCACCAAGCCAACAGAC-3' and
5'-TCAGCCCTACACCAGCACGCAGAC-3'.
RNA Isolation and Quantitative PCRSnap frozen adrenals from
NaCl- or LiCl-injected mice (see "Co-Immunoprecipitation Assay"
above) were manually disrupted using a mortar and pestle. Total cellular RNA
was isolated using an RNeasy RNA isolation kit (Qiagen, Valencia, CA). One
microgram of total cellular RNA was reverse-transcribed using the iScript
Reverse Transcription Kit (BD Biosciences, San Diego, CA). Quantitative PCR
for -inhibin transcript was performed on an Opticon Thermocycler (MJ
Research, Waltham, MA) using the QuantiTect SYBR Green PCR Kit (Qiagen). The
sequence of primers targeted against the
-inhibin cDNA were
5'-AAGACATGCCGTTGGGGGTTTCA-3' and
5'-CTATTGGCGGCTGCTGTGCTCTTC-3'. 18 S ribosomal RNA was used as an
internal standard using commercially available primers (Ambion, Austin,
TX).
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RESULTS |
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A Proximal 165-bp Cis-acting Element Is Sufficient for
-Catenin Transactivation of
-InhibinIn
order to determine the mechanism of activation of the
-inhibin promoter
by
-catenin, we systematically analyzed this promoter in an attempt to
identify putative TCF binding sites. A panel of serial deletions of the
-inhibin promoter fused to a luciferase reporter was utilized to locate
the region of the promoter required for functional synergy between SF-1 and
-catenin. Co-transfection of each reporter construct in the presence or
absence of SF-1 expression plasmid was performed with or without a maximal
dose of
-catenin. These experiments demonstrated that
-catenin was
capable of synergizing with SF-1 on all deletion constructs tested
(Fig. 1B). In
addition,
-catenin activated the 165-bp construct 6.5-fold over
SF-1-stimulated activation, similar to both the 1641 bp construct and
the full-length construct described in Fig.
1A. To further characterize this synergy, JEG3 cells were
co-transfected with increasing amounts of S33Y and 165 Inh-luc in the
presence or absence of SF-1. A dose-dependent increase in SF-1-mediated
transcriptional activation, up to
4.5-fold, was observed in response to
increasing quantities of S33Y (Fig.
1C). Similar to results obtained with full-length
-inhibin-luc, S33Y was barely able to activate transcription of the
reporter plasmid in the absence of SF-1, confirming that the cis-acting
element required for
-catenin synergy with SF-1 is located within the
proximal 165 bp of the
-inhibin promoter and that SF-1 is required for
-catenin transcriptional activation of the
-inhibin promoter.
The SF-1 RE Is Necessary and Sufficient for the Synergistic Activation
of the -Inhibin Promoter by
-Catenin and
SF-1 Since the SF-1 response element (RE) resides within the
proximal 165 bp of the
-inhibin promoter, we tested whether the SF-1 RE
was an absolute requirement for the synergistic effects of
-catenin on
the
-inhibin promoter. Previously, mutation of the SF-1 RE has been
shown to result in loss of binding of SF-1
(46). We therefore introduced
identical point mutations in this binding site in the context of the
full-length reporter. As expected, disruption of the SF-1 RE resulted in the
complete loss of SF-1-dependent activation of the reporter construct
(Fig. 2A). In
addition, S33Y was unable to transactivate the mutated promoter in either the
absence or presence of SF-1, indicating that sequences between 165 and
+1 are sufficient, and the SF-1 RE within this promoter region is necessary
for the synergistic activation of the
-inhibin promoter by
-catenin and SF-1.
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In order to test whether the SF-1 RE was sufficient for synergy between
SF-1 and -catenin, we utilized a reporter construct containing 5 tandem
copies of a consensus SF-1 RE driving the luciferase gene reporter (p-65-luc).
-catenin was able to transcriptionally synergize with SF-1 on this
reporter construct in a dose-dependent manner, showing a 9-fold activation
over SF-1 levels (Fig.
2B). As expected,
-catenin was unable to activate
this reporter in the absence of SF-1. These data demonstrate that the SF-1 RE
is a necessary and sufficient DNA promoter element for transcription synergy
between SF-1 and
-catenin.
TCF/Lef Is Not Required for the Synergy between SF-1 and
-Catenin on the
-Inhibin PromoterSince
-catenin transactivation is classically propagated through TCF/Lef
(lymphoid enhancer factor), we evaluated the potential role of TCF/Lef in the
synergy between SF-1 and
-catenin. However, sequence analysis of the
proximal 165 bases of the
-inhibin promoter did not reveal a strong
consensus TCF/Lef binding site, and the sufficiency of the SF-1 RE for the
synergy between SF-1 and
-catenin predicts a potential TCF-independent
mechanism of
-catenin synergy. Using TOPFLASH, an artificial promoter
containing three tandem consensus TCF binding sites as a positive control for
TCF/Lef-mediated transcriptional response, co-transfection assays were
performed. JEG3 cells were co-transfected with either (a) TOPFLASH,
S33Y, and increasing quantities of TCF-4 or (b) Inh-luc, SF-1, S33Y,
and increasing quantities of TCF-4. As expected, S33Y strongly activated the
TOPFLASH artificial promoter, and addition of TCF-4 led to further
dose-dependent transcriptional activation
(Fig. 3A), consistent
with a TCF-dependent
-catenin effect. However, when TCF-4 was titrated
with Inh-luc, a dose-dependent repression of
-inhibin transactivation
was observed, indicating that TCF is not required for the synergy between SF-1
and
-catenin. In addition, the repression predicts that TCF
sequestration of
-catenin might prevent transcriptional synergy with
SF-1 on the
-inhibin promoter. These data suggest that the interaction
between SF-1 and
-catenin is independent of the action of the TCF family
of transcription factors predicting a novel mechanism of action for
-catenin-mediated transcriptional activation by orphan receptors.
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A modified mammalian two-hybrid assay was performed to confirm whether the
functional synergy between SF-1 and -catenin was independent of TCF/Lef
DNA binding. In this assay, increasing amounts of S33Y
-catenin were
tested for the ability to increase expression of a LexA-RE-Luc reporter
plasmid in the presence and absence of the LexA-SF-1 fusion construct in which
the SF-1 DNA binding domain (DBD) was replaced with the LexA DBD. A
dose-dependent increase of LexA-RE-Luc transcription was observed in the
presence of increasing amounts of S33Y when co-transfected with LexA-SF-1. No
effect of S33Y was observed with LexA DBD alone, demonstrating that the
activation of LexA-Luc by S33Y was propagated through interactions with SF-1
(Fig. 3B). The ability
of
-catenin to activate LexA-RE-Luc transcription in the presence of
LexA-SF-1 confirms that the synergy between SF-1 and
-catenin is
independent of TCF/Lef DNA binding and suggests that the synergy is mediated
by a physical interaction between SF-1 and
-catenin.
-Catenin Is a Component of the SF-1 Transcription Complex
Assembled on the Endogenous
-Inhibin Promoter in the
AdrenalDirect evidence of a transcriptionally active protein
complex including SF-1 and
-catenin was explored through a combination
of co-immunoprecipitation and ChIP assays. To evaluate the significance of
protein complex formation in vivo, we utilized standard
co-immunoprecipitation assays on nuclear lysates from adrenals of mice
injected with either NaCl or LiCl, which is a potent inhibitor of GSK-3
and hence should result in a stabilization of
-catenin. Immunoblot
analysis revealed that endogenous
-catenin was elevated in the nuclear
fraction of adrenals from LiCl-injected mice when compared with adrenals from
mice injected with NaCl, while SF-1 levels were unaffected
(Fig. 4). Immunoprecipitation
was performed on nuclear lysates using
-SF-1 antibodies or
-HA
antibodies (negative control) followed by immunoblot analysis for
-catenin using polyclonal
-
-catenin antibodies. As
expected, no
-catenin was detected in
-HA immunoprecipitates.
While only minimal
-catenin was detected in
-SF-1
immunoprecipitates from nuclear extracts of adrenals from NaCl-injected mice,
a strong
-catenin signal was observed in
-SF-1 immunoprecipitates
following LiCl treatment, indicative of the dynamic assembly of a protein
complex that includes SF-1 and
-catenin following LiCl treatment. Such a
result is consistent with the activation of the canonical Wnt signaling
cascade in the adrenal cortex, inhibition of GSK-3
, stabilization of
-catenin and subsequent association with the SF-1 transcription complex
(Fig. 4).
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In order to test whether -catenin is a component of the SF-1
transcription complex on the endogenous
-inhibin promoter within the
adrenal, chromatin immunoprecipitation assays were performed. Y1 nuclear
lysates were immunoprecipitated with rabbit polyclonal IgG,
-
-catenin or
-SF-1 antibodies. PCR amplification was
performed using primers designed to amplify a fragment containing the proximal
SF-1 RE within the
-inhibin promoter. The predicted 422-bp PCR product
was amplified out of samples derived from
-
-catenin and
-SF-1 immunoprecipitates, but not from samples immunoprecipitated with
IgG (Fig. 5). No PCR
amplification was observed when primers targeted against the distal
-inhibin promoter were used, demonstrating that the DNA was
sufficiently sheared and the antibodies did not pull down large chromosomal
fragments that might interact with SF-1 and/or
-catenin at
additional/alternative contact points on chromatin. These data demonstrate
that
-catenin and SF-1 interact with sequences within the proximal
-inhibin promoter within the context of an in vivo,
chromatinized template.
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Injection of LiCl into Mice Increases -Inhibin
Transcription in the Adrenal CortexSince we observed an increase
in
-catenin stabilization together with an increase in the association
between SF-1 and
-catenin in response to LiCl injection, we examined
whether LiCl treatment would activate transcription of the endogenous
-inhibin gene within the adrenal cortex of mice. Using an identical
paradigm for LiCl treatment as utilized in co-immunoprecipitation studies,
quantitative RT-PCR reveals a relative 3.50 ± 0.95-fold increase in
-inhibin transcript levels after 3 h of LiCl treatment when compared
with NaCl-treated controls, with a relative expression level of 1.0 ±
0.26 (Fig. 6). These data are
consistent with a Wnt-mediated stabilization of nuclear
-catenin and
synergistic activation of SF-1-dependent transcription of the
-inhibin
gene in the mouse adrenal cortex.
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DISCUSSION |
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We demonstrate that -catenin is able to synergize with SF-1 in a
dose-dependent manner to activate transcription of the
-inhibin
promoter. The cis-acting element required for synergy between SF-1 and
-catenin maps to the proximal 165 bp of the
-inhibin promoter.
While Mizusaki et al.
(42) demonstrate a role for
TCF in the regulation of Dax-1, the lack of a TCF consensus binding site,
coupled with the absence of transactivation of the
-inhibin promoter by
-catenin alone (without SF-1), suggests a novel mechanism of action for
the regulation of
-inhibin by
-catenin. This is supported by the
loss of both SF-1-mediated transactivation of the
-inhibin promoter and
the synergy with
-catenin following introduction of a point mutant in
the SF-1 RE within the 165-bp region along with the sufficiency of a consensus
SF-1 RE to support the synergy between SF-1 and
-catenin. In addition,
titration of TCF-4B, while activating the synthetic TCF/Lef RE construct
TOPFLASH, disrupts the functional synergy between SF-1 and
-catenin on
the
-inhibin promoter, suggesting a sequestration of
-catenin
that prevents an association with SF-1 and supports a TCF-independent
-catenin interaction with SF-1.
A direct interaction between -catenin and nuclear receptors is not
unprecedented. Recent reports have characterized an interaction between
-catenin and AR that may serve as a common mechanism for interaction of
-catenin with nuclear receptors
(3739).
In prostate cancer cell lines, nuclear translocation of
-catenin was
dependent upon the presence of an AR ligand. AR does not interact with either
adenomatous polyposis coli or glycogen synthase kinase 3
, both
regulators of the canonical Wnt signaling pathway, suggesting a novel pathway
for the interaction between AR and
-catenin
(38). The ability of
-catenin to activate an AR-dependent promoter maps to the AR response
element (ARE), and
-catenin is able to activate a minimal promoter
containing just two copies of a consensus ARE, suggesting that the interaction
between AR and
-catenin is independent of TCF binding to DNA, which is a
novel mechanism of action for
-catenin
(37). Addition of liganded AR
was able to repress activation of a TCF/Lef dependent reporter construct,
further suggesting that AR competes with TCF for
-catenin binding
(39). Data presented in this
report support the participation of
-catenin in nuclear
receptor-mediated transcriptional activation and extend the finding to the
monomer binding class of orphan nuclear receptors.
The presence of a protein complex containing SF-1 and -catenin was
evaluated by modified mammalian two-hybrid, co-immunoprecipitation, and ChIP
assays. While the synergistic activation by SF-1 and
-catenin in the
mammalian two-hybrid suggests a physical interaction between the two proteins,
the ability of SF-1 to co-immunoprecipitate with
-catenin out of adrenal
cortical nuclear lysates serves as further evidence that these two factors
form a protein complex within the adrenal cortex. ChIP assays reveal that SF-1
and
-catenin antibodies are both able to immunoprecipitate the proximal
-inhibin promoter, indicating that the transcriptional complex seen in
the co-immunoprecipitation is active on the
-inhibin promoter in the
adrenal cortical cell. The ability of LiCl, which mimics canonical Wnt
signaling by inhibiting GSK-3
, to increase both the interaction between
SF-1 and
-catenin and
-inhibin expression within the adrenal
cortex further supports a model by which Wnt-activated signaling cascades
regulate
-inhibin expression.
Not surprisingly, additional factors participate in the regulation of
-inhibin gene transcription. CBP, CREB
(46), and GATA-1
(58) stimulate while WT-1
(59) and ICER
(60) inhibit
-inhibin
transcription. In addition, the binding of CREB to the
-inhibin CRE
(6163)
is dependent upon SF-1 binding to the SF-1 RE
(46). As such, future studies
that examine the inter-play between these factors and the regulation of
transcription complex formation by membrane signals will be essential. Our
preliminary studies indicate that adrenocorticotropic hormone results in a
significant attenuation of the co-immunoprecipitation of SF-1 and
-catenin in mouse Y1 adrenocortical
cells.2 How Wnt
ligands shown or predicted to be involved in adrenal growth and
differentiation (Wnt2b, Ref.
33; Wnt-4, Ref.
31; Wnt 11, Ref.
32) influence the
SF-1·
-catenin complex will provide important mechanistic insight
into the regulation of monomer binding orphan nuclear receptors by
membrane-induced signaling cascades.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Internal Medicine, Division of Endocrinology and Metabolism, University of Michigan, 1150 W. Medical Center Dr., 5560 MSRBII, Ann Arbor, MI 48109. Tel.: 734-763-3056; Fax: 734-936-6684; E-mail: ghammer{at}umich.edu.
1 The abbreviations used are: SF-1, steroidogenic factor-1; Dax-1,
dose-sensitive sex reversal adrenal hypoplasia congenital determining region
on the X-chromosome-1; TCF, T-cell factor; Lef, lymphoid enhancer factor;
CREB, CRE-binding protein; CBP, CREB-binding protein; AR, androgen receptor;
Inh-luc, -inhibin luciferase; DBD, DNA binding domain; ARE, AR response
element; SF-1 RE, SF-1 response element; ChIP, chromatin immunoprecipitation;
GSK-3
, glycogen synthase kinase-3
.
2 J. Winnay and G. Hammer, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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