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RECEPTORS |
Departments of Obstetrics and Gynecology (D.L., W.S.-E., M.D.S., M.P.M.) and Biochemistry and Molecular Biology (M.P.M.), University of South Florida, Tampa, Florida 33606
Address all correspondence and requests for reprints to: Dr. Mark P. McLean, Departments of Obstetrics and Gynecology, 4 Columbia Drive, Room 529, Tampa, Florida 33606. E-mail: mmclean{at}hsc.usf.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well known that negative factors also play a critical role in
gene regulation. One negative factor that appears to be important for
the regulation of the HDL-R gene is the dosage-sensitive sex adrenal
hypoplasia congenital critical region on the X chromosome, gene-1
(DAX-1). DAX-1 is an orphan nuclear receptor that has been shown to be
required for adrenal development (19, 20, 21, 22). This orphan
nuclear receptor displays a novel DNA-binding domain that lacks the
characteristic zinc finger motif that is highly conserved among true
nuclear receptors (23, 24, 25). Instead, the amino terminus of
DAX-1 consists of three and one-half repeats of an alanine- and
glycine-rich 65–67 amino acid motif that has been proposed to serve as
its DNA-binding domain (23, 24, 25). The findings that DAX-1
and SF-1 are colocalized to steroidogenic tissues (26) and
that targeted mutations of the SF-1 gene result in phenotypic features
similar to those seen with DAX-1 mutations (27) suggest
that DAX-1 and SF-1 may function together or in sequential steps during
embryonic development. In vitro studies indicate that DAX-1
can strongly repress steroidogenesis and antagonize SF-1-mediated
stimulation of genes such as steroidogenic acute regulatory protein
(StAR) (28, 29), P450 side-chain cleavage enzyme
(29), 3ß-hydroxysteroid dehydrogenase (29),
Müllerian-inhibiting substance (30), aldose
reductase-like protein (31), and P450 17
hydroxylase
(32). DAX-1 has also been shown to act as a corepressor of
the estrogen receptors and ß (33). Interestingly,
ovarian DAX-1 protein levels have been shown to increase in response to
PGF2 treatment (28), leading to a
decrease in HDL-R mRNA levels (34). These data suggest
that DAX-1 may be responsible for the reduction in HDL uptake following
PGF2 treatment.
Two mechanisms have been proposed by which DAX-1 represses gene
expression. The first mechanism involves the direct binding of DAX-1 to
DNA or RNA at stem-loop structures preventing gene expression
(25, 35). Stem-loop structures to which DAX-1 binds have
been identified in the mouse DAX-1 and human StAR promoters
(25). The second DAX-1 inhibitory mechanism suggests that
DAX-1 represses transcription by directly interacting with other
nuclear receptors such as SF-1 (36, 37). Furthermore, the
efficiency of DAX-1’s inhibitory effect on the P450 17 hydroxylase
gene depends on the number of SF-1-binding elements found in the
promoter (32). DAX-1 also acts as an adapter molecule that
recruits other corepressors to DNA-bound complexes like SF-1
(37) and in this way prevents the interaction between the
trans-acting factor and the transcription initiation complex
(38, 39). Corepressors reported to interact with DAX-1
include the nuclear receptor corepressor and Alien (37, 40).
In the present study, the effect of DAX-1 on the regulation of the HDL-R gene was examined. DAX-1 inhibition of the HDL-R gene was found to be coactivator (SREBP-1a or SF-1) dependent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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32P]Deoxy (d)-CTP (3000 Ci/mmol), the T7
Sequenase DNA sequencing kit, polydeoxyinosinic-deoxycytidylic acid,
and the glutathione S-transferase (GST)-fusion protein purification kit
were obtained from Amersham Pharmacia Biotech (Piscataway,
NJ). [35S]dATP (1000–1500 Ci/mmol) was
obtained from Dupont/NEN Life Science Products
(Wilmington, DE). The Fugene 6 transfection reagent was obtained from
Roche Molecular Biochemicals (Indianapolis, IN). DMEM:
nutrient mixture F-12 (DMEM/F12) was obtained from Life Technologies, Inc./BRL (Grand Island, NY). FBS was purchased
from Summit Biotechnology (Ft. Collins, CO). BioMax-MR
films were obtained from Fisher Scientific (Norcross, GA).
Rivastatin (Merck & Co., Inc., Rahway, NJ) was provided by
Dr. G. C. Ness (Department of Biochemistry and Molecular Biology,
University of South Florida, Tampa, FL). All other chemicals were
purchased from Fisher Scientific or Sigma
(St. Louis, MO).
Site-directed mutagenesis
Site-directed mutants were obtained using the QuickChange
site-directed mutagenesis kit (Stratagene) as previously
described (17). Oligonucleotides used for site-directed
mutagenesis to delete the hairpin in the p-2267 HDL-R promoter
construct were 5'-GTGCCAGGGATATCTGCCCTCTGTG-3',
5'-CTAGGCCAGCGATATCAGCAGGAAG-3', and their
respective complements. Following site-directed mutagenesis, the
mutated p-2267 construct was digested with EcoRV and
religated thereby generating the hairpin negative promoter construct
(p-2267 HN-HDL-R). To prepare truncated DAX-1 (tDAX-1),
oligonucleotides used were
5'-GATCCGCTGAATGATGAAGTGCCCTTTTCC-3' and its complement.
This mutation introduces two premature stop codons resulting in a
truncated protein that lacks 42 amino acids at the carboxy terminal.
The nucleotides in boldface letters correspond to the
mutated bases. Mutations were confirmed by sequencing using the T7
sequenase DNA sequencing kit and [35S]dATP.
Cell transfections and luciferase assays
The p-2267 and p-719 HDL-R promoter-luciferase gene constructs
were prepared as previously described (12). The hairpin
negative HDL-R promoter construct (p-2267 HN-HDL-R) was prepared as
described above. Y1, HTB-9, and HepG2 cells were transfected with the
specified HDL-R promoter-luciferase gene constructs either in the
presence or absence of DAX-1-pcDNA3.1 ± SREBP-1a-pCMV5 or
SF-1-pCMV using the Fugene 6 transfection method (Roche Molecular Biochemicals) according to the manufacturer’s instructions.
Cells were first plated in either 6-well tissue culture plates at a
density of 3 x 106 cells/well or 12-well
tissue culture plates at a density of 5 x
104 cells/well and incubated for 24 h at 37
C (5% CO2). Fresh DMEM/F12 medium + 10% FBS was
added before transfections. With the exception of DAX-1, one (for
6-well plates) or 0.5 µg (for 12-well plates) of each plasmid to be
transfected was incubated with Fugene 6 (2:1, Fugene 6:DNA) in 100 µl
of medium for 15 min. DAX-1 expression plasmid concentrations used in
these studies varied from 0.5 to 10 µg (see Figures). DNA-Fugene 6
complex was then added to the cells. Cells were allowed to incubate for
48 h at 37 C (5% CO2). Dibutyryl cAMP
([Bu]2AMP, 1 mM), 8-bromo cAMP
(8-Br-cAMP, 1 mM), or Rivastatin (Riv, 50 µM)
was added to plates 24 h before the end as per the study
design.
Cell lysate preparation and luciferase assays were performed as previously described (17). Cotransfection of a plasmid containing the renilla luciferase gene (0.5 µg for 6-well plates or 0.25 µg for 12-well plates) under control of the SV40 early enhancer/promoter region was used as a control to correct for differences in transfection efficiencies. Luciferase data were expressed as the mean ± SEM, in which the value of luciferase activity for the pGL3-basic empty vector was set to 1.0, unless otherwise specified. Each luciferase assay experiment was performed in triplicate and repeated for the indicated number of times in the figure legends. Data from the individual parameters were compared by ANOVA followed by Student-Newman-Keuls multiple comparison test when applicable (41). Values of P < 0.05 was considered significant for all tests.
For Western blot analysis, cells were lysed in PBS by repetitive freezing and thawing, and proteins were then concentrated by adding 8 vol of ice-cold acetone. Electrophoresis and transfer was performed as previously described (12).
Recombinant protein preparation
GST-DAX-1 or histidine-tagged SREBP-1a proteins were
overexpressed in Escherichia coli by induction of
midlogarithmic-phase cultures with 1 mM
isopropyl-ß-D-thiogalactopyranoside. After
incubating for 6 h at 27 C, cells were sedimented by centrifuging
at 7700x g for 10 min at 4 C. For GST-protein purification,
the cell pellet was resuspended in PBS and lysed by sonication. Triton
X-100 was then added to a final concentration of 1% and the sample
incubated for 30 min at 4 C. The suspension was centrifuged at 12,000
x g for 10 min at 4 C. Affinity purification of the fusion
protein was performed using the GST-fusion purification kit
(Pharmacia Biotech, Piscataway, NJ) as per the
manufacturer’s recommendations. After washing the column with PBS, the
fusion protein was digested with precision protease to remove the GST
moiety and used in gel mobility shift assay. For histidine-tagged
SREBP-1a protein purification, the cell pellet after the initial
centrifugation was resuspended in guanidinium lysis buffer provided in
the XPRESS system (Invitrogen, Carlsbad, CA). Purification
was performed using an immobilized metal affinity column according to
the manufacturer’s recommendations. Recombinant protein samples were
concentrated 4- to 6-fold using Centricon-10 concentrators
(Millipore Corp., Bedford, MA). Protein concentrations
were determined using the protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).
Preparation of Y1 nuclear extracts
Adrenal Y1 cells (5 x 107) were
treated with 0.25% trypsin in 1 mM EDTA and then
centrifuged. Nuclear extracts were prepared from the cell pellets as
previously described (12). Protein samples were
concentrated 4- to 6-fold using Centricon-10 concentrators
(Millipore Corp.), and concentrations were determined
using the protein assay (Bio-Rad Laboratories, Inc.).
Gel mobility shift assay
Oligonucleotides corresponding to the HDL-R promoter region from
-1966 to -1940 [distal SRE (dSRE):
5'-CTGCCCCCCTCACACCCTCCTCTGTAG-3'] and from -246 to -220 [proximal
SRE (pSRE): 5'-CCATCAGAGCACCGCCCACTCCCCGCC-3'], or from -655 to -628
[SFB: 5'-GACAGTGCATCAAGGCCGCGAGGGACA-3'] were synthesized and
annealed in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA,
25 mM NaCl, 10 mM MgCl2,
and 1 mM dithiothreitol. The oligonucleotide probe was then
labeled using the Klenow fragment of DNA polymerase and
[32P]dCTP (3000 Ci/mmol). Unlabeled
oligonucleotides were used as a competitor in some experiments. Five
micrograms nuclear protein (or 50 ng recombinant protein, unless
otherwise indicated) were incubated either in the presence or absence
of competitor for 30 min at room temperature in binding buffer [12
mM HEPES (pH 7.9), 12% glycerol, 60 mM KCl, 1
mM EDTA, 1 mM dithiothreitol, and 4
mM Tris-HCl (pH 8.0)], 2 µg
polydeoxyinosinic-deoxycytidylic acid, and 1 µg BSA. After
incubation, 50,000–100,000 cpm of the radiolabeled probe was added and
the mixture incubated for 20 min at 15 C. Where indicated, SREBP-1a or
SF-1 antibody was also added to the reaction for supershift analysis.
The DNA protein complexes were resolved on a 4% (or 6% in case of the
SF-1 protein) nondenaturing acrylamide gel at 4 C in 1x TGE (0.25
M Tris, 1.9 M glycine, and 10 mM
EDTA). Gels were then vacuum dried and exposed to BioMax-MR films at
-80 C for 12–24 h or developed using a Cyclone storage phosphor
system (Packard Instrument Co., Meriden, CT).
Densitometric quantitation of the autoradiograms was performed using
the UVP imaging system and Labworks software (UVP Laboratory Products, Upland, CA).
For Western blot analysis, unlabeled mobility shift assay gels were electroblotted onto nitrocellulose membranes (0.2-µm pore) in buffer containing 0.25 M Tris-HCl (pH 8.3), 1.92 M glycine, and 20% methanol at 4 C overnight using 200 mA.
Western blot analysis
Western blot analysis was carried out with a 1:1000 dilution of
the rabbit polyclonal antiserum to DAX-1, SREBP-1a, or SF-1 in 3%
milk. Immunoreactive proteins were then visualized using a 1:6000
dilution of horseradish peroxidase-conjugated goat antirabbit antisera
(Santa Cruz Biotechnology, Inc.) in 3% milk and the
SuperSignal ULTRA chemiluminescent substrate method (Pierce Chemical Co., Rockford, IL).
Mammalian two-hybrid assay
The plasmids pBIND, pACT, and pG5-luciferase used in these
assays were provided in the mammalian two-hybrid system (Promega Corp.). The plasmid SF-1-pBIND was constructed using full-length
mouse SF-1 cDNA inserted into the BamHI and NotI
sites. To construct the plasmid SREBP-1a-pBIND, the
NH2-terminal active segment of SREBP-1a was
inserted into the EcoRI and KpnI sites. The
DAX-1-pACT plasmid was prepared using full-length mouse DAX-1 cDNA
inserted into the XhoI/SalI and NotI
sites. The tDAX-1-pACT plasmid was prepared by site-directed
mutagenesis as described above. To examine luciferase activities, HTB-9
cells (plated in six-well plates) were cotransfected with the
pG5-luciferase vector either in the presence or absence of SF-1-pBIND
(or SREBP-1a-pBIND) ± DAX-1-pACT (or tDAX-1-pACT). For these
experiments, transfections were performed with 2 µg of each plasmid
using the Fugene 6 transfection reagent Roche Molecular Biochemicals according to the manufacturer’s instructions.
After transfection, cells were incubated for 48 h at 37 C before
harvesting. Transfection efficiencies were corrected using renilla
activity expressed by the pBIND plasmid. Statistical analysis was
performed as described in Cell transfections and luciferase
assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or the p-719 (Fig. 1C) HDL-R
promoter construct in the presence or absence of DAX-1-pcDNA3.1
plasmid. Figure 1A shows a schematic representation of both promoter
constructs. These studies were completed in HTB-9 cells because these
cells have undetectable levels of HDL-R, SF-1, SREBP-1a, or DAX-1.
DAX-1 reduced HDL-R promoter-driven luciferase activity of both
promoter constructs by 50% and 70%, respectively (P
< 0.01; Fig. 1, B and C). These data suggest that DAX-1 can inhibit
basal HDL-R promoter activity. The p-2267 promoter construct
consistently displayed basal activity levels similar to the pGL3-basic
(control) plasmid alone, whereas the p-719 promoter construct induced
luciferase activity 30-fold (P < 0.001) over
pGL3-basic activity (Fig. 1, B and C). This could be owing to the
presence of a negative regulatory region in the p-2267 promoter that
prevents maximal activation of the HDL-R promoter in HTB-9 cells. It
has been previously shown that this promoter region contains two Yin
Yang 1 (YY1) binding sites (42) that may be responsible
for decreased basal activity of the p-2267 HDL-R promoter construct in
the HTB-9 cells.
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, A and B, DAX-1
significantly reduced SF-1 + [Bu]2cAMP- or
SREBP-1a-dependent activation of the HDL-R promoter in a dose-dependent
manner. Ten micrograms DAX-1-pcDNA3.1 plasmid reduced SF-1 +
[Bu]2cAMP- and SREBP-1a-dependent activation of
the HDL-R promoter by 93% and 96% (P < 0.001),
respectively (Fig. 2, A and B). The DAX-1’s effect on SF-1 and
SREBP-1a-dependent activity of the HDL-R promoter were specific because
the total amount of DNA plasmid transfected per well was corrected
using pcDNA3.1 empty vector. In addition, Western blot analysis
demonstrated that DAX-1 protein levels increased as increasing amounts
of the DAX-1 expression plasmid were transfected into the cells,
whereas SREBP-1a protein levels were not significantly changed
(Fig. 2C).
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) and human liver HepG2 cells
(Fig. 3B). These cells endogenously express SF-1 and SREBP-1a,
respectively. Rivastatin, a cholesterol biosynthesis inhibitor, causes
a decrease in intracellular cholesterol levels with a corresponding
increase in mature SREBP levels. As shown in Fig. 3 (upper
panels), DAX-1 was able to significantly repress basal, 8-Br-cAMP,
and rivastatin-dependent activation of the HDL-R promoter. Likewise,
DAX-1 was able to repress SF-1 (±8-BrcAMP) and SREBP-1a
(±Riv)-dependent activation of the HDL-R promoter in Y1 and HepG2
cells, respectively (Fig. 3, lower panels).
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).
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) was involved in DAX-1
inhibition of HDL-R gene expression, a mutated promoter construct was
prepared as described in Materials and Methods. Figure 5, B and C, demonstrates that deleting
the DAX-1-binding site (p-2267 HN-HDL-R) did not significantly affect
basal or SF-1 ± [Bu]2cAMP or
SREBP-1a-dependent activation of the HDL-R promoter in HTB-9 cells. The
lack of the putative DAX-1 hairpin-binding structure did not prevent
DAX-1 repression of the HDL-R promoter in the presence or absence of
SF-1 or SREBP-1a (Fig. 5, B and C). Similar results were obtained in Y1
and HepG2 cells (data not shown). These results suggest that DAX-1 does
not mediate repression of the HDL-R gene by binding to the hairpin
structure found in this promoter. These results correlate with our
findings that recombinant DAX-1 protein did not bind to the HDL-R
promoter hairpin structure (data not shown) under conditions identical
to those used for the human StAR and mouse DAX-1 hairpin structures
(25).
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, 5 µg Y1 adrenal cell nuclear
proteins formed a major complex (lower arrow, lane 2) with
radiolabeled SFB oligonucleotide. The addition of 125-fold excess of
unlabeled SFB (lane 3) or SF-1 specific antibody (9.6 µg IgG, lane 4)
reduced protein/DNA complex formation demonstrating the specificity of
the complex formed and that SF-1 protein is part of that complex.
Adding recombinant DAX-1 to the binding reaction shifted the SF-1/SFB
complex to a higher molecular weight (upper arrow) in a
dose-dependent manner (Fig. 6A, lanes 5–6 and 7–8). Apparently, DAX-1
and SF-1 formed a complex with the SFB motif causing the supershift.
These effects appear to be specific for DAX-1 because the addition of
recombinant GST to the binding reaction before or after the addition of
DNA did not alter the protein/DNA complex (Fig. 6A, lanes 9 and 10).
DAX-1 did not bind to the SFB motif in the absence of Y1 nuclear
extracts (data not shown) suggesting that the supershifted complex
resulted from direct protein-protein interaction between DAX-1 and
SF-1.
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). The
presence of DAX-1 in the supershifted complex was demonstrated using
Western blot analysis (Fig. 6B, lanes 4–6), whereas the presence of
SF-1 in the supershifted complex was demonstrated by abrogation (Fig. 6A, lane 11) and by Western blot analysis (Fig. 6B, lanes 4–6). Using
our current conditions, SF-1 protein binding to the DNA prevents
antibody recognition. Therefore, the presence of SF-1 in the SF-1/SFB
complex when the binding reactions were performed in the absence of
DAX-1 or in the presence of GST was not detected by Western blot
analysis (Fig. 6B, lanes 2 and 7, respectively). It appeared that DAX-1
interaction with SF-1 induced a conformational change in the SF-1
protein that permitted DNA binding but also exposed that portion of the
SF-1 protein necessary for antibody recognition.
To examine whether DAX-1 affected SREBP-1a binding to the HDL-R SREs in
a similar fashion, recombinant SREBP-1a protein was incubated with
DAX-1 before the addition of dSRE or pSRE, and the results are shown in
Fig. 7A. In the absence of DAX-1,
SREBP-1a formed a single DNA/protein complex with both SREs, which was
specifically reduced by the addition of cold oligonucleotide (100x)
and supershifted with an SREBP-1a specific antibody (2 µg IgG; Fig. 7A, lanes 2–4 and 9–11, respectively). Surprisingly, rather than
supershifting the DNA/protein complex as in the case of SF-1 (Fig. 6),
DAX-1 prevented SREBP-1a binding to the DNA in a dose-dependent manner
(Fig. 7A). Recombinant DAX-1 protein (100 ng) reduced SREBP-1a binding
to dSRE more efficiently than binding to pSRE, whereas higher amounts
of DAX-1 (250–500 ng) reduced SREBP-1a binding to pSRE more
efficiently than to dSRE (Fig. 7A). The effects of DAX-1 on SREBP-1a
binding were not significantly influenced by the order in which the
radiolabeled dSRE oligonucleotide was added (i.e. before and
after adding the DNA to SREBP-1a containing binding reaction; Fig. 7B).
Similar results were obtained in the case of the pSRE (data not shown).
To rule out the possibility that a nuclear factor may be responsible
for causing the supershift seen with SF-1, binding reactions were also
performed using nuclear extracts containing SREBP-1a, and once again,
DAX-1 prevented SREBP-1a binding to the dSRE (data not shown). These
results suggested that the effects were factor dependent
(i.e. SF-1 vs. SREBP-1a).
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, cotransfecting DAX-1 and SREBP-1a with
the pG5-basic vector caused an 8-fold increase (P <
0.001) in luciferase activity, compared with cells cotransfected with
SREBP-1a-pBIND and pACT empty vector. These results demonstrated that
DAX-1 and SREBP-1a were able to interact in the mammalian two-hybrid
assays.
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. The lack of the
carboxy-terminal transrepression domain caused a loss of repression
relative to the wild-type DAX-1 in the presence of SF-1 or SREBP-1a.
Similar results were obtained in Y1 and HepG2 cells (data not shown).
Mammalian two-hybrid assays were performed to examine whether tDAX-1
could still interact with SF-1 and SREBP-1a. The data in Fig. 10 are represented as
fold-induction ± SEM where the value of
luciferase activity for the construct transfected in the presence of
the pACT/pBIND negative controls was set to 1.0 (data not shown). As
shown, the lack of the transrepression domain within the DAX-1 protein
also resulted in a loss of interaction between DAX-1 and SF-1 or
SREBP-1a. These results suggested that the DAX-1 transrepression domain
was involved in the interaction of DAX-1 with proteins such as SF-1 and
SREBP-1a.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that DAX-1 does not interfere with SF-1 binding to the DNA
has been previously reported (30, 36). However, these
groups were not able to show heterodimer formation between DAX-1 and
SF-1 in mobility shift assays (30, 36) perhaps owing to
the close proximity between the two complexes (SF-1/DNA vs.
SF-1/DAX-1/DNA). In the current study, we were able to separate the
complexes using a higher percentage acrylamide gel (6% vs.
4%). One interesting fact, however, is the Western blot result showing
that the SF-1 specific antibody was able to recognize the SF-1 protein
in the protein-DNA complexes only when DAX-1 protein was present,
perhaps owing to a conformation change in the SF-1 protein upon
interaction with DAX-1. This conformation change in the SF-1 protein
may prevent SF-1 interaction with its coactivator or allow the
interaction of SF-1 with a corepressor. DAX-1 has also been shown to
interact with estrogen receptors or ß on the DNA, forming a
higher molecular weight complex (33).
The most novel finding of the current study is that DAX-1 inhibited SREBP-1a-dependent activation of the HDL-R promoter by directly interacting with SREBP-1a to prevent SREBP-1a binding to the HDL-R SREs. DAX-1 was also able to reduce SREBP-1a/SRE binding even if SREBP-1a was allowed to bind the DNA first followed by DAX-1 addition, suggesting that the protein region of SREBP-1a that interacts with DAX-1 may be critical for SREBP-1a DNA binding. If the amount of SREBP-1a that was bound to the DNA was reduced owing to the presence of DAX-1, factors involved in the formation of the transcription initiation complex may not be properly recruited, causing a decrease in HDL-R gene transcription. Whether the recruitment of corepressors by DAX-1 is involved in DAX-1’s repression of SREBP-1a-dependent HDL-R promoter activity requires further study.
Another interesting finding was that DAX-1’s efficiency to prevent SREBP-1a binding to the DNA differs depending on the amount of DAX-1 present in the binding reaction and the type of SRE. Because both proteins used in these experiments, SREBP-1a and DAX-1, were purified to the homogeneity, the only other difference that could be responsible for this discrepancy would be the oligonucleotide sequence (both oligonucleotides are 30 bp long). The oligonucleotide containing the pSRE also contains a putative Sp1 site, whereas the oligonucleotide containing the dSRE does not contain any other known response elements. Whether this difference in DAX-1’s efficiency is critical to its action in vivo will require additional study.
We have recently reported that the repressor YY1 also inhibits HDL-R
gene transcription by preventing SREBP-1a binding to the DNA
(42). Interestingly, both DAX-1 (28) and YY1
(Nackley, A. C., W. Shea-Eaton, D. Lopez, and M. P. McLean,
unpublished observations) protein levels increase significantly in rat
ovary after PGF2 treatment, suggesting that
these negative transcriptional regulators may act together to inhibit
the expression of the HDL-R gene during luteolysis. The necessity for
two repressors to downregulate the HDL-R gene under these conditions is
still unknown.
Herein, it was also demonstrated that the hairpin structure found in
the HDL-R promoter is not involved in DAX-1 inhibition of this gene.
Similar results have been reported for the rat StAR gene
(28). However, in the case of the human StAR and mouse
DAX-1 genes, the hairpins were able to bind DAX-1, and deleting them
from the promoter prevented DAX-1 inhibition (25). Further
support for DAX-1 inhibition by mechanisms that do not require hairpin
secondary structures comes from experiments with the human CYP17 gene
that does not contain a hairpin structure, yet it is strongly inhibited
by DAX-1 (32). It has been proposed that efficient
repression of genes by DAX-1 may require multiple SF-1 elements
(32). Genes such as the type II 3ß-hydroxysteroid
dehydrogenase/
5-4 isomerase promoter, which contains a single
SF-1 binding site are not significantly repressed by DAX-1 (29, 43). In the case of the rat HDL-R gene, there is a single SF-1
binding site; however, the data indicate that DAX-1 strongly represses
this gene perhaps owing to the presence of the SREs. The finding that
deleting the HDL-R hairpin structure did not prevent DAX-1 inhibition
of the HDL-R promoter in the absence of SF-1 and SREBP-1a suggests that
DAX-1 may also inhibit this promoter through interaction with
additional factors. In addition to its inhibitory effects on
transcription, DAX-1 has also been found to inhibit translation by
directly binding RNA hairpin structures (35). Whether
DAX-1 is able to inhibit the translation of the HDL-R mRNA through RNA
hairpin structures needs to be examined.
Because mutations of human DAX-1 cause X-linked adrenal hypoplasia congenita (20, 21, 23), extensive studies have been done to identify regions within the DAX-1 protein involved in DAX-1’s inhibitory effects. Zhang et al. (33) have shown that DAX-1 and estrogen receptor interaction is mediated by the DAX-1 N-terminal repeat domain. The C-terminal transrepression domain has been implicated in SF-1-mediated transactivation (28, 36). In the current study, we demonstrated that deleting the last 42 amino acids of the DAX-1 protein (transrepression domain) also eliminated DAX-1 inhibition of the HDL-R promoter. These results are consistent with the findings that the C-terminal DAX-1 protein region was involved in nuclear receptor corepressor recruitment (37). Interestingly, most naturally occurring DAX-1 deletions eliminate the carboxy-terminal inhibitory domain (36). In contrast to other studies (30, 36), we have demonstrated that deleting the transrepression domain of the DAX-1 protein prevented DAX-1 interaction with SF-1 and SREBP-1a. The differences in these results could be owing to the techniques used to examine protein-protein interactions (GST pull-down vs. mammalian two-hybrid) or to the exact deletion analyzed.
In conclusion, the data shown here demonstrate that DAX-1 may use different repressive mechanisms to inhibit SF-1- and SREBP-1a-activation of the HDL-R gene. This investigation confirms that DAX-1 has an important role in regulating steroidogenesis by interfering with SREBP-1a- and SF-1-induction of a gene involved in the transport of cholesterol thereby limiting the amount of substrate available for steroid hormone production.
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Acknowledgments |
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Abbreviations: CMV, Cytomegalovirus; DAX-1, dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome, gene-1; dSRE, distal SRE; GST, glutathione S-transferase; HDL-R, high-density lipoprotein receptor; HP, hairpin-binding motif; pSRE, proximal SRE; SF-1, steroidogenic factor-1; SFB, SF-1-binding motif; SF-1-pCMV, CMV promoter; SRE, sterol regulatory elements; SREBP-1a, sterol regulatory element-binding protein-1a; SREBP-1a-pCMV5, SREBP-1a under control of the CMV promoter; StAR, steroidogenic acute regulatory protein; tDAX-1, truncated DAX-1; TGE, 0.25 M Tris, 1.9 M glycine, and 10 mM EDTA.
Received April 2, 2001.
Accepted for publication August 9, 2001.
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is modulated by the negative
transcription factor DAX-1. Endocrine 10:83–91[CrossRef][Medline]
5-4 isomerase promoter by the transcription factor
steroidogenic factor-1/adrenal 4-binding protein and phorbol ester.
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