Synergistic Activation of the Inhibin
-Promoter by Steroidogenic Factor-1 and Cyclic Adenosine 3',5'-Monophosphate
Masafumi Ito1,
Youngkyu Park1,
Jennifer Weck,
Kelly E. Mayo and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine
(M.I., Y.P., J.L.J.) Northwestern University Medical School
Chicago, Illinois 60611
Department of Biochemistry, Molecular
Biology, and Cell Biology (J.W., K.E.M.) Northwestern
University Evanston, Illinois 60208
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ABSTRACT
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The inhibin
-subunit gene is expressed in the
ovary, testis, adrenal, and pituitary. Because this pattern of
expression corresponds to that of the orphan nuclear receptor,
steroidogenic factor-1 (SF-1), we hypothesized that the inhibin
promoter might be regulated by SF-1. Expression of exogenous SF-1, in
an SF-1 deficient cell line, caused modest stimulation of the inhibin
promoter. However, activation of the cAMP pathway, which is known
to regulate inhibin
expression, greatly enhanced the actions of
SF-1. Coexpression of SF-1 with the catalytic subunit of cAMP-dependent
protein kinase A caused greater than 250-fold stimulation,
whereas only 4- or 7-fold stimulation was seen by the SF-1 or protein
kinase A pathway alone. Synergistic stimulation by SF-1 and the cAMP
pathway was also seen in GRMO2 granulosa cells, which express
endogenous SF-1. Deletion and site-directed mutagenesis localized a
novel SF-1 regulatory element (TCA GGGCCA; -137 to -129) adjacent to
a variant cAMP-response element (CRE; -120 to -114). The synergistic
property of SF-1 and cAMP stimulation was inherent within this
composite inhibin
fragment (-146 and -112), as it was
transferable to heterologous promoters. Mutations in either the CRE or
the SF-1 regulatory element completely eliminated synergistic
activation by these pathways. The binding of SF-1 and CRE binding
protein (CREB) to the inhibin
regulatory elements was relatively
weak in gel mobility shift assays, consistent with their deviation from
consensus binding sites. However, SF-1 was found to interact with CREB
using an assay in which epitope-tagged SF-1 was expressed in cells and
used to pull down in vitro translated CREB. Expression of
CREB binding protein (CBP), a coactivator that interacts with SF-1 and
CREB, further enhanced transcription by these pathways. Stimulation by
the SF-1 and cAMP pathways was associated with increased histone H4
acetylation, suggesting that chromatin remodeling accompanies their
actions. We propose a model in which direct interactions of SF-1, CREB,
and associated coactivators like CBP induce strongly cooperative
transactivation by pathways that individually have relatively weak
effects on transcription.
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INTRODUCTION
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Gonadotropins play a major role in the control of ovarian
follicular development and function [see review (1 )]. FSH and LH
increase intracellular cAMP levels through G protein coupled membrane
receptors (2 3 ) and modulate the downstream expression of many ovarian
genes. These include the gonadotropin receptors, steroidogenic
enzymes (aromatase, side chain cleavage enzyme, and 3ß-hydroxysteroid
dehydrogenase), and the paracrine peptides (inhibin and activin), among
others.
Inhibin was initially isolated and characterized as a factor that
suppresses the synthesis and secretion of pituitary FSH (4 5 6 7 ).
However, inhibin also acts locally in the ovary to enhance theca cell
androgen synthesis, which also leads to increased estrogen production
(8 ). Inhibin is a dimeric glycoprotein hormone consisting of a common
-subunit and either of two ß-subunits (
-ßA,
-ßB)
(9 10 11 12 13 ). In the ovary, inhibin is expressed in granulosa cells, and its
expression is modulated during the estrous cycle (14 15 ). FSH
stimulates the expression and secretion of inhibin from granulosa cells
(16 17 ). In addition, pharmacological agents that increase
intracellular cAMP levels can stimulate inhibin secretion in granulosa
cells (18 19 ). Consistent with these findings, the inhibin
common
-subunit promoter contains a functional cAMP response element
(CRE) (20 ). Previous studies have shown that the CRE-binding protein
(CREB) (21 ) and the inducible cAMP early repressor (ICER) (22 23 ) are
involved in the stimulation (20 ) and suppression (24 ), respectively, of
the inhibin
gene in granulosa cells. It is probable, however, that
many other transcription factors, in addition to CREB and ICER, are
involved in regulation of the inhibin
gene.
The orphan nuclear receptor steroidogenic factor 1 (SF-1) (25 26 ) is
expressed in the adrenal cortex, testis, ovary, pituitary gonadotrope
cells, and hypothalamus (27 ), and it plays an essential role in the
development of these tissues (28 ). In addition, SF-1 functions as a
transcriptional regulator of a variety of target genes including
aromatase (29 30 ), cholesterol side chain cleavage enzyme (31 ),
steroidogenic acute regulatory protein (32 ), LHß (33 ), and DAX-1
(dosage-sensitive sex reversal-adrenal hypoplasia congenita critical
region on the X chromosome, gene 1) (34 ).
SF-1 is a member of the nuclear receptor superfamily and it contains a
characteristic zinc finger DNA-binding domain and putative
ligand-binding/dimerization domain that are well conserved among
members of this family (35 ). SF-1 binds to a consensus DNA recognition
sequence (PyCA AGGTPyC or PuPu AGGTCA) as a monomer. Like other
nuclear receptors, an AF2 transactivation domain is present at the
carboxy terminus of SF-1. Recently, multiple coactivators and
corepressors that mediate the transcriptional activity of steroid
receptors have been identified and characterized [see review (36 )].
Steroid receptor coactivator-1 (SRC-1), one of the well known
coactivators, interacts with the AF2 domain of SF-1 and potentiates the
activity of SF-1 (37 ). CREB binding protein (CBP), originally cloned as
a coactivator for CREB (38 ), also interacts directly with SF-1 and has
been shown to enhance transcription of the cholesterol side chain
cleavage enzyme (CYP11A1) gene (39 ).
SF-1 is involved in the cAMP-regulated expression of various genes
including cholesterol side chain cleavage enzyme (40 ), aromatase (41 42 ), and StAR (steroidogenic acute regulatory protein) (43 ). It has
been suggested that SF-1 might be phosphorylated (41 44 ), and a
phosphorylation site (Ser 203) in SF-1 was identified recently (45 ).
This site, which modulates the transcriptional activity of SF-1, was
shown to be phosphorylated by the mitogen-activated kinase (MAPK)
pathway, but not by the cAMP-dependent pathway (45 ). Thus, the
molecular mechanism of SF-1 action in cAMP-dependent gene regulation
remains incompletely understood, despite its importance in the control
of many target genes.
In addition to direct actions of phosphorylation on the transcriptional
activity of SF-1, it is also possible that phosphorylation modifies
other proteins that interact with SF-1, or mediate its transcriptional
effects. In addition to transcriptional coactivators, SF-1 has been
shown to interact functionally with a variety of other transcription
factors. SF-1 enhances estrogen receptor-mediated stimulation of the
salmon gonadotropin II ß-subunit gene (46 ). SF-1 and Egr-1
synergistically stimulate promoter activity of the rat LHß gene (47 ).
Transcription of the anti-Müllerian hormone gene is cooperatively
stimulated by SF-1 and SOX9 (48 ), and by SF-1 in combination with WT-1
(49 ). SF-1 action can also be inhibited by a direct protein interaction
with DAX-1 (50 ).
Several lines of evidence have shown that posttranslational
modifications of histones, such as acetylation/deacetylation,
methylation, or phosphorylation, can alter gene expression [see review
(51 )]. Several coactivators, including CBP/P300 (52 53 ) and P/CAF
(54 55 ), contain histone acetyltransferase activity. Histone
acetylation is thought to open chromatin structure and allow additional
transcription factors to bind to DNA and activate transcription.
Although CBP is recruited by SF-1 (39 ), little is known about the role
of histone acetylation in SF-1-mediated transactivation.
In this report, we examined SF-1 regulation of the inhibin
promoter
as a model of synergistic actions of these pathways. A novel SF-1 site
was identified adjacent to the inhibin
CRE. We provide evidence for
direct protein interactions between SF-1 and CREB, with recruitment of
CBP, and increased histone acetylation as a mechanism for the strong
synergism between the SF-1 and cAMP pathways.
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RESULTS
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Synergistic Activation of the Inhibin
-Subunit Gene by SF-1 and
the cAMP Pathway
A number of promoters have both SF-1 and CRE sites, suggesting
transcriptional cross-talk between these two pathways. Several
promoters that are regulated by the SF-1 and cAMP pathway were analyzed
using transfection assays in SF-1-deficient human embryonic kidney tsa
201 cells. Promoter-luciferase constructs included the human
glycoprotein hormone
subunit, rat aromatase, and the inhibin
gene. An SF-1-expressing plasmid was cotransfected with, or without a
protein kinase A (PKA) expression vector. The human common
-subunit
gene contains a single SF-1 binding site (56 ) and two CREs (57 ) (Fig. 1
). Despite the presence of this binding
site, SF-1 did not stimulate the activity of this promoter, whereas PKA
induced strong transactivation (187-fold). Cotransfection of SF-1 with
PKA did not increase PKA-induced transactivation further (170-fold).
The rat aromatase gene has an SF-1 binding site (29 30 ) and a CRE
(58 ), and there is functional interaction between the SF-1 and
CRE-regulatory elements (41 ). SF-1 and PKA stimulated the promoter
activity by 8- and 105-fold, respectively. SF-1 increased the
PKA-induced transactivation by 2-fold. In the case of the inhibin
promoter, a single CRE site was previously identified (20 ), but SF-1
binding sites have not been described. SF-1 and PKA stimulated the
-2021 promoter by 5- and 40-fold, respectively. When both SF-1 and PKA
were cotransfected, transactivation was dramatically increased
(607-fold), suggesting that a SF-1 binding site may be present in the
promoter and that synergistic activation by SF-1 and the cAMP pathway
plays an important role in the regulation of inhibin
gene
expression. These findings illustrate that interactions between the
SF-1 and PKA pathways vary markedly, depending on the promoter being
studied.
The SF-1 regulatory element in the inhibin
promoter was localized
further using deletion mutagenesis. Deletion of the inhibin
reporter to -769 or -547 retained strong synergistic activation by
SF-1 and PKA (Fig. 2A
). Further deletion to
-311 or -146 also retained the synergistic activation by SF-1 and PKA
(Fig. 2B
), suggesting that a functional SF-1 site resides within the
proximal promoter. Deletion from -146 to -134 eliminated the
synergistic activation, suggesting that an SF-1 site resides within
this region. Consistent with these results, SF-1 stimulation was
observed with the -146 promoter (2.5-fold), but not with the -134
promoter.

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Figure 2. Deletion Mutagenesis of Inhibin Promoter to
Localize Sequences Involved in Synergistic Activation
A deletion series of the inhibin reporter constructs (A; -2021,
-769, and -547 to +68, B; -311, -236, -160, -146, -134, -115,
and -89 to +68) (0.5 µg) was transfected into tsa cells with a
mutant or wild -type PKA expression vector (25 ng) and an empty or SF-1
expression vector (20 ng). Forty eight hours after transfection, cell
extracts were prepared and luciferase assays were performed. Results
are the mean ± SEM of triplicate transfections. The
location of the CRE is shown.
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Synergistic Activation Is Dose Dependent
A wild-type or mutant PKA expression vector was cotransfected with
increasing amounts of the SF-1 expression vector (Fig. 3A
). In addition, an empty or SF-1 expression
vector was transfected with increasing amounts of wild-type PKA
expression vector (Fig. 3B
). In both experiments, synergistic
activation was increased in proportion to the dose of the PKA or SF-1
expression constructs.

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Figure 3. Dose-Dependent Effect Of SF-1 And PKA On
Synergistic Activation
A, The -146 wild-type reporter (0.5 µg) was transfected into cells
with wild-type or mutant PKA expression vector (25 ng) and increasing
amounts of SF-1 expression vector (0, 2, 5, 10, 20 ng). The total
amount of transfected plasmid was adjusted with an empty vector. B, The
-146 wild-type reporter (0.5 µg) was transfected with an empty or
SF-1 expression vector (20 ng) and increasing amounts of wild-type PKA
expression vector (0, 2, 5, 10, 25 ng). The total amount of transfected
plasmid was adjusted with the mutant PKA expression vector. Forty eight
hours after transfection, luciferase assays were performed. Results are
the mean ± SEM of triplicate transfections.
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Synergistic Activation Occurs in Granulosa Cells
The GRMO2 ovarian granulosa cells express inhibin, and
expression of the inhibin and activin ßA-subunit genes is known to be
regulated by cAMP (59 ). The level of inhibin
promoter activity in
GRMO2 cells is similar to that in other SF-1 expressing cells,
such as pituitary gonadotrope
T3 and adrenal cortical Y1 cells (data
not shown). The -146 wild-type reporter was cotransfected with either
an empty or SF-1 expression vector, and cells were treated with 1
mM 8-bromo-cAMP for 12 h to determine whether the
synergistic activation also occurs in the GRMO2 cells (Fig. 4
). SF-1 transfection and 8-bromo cAMP
treatment stimulated promoter activity by 2.4- and 5.4-fold,
respectively (lanes 2 and 3). When both were combined, SF-1 increased
the 8-bromo-cAMP-induced transactivation by an additional 2.5-fold
(lane 4). To interpret this data, it is necessary to take into account
that SF-1 is expressed endogenously in granulosa cells (60 ), and SF-1
mRNA was detected in GRMO2 cells (data not shown). Because of the
presence of endogenous SF-1, some element of synergistic activation may
occur after the addition of 8-bromo cAMP alone (lane 3). Further
enhancement of synergistic activation by overexpression of SF-1 was
relatively small (lanes 3 and 4), presumably due to the presence of
endogenous SF-1. These findings suggest that synergistic activation by
SF-1 and the cAMP pathway also occurs in granulosa cells.

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Figure 4. Synergistic Activation In Ovarian GRMO2 Granulosa
Cells
The -146 wild-type reporter (0.5 µg) was cotransfected with an empty
or SF-1 expression vector (50 ng), and cells were treated with 1
mM 8-bromo-cAMP for 12 h before the luciferase assay.
Luciferase activity is normalized to total cellular protein and
expressed as arbitrary light units (ALU)/mg protein. Results are
the mean ± SEM of triplicate transfections.
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Identification of the SF-1 Binding Site Responsible for the
Synergistic Activation
By inspection of the DNA sequence, three potential SF-1 binding
sites were found (A; ATA AGGCTC, B; CTC AGGGCC, C; TCA GGGCCA) between
-144 and -129 (Fig. 5
). Mutations were
introduced into the second and third positions of the putative nuclear
receptor half-sites in the -146 reporter constructs to identify
functional SF-1 regulatory elements. SF-1 and PKA stimulated the -146
wild-type reporter by 4- and 7-fold, respectively. When both were
transfected, synergistic activation was observed (276-fold).
Introduction of mutations into the putative binding sites A (m1; AGGCTC
ATTCTC) and B (m3; AGGGCC
ATTGCC) did not
affect synergistic activation. In contrast, the m4 mutation, which
disrupts the putative binding site C (GGGCCA
GTTCCA)
eliminated synergism completely. Also, SF-1 transactivation was
abolished by this mutation. These data indicate that the synergistic
activation is mediated by SF-1 binding to site C. Two additional
mutations introduced into site C (m2; TCA
AAA, m5;
GGGCCA
GGGAAA) reduced the synergistic activation
(~25% of wild type), whereas mutations adjacent to the SF-1 binding
site (m1 and m6) did not alter the synergistic activation.

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Figure 5. Localization of the SF-1 Binding Site and CRE
Responsible for the Synergistic Activation
The -146 wild-type or mutagenized reporters (0.5 µg) were
transfected into tsa cells with a mutant or wild type PKA expression
vector (25 ng) and an empty or SF-1 expression vector (20 ng). Forty
eight hours after transfection, cells were subjected into luciferase
assays. Results are the mean ± SEM of triplicate
transfections. Three putative SF-1 binding sites are shown by A, B, and
C.
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To confirm SF-1 binding to site C, electrophoretic mobility shift assay
(EMSA) was performed using radiolabeled probes (-146 to -122) and
nuclear extracts prepared from cells transfected with an empty or SF-1
expression vector (Fig. 6A
). SF-1 binding was
detected with extracts that contain expressed SF-1 (lane 3), but
binding was not detected using extracts from cells transfected with an
empty vector (lane 2). SF-1 binding was eliminated by 100-fold molar
excess of competitor oligonucleotides (lane 4) or by anti-Ad4BP
(anti-SF-1) antibody (lane 5). The m4 mutation at the second and third
positions of the nuclear receptor half-site completely eliminated SF-1
binding (lane 8). In contrast, SF-1 bound to the m3 probe, which
contains mutations at the first and second positions of the half-site
(lane 7), although its binding was reduced to 60% of the wild-type
probe (lane 3). The m2 and m5 mutations significantly reduced the
SF-1 binding (lanes 6 and 9), but very weak SF-1 binding was detected
when the films were overexposed (data not shown). Consistent with these
observations, SF-1 binding to the wild-type probe (lane 3) was not
abolished by 100-fold molar excess of the m2 (lane 10), m4 (lane 12),
or m5 (lane 13) competitor oligonucleotides. The m3 oligonucleotide
competed for SF-1 binding to the wild-type probe, consistent with its
weak binding activity (lane 11). The binding characteristics of SF-1 to
the mutated probes (Fig. 6
) were consistent with the promoter
activities observed using mutated SF-1 binding sites (Fig. 5
). Taken
together, these experiments identify an SF-1 binding site (TCA GGGCCA;
-137 to -129) that is responsible for the synergistic activation with
PKA.

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Figure 6. DNA Binding of SF-1 and CREB
A, EMSA was performed using nuclear extracts prepared from cells
transfected with an empty or SF-1 expression vector. Ten micrograms of
nuclear extracts were subjected into the binding reaction along with 20
fmol of wild-type or mutagenized probes (-146 to -122). The DNA
protein complexes were resolved on a 0.5x TBE polyacrylamide gel. B,
EMSA was performed as described above using nuclear extracts prepared
from cells transfected with an empty or CREB expression vector and the
wild- type or m7 mutant probe (-132 to -104).
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Involvement of CREB in the Synergistic Activation
It has been shown previously that CREB binds to the inhibin
CRE (TGCGTCA; -120 to -114) and that mutation of the CRE
(TGTATCA) eliminates its binding (20 ). EMSA using nuclear
extracts prepared from cells transfected with an empty vector (Fig. 6B
, left panel) showed binding of endogenous CREB to the
CRE-containing probe (-132 to -104) (lane 15). CREB binding was
eliminated by 100-fold molar excess of a consensus CRE oligonucleotide
(TGACGTCA) (lane 16) , and it was decreased by incubation
with anti-CREB antibody (lane 17). CREB did not bind to the CRE mutant
probe (m7) (lane 18), and the CREB binding was not eliminated by
100-fold molar excess of the m7 oligonucleotides (lane 19). The binding
of endogenous CREB to the CRE was relatively weak compared with SF-1
binding to its element. As shown later (Fig. 7
), weak CREB binding is explained by
deviation of the inhibin
CRE sequence (TGCGTCA) from the consensus
CRE sequence (TGACGTCA). In an effort to provide additional
evidence for CREB binding, EMSA was performed using nuclear extracts
prepared from cells transfected with a CREB expression vector (Fig. 6B
, right panel). CREB binding (lane 20), competition by CRE
oligonucleotides (lane 21), and its supershift with anti-CREB antibody
(lane 22) were clearly observed. Also, overexpressed CREB did not bind
to the m7 probe (lane 23), and CREB binding was not competed by the m7
oligonucleotides (lane 24). When the m7 mutation was introduced into
the CRE (Fig. 5
), synergistic activation was totally eliminated. These
results indicate that CREB binds to the CRE and is involved in the
synergistic interaction with SF-1.

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Figure 7. Effect of Enhanced Binding of SF-1 and CREB on the
Synergistic Activation
A, EMSA was performed using nuclear extracts with or without SF-1
expression. In addition to the wild-type probes (-146 to -122 and
-132 to -104), mutant probes containing consensus binding sites for
SF-1 (m8) and CREB (m9) were used. The consensus sequences introduced
into the probes are described below. B, The -146 wild-type or
mutagenized reporters (m8, m9, and m10) (0.5 µg) were transfected
with a mutant or wild-type PKA expression vector (25 ng) and an empty
or SF-1 expression vector (20 ng). Forty eight hours after
transfection, cell extracts were subjected into luciferase assays.
Results are the mean ± SEM of triplicate
transfections.
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PKA Does Not Increase Binding of SF-1 or CREB to Their
Response Elements
DNA binding affinities of SF-1 and CREB were examined to assess
whether enhanced binding might explain synergistic interactions.
Nuclear extracts were prepared in the presence of phosphatase
inhibitors from cells transfected with wild-type or mutant PKA
expression vector and empty or SF-1 expression vector. Western blot
analysis using antiphosphorylated CREB antibody demonstrated increased
phosphorylation of CREB and ATF-1 in cells transfected with a wild-type
PKA expression vector compared with cells transfected with a mutant
expression vector (data not shown). When EMSA was performed using these
nuclear extracts, PKA did not increase DNA binding of SF-1 and CREB
(data not shown). These results suggest that the synergistic activation
is not the consequence of increased binding of SF-1 or CREB to DNA.
Increased Binding of SF-1 and CREB Diminishes Synergistic
Activation.
The SF-1 binding site identified in the inhibin
promoter
(GGGCCA) differs from that of the consensus binding site (AGGTCA). It
was anticipated that replacement of the half-site with the consensus
half-site would increase SF-1 binding. Also, introduction of the
consensus CRE was expected to increase CREB binding. EMSA using
nuclear extracts that express SF-1 (Fig. 7A
) confirmed increased
binding to the consensus SF-1 binding site (m8) compared with the
inhibin
SF-1 site (lanes 1 and 2). Similarly, endogenous CREB bound
to the consensus CRE (m9) much better than to the native inhibin
CRE (lanes 3 and 4). The CREB binding complexes were supershifted by
anti-CREB antibody, but not by anti-ATF-1 antibody (data not shown).
Introduction of the SF-1 consensus binding site did not increase
the activation by SF-1 alone, whereas conversion of the CRE to a
consensus sequence increased the response to PKA alone (2.5 fold).
Despite increased binding of SF-1 (m8) and CREB (m9), synergistic
transactivation by SF-1 and PKA was not increased further (Fig. 7B
).
Rather, synergistic activation was reduced (m8; 57%, m9; 50%). These
results indicate that the relatively weak binding of SF-1 and CREB to
the natural sites in the inhibin
promoter may facilitate
synergistic activation. In addition, elimination of five bases (AGACA)
from the intervening sequence between the SF-1 binding site and
the CRE (m10) resulted in a marked decrease in synergistic
activation (15%), suggesting that the spacing between the regulatory
elements is important for optimal synergism.
Synergistic Activation Is Mediated through the Composite Enhancer
Element
The promoter regions containing the responsive elements (-146 to
-112, -146 to -80, and -146 to -40) were linked to the thymidine
kinase minimal promoter to assess whether the element containing the
SF-1 binding site and CRE is sufficient to mediate synergistic
activation (Fig. 8
). The degree of synergism
obtained with these heterologous reporters (280- to 350-fold) was
similar to that obtained with the native -146 native reporter
construct (276-fold). These data indicate that the minimal composite
element containing the SF-1 binding site and CRE (-146 to -112) is
sufficient to mediate synergistic activation. PKA activation was
stronger with these reporters (35- to 49-fold) than seen with the -146
wild-type reporter (5-fold) (Fig. 5
).

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Figure 8. Characterization of the SF-1 and CREB Composite
Enhancer Element
The promoter region containing the SF-1 binding site and CRE (-146 to
-112, -146 to -80, and -146 to -40) was linked to the thymidine
kinase minimal promoter and luciferase gene. The reporter genes (0.5
µg) were cotransfected with a mutant or wild-type PKA expression
vector (25 ng) and either an empty or SF-1 expression vector (20 ng).
Forty eight hours after transfection, luciferase assays were performed.
Results are the mean ± SEM of triplicate
transfections.
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SF-1 Interacts Directly with CREB
Because of the proximity of the SF-1 binding site and CRE in the
composite enhancer element, it is possible that SF-1 and CREB interact
directly with one another. Nuclear extracts were prepared from cells
transfected with either an empty or hemagglutinin (HA)-tagged SF-1
expression vector, and the expression of HA-tagged SF-1 was confirmed
by Western blot analysis using anti-HA antibody (Fig. 9A
). 35S-labeled CREB
was synthesized by in vitro translation and introduced into
the binding reaction along with nuclear extracts. Either nonimmune IgG
or anti-HA antibody was used to pull down the SF-1-CREB complexes (Fig. 9B
). Labeled CREB was detected only in the presence of HA-tagged
SF-1, demonstrating physical interaction between SF-1 and CREB.

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Figure 9. Protein Interaction Assay between SF-1 and CREB
A, Nuclear extracts were prepared from cells transfected with an empty
or HA-tagged SF-1 expression vector. The nuclear extracts were
separated on 10% SDS-PAGE followed by Western blot analysis using
anti-HA antibody. B, 35S-labeled CREB proteins translated
in vitro were introduced into the binding reaction
containing nuclear extracts with or without HA-tagged SF-1 expression.
The reaction was immunoprecipitated with either nonimmune IgG (NI) or
anti-HA antibody (HA). Immunoprecipitates were loaded on 10%
SDS-PAGE followed by autoradiography. Ten percent of the total input
was also run on the gel.
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CBP Enhances Synergistic Activation
CBP is a coactivator for both SF-1 (39 ) and CREB (38 ), and it is
capable of directly interacting with each protein. As shown above, SF-1
and CREB can interact with each other. Thus, it is possible that CBP is
involved in the synergistic activation through the formation of ternary
complexes with SF-1 and CREB. PKA or SF-1, or the combination of
proteins, was cotransfected with increasing amounts of CBP expression
vector (Fig. 10A
). CBP had little or no
effect on SF-1-mediated transcriptional activation, but it increased
PKA-induced transactivation by 2-fold when 50 ng of CBP expression
vector were cotransfected. Synergistic activation was increased (1.8-
and 2.9-fold) by cotransfection with increasing amounts of CBP (20 and
50 ng). CBP lacking the carboxy-terminal region (CBP 1460), or P/CAF,
had no effect on the synergistic activation (Fig. 10B
).

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Figure 10. Effect of CBP on the Synergistic Activation
A, The -146 wild-type reporter (0.5 µg), mutant, or wild-type PKA
expression vector (25 ng) and empty or SF-1 expression vector (20 ng)
were cotransfected with increasing amounts of CBP expression vector (0,
20, 50 ng). B, Empty, CBP, CBP 1460, or P/CAF expression vectors (50
ng) were transfected into cells along with the -146 wild-type reporter
(0.5 µg), and SF-1 (20 ng) and wild-type PKA expression vectors (25
ng). Forty eight hours after transfection, luciferase assays were
performed. Results are the mean ± SEM of triplicate
transfections.
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Synergistic Activation Involves Histone Acetylation
CBP can induce histone acetylation directly (52 53 ), or
indirectly, through the recruitment of P/CAF (54 55 ). Activation of
histone acetylation may, therefore, parallel or be involved in the
synergistic activation. Chromatin immunoprecipitation (CHIP) assays
were performed to assess the extent of histone acetylation specific for
the inhibin
promoter under various treatment conditions. The -311
inhibin
luciferase reporter construct was transfected into tsa 201
cells with mutant or wild-type PKA expression vector, and empty or SF-1
expression vector, and CHIP assays were performed 48 h after
transfection (Fig. 11
). PCR using
immunoprecipitates with antiacetylated histone H4 antibody showed a
significant increase of the 287-bp amplified products (3-fold) in cells
transfected with both SF-1 and PKA expression vectors (lane 4) compared
with the basal state (lane 1). SF-1 (lane 2) and PKA (lane 3) alone did
not increase the band intensity. As a control, there was no difference
in the PCR product using total DNA (lanes 58) before
immunoprecipitation, and immunoprecipitates with nonimmune IgG did not
amplify PCR products (data not shown).

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Figure 11. Association of Synergistic Activation with Histone
Acetylation
The inhibin -311 luciferase reporter (5 µg) was transfected with
either a mutant or wild-type PKA expression vector (10 µg) and either
an empty or SF-1 expression vector (5 µg) into tsa 201 cells. Forty
eight hours after transfection, the CHIP assay was performed. The DNA
fragments (287 bp) amplified by PCR using primers corresponding to the
promoter- and luciferase-coding sequences were separated on 6%
nondenaturing polyacrylamide gels followed by autoradiography. The data
obtained from immunoprecipitates with antiacetylated histone H4
antibody (lanes 14) and total DNA (lanes 58) are shown.
|
|
 |
DISCUSSION
|
---|
The SF-1 and cAMP pathways are involved in the regulation of many
genes in the gonads and in the adrenal gland. Because SF-1 is expressed
in a highly restricted manner, it has been suggested to play a pivotal
role in tissue-specific gene expression (61 ). On the other hand, the
cAMP pathway represents one of the dominant signal transduction
cascades in the gonads and adrenal, reflecting its activation by
trophic hormones like FSH, LH, and ACTH. Thus, it is perhaps not
surprising that these pathways converge in the control of many
different target genes in these tissues. Although previous studies have
shown that the inhibin
gene is regulated by cAMP (20 ), the
potential role of SF-1 has not been studied. It is notable that the
inhibin
gene is expressed primarily in the ovary, testis, adrenal
gland, and pituitary gonadotropesa pattern that largely coincides
with the expression profile of SF-1. Consistent with this pattern of
cell-specific expression, the inhibin
promoter is most active in
SF-1 expressing cell lines (data not shown). For this reason, we were
prompted to examine the role of SF-1 in control of inhibin
promoter
activity. Somewhat disappointingly, initial experiments using SF-1
alone revealed modest (<5-fold) stimulation of the inhibin
promoter. However, in the presence of cAMP or PKA treatment, SF-1
greatly stimulated the activity of this promoter, leading to the
recognition of potent synergy between these two pathways. Based on this
finding, we identified a novel SF-1 binding site (TCA GGGCCA) adjacent
to the imperfect CRE (TGCGTCA) in the proximal region of the inhibin
promoter.
There are several possible explanations for the synergistic
transactivation of inhibin
gene by SF-1 and CREB. One possibility
is that the cAMP pathway leads to the phosphorylation of SF-1 and
increases its transcriptional activity. Although SF-1 is also
phosphorylated by PKA in vitro (41 44 ), recent studies
suggest that phosphorylation on Ser 203 is mediated by the MAPK pathway
(45 ). Other evidence suggests that direct phosphorylation of SF-1 by
PKA is not the primary basis for synergy in the case of the inhibin
promoter. Mutation of the CRE completely eliminates synergy (and SF-1
stimulation), indicating a requirement for the CRE and its cognate
transcription factors. We also found that PKA did not stimulate the
activity of GAL4-SF-1 (data not shown), providing additional evidence
against a direct effect of phosphorylation on the transcriptional
activity of SF-1. An alternative mechanism for synergy could involve
PKA-mediated alterations in the DNA-binding affinity of SF-1 or CREB.
However, we found that under conditions in which PKA enhanced CREB
phosphorylation on Ser 133, there was no change in the amount of DNA
binding by SF-1 or CREB. Because mutations in either the SF-1 or the
CRE elements are sufficient to eliminate synergy, an attractive
mechanism would be for SF-1 and CREBs to act together to recruit
additional transcription factors or coactivators. Although this
mechanism is not assured by the current data, the inhibin
promoter
appears to provide a particularly robust system for dissecting the
molecular basis of transcriptional synergy.
As an initial step toward understanding the basis of synergy between
the SF-1 and PKA pathways, we localized the SF-1- and cAMP-responsive
regulatory elements. Deletion mutagenesis narrowed the SF-1-responsive
region to the proximal -146 bp of the inhibin
promoter. Although
there are no consensus SF-1 sequences in this region, several closely
adjacent sequences were considered as potential SF-1 sites. A series of
point mutations indicated that the sequence, TCA
GGGCCA, which deviates somewhat from the
consensus sequence (TCA AGGTCA), represents the functional SF-1 site.
In addition, EMSA studies confirmed SF-1 binding to this site, and
there was good correlation between the functional effects of various
mutations and the effects of the mutations on SF-1 binding. We also
confirmed CREB binding to the adjacent CRE (TGCGTCA), even though it
deviates from the consensus octameric CRE sequence (TGACGTCA). It is
probable that other members of the B-Zip transcription factor family
might also bind to this variant CRE site, although CREB appears to be
the major binding protein in the tsa cells used in this study, as well
as in granulosa cells (20 ). The SF-1 and CRE sites are closely spaced
between -137 to -114 bp, but it remained possible that other
sequences in the proximal inhibin promoter might be involved in the
synergistic regulation by the SF-1 and PKA pathways. However, the
synergistic properties of the composite SF-1/CRE element were retained
after transfer to a heterologous promoter, suggesting that this feature
is inherent in the SF-1/CRE element.
It is notable that there is a broad spectrum of interplay between the
SF-1 and PKA pathways among the promoters that we tested. For example,
although the glycoprotein hormone
-subunit promoter contains both
SF-1 and CRE sites, it is controlled predominantly by the cAMP pathway,
and SF-1 exerts little effect alone, or in combination with PKA. The
rat aromatase promoter exhibits strong stimulation by PKA. SF-1 also
activates this promoter, and it enhances PKA stimulation, although less
so than seen with the inhibin
promoter. Why is synergism between
the SF-1 and CRE elements so strong in the case of the inhibin
promoter? One possibility is that the inhibin
sites for both SF-1
and the CRE are imperfect binding sites. In fact, revertent mutations
to optimized consensus sequences did not improve synergy, but instead
reduced the ability of these two pathways to functionally interact. In
addition, alteration in the spacing between the SF-1 and CRE sites
diminished synergy, suggesting that DNA topology, or the physical
relationship of the bound transcription factors, is critical for
functional interactions to occur.
Based on these findings, we hypothesized that SF-1 might interact
directly with CREB to stabilize the binding of one or both
transcription factors. EMSA using the probe containing both the SF-1
binding site and the CRE (-146 to -112) did not show the formation of
higher order SF-1 and CREB complexes (data not shown). However, the
physical interaction between these two factors was apparent in protein
interaction assays. It is not surprising that SF-1-CREB interactions
are not apparent in EMSA assays, as other transcription factors that
interact directly with SF-1 are not detected by gel mobility shifts.
For example, DAX-1 (50 ), Egr-1 (early growth response protein 1)
(47 ), WT-1 (49 ), and SOX9 (48 ) have each been shown to interact with
SF-1, even though there is no evidence of heterodimer formation when
studied by EMSA. It is notable that a specific, but weaker, physical
interaction was detected when a glutathione S-transferase
(GST) pull-down assay was performed using bacterially expressed GST
SF-1 fusion protein and radiolabeled CREB (data not shown), suggesting
that the presence of other factors in the cellular lysates may
stabilize the physical interaction between SF-1 and CREB.
It is possible that the functional importance of the SF-1-CREB
interaction is not so much to stabilize their binding to DNA as to
facilitate the formation of an effective ternary complex with
coactivators. Phosphorylation of CREB allows interaction with
coactivator CBP, which also binds to components of the basal
transcription machinery (62 63 64 ). In addition, CBP has been shown to
interact directly with a variety of nuclear hormone receptors including
the retinoic acid receptor, glucocorticoid receptor, thyroid hormone
receptor, estrogen receptor, and SF-1 (39 65 66 67 ). The ability of CBP
to interact with both SF-1 and CREB raises the possibility that it may
serve as a signal integrator for these two factors. Our results show
that the synergistic activation of the inhibin
gene was enhanced by
the addition of exogenous CBP. However, CBP 1460, which has an
interaction domain for nuclear hormone receptors (codon 1101), but
lacks an interaction domain for CREB (codon 590669), did not increase
synergism.
CBP possesses histone acetyltransferase (HAT) activity and it also
recruits other proteins with HAT activity (52 53 54 55 ). Histone acetylation
is often associated with gene activation, probably because of
alterations in chromatin structure (51 ). Transfected plasmids generate
a typical nucleosome ladder in cells (68 ), allowing analysis of the
relationship between histone acetylation status and transcription
of the transiently transfected inhibin
gene. Consistent with the
transient expression studies, we found that treatment with the
combination of PKA and SF-1 increased the state of histone H4
acetylation associated with the inhibin
promoter. Although both
SF-1 and CREB can interact with and recruit CBP, histone acetylation
was not significantly increased with each factor alone, emphasizing
that the presence of both SF-1 and CREB are necessary for the effective
recruitment of CBP and activation of histone acetylation associated
with the inhibin
promoter. Mutations of either the SF-1 binding
site or CRE also diminished histone H4 acetylation (data not shown).
The extent of synergism observed with respect to histone acetylation
was not as great as that seen using luciferase reporter genes. However,
we find consistently that changes of reporter gene activity are much
greater than changes seen in the CHIP assay, perhaps because the
activated templates allow multiple rounds of transcription and
amplification of the signal by the reporter enzyme. These studies of
histone acetylation support the hypothesis that recruitment of CBP and
other HAT enzymes may be involved in synergistic activation of the
inhibin
promoter.
Based on these studies, we propose a model for the synergistic
activation (Fig. 12
). Because the binding
of endogenous CREB to the imperfect CRE is relatively weak, CREB does
not effectively mediate the signal from the cAMP pathway in SF-1
deficient cells. When SF-1 is present, it binds to the promoter,
allowing interactions between CREB and SF-1. In combination, SF-1 and
phosphorylated CREB recruit CBP and other cofactors, which may further
stabilize their interactions. Then, CBP, in conjunction with other
HATs, may induce histone acetylation and gene transactivation. In
granulosa cells, endogenous SF-1 and CREB may form complexes on the
composite enhancer element. Without SF-1, a robust increase of inhibin
gene expression by cAMP would not be attained. Thus, SF-1 may play
a key role in mediating FSH signaling in granulosa cells. This model
does not exclude the interaction of SF-1 with other transcription
factors, or with other coactivators, aside from CBP. It is tempting to
speculate that similar mechanisms involving direct protein interaction
with SF-1, recruitment of cofactors, and increased histone acetylation
are used in the cAMP regulation of other SF-1 responsive genes.
Moreover, the functional properties of the composite regulatory element
in the inhibin
promoter appear to share certain features in common
with the interaction of SF-1 with Egr-1 in the context of the rat LHß
promoter. In this case, there is strong interdependence between SF-1
and Egr-1 for promoter activity (47 ). Thus, it is possible that a
recurring feature of regulation by SF-1 will be its integrated action
with other transcription factors, some of which may be regulated
dynamically (e.g. Egr-1, WT-1) (49 69 70 71 ), whereas others
may be subject to posttranslational control (e.g. CREB)
(62 ).

View larger version (19K):
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|
Figure 12. Proposed Model for the Synergistic Activation
SF-1 and CREB directly interact with each other on the composite
regulatory element. The formation of complexes may stabilize the weak
binding of CREB to the imperfect CRE. After activation of the cAMP
pathway by FSH, SF-1 and phosphorylated CREB recruit CBP, which may
further stabilize their interactions. CBP may induce histone
acetylation and gene transactivation. GTF, General transcription
factors; Pol II, RNA polymerase II.
|
|
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
The pA3 luciferase reporter construct for the human gonadotropin
common
- subunit gene (-420 to +44) was previously described (72 ).
The rat aromatase promoter region (-294 to +20) was amplified by PCR
and cloned into the pA3 vector. The pA3 reporter constructs for the rat
inhibin common
-subunit gene (-2021, -769, and -547 to +68) were
previously described (20 ). The rat inhibin
promoter regions (-311,
-236, -160, -146, -134, -115, and -89 to +68) were amplified by
PCR and subcloned into the pGL3 basic luciferase reporter vector
(Promega Corp., Madison, WI). The inhibin
-146 mutant
reporters (m1, m2, m3, m4, m5, m6, m7, m8, m9, and m10) (see
Results for locations of mutations) were constructed by
overlapping PCR. The inhibin
enhancer elements (-146 to -112,
-146 to -80, and -146 to -40) were fused to the thymidine kinase
minimal promoter (TK81) and luciferase gene. Murine SF-1 cDNA (50 ) was
cloned into the pCMX mammalian expression vector (73 ). In most of the
experiments, the pCMX SF-1 expression vector was used along with the
pCMX vector without insert (empty vector). The wild -type and mutant
expression vectors for the catalytic subunit of cAMP-dependent protein
kinase A (PKA) were provided by R. A. Maurer. The CREB cDNA was
provided by Dr. J. Leiden. CBP, CBP 1460 (provided by R. H.
Goodman), and P/CAF (provided by Y. Nakatani) cDNAs were cloned into
the pCMX expression vector. The influenza hemagglutinin (HA) epitope
tag was introduced immediately after the last codon of SF-1 by PCR
(74 ), and the HA-tagged SF-1 cDNA was subcloned into the pCMX vector.
After PCR and subcloning, DNA sequence was confirmed using a dRhodamine
terminator cycle sequencing kit (Perkin Elmer Corp.,
Norwalk, CT) and an ABI377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA).
Cell Culture, Transfection, and Luciferase Assay
Human embryonic kidney tsa 201 cells (75 ) were grown in DMEM
supplemented with 10% FBS. Cells were transfected by the calcium
phosphate method (76 ). The immortalized granulosa cell line,
GRMO2, was provided by Innogenetics N.V. (Ghent, Belgium).
GRMO2 cells were maintained in Hams F-12/DMEM containing 2%
FBS, 10 µg/ml transferrin, 5 µg/ml insulin, 30 nM
sodium selenite, and 3 µg/ml BSA (77 ). Transfection was performed
using a modified lipofectin-mediated method (78 ). Cells were treated
with 8-bromo-cAMP for 12 h before harvest. Forty eight hours after
transfection, cell extracts were prepared and luciferase assays were
performed (79 ).
Preparation of Nuclear Extracts and EMSA
Nuclear extracts were prepared from transfected cells (80 ). In
some experiments, nuclear extracts were prepared in the presence of
phosphatase inhibitors (25 mM sodium fluoride, 2
mM sodium orthovanadate). EMSA was performed as described
previously (50 ). Briefly, nuclear extracts (10 µg) were incubated
with 20 fmol of 32P-labeled oligonucleotides, and
the DNA protein complexes were resolved on 4% native polyacrylamide
gels in 0.5x Tris-buffered EDTA (TBE ) buffer. Antibodies used
in the supershift assay were obtained from Dr. Morohashi (anti-Ad4BP)
and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)
(anti-CREB).
Western Blot Analysis
Nuclear extracts were separated by 10% SDS-PAGE and were
electrotransferred to nitrocellulose membranes. For detection of
phosphorylated CREB, membranes were incubated with antiphosphorylated
CREB antibody (Upstate Biotechnology, Inc., Lake Placid,
NY) followed by incubation with horseradish peroxidase- conjugated
secondary antibody. For detection of HA-tagged SF-1, membranes were
probed with anti-HA antibody conjugated to horseradish peroxidase
(Roche Molecular Biochemicals, Indianapolis, IN). Protein
detection was performed using an enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech Inc, Piscataway,
NJ).
Protein Interaction Assay
In vitro translation of CREB was performed with the
TNT reticulocyte lysate system (Promega Corp.) in the
presence of 35S-methionine. Nuclear extracts were
prepared from cells transfected with an empty vector or HA-tagged SF-1
expression vector as described above and were incubated with the
labeled proteins in the presence of 2 mM
dithiobis succinimidyl propionate for 15 min at room
temperature. The reactions were then immunoprecipitated with either rat
nonimmune IgG or rat anti-HA high affinity antibody (Roche Molecular Biochemicals) for 2 h at 4 C. Immunoprecipitates
were recovered by incubation with protein G agarose. After extensive
washing, bound proteins were eluted from the agarose beads and
separated by 10% SDS-PAGE followed by autoradiography.
CHIP Assay
The CHIP assay was performed as described previously with minor
modifications (81 ). Forty eight hours after transfection, tsa 201 cells
were harvested and incubated in 1% formaldehyde for 15 min at room
temperature. After cross-linking, cells were sonicated in lysis buffer
(1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.0, 1
mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1
µg/ml leupeptin, 1 µg/ml pepstatin A). One tenth of the total
lysate was used for purification of total DNA. The rest of the lysate
was incubated with either nonimmune IgG or antiacetylated histone H4
antibody (Upstate Biotechnology, Inc.) at 4 C for 18
h. Immunoprecipitates were recovered using protein A agarose pretreated
with BSA and sonicated salmon sperm DNA. DNA was extracted from
immunoprecipitates by phenol/chloroform extraction and ethanol
precipitation. PCR was performed using either total DNA or
immunoprecipitated DNA in the presence of
-P32-dCTP with a temperature cycle of 1 min at
94 C, 1 min at 55 C, and 1 min at 72 C. After 30 cycles, PCR products
were separated on 6% nondenaturing polyacrylamide gels followed by
autoradiography. Primers used for PCR correspond to the sequence within
the inhibin
promoter region (-160 to -141)
(5'-TTGGCGGGAGTGGGAGATAA-3') and luciferase coding sequence
(5'-GAAATACAAAAACCGCAGAAGGTA-3').
 |
ACKNOWLEDGMENTS
|
---|
We thank R. H. Goodman for the CBP and CBP 1460 cDNAs, Y.
Nakatani for the P/CAF cDNA, R. A. Maurer for wild-type and mutant
PKA catalytic subunit cDNAs, J. Leiden for CREB cDNA, K. Morohashi for
anti-Ad4BP antibody, R. N. Yu and J. Weiss for helpful
discussions, and T. Kotlar and L. Sabacan for assistance with
sequencing.
 |
FOOTNOTES
|
---|
Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15709, 303 East Chicago Avenue, Chicago Illinois 60611.
This work was performed as part of the National Cooperative Program for
Infertility Research (NIH Grant U54-HD-29164) and PO1 HD-21921.
1 These authors contributed equally to this work. 
Received for publication August 3, 1999.
Revision received September 23, 1999.
Accepted for publication October 1, 1999.
 |
REFERENCES
|
---|
-
Richards JS 1980 Maturation of ovarian follicles: actions
and interactions of pituitary and ovarian hormones on follicular cell
differentiation. Physiol Rev 60:5189[Free Full Text]
-
McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N,
Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin
receptor: an unusual member of the G protein-coupled receptor family.
Science 245:494499[Medline]
-
Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and
functional expression of cloned cDNA. Mol Endocrinol 4:525530[Abstract]
-
Ling N, Ying SY, Ueno N, Esch F, Denoroy L, Guillemin R 1985 Isolation and partial characterization of a Mr 32,000 protein with
inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 82:72177221[Abstract]
-
Robertson DM, Foulds LM, Leversha L, Morgan FJ, Hearn MT,
Burger HG, Wettenhall RE, de Kretser DM 1985 Isolation of inhibin from
bovine follicular fluid. Biochem Biophys Res Commun 126:220226[Medline]
-
Miyamoto K, Hasegawa Y, Fukuda M, Nomura M, Igarashi M,
Kangawa K, Matsuo H 1985 Isolation of porcine follicular fluid inhibin
of 32K daltons. Biochem Biophys Res Commun 129:396403[Medline]
-
Rivier J, Spiess J, McClintock R, Vaughan J, Vale W 1985 Purification and partial characterization of inhibin from porcine
follicular fluid. Biochem Biophys Res Commun 133:120127[Medline]
-
Findlay JK 1993 An update on the roles of inhibin, activin,
and follistatin as local regulators of folliculogenesis. Biol Reprod 48:1523[Abstract]
-
Mason AJ, Hayflick JS, Ling N, Esch F, Ueno N, Ying SY,
Guillemin R, Niall H, Seeburg PH 1985 Complementary DNA sequences of
ovarian follicular fluid inhibin show precursor structure and homology
with transforming growth factor-ß. Nature 318:659663[Medline]
-
Forage RG, Ring JM, Brown RW, McInerney BV, Cobon GS, Gregson
RP, Robertson DM, Morgan FJ, Hearn MT, Findlay JK 1986 Cloning and
sequence analysis of cDNA species coding for the two subunits of
inhibin from bovine follicular fluid. Proc Natl Acad Sci USA 83:30913095[Abstract]
-
Mayo KE, Cerelli GM, Spiess J, Rivier J, Rosenfeld MG, Evans
RM, Vale W 1986 Inhibin A-subunit cDNAs from porcine ovary and human
placenta. Proc Natl Acad Sci USA 83:58495853[Abstract]
-
Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying SY,
Ueno N, Ling N 1987 Complementary deoxyribonucleic acid (cDNA) cloning
and DNA sequence analysis of rat ovarian inhibins. Mol Endocrinol 1:388396[Abstract]
-
Woodruff TK, Meunier H, Jones PB, Hsueh AJ, Mayo KE 1987 Rat
inhibin: molecular cloning of
- and ß-subunit complementary
deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1:561568[Abstract]
-
Woodruff TK, DAgostino J, Schwartz NB, EMK 1988 Dynamic
changes in inhibin messenger RNAs in rat ovarian follicles during the
reproductive cycle. Science 239:12961299[Medline]
-
Meunier H, Cajander SB, Roberts VJ, Rivier C, Sawchenko PE,
Hsueh AJ, Vale W 1988 Rapid changes in the expression of inhibin
-,
ßA-, and ßB-subunits in ovarian cell types during the rat estrous
cycle. Mol Endocrinol 2:13521363[Abstract]
-
Zhang ZW, Carson RS, Herington AC, Lee VW, Burger HG 1987 Follicle-stimulating hormone and somatomedin-C stimulate inhibin
production by rat granulosa cells in vitro. Endocrinology 120:16331638[Abstract]
-
Turner IM, Saunders PT, Shimasaki S, Hillier SG 1989 Regulation of inhibin subunit gene expression by FSH and estradiol in
cultured rat granulosa cells. Endocrinology 125:27902792[Abstract]
-
Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W,
Hsueh AJ 1986 Hormonal regulation of granulosa cell inhibin
biosynthesis. Endocrinology 119:27112719[Abstract]
-
Suzuki T, Miyamoto K, Hasegawa Y, Abe Y, Ui M, Ibuki Y,
Igarashi M 1987 Regulation of inhibin production by rat granulosa
cells. Mol Cell Endocrinol 54:185195[CrossRef][Medline]
-
Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE 1991 Regulation of the
inhibin gene by cyclic adenosine
3',5'-monophosphate after transfection into rat granulosa cells. Mol
Endocrinol 5:521534[Abstract]
-
Hoeffler JP, Meyer TE, Yun Y, LJJ, Habener JF 1988 Cyclic
AMP-responsive DNA-binding protein: structure based on a cloned
placental cDNA. Science 242:14301433[Medline]
-
Stehle JH, Foulkes NS, Molina CA, Simonneaux V, Pevet P,
Sassone-Corsi P 1993 Adrenergic signals direct rhythmic expression of
transcriptional repressor CREM in the pineal gland. Nature 365:314320[CrossRef][Medline]
-
Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and negative autoregulation of CREM: an alternative
promoter directs the expression of ICER, an early response repressor.
Cell 75:875886[Medline]
-
Mukherjee A, Urban J, Sassone-Corsi P, Mayo KE 1998 Gonadotropins regulate inducible cyclic adenosine 3',5'-monophosphate
early repressor in the rat ovary: implications for inhibin alpha
subunit gene expression. Mol Endocrinol 12:785800[Abstract/Free Full Text]
-
Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor I, a key
regulator of steroidogenic enzyme expression, is the mouse homolog of
fushi tarazu-factor I. Mol Endocrinol 6:12491258[Abstract]
-
Honda S, Morohashi K, Nomura M, Takeya H, Kitajima M, Omura T 1993 Ad4BP regulating steroidogenic P-450 gene is a member of steroid
hormone receptor superfamily. J Biol Chem 268:74947502[Abstract/Free Full Text]
-
Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key
regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852860[Abstract]
-
Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear
receptor is essential for adrenal and gonadal development and sexual
differentiation. Cell 77:481490[Medline]
-
Fitzpatrick SL, Richards JS 1993 cis-acting elements of the
rat aromatase promoter required for cyclic adenosine
3',5'-monophosphate induction in ovarian granulosa cells and
constitutive expression in R2C Leydig cells. Mol Endocrinol 7:341354[Abstract]
-
Lynch JP, Lala DS, Peluso JJ, Luo W, Parker KL, White BA 1993 Steroidogenic factor 1, an orphan nuclear receptor, regulates the
expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 7:776786[Abstract]
-
Clemens JW, Lala DS, Parker KL, Richards JS 1994 Steroidogenic
factor-1 binding and transcriptional activity of the cholesterol
side-chain cleavage promoter in rat granulosa cells. Endocrinology 134:14991508[Abstract]
-
Sugawara T, Holt JA, Kiriakidou M, Strauss Jr JF 1996 Steroidogenic factor 1-dependent promoter activity of the human
steroidogenic acute regulatory protein (StAR) gene. Biochemistry 35:90529059[CrossRef][Medline]
-
Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of
luteinizing hormone beta gene promoter activity by the orphan nuclear
receptor, steroidogenic factor-1. J Biol Chem 271:66456650[Abstract/Free Full Text]
-
Yu RN, Ito M, Jameson JL 1998 The murine Dax-1 promoter is
stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF
(chicken ovalbumin upstream promoter-transcription factor) via a
composite nuclear receptor-regulatory element. Mol Endocrinol 12:10101022[Abstract/Free Full Text]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg B, Kastner P, Mark M, Chambon PEvans
RM 1995 The nuclear receptor superfamily: the second decade.
Cell 83:835839[Medline]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Ito M, Yu RN, Jameson JL 1998 Steroidogenic factor-1 contains
a carboxy-terminal transcriptional activation domain that interacts
with steroid receptor coactivator-1. Mol Endocrinol 290301
-
Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman
RH 1993 Phosphorylated CREB binds specifically to the nuclear protein
CBP. Nature 365:855859[CrossRef][Medline]
-
Monte D, DeWitte F, Hum DW 1998 Regulation of the human
P450scc gene by steroidogenic factor 1 is mediated by CBP/p300. J
Biol Chem 273:45854591[Abstract/Free Full Text]
-
Liu Z, Simpson ER 1997 Steroidogenic factor 1 (SF-1) and SP1
are required for regulation of bovine CYP11A gene expression in bovine
luteal cells and adrenal Y1 cells. Mol Endocrinol 11:127137[Abstract/Free Full Text]
-
Carlone DL, Richards JS 1997 Functional interactions,
phosphorylation, and levels of 3',5'-cyclic adenosine
monophosphate-regulatory element binding protein and steroidogenic
factor-1 mediate hormone-regulated and constitutive expression of
aromatase in gonadal cells. Mol Endocrinol 11:292304[Abstract/Free Full Text]
-
Fitzpatrick SL, Carlone DL, Robker RL, Richards JS 1997 Expression of aromatase in the ovary: down-regulation of mRNA by the
ovulatory luteinizing hormone surge. Steroids 62:197206[CrossRef][Medline]
-
Sugawara T, Kiriakidou M, McAllister JM, Kallen CB, Strauss J
Fr 1997 Multiple steroidogenic factor 1 binding elements in the human
steroidogenic acute regulatory protein gene 5'-flanking region are
required for maximal promoter activity and cyclic AMP responsiveness.
Biochemistry 36:72497255[CrossRef][Medline]
-
Zhang P, Mellon SH 1996 The orphan nuclear receptor
steroidogenic factor-1 regulates the cyclic adenosine
3',5'-monophosphate-mediated transcriptional activation of rat
cytochrome P450c17 (17
-hydroxylase/c1720 lyase). Mol Endocrinol 10:147158[Abstract]
-
Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson K, Weigel
NL, Ingraham HA 1999 Phosphorylation of the nuclear receptor SF-1
modulates cofactor recruitment: integration of hormone signaling in
reproduction and stress. Mol Cell 3:521526[Medline]
-
Drean YL, Liu D, Wong AOL, Xiong F, Hew CL 1996 SF-1 and
estradiol receptor act in synergism to regulate the expression of the
salmon gonadotropin II ß subunit gene. Mol Endocrinol 10:217229[Abstract]
-
Halvorson LM, Ito M, Jameson JL, Chin WW 1998 Steroidogenic
factor-1 and early growth response protein 1 act through two composite
DNA binding sites to regulate luteinizing hormone ß-subunit gene
expression. J Biol Chem 273:1471214720[Abstract/Free Full Text]
-
De Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M,
Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P 1998 Direct
interaction of SRY-related protein SOX9 and steroidogenic factor 1
regulates transcription of the human anti-Mullerian hormone gene. Mol
Cell Biol 18:66536665[Abstract/Free Full Text]
-
Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN,
Hammer GD, Ingraham HA 1998 Wilms tumor 1 and Dax-1 modulate the
orphan nuclear receptor SF-1 in sex specific gene expression. Cell
445454
-
Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated
transactivation via a carboxy-terminal domain that is deleted in
adrenal hypoplasia congenita. Mol Cell Biol 17:14761483[Abstract]
-
Struhl K 1998 Histone acetylation and transcriptional
regulatory mechanisms. Genes Dev 12:599606[Free Full Text]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953959[Medline]
-
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a
histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
-
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A
p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Blanco JC, Minucci S, Lu J, Yang XJ, Walker KK, Chen H, Evans
RM, Nakatani Y, Ozato K 1998 The histone acetylase PCAF is a nuclear
receptor coactivator. Genes Dev 12:16381651[Abstract/Free Full Text]
-
Barnhart KM, Mellon PL 1994 The orphan nuclear receptor,
steroidogenic factor-1, regulates the glycoprotein hormone
-subunit
gene in pituitary gonadotropes. Mol Endocrinol 8:878885[Abstract]
-
Jameson JL, Jaffe RC, Deutsch PJ, Albanese C, Habener JF 1988 The gonadotropin
-gene contains multiple protein binding domains
that interact to modulate basal and cAMP-responsive transcription.
J Biol Chem 263:98799886[Abstract/Free Full Text]
-
Fitzpatrick SL, Richards JS 1994 Identification of a cyclic
adenosine 3',5'-monophosphate-response element in the rat aromatase
promoter that is required for transcriptional activation in rat
granulosa cells and R2C Leydig cells. Mol Endocrinol 8:13091319[Abstract]
-
Ardekani AM, Romanelli JC, Mayo KE 1998 Structure of the rat
inhibin and activin ßA-subunit gene and regulation in an ovarian
granulosa cell line. Endocrinology 139:32713279[Abstract/Free Full Text]
-
Takayama K, Sasano H, Fukaya T, Morohashi K, Suzuki T, Tamura
M, Costa MJ, Yajima A 1995 Immunohistochemical localization of
Ad4-binding protein with correlation to steroidogenic enzyme expression
in cycling human ovaries and sex cord stromal tumors. J Clin
Endocrinol Metab 80:28152821[Abstract]
-
Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key
determinant of endocrine development and function. Endocr Rev 18:361377[Abstract/Free Full Text]
-
Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP,
Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP
is a coactivator for the transcription factor CREB. Nature 370:223226[CrossRef][Medline]
-
Bisotto S, Minorgan S, Rehfuss RP 1996 Identification and
characterization of a novel transcriptional activation domain in the
CREB-binding protein. J Biol Chem 271:1774617750[Abstract/Free Full Text]
-
Kee BL, Arias J, Montminy MR 1996 Adaptor-mediated recruitment
of RNA polymerase II to a signal-dependent activator. J Biol Chem 271:23732375[Abstract/Free Full Text]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
S-C, Heyman RA, Rose DW, Glass CKRosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1
inhibition by nuclear receptors. Cell 85:403414[Medline]
-
Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG,
Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear
receptor signalling. Nature 383:99103[CrossRef][Medline]
-
Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B,
Kurokawa R, Brown M 1996 p300 is a component of an estrogen receptor
coactivator complex. Proc Natl Acad Sci USA 93:1154011545[Abstract/Free Full Text]
-
Jeong S, Stein A 1994 Micrococcal nuclease digestion of nuclei
reveals extended nucleosome ladders having anomalous DNA lengths for
chromatin assembled on non-replicating plasmids in transfected cells.
Nucleic Acids Res 22:370375[Abstract]
-
Wolfe MW, Call GB 1999 Early growth response protein 1 binds
to the luteinizing hormone-beta promoter and mediates
gonadotropin-releasing hormone-stimulated gene expression. Mol
Endocrinol 13:752763[Abstract/Free Full Text]
-
Dorn C, Ou Q, Svaren J, Crawford PA, Sadovsky Y 1999 Activation of luteinizing hormone beta gene by gonadotropin-releasing
hormone requires the synergy of early growth response-1 and
steroidogenic factor-1. J Biol Chem 274:1387013876[Abstract/Free Full Text]
-
Tremblay JJ, Drouin J 1999 Egr-1 is a downstream effector of
GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance
luteinizing hormone ß gene transcription. Mol Cell Biol 19:25672576[Abstract/Free Full Text]
-
Chatterjee VKK, Lee JK, Rentoumis A, Jameson JL 1989 Negative
regulation of the thyroid-stimulating hormone
gene by thyroid
hormone: receptor interaction adjacent to the TATA box. Proc Natl Acad
Sci USA 86:91149118[Abstract]
-
Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct
repeats as selective response elements for the thyroid hormone,
retinoic acid, and vitamin D3 receptors. Cell 65:12551266[Medline]
-
Ito M, Yu RN, Jameson JL, Ito M 1999 Mutant vasopressin
precursors that cause autosomal dominant neurohypophyseal diabetes
insipidus retain dimerization and impair the secretion of wild type
proteins. J Biol Chem 274:90299037[Abstract/Free Full Text]
-
Margolskee RF, McHendry-Rinde B, Horn R 1993 Panning
transfected cells for electrophysiological studies. Biotechniques 15:906911[Medline]
-
Graham FL, van der Eb AJ 1973 Transformation of rat cells by
DNA of human adenovirus 5. Virology 52:456487[Medline]
-
Vanderstichele H, Delaey B, de Winter J, de Jong F, Rombauts
L, Verhoeven G, Dello C, van de Voorde A, Briers T 1994 Secretion of
steroids, growth factors, and cytokines by immortalized mouse granulosa
cell lines. Biol Reprod 50:11901202[Abstract]
-
Campbell MJ 1995 Lipofection reagents prepared by a simple
ethanol injection technique. Biotechniques 18:10271032[Medline]
-
de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol 7:725737[Medline]
-
Shapiro DJ, Sharp PA, Wahli WW, Keller MJ 1988 A
high-efficiency HeLa cell nuclear transcription extract. DNA 7:4755[Medline]
-
Luo RX, Postigo AA, Dean DC 1998 Rb interacts with histone
deacetylase to repress transcription. Cell 92:463473[Medline]