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-Promoter by Steroidogenic Factor-1 and Cyclic Adenosine 3',5'-Monophosphate
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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Subunit Gene by SF-1 and
the cAMP Pathway 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.
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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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). 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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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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). 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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). 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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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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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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). 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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). 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 1–460), or P/CAF,
had no effect on the synergistic activation (Fig. 10B).
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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 5–8) before
immunoprecipitation, and immunoprecipitates with nonimmune IgG did not
amplify PCR products (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 gonadotropes—a 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 1–460, which has an
interaction domain for nuclear hormone receptors (codon 1–101), but
lacks an interaction domain for CREB (codon 590–669), 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 ).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- 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 1–460 (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 Ham’s 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 |
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FOOTNOTES |
|---|
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.
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H. Pincas, J.-N. Laverriere, and R. Counis Pituitary Adenylate Cyclase-activating Polypeptide and Cyclic Adenosine 3',5'-Monophosphate Stimulate the Promoter Activity of the Rat Gonadotropin-releasing Hormone Receptor Gene via a Bipartite Response Element in Gonadotrope-derived Cells J. Biol. Chem., June 22, 2001; 276(26): 23562 - 23571. [Abstract] [Full Text] [PDF]
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