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Division of Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, Illinois 60611
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In mammals, SF-1 plays a key role in the development and differentiated function of the adrenal gland and gonads. Disruption of the FTZ-F1 locus in mice precludes the development of the adrenal gland and gonads (8, 9, 10). Genetic males appear sex reversed because of the absence of male external genitalia and the preservation of Müllerian structures (8). These mice also have abnormal gonadotropin production, apparently reflecting a role for SF-1 in the development of the ventromedial hypothalamus (11) and function of the pituitary gonadotropes (11, 12). This array of physiological effects of SF-1 parallels its expression in the adrenal cortex, testis, ovary, ventromedial nucleus of the hypothalamus, and gonadotrope cells in the pituitary (8, 11, 12, 13, 14).
In addition to its role during development, SF-1 functions as a
transcription factor for a variety of different target genes that
characterize the differentiated cells in which it is expressed (for
review, see Ref.15). These include steroidogenic enzyme genes in the
adrenal gland and gonads (2, 3, 16, 17, 18, 19, 20), the Müllerian inhibiting
substance (21), and DAX-1 (dosage-sensitive sex reversal-adrenal
hypoplasia congenita critical region on X chromosome, gene 1) promoters
(22), and the gonadotropin 
- and ß-subunit promoters in
gonadotrope cells (14, 23, 24).
Several naturally occurring variants of SF-1 are produced by the FTZ-F1 locus (25, 26, 27). A second FTZ-F1 homolog, termed embryonal long terminal repeat-binding protein (ELP), was isolated from murine embryonal carcinoma cells (27). Recently, additional isoforms of ELP-1 (the original ELP isolate), ELP-2, and ELP-3, have been cloned from the same cell line (28). It is now recognized that each of the ELP isoforms, along with SF-1, are transcribed from a single FTZ-F1 gene as a result of alternative promoter usage and differential splicing (28). The transcripts of ELP-3 and SF-1 differ in their 5'-untranslated regions, but they encode an identical SF-1 protein. In contrast, ELP-1 and ELP-2 contain an additional 77 amino-terminal amino acids relative to SF-1. ELP-2 and SF-1 are otherwise identical, whereas the carboxy terminus of ELP-1 is 74 amino acids shorter reflecting alternate splicing. In Xenopus, variants that resemble SF-1 (xFF1rA) and ELP-1 (xFF1rAshort) have been shown to differ in their functional properties; the carboxy-terminally truncated xFF1rAshort is less active and inhibits the function of xFF1rA in transient expression assays (26). These studies suggest the possibility that the carboxy terminus of SF-1 might possess a transactivation domain. Consistent with this idea, deletion of 128 amino acids from the carboxy terminus of SF-1 was recently shown to eliminate SF-1-mediated transactivation (29).
Recently, SF-1 has been shown to interact functionally with other transcription factors. FTZ-F1 has been shown to interact with the homeodomain protein FTZ, and the two proteins function together as mutually dependent cofactors in the activation of the Drosophila-engrailed gene (30, 31). SF-1 synergizes with the estrogen receptor to stimulate expression of the salmon gonadotropin II ß-subunit gene (32). A synergistic interaction has also been reported between the cAMP responsive element and the SF-1 binding sites of the aromatase (Cyp19) gene (33). SF-1 and Sp1 also function cooperatively in the transactivation of cholesterol side-chain cleavage (Cyp11A1) promoter (34). DAX-1, an orphan nuclear receptor that is coexpressed with SF-1 (35, 36), has been shown to inhibit SF-1-mediated transactivation (37).
Until recently, there was no known ligand for SF-1. However, several different oxysterols (e.g. 25-, 26-, or 27-hydroxycholesterol) have been shown to activate SF-1-dependent transcription (29). Inspection of carboxy terminus of SF-1 suggests the presence of a potential transactivation (AF-2) domain that is homologous to that found in certain other nuclear hormone receptors (4). In other receptors, this region has been shown to interact with transcriptional coactivators such as steroid receptor coactivator-1 (SRC-1) (for review, see Ref.38). The interaction with SRC-1 occurs in a ligand-dependent manner and has not been documented for orphan nuclear receptors such as SF-1. In this study, we characterized a carboxy-terminal transactivation domain in SF-1 and examined the biochemical and functional interactions between SF-1 and SRC-1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The
amino-terminal region of ELP-1 and ELP-2 is longer than SF-1 by 77
amino acid residues. The carboxy-terminal region is shared by ELP-2 and
SF-1 and consists of 131 residues, whereas the ELP-1-specific carboxy
terminus is 57 residues in length. An artificial mutant of the mouse
FTZ-F1 proteins, ELP-1 del 1–77, was constructed to delete the
ELP-1-specific amino terminus (Fig. 1A).
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Because of the differences in DNA binding among the FTZ-F1 variants, it
is necessary to separate the binding characteristics from
transcriptional activities to define the transactivation domains. The
zinc finger region and FTZ-F1 box of these proteins were replaced with
the DBD of yeast GAL4 (40), yielding ELP-1-GAL4, ELP-2-GAL4, SF-1-GAL4,
and ELP-1-GAL4 del 1–77 (Fig. 1B
). The expressed proteins extracted
from transfected tsa 201 cells (which express proteins at high levels)
and in vitro translated proteins were used to assess binding
to DNA (Fig. 1D). Each of the GAL4 fusion proteins formed DNA-protein
complexes with the radiolabeled GAL4-binding site (also known as UAS-;
see Materials and Methods). Similar amounts of ELP-1-GAL4,
ELP-2-GAL4, SF-1-GAL4 complexes were observed. Although the ELP-1-GAL4
del 1–77 complex was less abundant than other SF-1-related proteins
using extracts from transfected cells, its binding was similar to that
of other constructs using in vitro translated proteins.
These results raise the possibility that this deletion mutant may be
less stable in transfected cells, but otherwise, the GAL4 fusion
proteins appear to be expressed similarly and retain DNA-binding
activity.
JEG-3 cells, which lack endogenous SF-1 (37), were used to examine the
functional properties of these GAL4 fusion proteins. Using UAS TK109
luc as a reporter gene, ELP-2-GAL4 (14-fold) and SF-1-GAL4 (30-fold)
fusion proteins caused transcriptional activation relative to the GAL4
DBD alone (Fig. 1C). In contrast, ELP-1-GAL4 and ELP-1-GAL4 del 1–77
were inactive. Because ELP-2 and SF-1 share a common carboxy terminus
that is distinct from ELP-1 (or ELP-1 del 1–77), these results suggest
that a transactivation domain resides within the unique
carboxy-terminal 131 amino acids of SF-1/ELP-2.
AF-2 Domain at the Carboxy Terminus Is Essential for
Transactivation
Nuclear hormone receptors such as the thyroid hormone receptor
(TR), estrogen receptor (ER), and retinoic acid receptor (RAR) are
known to contain a transactivation domain (referred to as AF-2) within
the carboxy-terminal region of the receptors (4, 41, 42). The carboxy
terminus of SF-1 also contains hydrophobic regions and an invariant
glutamate residue that is commonly found in the AF-2 domain (Fig. 2A). Two deletion mutants were
constructed to characterize a potential role of the AF-2 domain of
SF-1. SF-1-GAL4 del 458–462 and SF-1-GAL4 del 443–462 lack the last
five residues and 20 amino acid residues, respectively. The activities
of these constructs were examined in JEG-3 cells using UAS TK109 luc as
a reporter gene. Deletion of the last five amino acids (SF-1-GAL4 del
458–462) reduced transactivation partially (20-fold stimulation)
compared with full-length SF-1-GAL4 (30-fold stimulation) (Fig. 2B).
Using extracts from transfected Tsa 201 cells or in vitro
translation products (Fig. 2C), each of the carboxy-terminal deletion
mutants was shown to retain binding to the GAL4 DNA-recognition
element. In contrast, deletion of 20 amino acids (SF-1-GAL4 del
443–462) eliminated transactivation, even though the amount of the
DNA-protein complex was similar to the five-amino acid deletion.
These findings indicate that the AF-2 domain of SF-1 is localized
between amino acids 443 and 457.
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). The transcriptional
activities of the SF-1-GAL4 and GAL4-SF-1 constructs were similar,
indicating that the first 19 amino acid residues of SF-1 are not
critical for transactivation (data not shown).
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). Cotransfection with SRC-1 increased
transactivation to 32-fold, whereas the SRC-1 d.n. construct decreased
activity to 6-fold. The dominant negative activity of the SRC-1 d.n.
construct was evaluated further by transfecting increasing amounts of
the SRC-1 d.n. expression vector in the presence of a constant amount
of the SRC-1 expression vector. In the absence of SRC-1, 13-fold
transactivation was observed with GAL4-SF-1 (Fig. 3C). Cotransfection
with full-length SRC-1 enhanced the GAL4-SF-1-induced transactivation
to 27-fold, and this activity was inhibited in a dose-dependent manner
by increasing amounts of SRC-1 d.n. Cotransfection with 50 ng SRC-1
d.n. vector essentially eliminated the SRC-1-potentiated
transactivation (15-fold).
SRC-1 Interactions with SF-1 Are Mediated through the AF-2
Domain
The functional interactions between SRC-1 and SF-1 were examined
further by using a version of the mammalian two-hybrid system, which
allows the detection of protein interactions even after the
introduction of inactivating mutations. The interaction domain of SRC-1
was shown to be sufficient for binding to SF-1 in protein pull-down
assays (data not shown) and to function as a dominant negative mutant
(Fig. 3, B and C), and it was fused to the VP16 transactivation domain,
yielding VP16-SRC-1 (Fig. 4A). Using UAS
TK109 luc as a reporter gene, either the VP16 or VP16-SRC-1 expression
vectors were transfected with GAL4, SF-1-GAL4, or the SF-1-GAL4 del
443–462 construct (Fig. 4B). In the presence of VP16 alone, SF-1-GAL4
transcriptional activation was relatively low (14-fold). Cotransfection
with VP16-SRC-1 enhanced the SF-1-GAL4-mediated transactivation from
14-fold to 43-fold, indicating recruitment of the VP16 domain by an
interaction between SF-1 and the SRC-1 interaction domain. In contrast,
VP16-SRC-1 had no effect on the transcriptional activity of the
SF-1-GAL4 del 443–462 protein, which lacks the AF-2 domain.
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). However, the greatest
amount (500 ng) of VP16-SRC-1 was inhibitory, perhaps reflecting
squelching of other transcription factors (Fig. 4C). Cotransfection
with increasing amounts of the control VP16 cDNA (0, 100, 200, 500 ng)
did not alter GAL4-SF-1-induced transactivation (10-fold). The
potentiation of GAL4-SF-1-induced transactivation by VP16-SRC-1 (3- to
5-fold) was somewhat greater than that obtained with native full-length
SRC-1 (2-fold) (Fig. 3B).
Functional Interactions of SF-1 and SRC-1 in SF-1-Containing Cell
Lines
The previous experiments were performed in SF-1-deficient JEG-3
cells because they support relatively large transcriptional responses
to exogenous SF-1. Additional experiments were performed in cell lines
that contain endogenous SF-1 to confirm that the functional
interactions with SRC-1 are not unique to JEG-3 cells. RT-PCR analyses
were used to assess expression of SF-1 and to document SRC-1 expression
in various cell lines (Fig. 5A). As
expected, SF-1 expression was seen in human testis, human H295R adrenal
cells, murine T3 gonadotrope cells, and murine Y1 adrenal cells.
SF-1 expression was absent in human JEG-3 choriocarcinoma cells, human
Tsa 201 kidney fibroblast cells, murine neuro 2A neuronal cells, and
monkey kidney CV-1 cells. SRC-1 and a splicing variant, SRC-1E (43),
were expressed in each of the cell types. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) mRNA was amplified as a positive control, and
reverse transcriptase was omitted from a set of reactions as a negative
control.
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). GAL4-SF-1
conferred 2.4-fold activation relative to the GAL4-DBD alone. The
addition of VP16-SF-1 increased activation to 6.4-fold. Similar results
were seen in the SF-1-containing T3 gonadotrope cells (data not
shown). Thus, although the magnitude of GAL-4-SF-1 responses are lower
in Y1 and T3 cells than in JEG-3 cells, VP16-SRC-1 causes a similar
degree of enhancement (
3-fold).
CBP and p300 Potentiate SRC-1 Stimulation of SF-1-Mediated
Transactivation
SRC-1 and CBP have been shown to synergistically stimulate
transcription by the estrogen and progesterone receptors (44). As a
control, GAL4-ER was used to test the interactions of SRC-1 and CBP
under the current experimental conditions. In JEG-3 cells, there is
basal estrogen production, and GAL4-ER exhibited 12-fold
transactivation of UAS TK109 luc relative to GAL4 in the absence of
added estradiol (E2) (Fig. 6B). CBP and p300 had little or no effect
on GAL4-ER-induced transactivation, but SRC-1 increased the
transcriptional activity 25-fold. Cotransfection with CBP and p300
cDNAs doubled the SRC-1-induced activity of GAL4-ER-induced
transactivation (54- and 50-fold, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Multiple transcripts and protein isoforms are derived from the FTZ-F1
locus, and the presence of these gene products is conserved across
species (15, 28). Comparison of the transcriptional properties of
murine ELP-1, ELP-2, and SF-1 revealed that only ELP-2 and SF-1 were
functional, at least as assessed in transient expression assays in
JEG-3 cells (Fig. 1). These experiments were useful for localizing the
transactivation domain but also have implications regarding the
functional roles of these isoforms. In NIH3T3 cells, it has been
reported that ELP-1 functions as a repressor using a reporter construct
containing eight copies of an SF-1 binding site (39). In JEG-3 cells,
however, little or no inhibitory activity was detected when a similar
reporter construct containing two copies of SF-1 binding sites was used
(data not shown). Because ELP-1 binds to DNA relatively weakly,
ELP-1-GAL4 fusion proteins were created but also did not show any
repression (Fig. 1). Although it is possible that the zinc finger
region and FTZ-F1 box that were replaced with the GAL4-DBD are
necessary for the inhibitory activity of ELP-1, these experiments
suggest that it may be a relatively weak inhibitor or that its effects
may be cell type specific. In this regard, the silencing properties of
other nuclear receptors, such as the unliganded thyroid hormone
receptor, are less pronounced in JEG-3 cells than in other cell lines
(46), perhaps reflecting the levels or compositions of
corepressors.
Overexpression of SF-1 in JEG-3 cells resulted in decreased
transactivation by SF-1-GAL4 (data not shown). This result suggested
that titratable coactivators might be involved in the transcription
mediated by SF-1. This observation, in conjunction with the
conservation of the AF-2 domain in SF-1, led us to examine potential
interactions between SF-1 and SRC-1, a coactivator that is known to
interact with many other nuclear hormone receptors and to augment
ligand-dependent transactivation by the receptors (47, 48). Using
in vitro protein interaction assays, a relatively weak, but
specific, physical interaction was seen between SF-1 and SRC-1 (data
not shown). In view of additional evidence that SF-1 and SRC-1 interact
in functional assays (Figs. 3 and 4), it will be of interest to examine
the in vitro interactions further using the recently
identified candidate ligands for SF-1 (29). Full-length SRC-1 enhanced
GAL4-SF-1-induced transactivation, whereas the interaction domain of
SRC-1 inhibited SF-1-mediated transactivation (Fig. 3). The dominant
negative effect of the SRC-1 interaction domain was demonstrated
further by showing dose-dependent inhibition by cotransfection of a
constant amount of SRC-1 in the presence of increasing amounts of SRC-1
interaction domain. These findings indicate that exogenous SRC-1
enhances SF-1 transcriptional activity and that endogenous SRC-1 may be
involved in SF-1-induced transactivation in JEG-3 cells.
Experiments using carboxy-terminal deletion mutants of SF-1-GAL4 and
the VP16 transactivation domain fused to the interaction domain of
SRC-1 provides another line of evidence for interactions between SF-1
and SRC-1 and indicates that the SRC-1 effect is mediated through the
AF-2 domain in the carboxy terminus of SF-1 (Fig. 4). GAL4-SF-1-induced
transactivation was not augmented by VP16-NCoR or VP16-SMRT, which were
shown to interact with and stimulate the transcriptional activity of
GAL4-TR (data not shown). Thus, it appears that corepressors, NCoR
(nuclear corepressor) and SMRT (silencing mediator corepressor for
retinoid and thyroid hormone receptors), do not interact with SF-1, at
least in this cell line (which may produce SF-1 ligands).
While this work was in progress, SF-1 was shown to be activated by
several different oxysterols (e.g. 25-hydroxycholesterol)
that are compounds generated by P450c27 (29). GAL4-SF-1 activity
relative to GAL4 alone was much greater in JEG-3 cells among the cell
lines tested (human placental JEG-3 cells, murine gonadotrope T3
cells, murine adrenal cortical Y1 cells, and human embryonic kidney tsa
201 cells) (data not shown). JEG-3 cells are known to produce a variety
of steroid hormones including progesterone and estrogens (49), and it
is plausible that they may also produce ligands that activate SF-1.
This feature would account for the relatively high transcriptional
activity of SF-1 in JEG-3 cells as well as the ability of SF-1 to
interact efficiently with SRC-1 in the absence of exogenous ligand. By
analogy, in the experiment in which GAL4-ER was used, no exogenous
estradiol was required for activation because it is already produced by
this cell line.
Recently, it has been reported that CBP/p300 and SRC-1 synergistically
potentiate nuclear receptor transcriptional activity through direct
interactions between CBP/p300 and SRC-1 (44, 48). In the present study,
we extended this observation by showing that SRC-1 and CBP/p300
synergistically enhance the transcriptional activity of SF-1 (Fig. 6).
Although CBP/p300 has been shown to interact directly with some nuclear
hormone receptors (44, 48), SF-1-mediated transactivation was not
altered by cotransfection with CBP/p300 alone. These results favor a
model in which CBP/p300 interacts with SF-1 indirectly through SRC-1.
However, potential protein interactions between SF-1 and CBP/p300
remain to be studied.
The involvement of SRC-1 and CBP/p300 in the SF-1 coactivator complex is consistent with studies of the cholesterol side-chain cleavage enzyme (Cyp11A1), which is regulated by a synergistic interaction between CREB and SF-1 (34). In fact, many of the steroidogenic enzyme genes are regulated by cAMP as well as SF-1 (15). We have previously shown that DAX-1 inhibits the activity of SF-1 (37), and this may involve competition for shared coactivators such as SRC-1 or CBP/p300.
Although we have shown interactions between SF-1, SRC-1, and CBP/p300 in the regulation of SF-1-mediated transcription, it is likely that other coactivators will be identified for SF-1. The list of known transcriptional coactivators for nuclear receptors is growing rapidly (38) and promises to increase further in the next several years. In addition to its role in the transcriptional regulation of genes that characterize differentiated tissues, SF-1 is also critical for the development of adrenal gland and gonad (2). Cofactors involved in cell survival may be different from those involved in the regulation of target genes such as the steroidogenic enzymes. The present study indicates that SF-1 utilizes coactivators and emphasizes the need to search for other such proteins to better understand the biological actions of SF-1. It is also likely that other orphan nuclear receptors, particularly those with apparent AF-2 domains, will be shown to interact with transcriptional coactivators such as SRC-1 and CBP/p300.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The
DNA segment encoding the zinc finger region and FTZ-F1 box of ELP-1,
ELP-2, SF-1, and ELP-1 del 1–77 was removed by digestion with Eam1105I
and BcgI and replaced with PCR-amplified DNA corresponding to the GAL4
DBD (1–147), yielding ELP-1-GAL4, ELP-2-GAL4, SF-1-GAL4, and
ELP-1-GAL4 del 1–77 (Fig. 1B). The 3'-primer was designed to introduce
a BsrDI site at the end of PCR products (which is compatible
with the BcgI site). A termination codon was introduced by
PCR into the SF-1-GAL4 construct to create deletion mutants (SF-1-GAL4
del 458–462, SF-1-GAL4 del 443–462) (Fig. 2B). As shown in Fig. 3A, an alternative GAL4-SF-1 construct was also used (37). Briefly, this
construct contains the carboxy terminus of SF-1 (residues 133–462)
downstream of the GAL4 DBD in the pSG424 vector. The GAL4-ER construct
was synthesized similarly (Fig. 6A) and contains human estrogen
receptor (ER) (50) residues 282–595 subcloned downstream of the GAL4
DBD in the pSG424 vector. The pBKCMV vector containing full-length
SRC-1 (47) was provided by Bert O’Malley (Baylor College of Medicine,
Houston, TX). An expression vector for a dominant negative form of
SRC-1 (SRC-1 d.n.) was created by amplifying the interaction domain of
SRC-1 (residues 865-1061) and inserting it into the pBKCMV vector (Fig. 3A). The 5'-primer was designed to introduce a Kozak sequence and ATG
codon at the amino-terminal end of the product. The SRC-1 interaction
domain was fused to VP16 (40) by inserting the interaction domain
downstream of the cDNA encoding the VP16 transactivation domain,
yielding VP16-SRC-1 (Fig. 4A). Expression vectors for VP16 fused to the
interaction domain of NCoR (51) and SMRT (52) (VP16-NCoR, VP16-SMRT)
are described elsewhere (46). CBP (53) and p300 (54) (provided by
R. H. Goodman, Vollum Institute, Portland, OR) were expressed
using the pRCRSV vector (Invitrogen). The reporter gene plasmid used in
this study contains two copies of GAL4-binding sites (UAS) upstream of
the thymidine kinase promoter (TK109) linked to the luciferase gene
(UAS TK109 luc) (37).
Cell Culture and Transient Expression Assays
JEG-3 human placental choriocarcinoma cells, human embryonic
kidney tsa 201 cells (37), and murine T3 gonadotrope cells (55) were
grown in DMEM supplemented with 10% FBS in a 5% CO2
atmosphere at 37 C. Murine Y1 adrenal cells were grown in Ham’s F-10
supplemented with 15% horse serum and 2.5% FBS. Triplicate wells of
cells were transfected by the calcium phosphate method (56). Luciferase
assays were performed 48 h after transfection (57). Luciferase
activity is expressed as mean ± SEM of triplicate
transfections. Each experiment was repeated at least three times with
similar results, and a representative experiment is shown.
EMSA
Expression vectors (10 µg) for the GAL4 fusion proteins were
transfected into tsa 201 cells by the calcium phosphate method (56).
Whole cell extracts were prepared 48 h after transfection by three
cycles of freeze-thaw lysis in 20 mM Tris HCl pH 7.5, 0.5
M KCl, 2 mM dithiothreitol, 20% glycerol, and
1 mM phenylmethylsulfonyl fluoride. Cell extracts were
prepared by centrifugation at 10,000 x g for 30 min at
4 C, and supernatants were stored at -20 C (58). Protein
concentrations were determined using the Bio-Rad (Hercules, CA) protein
assay. In vitro translation was performed using the TNT
reticulocyte lysate system (Promega, Madison, WI). The synthetic
oligonucleotides for GAL4 binding site (UAS) (CTA GAG GTC GGA GTA CTG
TCC TCC GAC T) were labeled with [32P]dCTP by using
Klenow polymerase. Whole cell extracts (6 µg) were incubated with 20
fmol labeled oligonucleotides for 30 min at room temperature in 20 µl
of the binding buffer (10 mM Tris, pH 7.5, 10% glycerol,
100 mM KCl, 1 mM dithiothreitol) containing 6
µg poly(deoxyinosinic-deoxycytidylic)acid and 6 µg salmon sperm
DNA. The binding reaction with in vitro translation products
was performed in the same binding buffer with 1 µg
poly(deoxyinosinic-deoxycytidylic)acid and 1 µg salmon sperm DNA. The
DNA-protein complexes were resolved on 4% native polyacrylamide gels
in 0.5x Tris-borate-EDTA buffer.
RT-PCR Assays
RNA was isolated from cell lines using a Qiagen extraction kit
(Chatsworth, CA). Total RNA (1 µg) was reverse transcribed (42 C, 30
min) by the addition of 15 U reverse transcriptase (Promega) in the
presence of 10 pmol random hexamer primers, 25 mM
deoxynucleoside triphosphates (dNTPs) as described previously (59). PCR
reactions included specific primers for SF-1, SRC-1, or the controls
GAPDH. Primers were designed to span exon-intron boundaries to avoid
amplification of genomic DNA. The primers include: mouse SF-1 sense
strand 5'-CCC TGG TGT CCA GTG TCC ACC CTT ATC CGG-3', SF-1 antisense
5'-CTC GCA CGT GAG CAG CCC GTA GTG GTA GCC-3', product 160 bp; human
SRC-1 sense 5'-ACT GAG ACA CAC AGG CCT CTA CTG CAA CCA-3', SRC-1
antisense 5'-TTC AGT CAG TAG CTG CTG AAG GAG GCT CTT-3', products 244
bp (SRC-1) and 299 bp (SRC-1E); human GAPDH sense 5'-CCC TTC ATT GAC
CTC AAC TA-3', GAPDH antisense 5'-CCA AAG TTG TCA TGG ATG AC-3',
product 399 bp. Cycle conditions were 96 C for 4 min, 94 C for 1 min,
55 C for 1 min, and 72 C for 1 min.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was performed as part of the National Cooperative Program for Infertility Research and was supported by NIH Grant U54-HD-29164.
Received for publication June 27, 1997. Revision received October 9, 1997. Accepted for publication October 31, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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S. H. Mellon, S. R. Bair, C. Depoix, J.-L. Vigne, N. B. Hecht, and P. B. Brake Translin Coactivates Steroidogenic Factor-1-Stimulated Transcription Mol. Endocrinol., January 1, 2007; 21(1): 89 - 105. [Abstract] [Full Text] [PDF]
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 T. Sugawara, N. Sakuragi, and H. Minakami CREM confers cAMP responsiveness in human steroidogenic acute regulatory protein expression in NCI-H295R cells rather than SF-1/Ad4BP. J. Endocrinol., October 1, 2006; 191(1): 327 - 337. [Abstract] [Full Text] [PDF]
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 T. N. Parakh, J. A. Hernandez, J. C. Grammer, J. Weck, M. Hunzicker-Dunn, A. J. Zeleznik, and J. H. Nilson Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin PNAS, August 15, 2006; 103(33): 12435 - 12440. [Abstract] [Full Text] [PDF]
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J. Weck and K. E. Mayo Switching of NR5A Proteins Associated with the Inhibin {alpha}-Subunit Gene Promoter after Activation of the Gene in Granulosa Cells Mol. Endocrinol., May 1, 2006; 20(5): 1090 - 1103. [Abstract] [Full Text] [PDF]
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 J. N. Winnay, J. Xu, B. W. O'Malley, and G. D. Hammer Steroid Receptor Coactivator-1-Deficient Mice Exhibit Altered Hypothalamic-Pituitary-Adrenal Axis Function Endocrinology, March 1, 2006; 147(3): 1322 - 1332. [Abstract] [Full Text] [PDF]
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J. N. Winnay and G. D. Hammer Adrenocorticotropic Hormone-Mediated Signaling Cascades Coordinate a Cyclic Pattern of Steroidogenic Factor 1-Dependent Transcriptional Activation Mol. Endocrinol., January 1, 2006; 20(1): 147 - 166. [Abstract] [Full Text] [PDF]
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S. N Kelly, T J. McKenna, and L. S Young Coregulatory protein-orphan nuclear receptor interactions in the human adrenal cortex J. Endocrinol., July 1, 2005; 186(1): 33 - 42. [Abstract] [Full Text] [PDF]
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 J. Simard, M.-L. Ricketts, S. Gingras, P. Soucy, F. A. Feltus, and M. H. Melner Molecular Biology of the 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Gene Family Endocr. Rev., June 1, 2005; 26(4): 525 - 582. [Abstract] [Full Text] [PDF]
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W. Zheng and C. R. Jefcoate Steroidogenic Factor-1 Interacts with cAMP Response Element-Binding Protein to Mediate cAMP Stimulation of CYP1B1 via a Far Upstream Enhancer Mol. Pharmacol., February 1, 2005; 67(2): 499 - 512. [Abstract] [Full Text] [PDF]
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T. Komatsu, H. Mizusaki, T. Mukai, H. Ogawa, D. Baba, M. Shirakawa, S. Hatakeyama, K. I. Nakayama, H. Yamamoto, A. Kikuchi, et al. Small Ubiquitin-Like Modifier 1 (SUMO-1) Modification of the Synergy Control Motif of Ad4 Binding Protein/Steroidogenic Factor 1 (Ad4BP/SF-1) Regulates Synergistic Transcription between Ad4BP/SF-1 and Sox9 Mol. Endocrinol., October 1, 2004; 18(10): 2451 - 2462. [Abstract] [Full Text] [PDF]
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J. Qin, D.-m. Gao, Q.-F. Jiang, Q. Zhou, Y.-Y. Kong, Y. Wang, and Y.-H. Xie Prospero-Related Homeobox (Prox1) Is a Corepressor of Human Liver Receptor Homolog-1 and Suppresses the Transcription of the Cholesterol 7-{alpha}-Hydroxylase Gene Mol. Endocrinol., October 1, 2004; 18(10): 2424 - 2439. [Abstract] [Full Text] [PDF]
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 W.-Y. Chen, W.-C. Lee, N.-C. Hsu, F. Huang, and B.-c. Chung SUMO Modification of Repression Domains Modulates Function of Nuclear Receptor 5A1 (Steroidogenic Factor-1) J. Biol. Chem., September 10, 2004; 279(37): 38730 - 38735. [Abstract] [Full Text] [PDF]
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Z. Chen, D. J. Eastburn, and M. Han The Caenorhabditis elegans Nuclear Receptor Gene nhr-25 Regulates Epidermal Cell Development Mol. Cell. Biol., September 1, 2004; 24(17): 7345 - 7358. [Abstract] [Full Text] [PDF]
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P.-L. Xu, Y.-Q. Liu, S.-F. Shan, Y.-Y. Kong, Q. Zhou, M. Li, J.-P. Ding, Y.-H. Xie, and Y. Wang Molecular Mechanism for the Potentiation of the Transcriptional Activity of Human Liver Receptor Homolog 1 by Steroid Receptor Coactivator-1 Mol. Endocrinol., August 1, 2004; 18(8): 1887 - 1905. [Abstract] [Full Text] [PDF]
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R. V. Correa, S. Domenice, N. C. Bingham, A. E. C. Billerbeck, W. E. Rainey, K. L. Parker, and B. B. Mendonca A Microdeletion in the Ligand Binding Domain of Human Steroidogenic Factor 1 Causes XY Sex Reversal without Adrenal Insufficiency J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1767 - 1772. [Abstract] [Full Text] [PDF]
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J.-F. Mouillet, C. Sonnenberg-Hirche, X. Yan, and Y. Sadovsky p300 Regulates the Synergy of Steroidogenic Factor-1 and Early Growth Response-1 in Activating Luteinizing Hormone-{beta} Subunit Gene J. Biol. Chem., February 27, 2004; 279(9): 7832 - 7839. [Abstract] [Full Text] [PDF]
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B. Borud, G. Mellgren, J. Lund, and M. Bakke Cloning and Characterization of a Novel Zinc Finger Protein that Modulates the Transcriptional Activity of Nuclear Receptors Mol. Endocrinol., November 1, 2003; 17(11): 2303 - 2319. [Abstract] [Full Text] [PDF]
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J. J. Tremblay and R. S. Viger A Mutated Form of Steroidogenic Factor 1 (SF-1 G35E) That Causes Sex Reversal in Humans Fails to Synergize with Transcription Factor GATA-4 J. Biol. Chem., October 24, 2003; 278(43): 42637 - 42642. [Abstract] [Full Text] [PDF]
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B. M. Gummow, J. N. Winnay, and G. D. Hammer Convergence of Wnt Signaling and Steroidogenic Factor-1 (SF-1) on Transcription of the Rat Inhibin {alpha} Gene J. Biol. Chem., July 11, 2003; 278(29): 26572 - 26579. [Abstract] [Full Text] [PDF]
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H. Sadie, G. Styger, and J. Hapgood Expression of the Mouse Gonadotropin-Releasing Hormone Receptor Gene in {alpha}T3-1 Gonadotrope Cells Is Stimulated by Cyclic 3',5'-Adenosine Monophosphate and Protein Kinase A, and Is Modulated by Steroidogenic Factor-1 and Nur77 Endocrinology, May 1, 2003; 144(5): 1958 - 1971. [Abstract] [Full Text] [PDF]
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M. Maira, C. Martens, E. Batsche, Y. Gauthier, and J. Drouin Dimer-Specific Potentiation of NGFI-B (Nur77) Transcriptional Activity by the Protein Kinase A Pathway and AF-1-Dependent Coactivator Recruitment Mol. Cell. Biol., February 1, 2003; 23(3): 763 - 776. [Abstract] [Full Text]
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T. Suzuki, M. Kasahara, H. Yoshioka, K.-i. Morohashi, and K. Umesono LXXLL-Related Motifs in Dax-1 Have Target Specificity for the Orphan Nuclear Receptors Ad4BP/SF-1 and LRH-1 Mol. Cell. Biol., January 1, 2003; 23(1): 238 - 249. [Abstract] [Full Text]
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X. Yan, J.-F. Mouillet, Q. Ou, and Y. Sadovsky A Novel Domain within the DEAD-Box Protein DP103 Is Essential for Transcriptional Repression and Helicase Activity Mol. Cell. Biol., January 1, 2003; 23(1): 414 - 423. [Abstract] [Full Text]
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X. Wei, M. Sasaki, H. Huang, V. L. Dawson, and T. M. Dawson The Orphan Nuclear Receptor, Steroidogenic Factor 1, Regulates Neuronal Nitric Oxide Synthase Gene Expression in Pituitary Gonadotropes Mol. Endocrinol., December 1, 2002; 16(12): 2828 - 2839. [Abstract] [Full Text] [PDF]
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F. Gizard, B. Lavallee, F. DeWitte, E. Teissier, B. Staels, and D. W. Hum The Transcriptional Regulating Protein of 132 kDa (TReP-132) Enhances P450scc Gene Transcription through Interaction with Steroidogenic Factor-1 in Human Adrenal Cells J. Biol. Chem., October 11, 2002; 277(42): 39144 - 39155. [Abstract] [Full Text] [PDF]
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B. Borud, T. Hoang, M. Bakke, A. L. Jacob, J. Lund, and G. Mellgren The Nuclear Receptor Coactivators p300/CBP/Cointegrator-Associated Protein (p/CIP) and Transcription Intermediary Factor 2 (TIF2) Differentially Regulate PKA-Stimulated Transcriptional Activity of Steroidogenic Factor 1 Mol. Endocrinol., April 1, 2002; 16(4): 757 - 773. [Abstract] [Full Text] [PDF]
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R. AEsoy, G. Mellgren, K.-I. Morohashi, and J. Lund Activation of cAMP-Dependent Protein Kinase Increases the Protein Level of Steroidogenic Factor-1 Endocrinology, January 1, 2002; 143(1): 295 - 303. [Abstract] [Full Text] [PDF]
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Z. J. Wang, B. Jeffs, M. Ito, J. C. Achermann, R. N. Yu, D. B. Hales, and J. L. Jameson Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1 PNAS, June 20, 2001; (2001) 141543298. [Abstract] [Full Text] [PDF]
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H. Pincas, K. Amoyel, R. Counis, and J.-N. Laverrière Proximal cis-Acting Elements, Including Steroidogenic Factor 1, Mediate the Efficiency of a Distal Enhancer in the Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene Mol. Endocrinol., February 1, 2001; 15(2): 319 - 337. [Abstract] [Full Text]
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Q. Ou, J.-F. Mouillet, X. Yan, C. Dorn, P. A. Crawford, and Y. Sadovsky The DEAD Box Protein DP103 Is a Regulator of Steroidogenic Factor-1 Mol. Endocrinol., January 1, 2001; 15(1): 69 - 79. [Abstract] [Full Text]
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J. Levallet, P. Koskimies, N. Rahman, and I. Huhtaniemi The Promoter of Murine Follicle-Stimulating Hormone Receptor: Functional Characterization and Regulation by Transcription Factor Steroidogenic Factor 1 Mol. Endocrinol., January 1, 2001; 15(1): 80 - 92. [Abstract] [Full Text]
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 L Zhao, M Bakke, Y Krimkevich, L. Cushman, A. Parlow, S. Camper, and K. Parker Steroidogenic factor 1 (SF1) is essential for pituitary gonadotrope function Development, January 1, 2001; 128(2): 147 - 154. [Abstract] [PDF]
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D. Boerboom, N. Pilon, R. Behdjani, D. W. Silversides, and J. Sirois Expression and Regulation of Transcripts Encoding Two Members of the NR5A Nuclear Receptor Subfamily of Orphan Nuclear Receptors, Steroidogenic Factor-1 and NR5A2, in Equine Ovarian Cells during the Ovulatory Process Endocrinology, December 1, 2000; 141(12): 4647 - 4656. [Abstract] [Full Text] [PDF]
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L. Q. Nguyen, P. Kopp, F. Martinson, K. Stanfield, S. I. Roth, and J. L. Jameson A Dominant Negative CREB (cAMP Response Element-Binding Protein) Isoform Inhibits Thyrocyte Growth, Thyroid-Specific Gene Expression, Differentiation, and Function Mol. Endocrinol., September 1, 2000; 14(9): 1448 - 1461. [Abstract] [Full Text]
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U. B. Kaiser, L. M. Halvorson, and M. T. Chen Sp1, Steroidogenic Factor 1 (SF-1), and Early Growth Response Protein 1 (Egr-1) Binding Sites Form a Tripartite Gonadotropin-Releasing Hormone Response Element in the Rat Luteinizing Hormone-{beta} Gene Promoter: an Integral Role for SF-1 Mol. Endocrinol., August 1, 2000; 14(8): 1235 - 1245. [Abstract] [Full Text]
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T. Sugawara, M. Saito, and S. Fujimoto Sp1 and SF-1 Interact and Cooperate in the Regulation of Human Steroidogenic Acute Regulatory Protein Gene Expression Endocrinology, August 1, 2000; 141(8): 2895 - 2903. [Abstract] [Full Text] [PDF]
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C. Frigeri, J. Tsao, W. Czerwinski, and B. P. Schimmer Impaired Steroidogenic Factor 1 (NR5A1) Activity in Mutant Y1 Mouse Adrenocortical Tumor Cells Mol. Endocrinol., April 1, 2000; 14(4): 535 - 544. [Abstract] [Full Text]
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E. Barnea and Y. Bergman Synergy of SF1 and RAR in Activation of Oct-3/4 Promoter J. Biol. Chem., February 25, 2000; 275(9): 6608 - 6619. [Abstract] [Full Text] [PDF]
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L.-A. Li, E. F-L. Chiang, J.-C. Chen, N.-C. Hsu, Y.-J. Chen, and B.-c. Chung Function of Steroidogenic Factor 1 Domains in Nuclear Localization, Transactivation, and Interaction with Transcription Factor TFIIB and c-Jun Mol. Endocrinol., September 1, 1999; 13(9): 1588 - 1598. [Abstract] [Full Text]
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H. Hong, L. Yang, and M. R. Stallcup Hormone-independent Transcriptional Activation and Coactivator Binding by Novel Orphan Nuclear Receptor ERR3 J. Biol. Chem., August 6, 1999; 274(32): 22618 - 22626. [Abstract] [Full Text] [PDF]
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D. Lopez, T. W. Sandhoff, and M. P. McLean Steroidogenic Factor-1 Mediates Cyclic 3',5'-Adenosine Monophosphate Regulation of the High Density Lipoprotein Receptor Endocrinology, July 1, 1999; 140(7): 3034 - 3044. [Abstract] [Full Text]
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A. J. Reinhart, S. C. Williams, B. J. Clark, and D. M. Stocco SF-1 (Steroidogenic Factor-1) and C/EBP{beta} (CCAAT/Enhancer Binding Protein-{beta}) Cooperate to Regulate the Murine StAR (Steroidogenic Acute Regulatory) Promoter Mol. Endocrinol., May 1, 1999; 13(5): 729 - 741. [Abstract] [Full Text]
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 A. Swain and R. Lovell-Badge Mammalian sex determination: a molecular drama Genes & Dev., April 1, 1999; 13(7): 755 - 767. [Full Text]
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H. Hong, B. D. Darimont, H. Ma, L. Yang, K. R. Yamamoto, and M. R. Stallcup An Additional Region of Coactivator GRIP1 Required for Interaction with the Hormone-binding Domains of a Subset of Nuclear Receptors J. Biol. Chem., February 5, 1999; 274(6): 3496 - 3502. [Abstract] [Full Text] [PDF]
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N. Pilon, R. Behdjani, I. Daneau, J. G. Lussier, and D. W. Silversides Porcine Steroidogenic Factor-1 Gene (pSF-1) Expression and Analysis of Embryonic Pig Gonads during Sexual Differentiation Endocrinology, September 1, 1998; 139(9): 3803 - 3812. [Abstract] [Full Text] [PDF]
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M. Ito, J. C. Achermann, and J. L. Jameson A Naturally Occurring Steroidogenic Factor-1 Mutation Exhibits Differential Binding and Activation of Target Genes J. Biol. Chem., October 6, 2000; 275(41): 31708 - 31714. [Abstract] [Full Text] [PDF]
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F. Gizard, B. Lavallee, F. DeWitte, and D. W. Hum A Novel Zinc Finger Protein TReP-132 Interacts with CBP/p300 to Regulate Human CYP11A1 Gene Expression J. Biol. Chem., August 31, 2001; 276(36): 33881 - 33892. [Abstract] [Full Text] [PDF]
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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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A. L. Jacob, J. Lund, P. Martinez, and L. Hedin Acetylation of Steroidogenic Factor 1 Protein Regulates Its Transcriptional Activity and Recruits the Coactivator GCN5 J. Biol. Chem., September 28, 2001; 276(40): 37659 - 37664. [Abstract] [Full Text] [PDF]
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Z. J. Wang, B. Jeffs, M. Ito, J. C. Achermann, R. N. Yu, D. B. Hales, and J. L. Jameson Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1 PNAS, July 3, 2001; 98(14): 7988 - 7993. [Abstract] [Full Text] [PDF]
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