Steroidogenic Factor-1 Contains a Carboxy-Terminal Transcriptional Activation Domain That Interacts with Steroid Receptor Coactivator-1
Masafumi Ito,
Richard N. Yu and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine
Northwestern University Medical School Chicago, Illinois 60611
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ABSTRACT
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The orphan nuclear receptor, steroidogenic
factor-1 (SF-1), plays an important role in the development of the
adrenal gland and in sexual differentiation. SF-1 regulates the
transcription of variety of genes, including several steroidogenic
enzymes, Müllerian inhibiting substance, and gonadotropin genes.
In this report, we sought to identify domains in SF-1 that are required
for transactivation and to determine whether SF-1 interacts with a
subset of known coactivators. Natural variants of the FTZ-F1 locus
include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2,
and SF-1, which share the DNA-binding domain. Analyses of the
transcriptional activity of these variants revealed that the activity
of ELP-2 and SF-1 was much greater than ELP-1, which contains a
distinct carboxy terminus. Further studies were performed using
GAL4-SF-1 fusion proteins that were constructed by replacement of the
zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain
of GAL4. Elimination of the putative AF-2 domain at the carboxy
terminus of GAL4-SF-1 proteins resulted in a complete loss of
transactivation. Several lines of evidence demonstrated that SF-1
interacts with steroid receptor coactivator-1 (SRC-1). Full-length
SRC-1 enhanced GAL4-SF-1-mediated transactivation, whereas a dominant
negative form of SRC-1, consisting of its interaction domain alone,
inhibited the activity of GAL4-SF-1. In mammalian two-hybrid assays,
fusion of the VP16 activation domain to the interaction domain of SRC-1
confirmed the interaction between SRC-1 and GAL4-SF-1 and demonstrated
that the AF-2 domain is required for interaction with SRC-1.
Furthermore, SRC-1, together with the cAMP responsive element binding
protein (CBP) or a closely related factor, p300, synergistically
enhanced transcriptional activity of GAL4-SF-1. We conclude that the
carboxy-terminal AF-2 region of SF-1 functions as an activation domain
and that SRC-1 and CBP/p300 are components of the coactivator complex
with SF-1.
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INTRODUCTION
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Steroidogenic factor 1 (SF-1) is a homolog of the
Drosophila orphan nuclear receptor, fushi tarazu factor 1
(FTZ-F1) (1), a transcription factor that regulates fushi tarazu
homeobox gene expression during early development (2, 3). Like other
members of the nuclear receptor superfamily, SF-1 possesses a
characteristic zinc finger DNA-binding domain (DBD) and putative
carboxy-terminal ligand-binding/dimerization domain (4). A consensus
DNA recognition site (PyCA AGGTPyC or PuPuAGGTCA) for SF-1 has been
identified, and it appears to bind to DNA as a monomer. Like a subset
of other orphan nuclear receptors (e.g. nerve growth
factor-induced gene-B), monomeric DNA binding involves interactions of
the A-box (FTZ-F1 box), which is adjacent to the zinc finger domains
(5, 6, 7). It is unclear whether SF-1 can also form homo- or heterodimeric
complexes.
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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ELP-2 and SF-1 Contain a Transactivation Domain
All the naturally occurring proteins that are derived from the
FTZ-F1 locus contain the zinc finger region and a FTZ-F1 box, which
together function as a DBD (Fig. 1A
). 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 177, was constructed to delete the
ELP-1-specific amino terminus (Fig. 1A
).

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Figure 1. Identification of a Transactivation Domain among
FTZ-F1 Homologs
A, Structures of naturally occurring (ELP-1, ELP-2, SF-1) and
artificially generated (ELP-1 del 177) FTZ-F1 proteins. All the
proteins share a common region including the zinc finger domain and
FTZ-F1 box. ELP-1 and ELP-2 contain specific sequences in the
amino-terminal region (77 amino acid residues). The different
carboxy-terminal regions specific for ELP-1 and ELP-1 del 177 and for
ELP-2 and SF-1 are shown. B, The zinc finger region and FTZ-F1 box were
replaced with the GAL4 DBD to create the indicated chimeric proteins.
C, JEG-3 cells were transfected with UAS TK109 luc (500 ng) and GAL4
fusion protein expression vectors described above (100 ng). Forty eight
hours after the transfection, cells were harvested for luciferase
assays. Results are the mean ± SEM from triplicate
transfections. D, Tsa 201 cells were transfected with expression
vectors for the GAL4 fusion proteins described above. Forty eight hours
after transfection, whole cell extracts were prepared. Whole cell
extract proteins (6 µg) or in vitro translated
proteins (3 µl) were incubated with a 32P-labeled probe
for the GAL4-binding site (20 fmol). After the binding reaction, the
DNA and protein complexes were resolved on 4% native polyacrylamide
gels. In lane 6, unprogrammed lysate was included as a control.
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Transient expression assays were performed in JEG-3 cells using a
reporter construct (2xSF-1 TK81luc) containing two copies of an
SF-1-regulatory element upstream of TK81 linked to the luciferase gene
(37). SF-1 and ELP-2 induced transactivation (6- to 8-fold
stimulation), but no transcriptional stimulation was observed with
ELP-1 or ELP-1 del 177 (data not shown). Electrophoretic mobility
shift assays (EMSA) were performed with each of these proteins to
assess whether the decreased transcriptional activity of ELP-1 and
ELP-1 del 177 might be caused by diminished binding to DNA.
Comparable amounts of in vitro translated proteins were
bound to a consensus SF-1 binding site (ACA AGGTCA). However, as
previously reported (39), very little DNA binding was seen with ELP-1,
and the rank order of the binding was SF-1 > ELP-1 del 177
> ELP-2 > ELP-1 (data not shown).
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 177 (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 177 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 177
were inactive. Because ELP-2 and SF-1 share a common carboxy terminus
that is distinct from ELP-1 (or ELP-1 del 177), 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 458462 and SF-1-GAL4 del 443462 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
458462) 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
443462) 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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Figure 2. Characterization of the AF-2 Domain of SF-1
A, SF-1 contains a putative AF-2 domain at the carboxy terminus. The
AF-2 domain of SF-1 is compared with that of other nuclear hormone
receptors (human TRß, ER, RAR ). Conserved hydrophobic regions
(bold) and an invariant glutamate residue
(underlined) are shown. Carboxy-terminal deletion
mutants of SF-1 GAL4 were constructed (SF-1-GAL4 del 458462,
SF-1-GAL4 del 443462) and used along with the SF-1-GAL4 construct in
the experiments described below. B, JEG-3 cells were transfected with
UAS TK109 luc (500 ng) and the GAL4 fusion protein expression vectors
(100 ng). Forty eight hours after the transfection, cells were
harvested for luciferase assays. Results are the mean ±
SEM from triplicate transfections. C, Tsa 201 cells were
transfected with expression vectors for the GAL4 fusion proteins. Whole
cell extracts or in vitro translated proteins were
analyzed by EMSA as described in Fig. 1 . A binding reaction using
unprogrammed lysate is shown in lane 5.
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Functional Interactions between SF-1 and SRC-1
Functional interactions between SF-1 and SRC-1 were assessed by
examining the effect of SRC-1 on SF-1-mediated transcription
and by determining whether the interaction domain of SRC-1 (SRC-1
d.n.) inhibited transcription. In these experiments, a GAL4-SF-1
construct in the pSG424 vector (37) was created to eliminate the
amino-terminal sequences of SF-1 and to allow more direct comparisons
with analogous fusions of GAL4 to the carboxy-terminal regions of
nuclear receptors (e.g. GAL4-ER, see below) (Fig. 3A
). 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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Figure 3. Functional Interaction between SF-1 and SRC-1
A, Comparison of the SF-1-GAL4 construct in the pBKCMV vector with the
GAL4-SF-1 construct in the pSG424 vector. Both constructs contain the
region corresponding to residues 133 to 462 of SF-1, but the SF-1-GAL4
construct also contains the first 19 amino acid residues of SF-1. B,
UAS TK109 luc (500 ng) and the GAL4 or GAL4 SF-1 construct (50 ng) were
transfected into JEG-3 cells with either empty, SRC-1, or SRC-1 d.n.
vectors (200 ng). Forty eight hours after the transfection, cells were
harvested for luciferase assays. C, The SRC-1 expression vector (100
ng) and increasing amounts of SRC-1 d.n. expression vector (0, 10, 20,
50, 100 ng) were cotransfected into JEG-3 cells along with UAS TK109
luc (500 ng), GAL4, or GAL4 SF-1 constructs (50 ng). The total amount
of the transfected pBKCMV vector was kept constant in each reaction.
Forty eight hours after the transfection, cells were harvested for
luciferase assays. The data are expressed as the ratio of the reporter
activity with GAL4-SF-1 relative to the basal reporter activity with
GAL4.
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When cotransfected with a control empty expression vector, GAL4-SF-1
caused 15-fold transactivation of UAS TK109 luc compared with the GAL4
DBD alone (Fig. 3B
). 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
443462 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 443462 protein, which lacks the AF-2 domain.

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Figure 4. Interaction of SF-1 with VP16 Fusion Proteins with
SRC-1
A, Construction of VP16 fusion proteins with the interaction domain of
SRC-1. The interaction domain was subcloned downstream of the VP16
transactivation domain. B, UAS TK109 luc (500 ng) and GAL4, SF-1 GAL4,
or SF- GAL4 del 443462 constructs (100 ng) were transfected into
JEG-3 cells along with either VP16 or VP16 SRC-1 (200 ng). Forty eight
hours after the transfection, cells were harvested for luciferase
assays. C, Increasing amounts of VP16 or VP16 SRC-1 expression vectors
(0, 100, 200, 500 ng) were cotransfected into JEG-3 cells with UAS
TK109 luc (500 ng) and GAL4 or GAL4 SF-1 constructs (50 ng). Forty
eight hours after the transfection, cells were harvested for luciferase
assays. The data are expressed as ratio of the reporter activity with
GAL4-SF-1 relative to the basal reporter activity with GAL4.
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The potentiating effect of VP16-SRC-1 on GAL4-SF-1-induced
transactivation was dose-dependent (Fig. 4C
). 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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Figure 5. Functional Interactions of SF-1 and SRC-1 in
SF-1-Containing Cells
A, RT-PCR analyses of SF-1 and SRC-1 in various cell lines. An ethidium
bromide-stained gel of PCR products is shown, and the tissues and cell
lines are indicated at the top of the figure. Specific
bands for SF-1 (160 bp), SRC-1 (244 bp), and the SRC-1E variant (299
bp) are indicated. GAPDH (399 bp) is included as a positive control and
a set of reactions without reverse transcriptase (-RT) is included as
a negative control. B, Interaction of GAL4-SF-1 and VP16 SRC-1 in Y1
cells. UAS TK109 luc (500 ng) and GAL4 (50 ng) or GAL4-SF-1 (50 ng)
were transfected into Y1 cells along with either VP16 or VP16 SRC-1
(200 ng). Forty eight hours after the transfection, cells were
harvested for luciferase assays.
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Transient expression assays using GAL4-SF-1 and VP16-SF-1 were also
performed in the SF-1-containing Y1 adrenal cells (Fig. 5B
). 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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Figure 6. Functional Interactions among SF-1, SRC-1, and
CBP/p300
A, The AF-2 domains are shown for SF-1 and ER. Hydrophobic regions
(bold) and an invariant glutamate residue
(underlined) within the AF-2 domain are indicated. B,
UAS TK109 luc (500 ng) and GAL4, GAL4 SF-1, or GAL4 ER constructs (50
ng) were transfected into JEG-3 cells along with 100 ng of the
indicated vectors (SRC-1, CBP, p300). Forty eight hours after the
transfection, cells were harvested for luciferase assays.
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In parallel, GAL4-SF-1 stimulated transcription 14-fold compared with
GAL4 alone. Similar to the results with GAL4-ER, SRC-1 increased the
transcriptional activity of GAL4-SF-1 (28-fold), whereas CBP and p300
showed little or no effect. Cotransfection of SRC-1 with CBP or p300
potentiated transcription, resulting in 66- and 55-fold induction,
respectively.
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DISCUSSION
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Recently, mechanisms of transcriptional activation by nuclear
receptors have been advanced considerably by the identification of
transactivation domains and so-called coactivators that interact with
these regions of the receptors (4, 38). Most progress has been made
with classic ligand-activated receptors such as the steroid receptors
(e.g. progesterone receptor, glucocorticoid receptor,
estrogen receptor, androgen receptor) and receptors that heterodimerize
with retinoid X receptor (e.g. RAR, TR, vitamin D receptor,
peroxisone proliferator-activated receptor). In these cases, ligand
binding is proposed to induce conformational changes that allow
receptor association with coactivators, which in turn make contacts
with other proteins that ultimately cause increased transcription.
There is less information about how orphan nuclear receptors activate
transcription (45). In this report, we demonstrate that SF-1 functions
in a manner reminiscent of other classes of nuclear receptors. A
transactivation domain that is structurally similar to the AF-2 domain
in other nuclear receptors is localized in the carboxy terminus of
SF-1.
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.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
Murine ELP-1 and ELP-2 cDNAs (provided by K. Ohtsura Niwa,
Hiroshima University, Hiroshima, Japan) were subcloned into the pBKCMV
expression vector (Stratagene, La Jolla, CA). The cloning of murine
SF-1 cDNA was described previously (37). A deletion mutant of ELP-1
lacking amino-terminal 77 amino acids (ELP-1 del 177) was constructed
by replacement of the SalI-XhoI fragment encoding
the carboxy-terminal region of SF-1 with that of ELP-1 (Fig. 1A
). The
DNA segment encoding the zinc finger region and FTZ-F1 box of ELP-1,
ELP-2, SF-1, and ELP-1 del 177 was removed by digestion with Eam1105I
and BcgI and replaced with PCR-amplified DNA corresponding to the GAL4
DBD (1147), yielding ELP-1-GAL4, ELP-2-GAL4, SF-1-GAL4, and
ELP-1-GAL4 del 177 (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 458462, SF-1-GAL4 del 443462) (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 133462)
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 282595 subcloned downstream of the GAL4
DBD in the pSG424 vector. The pBKCMV vector containing full-length
SRC-1 (47) was provided by Bert OMalley (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 Hams 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.
 |
ACKNOWLEDGMENTS
|
---|
We would like to acknowledge Ohtsura Niwa for providing ELP-1
and ELP-2 cDNAs, Bert O Malley for human SRC-1, Richard Goodman for
CBP and p300, Ron Evans for SMRT, Geoff Rosenfeld for NCoR, and Pierre
Chambon for the human ER cDNA. Tetsuya Tagami provided the VP16-NCoR
and VP16-SMRT constructs.
 |
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. Email:ljameson@nwu.edu.
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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