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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}- 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1AGo).



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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 1–77) 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 1–77 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.

 
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 1–77 (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 1–77 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 1–77 > 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 1–77 (Fig. 1BGo). 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. 1DGo). 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. 1CGo). 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. 2AGo). 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. 2BGo). Using extracts from transfected Tsa 201 cells or in vitro translation products (Fig. 2CGo), 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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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{alpha}). 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 458–462, SF-1-GAL4 del 443–462) 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. 1Go. A binding reaction using unprogrammed lysate is shown in lane 5.

 
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. 3AGo). 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.

 
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. 3BGo). 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. 3CGo). 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. 3Go, B and C), and it was fused to the VP16 transactivation domain, yielding VP16-SRC-1 (Fig. 4AGo). 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. 4BGo). 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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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 443–462 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.

 
The potentiating effect of VP16-SRC-1 on GAL4-SF-1-induced transactivation was dose-dependent (Fig. 4CGo). However, the greatest amount (500 ng) of VP16-SRC-1 was inhibitory, perhaps reflecting squelching of other transcription factors (Fig. 4CGo). 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. 3BGo).

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. 5AGo). As expected, SF-1 expression was seen in human testis, human H295R adrenal cells, murine {alpha}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.

 
Transient expression assays using GAL4-SF-1 and VP16-SF-1 were also performed in the SF-1-containing Y1 adrenal cells (Fig. 5BGo). 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 {alpha}T3 gonadotrope cells (data not shown). Thus, although the magnitude of GAL-4-SF-1 responses are lower in Y1 and {alpha}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. 6BGo). 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.

 
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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1Go). 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. 3Go and 4Go), 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. 3Go). 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. 4Go). 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 {alpha}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. 6Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1–77) was constructed by replacement of the SalI-XhoI fragment encoding the carboxy-terminal region of SF-1 with that of ELP-1 (Fig. 1AGo). 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. 1BGo). 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. 2BGo). As shown in Fig. 3AGo, 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. 6AGo) 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. 3AGo). 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. 4AGo). 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 {alpha}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.


    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 15–709, 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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 MATERIALS AND METHODS
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