The Activation Function-2 Hexamer of Steroidogenic Factor-1 Is Required, but Not Sufficient for Potentiation by SRC-1

Peter A. Crawford, Jeffrey A. Polish, Gauri Ganpule and Yoel Sadovsky

Department of Obstetrics and Gynecology (J.A.P., G.G., Y.S.) and Pathology (P.A.C.), Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The orphan receptor steroidogenic factor 1 (SF-1) plays a central role in development and differentiation of the adrenal gland and gonads. It also regulates the expression of several pivotal steroidogenic enzymes and other proteins that are essential for reproductive function. Its mechanism of target gene activation that directs these intricate processes has not been previously established. We demonstrate here that the activation function-2 (AF-2) activation hexamer (AF-2-AH) of SF-1, located within its carboxy-terminal region, is required for reporter gene activation by SF-1, as well as for SF-1-mediated induction of a steroidogenic phenotype in embryonic stem cells. We further demonstrate that SF-1’s AF-2-AH is not sufficient for gene activation, requiring an additional, proximally located domain of SF-1, positioned between residues 187–245. Correspondingly, we show that the coactivator SRC-1 potentiates the activity of SF-1 and that the interaction between SF-1 and SRC-1 requires both AF-2-AH and the proximal activation domain. We conclude that SF-1 harbors at least two activation domains within its carboxy terminus and that both are required for its transcriptional activation function and for direct interaction with SRC-1. It is likely that SRC-1 plays a key role in gene regulation by SF-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenic factor 1 (SF-1) is an orphan member of the steroid receptor superfamily of proteins, which are essential for regulation of embryonic development, differentiation, and cellular function (1, 2, 3, 4, 5, 6). SF-1 is highly expressed in all three layers of the adrenal cortex, in granulosa and theca cells in the ovary, and in Sertoli and Leydig cells in the testis (7). Consistent with its anatomical distribution, SF-1 functions as a potent transcriptional activator of several steroidogenic enzymes, including cytochrome P450scc, the key rate-limiting enzyme in the steroid hormone biosynthetic pathway, as well as P450c17, P450c21, P450c11, P450arom, 3ß-hydroxysteroid dehydrogenase, and steroidogenic acute regulatory protein (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Significantly, SF-1 is also expressed in pituitary gonadotrophs, where it is essential for production of the ß-subunit of LH and for expression of the GnRH receptor (20, 21, 22, 23).

Developmentally, SF-1 is expressed as early as E9 in the mouse urogenital ridge, before its morphological differentiation into adrenal and gonadal tissues (24). In the brain, SF-1 expression is detected in hypophyseal precursors, as well as in prosencephalic regions that give rise to the hypothalamus (24). To study the developmental and differentiated functions of SF-1 in vivo, Luo et al (25) and our group (26) generated SF-1 -/- mice. Intriguingly, these mice lacked gonads, resulting in the persistence of Mullerian structures in genotypically male and female mice. They also lacked adrenal glands, which led to their early neonatal death. Furthermore, SF-1 -/- mice demonstrated a developmental defect in the ventromedial hypothalamus, an area implicated in control of sexual behavior (27, 28). Together, these results demonstrate that SF-1 is expressed in tissues that are essential for reproductive and endocrine homeostasis and that it is required for intact development of the adrenal glands and gonads.

Members of the steroid receptor superfamily of proteins, like other transcription factors, are composed of modular functional domains (6). These include a DNA-binding domain, which is flanked by amino and carboxy termini. A ligand-independent activation function (AF-1) is commonly located at the amino terminus. The carboxy terminus harbors a hinge region and a ligand-binding domain, which commonly includes a second, ligand-dependent activation function (AF-2) domain (1, 29, 30, 31, 32). Transcriptional activity by these domains is either constitutive or ligand-dependent and determines the function of the nuclear receptors in development and differentiation.

Transcriptional activation domains are presumed to mediate an interaction with either components of the basal transcription machinery (33, 34, 35) or with cellular proteins distinct from basal transcription factors, termed coactivators. The interaction with coactivators may be of paramount importance in regulating physiological functions (2, 3). Several of these interacting proteins have been cloned recently and found to interact with the conserved AF-2 domain at the carboxy terminus of ligand-activated receptors. Several examples includes receptor-interacting protein 140, a coactivator of estrogen receptor [ER (36)], transcriptional intermediary factor 1, a regulator of ER, progesterone receptor (PR), retinoid X receptor (RXR), retinoic acid receptor (RAR), and vitamin D receptor (37), and cAMP response element (CREB)-binding protein (CBP), a coregulator of RXR and thyroid hormone receptor (TR) (38, 39). An additional coactivator, termed steroid receptor coactivator (SRC)-1, functions as a coactivator for ER, PR, glucocorticoid receptor (GR), TR, and RXR, as well as for several nonsteroid receptor activators such as Sp1 (40).

While the role of SF-1 in development and differentiation has been demonstrated, the mechanism of transcriptional activation by SF-1 has not been previously analyzed. Despite its ability to be activated by certain oxysterols (41), SF-1 differs from classic ligand-dependent steroid receptors, which have known high-affinity ligands that are essential for AF-2-mediated activation (42, 43, 44). Furthermore, SF-1 does not harbor a transcriptionally active amino terminus and, unlike many receptors that bind their response element as homo- or heterodimers, SF-1 binds DNA half-site sequences as a monomer (45). To identify the activation domains of SF-1, we initially characterized its AF-2 activation domain. While required for full SF-1 activity, this domain is not sufficient. An additional domain, located carboxy-terminal to the DNA-binding domain of SF-1, is also needed for gene activation. Moreover, we demonstrate that SRC-1 potentiates SF-1 activity in mammalian cells, and this interaction also requires both activation domains of SF-1.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The AF-2 Activation Hexamer (AF-2-AH) Is Critical for Transcriptional Activation by SF-1
The amino terminus of SF-1 contains only 12 nonconserved residues amino-terminal to the first zinc module of the DNA-binding domain. Therefore, to identify the activation domain of SF-1, we focused our attention on the carboxy terminus, searching for domains that are conserved between SF-1 and the carboxy-terminal region of other orphan or ligand-dependent members of the steroid receptor superfamily. At the carboxy-terminal end we found a region of six amino acids (AF-2 hexamer, see Fig. 1AGo), previously identified as a conserved activation domain in several steroid receptors (42, 43, 44, 46, 47, 48). This motif is composed of a glutamate, preceded by a variable amino acid, flanked by two pairs of hydrophobic amino acids. To test whether or not this hexamer confers transcriptional activity to SF-1, we initially deleted the entire hexamer and compared the transcriptional activity of the truncated protein (SF-1{Delta}AF-2) to the activity of wild type SF-1, using the S25 reporter gene. As shown in Fig. 1BGo, removal of the 11 carboxy-terminal amino acids of SF-1 diminished its activation capacity by 75% in both JEG3 and CV-1 cells. Interestingly, removal of the terminal 183 residues (SF-1{Delta}279) did not cause further diminution of the activation capacity of SF-1 (Fig. 1BGo), suggesting that the AF-2 hexamer is the most significant activation domain in this region of SF-1.



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Figure 1. AF-2-AH Is Required for the Transcriptional Activation Function of SF-1

A, Mutations of the SF-1 AF-2-AH and corresponding mutagenic oligonucleotides (see Materials and Methods). The conserved hexamer is highlighted and underlined. B, Transcriptional activation of an SF-1 reporter gene (S25, 0.5 µg) by transfected SF-1 constructs (0.1 µg) that harbor a mutant AF-2-AH. Deleted AF-2-AH is labeled {Delta}AF-2. Results are expressed as fold luciferase activity over baseline (mean ± SD) and represent four independent experiments, each performed in duplicate. C, Mutations of AF-2-AH or a deletion of this domain do not alter the DNA-binding capacity of SF-1. Electromobility shift assay was performed as described in Materials and Methods. Lysate is unprogrammed reticulocyte lysate, and SF-1 WT AS is an SF-1 construct cloned into pBS-KS in reverse orientation. Results represent two independent experiments.

 
Next, we determined the role of each pair of hydrophobic amino acids (leucine-leucine or methionine-leucine), as well as the conserved glutamate, in transcriptional activation by SF-1 (Fig. 1AGo). As shown in Fig. 1BGo, we found that mutation of either pair of hydrophobic amino acids (M1, M4) diminished the activation capacity of SF-1 to a level similar to SF-1{Delta}AF-2, suggesting that each pair of hydrophobic residues is essential for AF-2 function, as demonstrated for other steroid receptors (42, 44, 46). In contrast, mutation of the conserved glutamate (M3) had a weaker effect on the activation function of SF-1. Not surprisingly, mutation of the nonconserved isoleucine (M2) had no effect on activation by SF-1. A mutation of the glutamine at position 458 to either glutamate or alanine (M5) had no effect on activation by SF-1. Similar results were obtained using a human P450scc reporter construct (data not shown). These results indicate that, unlike other steroid receptors such as ER, where mutation of either pair of hydrophobic amino acids entirely abolish the activation capacity of the protein, mutation of AF-2 activation hexamer (AF-2-AH) or even a complete deletion of the hexamer renders SF-1 partly active, albeit at a low level (20–30% of wild type SF-1). This suggests the presence of an additional activation domain in SF-1.

To confirm that mutations in AF-2-AH did not alter the ability of SF-1 to bind its response element half-site, we tested the capacity of wild type and mutant SF-1 constructs to bind a double-stranded oligonucleotide probe that contains an SF-1 response element (45). As shown in Fig. 1CGo, we observed a comparable level of DNA binding by wild type and mutant receptors in an electromobility shift assay. Taken together, these results indicate that the AF-2-AH is an important activation domain of SF-1.

The AF-2-AH Is Required for SF-1-Dependent Induction of Steroidogenic Phenotype
Because SF-1 is required for development and differentiation of steroidogenic tissues, we determined whether or not AF-2-AH is important for this action of SF-1. For this purpose we used embryonic stem (ES) cells, which differentiate into steroid-producing cells when expressing SF-1 via stable transfection (49). Upon selection of SF-1-transfected ES cells, we found that they expressed cytochrome P450scc, a key enzyme in steroidogenic pathways (Fig. 2AGo), and released progesterone into the medium (Fig. 2BGo). Remarkably, neither of these phenotypic changes was observed when the SF-1 M4 mutant of AF-2-AH (Fig. 1AGo) was introduced (Fig. 2AGo-B). These data suggest that SF-1’s AF-2-AH is required for its physiological function in induction of a steroidogenic phenotype.



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Figure 2. SF-1 Requires AF-2-AH Activity for Induction of a Steroidogenic Phenotype in ES Cells

A, Northern blot demonstrating basal as well as 8-bromo-cAMP-induced expression of P450scc (SCC) in ES cells that stably express either wild type SF-1, native ES cells, or ES cells that express an AF-2-AH mutant SF-1 mAF-2 (M4, Fig. 1AGo). The 18S ribosomal RNA band is presented to illustrate equal loading. B, Progesterone production by ES cells that express SF-1 constructs according to the paradigms described in panel A. Results, expressed as mean ± SD, represent four independent experiments, using three independent ES cell clones.

 
AF-2-AH Is Required, but not Sufficient for the Activation Function of SF-1
The AF-2-AH bestows SF-1 with the ability to activate transcription. To test whether AF-2–AH is sufficient for this activity, we tested for the independent activation capacity of the carboxy-terminal region of SF-1, which contains the AF-2-AH. For this purpose we fused the carboxy-terminal 35 amino acids, which include AF-2-AH, to the DNA-binding domain of GAL4 (GAL41-147, Fig. 3AGo) and tested for its transcriptional activation capacity in JEG3 cells using a reporter ({Delta}GKI) that contains GAL4- binding sites. As shown in Fig. 3BGo, the activity of GAL4-SF-1428 was similar to that of GAL4 alone, suggesting that the AF-2-AH containing SF-1428 was not functional as an independent activation domain. We obtained a similar result when longer fragments of SF-1 (residues 279–462 or 245–465) were fused to GAL4. In contrast, we observed a strong activation of the GAL4 reporter gene by GAL4-SF-1187 and GAL4-SF-1119. As control, we identified a similar activation by GAL4-ER282-595 (which contains the carboxy-terminal region of ER) in the presence of E2. Thus, in addition to AF-2-AH, there is a second critical component of SF-1 activation function, located between residues 187–245. Next, to determine whether AF-2-AH is required for the activity of this domain, we tested for the activation capacity of GAL4-SF-1119{Delta}AF-2, or GAL4-SF-1119mAF-2, which contain a mutation in the terminal pair of hydrophobic residues within AF-2-AH (M4, Fig. 1AGo). The activation of the GAL4 reporter by either mutant was diminished by 70–80% (Fig. 3BGo). Taken together, these results indicate that AF-2–AH is required for the activation capacity of SF-1, but is not sufficient for this function, and requires an additional activation domain, which is located further upstream in the carboxy-terminal domain of SF-1 (between residues 187–245).



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Figure 3. Transcriptional Activation by SF-1 Requires AF-2-AH and an Additional Activation Domain

A, A schematic diagram depicting deletion fragments of the carboxy-terminal domain of SF-1, fused to the DNA-binding domain of GAL4 (residues 1–147). Deleted AF-2-AH is labeled {Delta}AF-2. B, Activation of a GAL4 reporter construct ({Delta}GKI, 0.5 µg) in JEG3 cells by transfected GAL4-SF-1 fusion constructs, depicted in panel A. Activation by GAL4-ER is used as a positive control. Results are expressed as relative luciferase activity (mean ± SD) and represent three independent experiments, each performed in duplicate.

 
SRC-1 Interacts with SF-1 and Potentiates Its Activity through AF-2-AH and the Proximal Activation Domain
SRC-1 has been recently identified as a ligand-dependent coactivator of several ligand-dependent steroid receptors [such as ER and PR (40, 50)]. To determine whether or not SF-1 interacts with SRC-1 in a manner dependent upon AF-2-AH, we used the two-hybrid system in mammalian cells to identify the domains within SF-1 that are required for interaction with SRC-1. For this purpose, we transiently transfected CV-1 cells with GAL4-SF-1119, along with the interacting domain of SRC-1 (residues 857-1061, 40 , fused to the activation domain of VP16 (SRC-1-VP16). Using a coexpressed GAL4 reporter, we observed a dramatic enhancement of GAL4-SF-1119-dependent activity, as the concentration of SRC-1-VP16 was increased (Fig. 4AGo). This increase was similar to that seen with a fusion of GAL4 and the carboxy-terminal region (residues 282–595) of ER (not shown). This result indicates that SRC-1 interacts with the carboxy-terminal, autonomously active region of SF-1.



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Figure 4. The Interaction between SF-1 and SRC-1 Requires Both Activation Domains of SF-1

A, The concentration-dependent interaction between SF-1 and SRC-1 (residues 857-1016, fused to the activation domain of VP16) is detected using a two-hybrid system in CV-1 cells. The GAL4 reporter gene ({Delta}GKI, 0.5 µg) is activated by transfected GAL4-SF-1119 (0.2 µg). B, The interaction between SF-1 and SRC-1-VP16 (0.7 µg) requires SF-1’s AF-2-AH. C, The interaction between SF-1 and SRC-1-VP16 in a two-hybrid system requires the proximal activation domain of SF-1, located between residues 187–245. Results are expressed as relative luciferase activity (mean ± SD) and represent three independent experiments, each performed in duplicate. D, Schematic diagram depicting currently identified domains within SF-1.

 
Because we previously determined that AF-2-AH is required for activation by SF-1, we tested whether or not AF-2-AH is required for the interaction between SF-1 and SRC-1. We transfected CV-1 cells with either GAL4-SF-1119 or a similar construct that contains a mutated AF-2-AH (M4, Fig. 1AGo), along with the SRC-1-VP16 vector, and found that the interaction between SRC-1 and SF-1 absolutely requires an intact AF-2-AH (Fig. 4BGo). Similarly, to test whether the proximal activation domain is required for the interaction between SF-1 and SRC-1, we transfected CV-1 cells with SRC-1-VP16, along with a series of SF-1 fragments, fused to GAL4. We observed that the interaction of SF-1 with SRC-1 in the two-hybrid system was abrogated when residues between 187–245 were deleted from the SF-1 construct (Fig. 4CGo). Together, these results indicate that both AF-2-AH and the proximal activation domain (between residues 187–245) are required for the activation capacity of SF-1, as well as for interaction with the coactivator SRC-1. The relative positions of these domains are indicated in Fig. 4DGo.

To determine whether these activation domains of SF-1 are required for potentiation by SRC-1, we transiently transfected CV-1 cells with SF-1, in the presence or absence of human SRC-1. For a positive control we used ER (and its cognate reporter, Vit2P36-LUC), as SRC-1 has been previously shown to synergize with ER in regulation of estrogen-responsive reporter genes (50). As shown in Fig. 5AGo, the addition of SRC-1 potentiated the transcriptional activity of holo-SF-1 on its reporter construct S25, while SRC-1 alone had no effect on promoter activity. Therefore, SRC-1 is a coactivator for SF-1. Next, we determined whether AF-2-AH and the proximal activation domain are both required for potentiation of SF-1 activity by SRC-1. Using GAL41-147 fusions of the SF-1 carboxy terminus we found that the same SF-1 domains required for autonomous activation capacity, and for two-hybrid interaction with SRC-1-VP16, were also required for potentiation by intact SRC-1 (Fig. 5BGo). Therefore, the transcriptional activity of SF-1 depends on both the proximal activation domain and AF-2-AH. The interaction of these domains with SRC-1 may provide the mechanism for regulation of SF-1 signaling by SRC-1 in vivo.



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Figure 5. SRC-1 Potentiates Transcriptional Activation by Wild Type SF-1

A, CV-1 cells were transfected with CMV-SF-1 expression vector (0.1 µg), in the presence or absence of a human SRC-1, cloned in a pCR vector (1 µg), and S25 reporter (0.5 µg). ER, in the presence of E2 (10-8 M), was used as a positive control (using the ER reporter Vit2P36-LUC). B, Potentiation of SF-1 activity by SRC-1 requires AF-2-AH and the proximal activation domain. CV-1 cells were transfected with GAL4 reporter gene and GAL41-147 fusions to SF-1 carboxy terminus as above. SRC-1 expression vector (1 µg) was cotransfected where indicated. Results are expressed as relative luciferase activity (mean ± SD) and represent two independent experiments, each performed in duplicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we have dissected the transcriptional activation function of SF-1. Like other members of the steroid receptor superfamily, SF-1 is composed of modular functional domains. The DNA-binding domain is highly homologous to other steroid receptors, and its interaction with half-site sequences has been previously described (45). In the absence of a functional AF-1 region in the amino terminus, our analysis of the activation function of SF-1 focused on its carboxy terminus, which harbors a conserved AF-2-AH sequence of six amino acids. We have clearly shown that this AF-2 hexamer is required for transcriptional activation by SF-1. This was demonstrated using a synthetic SF-1 reporter gene, P450scc promoter, as well as induction of differentiation in ES cells (49), where SF-1 requires AF-2-AH to promote the expression of P450scc and progesterone production.

Although SF-1’s AF-2-AH is conserved among other steroid receptors (42, 43, 44, 46, 47, 48), there are two features that distinguish the AF-2-AH in SF-1: 1) Unlike the AF-2 of other ligand-dependent steroid receptors (such as ER) where mutations of either pair of hydrophobic amino acids within AF-2-AH entirely abolished the transcriptional activity of the protein, mutations in SF-1’s AF-2-AH only diminished its activity to 25–30% of wild-type SF-1. 2) A replacement of the conserved glutamate with glutamine completely disrupts the activity of the chick T3R{alpha} (43), yet a similar replacement in SF-1 only diminished its activity by 50%. Interestingly, a replacement of this pivotal residue with alanine had no effect on the activation capacity of ER (42). As shown for other steroid receptors, none of these mutations altered the DNA- binding capacity of SF-1. These differences between SF-1 and ligand-dependent steroid receptors may be related to the ligand independence of SF-1. Alternatively, these differences may specify the interaction of AF-2-AH with other domains within SF-1 or with a distinct repertoire of coactivators.

While essential for transcriptional regulation by SF-1, the AF-2-AH of SF-1 does not exhibit an independent activation domain when fused to a GAL4 DNA-binding domain. In contrast, a region of 35 amino acids that span the AF-2 hexamer of T3R{alpha} exhibits a strong transcriptional activity when fused to GAL4 (43). Our results establish that the AF-2-AH of SF-1 is necessary, but insufficient, for transcriptional activation, and domains located further upstream (residues 187–245) are essential for the transcriptional activity of SF-1. Several steroid receptors contain additional activation domains amino-terminal to the ligand-binding domain. For example, two proximal activation domains ({tau}2, {tau}3) were identified in TR{alpha}, and homologous sequences have been found in RAR and RXR (46). Similarly, a proximal transcriptional activation function resides between residues 181–310 in the SF-1 homolog xFF1rA (51). These activation domains are capable of independent transcriptional activation when fused to GAL41-147. The proximal activation domain of SF-1 is also capable of activating a reporter gene even in the absence of the AF-2-AH, albeit at a markedly reduced potency. These results suggest that AF-2-AH and the proximal activation domains synergize in their activation function. This synergy may be achieved through intramolecular interaction between complementary domains or may furnish a platform for complex interaction with coactivators. Importantly, the proximal activation domain of SF-1 (amino acids 187–245) does not appear to share significant homology with any protein other than the closest relatives of SF-1, such as LRH-1 (FTF, PHR) and xFF1rA.

It has been proposed that coactivators are needed for bridging between the steroid receptor and components of the basal transcriptional machinery (52). This has been shown for RAR, which requires a E1A-like factor to interact with transcription factor IID (53). Similarly, the SWI/SNF family of yeast proteins are components of the basal transcription machinery that enhance transcription by the steroid receptors (54). SRC-1 is a recently identified coactivator for several steroid receptors that can associate with nuclear receptor-CBP complexes (see below), but the exact mechanism by which it potentiates transcriptional activation by steroid receptors is presently unknown. SRC-1 interacts with PR and ER and enhances the transcriptional activation of PR, ER, TR, GR, and RXR in the presence of their respective agonists (40, 52). It forms a family of proteins with ER-associated protein 160, which interacts with ER, RARß, and RXR{alpha} (55), as well as GR-interacting protein 1 (transcriptional intermediary factor 2), a protein that interacts with GR, ER, AR, RAR, and RXR in a ligand-dependent fashion in yeast and mammalian cells (56, 57). Unlike the SRC-1 association with most of these receptors, the interaction between SF-1 and SRC-1 may be ligand-independent, as seen with some steroid receptor coactivators (58). Our results indicate that SRC-1 potentiates the activation function of SF-1 in mammalian cells. The AF-2-AH of SF-1 is required for SF-1 interaction with SRC-1, as the interaction is abolished in the presence of a mutated AF-2-AH. However, AF-2-AH is not sufficient for this interaction and requires an additional, amino-terminal region of SF-1, located between residues 187–245. These data suggest that SRC-1 requires the correct conformation of the two activation domains to interact with SF-1, allowing signaling to the basal machinery or to additional coactivators. Interestingly, a GAL4-SF-1119 construct that is devoid of functional AF-2-AH does not interact with SRC-1, yet it retains some transcriptional activity (Fig. 3BGo). This suggests that additional coactivators may interact with proximal regions of SF-1.

The two-hybrid data presented herein demonstrate a functional interaction between SF-1 and SRC-1. However, our data do not prove that the interaction of the two proteins is direct, and it is possible that a complex of coactivating proteins is required for the functional interaction between SF-1 and SRC-1 to occur. Indeed, the coactivator CBP/p300 has been shown to participate in nuclear receptor-SRC-1 complexes and, in this case, could bridge SF-1 to SRC-1 (39, 59, 60, 61). Nevertheless, two lines of evidence argue for the direct interaction between SF-1 and SRC-1 in our two-hybrid assays. First, the interactive fragment of SRC-1 we employed for our two-hybrid assays contains none of the CBP/p300 interactive region (60). Second, using residues 1–450 of CBP in an analogous two-hybrid assay, we found that SF-1 and CBP do not interact in CV-1 cells (data not shown). Thus, because CBP/p300 does not appear to directly interact with SF-1, endogenous CBP probably does not bridge SF-1 to SRC-1 in the CV-1 two-hybrid assay. While CBP and SF-1 do not directly interact in CV-1 cells, it is still possible that an SF-1-SRC-1 interaction permits a ternary complex among SF-1, SRC-1, and CBP in vivo.

That SRC-1 plays a role in SF-1-mediated differentiation or function of steroidogenic cells remains to be determined. This is plausible, as a naturally occurring mutation within the AF-2-AH of human TRß disrupts interaction with SRC-1, implying that SRC-1 transduces the biological activity of TR (62). The results presented here demonstrate a clear correlation between the activation function of SF-1 and its interaction with SRC-1, implying that SRC-1 may be a primary determinant of SF-1 activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The SF-1 expression vector [cytomegalovirus(CMV)-SF-1] was generated by sequential subcloning of the SF-1 cDNA (a gift from K. Parker, Duke University, Durham NC) into the EcoRI sites of pBS-KS (generating pBS-SF-1 and pBS-revSF-1, cloned in reverse orientation), then using the KpnI and XbaI sites from the BS polylinker to clone into pCMV-Neo. Using PCR, we generated SF-1{Delta}AF-2 by placing a stop codon flanked by a 3'-EcoRI site downstream from residue 451, thereby terminating SF-1 immediately upstream from the AF-2-AH. The amplified EcoRI fragment was cloned into pBS-KS and pCMV-Neo as described above for full-length SF-1. CMV-SF-1{Delta}279 was generated by SalI digestion of pBS-revSF-1 and subcloning the EcoRI fragment of the truncated SF-1 cDNA back into pBS, then using the KpnI and XbaI sites as detailed above to clone into pCMV-Neo.

For mutagenesis of the SF-1’s AF-2-AH we used an inverse PCR-based site-directed mutagenesis, using pBS-SF-1 as a template. The forward primer harbored the mutations depicted in Fig. 1AGo and abutted a reverse primer that encoded a wild type sequence. Using T4 kinase we phosphorylated the reverse primer, then used KlenTaq (63) in ten cycles of amplification with the following parameters: 94 C for 0.5 min, 56 C for 2 min, 72 C for 5 min. The product was treated with 200 µg/ml proteinase K for 30 min at 56 C, then phenol-chloroform-extracted, ethanol-precipitated, treated with Pfu polymerase (Stratagene, La Jolla, CA) at 37 C for 30 min, then ligated overnight. To remove the original template, the ligation mix was treated with DpnI for 1 h and transformed into XL-1 blue bacteria. The authenticity of the mutant constructs was confirmed using the dideoxynucleotide sequencing method on an Applied Biosystems (Foster City, CA) model 373A DNA sequencer. All SF-1 constructs used for transfection of ES cells were cloned as an EcoRI fragment (from pBS) into the vector pCAGGS, which uses the cytomegalovirus immediate early enhancer and the chicken ß-actin promoter and first intron enhancer, as described elsewhere (49).

We used PCR to generate fusion proteins between the DNA-binding domain of GAL4 (1–147) and carboxy-terminal fragments of SF-1. Utilizing a BamHI-linked forward primer, a T3 reverse primer, and pBS-revSF-1 as a template, we amplified the desired fragments of SF-1 (residues 428–462, 279–462, 245–462, 187–462, 119–462, and 119–452), digested with BamHI and HindIII, and cloned downstream from GAL4 in pM2 vector (64). The correct frame of each chimeric protein was verified by sequencing. GAL4-ER282-595 was cloned in a similar fashion, using pBS-ER (a gift from Stuart Adler, Washington University) as a template.

A human SRC-1 expression vector (pBK-SRC1) was kindly provided by M. J. Tsai and B. W. O’Malley (Baylor College of Medicine, Houston, TX). To generate the SRC-1-VP16 fusion we amplified the receptor-interacting region of SRC-1 [corresponding to amino-acids 857-1061 (see Refs. 40 and 57); amino acids 1243-carboxy-terminus (see 60 ], and cloned it in frame upstream of the activation domain of VP16 (amino acids 413–440).

The SF-1 reporter construct (S25) contains the PRL minimal promoter downstream from two SF-1-binding elements (TCAAGGTCA) separated by five nucleotides, upstream from the luciferase gene. The hP450scc-luciferase construct, which includes 2327 bp of the human cytochrome P450scc promoter (65), was kindly provided by W. L. Miller (University of California, San Francisco). The GAL4 reporter gene ({Delta}GKI), which contains five GAL4- binding sites upstream of an E1B-TATA box, linked to luciferase, was kindly provided by P. Webb and P. J. Kushner (University of California, San Francisco). The ER reporter construct (Vit2P36-LUC) was previously described (66).

Cell Culture and Transfection
CV-1 cells are maintained in DMEM that contains 10% FBS and antibiotics at 37 C and 10% CO2. JEG3 cells are maintained in MEM that contains 10% FBS and antibiotics at 37 C and 5% CO2. All tissue culture reagents were obtained from the Tissue Culture Support Facility, Washington University School of Medicine (St. Louis, MO). RW4 mouse strain 129/SvJ embryonic stem (ES) cells are maintained on a feeder layer of murine embryonic fibroblasts, as described elsewhere (49).

One day before transfection, CV-1 or JEG3 cells were plated in either six- well plates at a density of 125,000 cells per well, or in 12-well plates at a density of 60,000 cells per well. Four hours before transfection the standard growth medium was replaced by fresh DMEM that contained additives as above, and the cells were incubated in 10% CO2 at 37 C. Transfection was performed using the standard calcium-phosphate precipitation method previously described (67) in six- and 12-well plates using 2.5 and 0.6 µg total DNA, respectively, which included 0.1 µg CMV ß-gal plasmid (to normalize for cell viability and transfection efficiency). Luciferase assay was performed 40–48 h after transfection. Cells were lysed in a lysis buffer that contains 50 mM Tris-2-(4-morpholino)-ethane sulfonic acid (pH 7.8), 1 mM dithiothreitol, and 1% Triton X-100. Lysates were assayed for luciferase using a luminometer (Monolight 2010 Analytical Luminescence Laboratory, San Diego, CA), and for ß-GAL using a 96-well plate reader (anthos htIII, Anthos Labtec, Salzburg, Austria). All experiments were performed in duplicate and repeated at least three times. Results (mean ±SD), normalized to ß-GAL activity, were expressed as relative luciferase units. In experiments in which ER was used we used phenol-red free medium with serum that contains negligible levels of E2.

For stable transfection of ES cells, 5 x 106 cells were electroporated with 25 µg plasmid DNA as described elsewhere (49). Transfected cells were plated on 100-mm tissue culture dishes coated with a confluent layer of murine embryonic fibroblasts, and 300 µg/ml G418 or 1 µg/ml puromycin were administered the following day for 5 days. On the sixth day, colonies were picked and expanded. For stimulation experiments, ES cells were plated at 5 x 104 cells per gelatinized well of 24-well plates in 0.5 ml ES media. After 1 day, cells were given 5 µg/ml 20{alpha}-hydroxycholesterol in the presence or absence of 1 mM 8-bromo-cAMP.

Electromobility Shift Assay
Wild type or mutant SF-1 cDNA was transcribed and translated from a pBSKS expression vector, using a TnT reticulocyte lysate system (Promega, Madison, WI). A double-stranded oligonucleotide (100 ng) that contained the SF-1 response element [TCAAGGTCA in tandem (45)], was end-labeled by 20 µCi [{gamma}-32P]ATP, using polynucleotide kinase. For each binding reaction 4 µl of the translation mixture were mixed with 1 ng labeled probe in a binding buffer (68) and incubated for 30 min at 25 C. Each binding reaction was loaded onto a 5% polyacrylamide gel, run in 0.5x TBE buffer at 150 V for 3 h. The gel was then dried and exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) screen or film.

RNA Analysis and Progesterone Measurement
RNA was extracted by the guanidinium thiocyanate-acid phenol method (69). Total RNA (15 µg) was electrophoresed on a denaturing gel of 1% agarose and 1.5% formaldehyde and blotted onto Zeta-Probe GT membranes (Bio-Rad, Hercules, CA). The blot was probed with a P450scc probe (a PCR product that spans the first 300 bp of the mouse coding sequence), which was labeled with [{alpha}-32P]-dCTP, as previously described (70). Membranes were hybridized for 16 h at 45 C, then washed with 0.2x SSC (1xSSC is 0.15 M NaCl, and 0.075 M sodium citrate) and 0.1% SDS for 20 min at 68 C, and analyzed by PhosphorImager.

For progesterone assay, media were harvested 24 h after the addition of hormones, and progesterone was determined by RIA using the Coat-a-Count system (Diagnostic Products Corporation, Los Angeles, CA). Inter- and intraassay coefficients of variation were 5.1% and 2.6%, respectively.


    ACKNOWLEDGMENTS
 
We thank K. L. Parker (Duke University, Durham, NC), M. J. Tsai, and B. W. O’Malley (Baylor University, Houston, TX), P. Webb, P. J. Kushner, W. L. Miller (University of California, San Francisco, CA), and S. Adler (Washington University, St. Louis, MO) for plasmids, and E. Sadovsky and J. Willand (Washington University) for technical assistance. We also thank J. Milbrandt for insightful discussions and suggestions in preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Yoel Sadovsky, M.D., Department of Obstetrics and Gynecology, Washington University School of Medicine, Box 8064, St. Louis, Missouri 63110.

This work was supported, in part, by NIH Grant HD-34110 and the Berlex Scholar Award (both to Y.S.).

Received for publication May 7, 1997. Revision received July 7, 1997. Accepted for publication July 17, 1997.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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