Characterization of the Promoter of SF-1, an Orphan Nuclear Receptor Required for Adrenal and Gonadal Development

Karen G. Woodson1, Peter A. Crawford1, Yoel Sadovsky1 and Jeffrey Milbrandt

Division of Laboratory Medicine Departments of Pathology and Internal Medicine (K.G.W., P.A.C., J.M.) Department of Obstetrics and Gynecology (Y.S.) Washington University School of Medicine St. Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenic factor 1 (SF-1) is a transcription factor shown to be critical for regulation of adrenal and gonadal development and function. To dissect the mechanisms that direct expression of this regulator, we have studied the promoter of the SF-1 gene and have identified cis-acting elements that recognize a basic-helix-loop-helix transcription factor; the CAAT binding factor; and Sp1. We demonstrate in Y1 adrenocortical cells that a 90-bp proximal promoter fragment is sufficient to direct steroidogenic-specific expression and that all three elements are required for activity of the SF-1 promoter. Functional analysis of the binding sites on a heterologous TATA box-containing promoter demonstrates that the CAAT box and Sp1 site are not essential for promoter activity when a TATA box is present, whereas the E box is absolutely required for gene expression and is most likely the steroidogenic cell-specific element. We also demonstrate that SF-1 itself does not significantly affect the transcription of its own gene, and thus conclude that the E box, CAAT box, and Sp1 site of the proximal promoter direct expression of the SF-1 gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenic factor 1 (SF-1) is a member of the nuclear hormone receptor superfamily of transcription factors. Members of this superfamily, which coordinate aspects of growth, development, and metabolism, include receptors for steroid and thyroid hormones, vitamin D, and retinoic acid. These receptors bind to specific cis-acting DNA sequences called hormone response elements and modulate gene expression, usually in the presence of their respective ligands (1). Because no ligand has been isolated for SF-1, it is termed an orphan nuclear receptor. SF-1 binds DNA as a monomer to a sequence element comprised of an estrogen receptor half-site (5'-AGGTCA) and three specific 5'-adjacent nucleotides (2). In the adult mouse, SF-1 is constitutively expressed in all steroidogenic tissues including the cortical cells of the adrenal gland, Leydig cells of the testis, and thecal and granulosa cells of the ovary (3, 4). It is thought to be a key regulator of steroid hormone biosynthesis as it has been shown to bind and transactivate the promoters of cytochrome P450 steroid hydroxylases that convert cholesterol to the various steroids (2, 3, 5, 6).

SF-1 is the murine homolog of the Drosophila orphan nuclear receptor FTZ-F1, a putative regulator of the fushi tarazu homeobox gene during embryonic development (7, 8). Similarly, SF-1 expression is developmentally regulated in mammals. SF-1 is expressed in the urogenital ridge before its differentiation into kidneys, adrenal glands, and gonads (9), and it is also expressed in the developing hypothalamus and in the gonadotropes of the anterior pituitary, where it regulates gonadotropin synthesis (10, 11). The developmental function of SF-1 was established when mice with a targeted disruption of the SF-1 gene were found to lack adrenal glands and gonads, resulting in neonatal lethality presumably due to adrenocortical insufficiency (11, 12, 13). Moreover, analysis of staged embryos of SF-1-deficient mice showed that gonadal precursors were initially present, but later regressed by programmed cell death (12).

Taken together, these observations extend SF-1’s role beyond steroid hormone biosynthesis to that of a key regulator of adrenal gland and gonadal development. However, little is known regarding the molecular mechanisms that control the tissue and developmental, stage-specific expression of the SF-1 gene. Here we show that the promoter of SF-1 contains at least three elements that are essential for promoter activity. The first element is an E box, which is recognized by factors that belong to the basic-helix-loop-helix (bHLH) family of transcription factors (14). These factors are thought to regulate many developmental programs such as muscle differentiation, neurogenesis, hematopoiesis, and sex determination (15, 16). The second element is a CAAT box, a common proximal promoter element that is recognized by a number of proteins (17, 18). The third is a GA-rich element, which is shown to bind the transcription factor Sp1.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence Analysis of the Mouse and Human SF-1 Promoters
To identify sequence elements important for the expression of SF-1, genomic clones containing either the mouse or human SF-1 genes were isolated. The nucleotide sequences of the human and mouse 5'-flanking regions, which are very similar, were analyzed for potential transcription factor binding sites (Fig. 1Go). In particular, the sequence of the -93 to -38 region is almost identical (54/55 bases). This region harbors a potential E box (5'-CANNTG) (19), as well as an element that could function as an E box or CAAT box, and a GA-rich region that contains potential sites for Ets (20), GAGA (47), or the Sp1 (22) families of transcription factors. Although no recognizable TATA element exists, there is a putative transcription initiator element (23) (5'-PyPyA+1NT/APyPy), which overlaps the transcription start site as previously determined (24).



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Figure 1. Comparison of Nucleotide Sequences of SF-1 5'-Flanking Region between Human and Mouse

Nucleotides that are identical between the mouse and human 5'-flanking sequences are indicated by vertical bars, and nucleotide deletions (alignment gaps) are indicated by dashes. The transcriptional start site (+1) (24) is indicated by the arrow, and the nucleotides are numbered relative to this position. Potential regulatory elements (an E box, an E and/or CAAT box, and a GA-rich element) are boxed. The sequence contains a potential initiator element spanning (+1) (23), but not a recognizable TATA box.

 
Deletion Analysis of the SF-1 Promoter
To localize the functional regions of the mouse SF-1 promoter, we generated a series of luciferase reporter constructs that contained deletions of the 5'-flanking regions with 5'-termini at nucleotide (nt) -1885, -712, -190, -90, and -12 and a common 3'-terminus at +140. The promoter activities of the 5'-deletion mutants were assessed by luciferase assays after transient transfection into two separate steroidogenic cell lines, Y1 (mouse adrenal cortex) and DC3 (rat ovarian granulosa), both of which express SF-1 (our unpublished data). A promoter fragment spanning nucleotide (nt) -90 to +140 (SF-1-90Luc) was sufficient to drive transcription in Y1 and DC3 cell lines at levels of ~100-fold over the promoter-less luciferase reporter (Fig. 2Go). Addition of further upstream flanking sequences up to nt -1885 did not significantly increase the activity of the -90 fragment, whereas deletion of sequences to nt -12 abolished activity. In contrast, when the promoter fragments were tested in NIH-3T3 fibroblasts, which are nonsteroidogenic and do not express SF-1, no promoter activity was observed (Fig. 2Go). These results indicate that the region between nt -90 and -12 contains regulatory elements that are required for steroidogenic cell-specific expression of SF-1.



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Figure 2. Deletion Analysis of the SF-1 Promoter

A schematic representation of the 5'-flanking region of the mouse SF-1 gene is shown. The locations of the relevant restriction sites used for construction of the serial deletion mutants are indicated. The SF-1 promoter/luciferase reporter constructs were named according to the nucleotide positions of their 5'-termini. All constructs were transiently transfected into Y1 adrenocortical cells, DC3 granulosa cells, and NIH-3T3 fibroblasts; luciferase assays were performed a minimum of three times as described in Materials and Methods. Mean luciferase activity (± SD) of each construct in all three cell lines is given relative to the promoter-less vector (pLuc).

 
Identification of cis-Acting Functional Elements in the SF-1 Promoter
Deletion analysis of the SF-1 promoter demonstrated that the region between nt -90 and -12 was essential for promoter activity. To functionally identify cis-acting regulatory elements, we generated a series of substitution mutations spanning this region within the context of the -90Luc construct (Fig. 3Go). We found that mutations in three different regions, at nt -84 to -75 (m1, a potential E box), nt -68 to -59 (m2, a potential E or CAAT box), and nt -30 to -24 (m4, GA-rich region), reduced promoter activity to 10% to 20% of wild type levels, whereas mutation of nt -40 to -31 (m3) and nt -26 to -17 (m5) appeared to have no effect. These mutations yielded a similar effect when incorporated into the -1885-Luc construct (Fig. 3Go).



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Figure 3. Mutational Analysis of the SF-1 Promoter

Substitution mutations of the -90 to -17 region were made in the SF1-90Luc construct. The indicated mutants are identical to the wild type sequence with the exception of the sequences shown. The effect of these mutations in the context of the larger promoter fragment is also shown. Constructs were transiently transfected into Y1 adrenocortical cells, and luciferase activities were measured. Results shown are the mean (± SD) of three independent experiments, calculated as above.

 
Because many promoters associated with steroidogenesis are regulated by SF-1 in cultured cells, we assessed the ability of SF-1 to transactivate two mouse SF-1 promoter fragments in CV-1 monkey kidney cells and Y1 adrenocortical cells: -1885-Luc, which spans nt -1885 to +140; and -3100-Luc, which spans nt -3100 to +3600. The latter fragment traverses the gene’s first intron and extends to the natural ATG of the mouse SF-1 gene, thus containing the SF-1-binding site within the first intron (25). Although previous work on the rat SF-1 promoter has shown autoregulation of the SF-1 promoter by its own product via this SF-1 binding site (25), neither of the SF-1 promoter fragments tested here was activated by coexpression of SF-1 in CV-1 cells (Fig. 4Go). Cotransfection of the SF-1 expression vector with an SF-1 responsive luciferase reporter in CV-1 cells yields more than 20-fold activation (data not shown), indicating the functional integrity of our transfected SF-1. In addition, transfection of these two SF-1 promoter constructs into Y1 cells, which constitutively express SF-1, yielded equivalent results, despite the presence of an SF-1-binding site in the -3100-Luc construct (Fig. 4Go). These data further indicate that SF-1 does not autoregulate its expression.



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Figure 4. Effect of Additional 5'-Flanking Sequences, Including an SF-1 Site, on Activity of the SF-1 Promoter

Diagrams schematically represent the promoter/luciferase reporter construct used in transient transfection of CV-1 and Y1 cells. In CV-1 transfections, 50 ng of the nonrecombinant or CMV-SF-1 expression vector were cotransfected with the reporter. In Y1 transfections, reporters were transfected in the absence of any expression vector. Results presented (mean ± SD) are representative of two independent experiments, calculated as above.

 
The Functional Element at nt -83 to -74 Is an E Box
To identify potential transcription factors that interact with the identified elements, we performed gel mobility shift analysis. Inspection of the functionally important regions reveals that the -83 to -74 region contains a putative E box; the -68 to -59 region contains a palindromic (5'-CCAATTGG) sequence, which could potentially be an E box, CAAT box, or both; and a GA-rich region at nt -30 to -24 (see below). Gel mobility shift assays using Y1 nuclear extracts and an oligonucleotide containing nt -87 to -49 shows two specific complexes (Fig. 5AGo, lane 1, designated Complex I and II). Complex I is formed by an interaction with the -83 to -74 region as a similar complex was observed using an oligonucleotide probe spanning only nt -87 to -69 (lane 5). In addition, formation of Complex I was abolished when competed with the -87 to -69 region oligonucleotide (lane 3). Complex II was formed by an interaction with the -68 to -59 region, as an oligonucleotide spanning nt -73 to -49 effectively competed for the species (lane 2), and when used as a probe, a similar complex was observed (lane 4).



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Figure 5. Gel Shift Analysis of the E Box in the SF-1 Promoter

The DNA sequences of all oligonucleotides are shown in Table 1Go. A, 32P-labeled oligonucleotides corresponding to SF-1 nucleotides (nt) -87 to -49 (lane 1), nt -73 to -49 (lane 4), and nt -87 to -69 (lane 5) were incubated with nuclear extract prepared from Y1 cells, as well as 100-fold molar excess of unlabeled SF-1 nt -73 to -49 oligonucleotide (lane 2), or SF-1 nt -87 to -69 (lane 3) oligonucleotide, as indicated. B, Gel shift analysis of the SF-1 -87 to -49 region probe using Y1 nuclear extracts incubated in the presence of the 100-fold molar excess of the indicated E box mutant. C, Gel shift analysis of the SF-1 -87 to -49 region probe using nuclear extracts from Y1 and NIH-3T3 cells. The free probe did not remain on the gel due to the longer electrophoresis time required to achieve appropriate resolution.

 
To confirm that Complex I interacts as an E box, we performed competition analysis with mutant E box competitors (Fig. 5BGo). Complex I is effectively competed with the nt -87 to -69 mutant oligonucleotides that contain mutations flanking immediately outside of the E box (E box Em3, E box Em4) but not with those in which the E box motif has been mutated (E box Em1, E box Em2) (Table 1Go and Fig. 5BGo). Furthermore, when the mutant E box oligonucleotides were labeled and used in gel shift assays, no specific complex was observed (data not shown).


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Table 1. Sequences of Oligonucleotides Used in This Study

 
To determine whether proteins that bind the SF-1 promoter are specific to steroidogenic cells, we also used nuclear extracts from NIH-3T3 cells in mobility shift assays. Despite the complete inactivity of this promoter fragment in NIH-3T3 cells (Fig. 2Go), we found two species corresponding in mobility to those obtained from Y1 nuclear extracts (Fig. 5CGo). Thus, the presence of proteins that bind these sequences within the SF-1 promoter does not imply that the SF-1 gene is active in these cells.

The Functional Element at nt -68 to -59 Binds the CAAT-Binding Factor (CBF)
The region between nt -68 to -61 (5'-CCAATTGG) could function as an E box, a CAAT box, or both. To distinguish these possibilities, gel shift assays were performed using mutant oligonucleotides that would distinguish between these two motifs (Fig. 6Go). The first mutant oligonucleotide tested altered 5'-CCAATTG to 5'-ACAATTG, thus maintaining the E box but destroying the CAAT box (E box +). A second mutant was tested in which the 5'-CCAATTG was changed to 5'-CCAATCG, thus maintaining the CAAT box but mutating the E box (CAAT+). Using the wild type nt -73 to -49 probe, Y1 nuclear extracts, and these mutant oligonucleotides as competitors, it is clear that complex II binds as a CAAT box rather than as an E box (Fig. 6Go). To determine whether the factor responsible for the shift of the CAAT box was a previously characterized protein, we performed competition analysis with oligonucleotides containing consensus binding sites for the CAAT box-binding proteins C/EBP, NF-1/CTF, and CBF/CP1 (26, 27, 28). As shown in Fig. 6Go, the CBF site was an effective competitor that abolishes formation of this complex, whereas the consensus CTF and C/EBP oligonucleotide competitors did not compete even at levels of 100-fold molar excess (Fig. 6Go). Finally, we performed a supershift assay with an anti-CBF antibody, which completely supershifted complex II, thus confirming that CBF (or a related protein) interacts with the nt -68 to -59 element of the SF-1 promoter. The preimmune serum control had no effect upon the complex, and the specificity of the antiserum was demonstrated by its nonreactivity against an unrelated complex (data not shown).



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Figure 6. Gel Shift Analysis of the CAAT Box in the SF-1 Promoter

The DNA sequences of all oligonucleotides are shown in Table 1Go. A 32P-labeled oligonucleotide corresponding to SF-1 nt -73 to -49 was incubated with Y1 nuclear extract in the presence or absence of either 10- or 50-fold molar excess of the indicated competitor oligonucleotides: control without competitor; E box +, SF-1 nt -73 to -49 mutant in which the E box was maintained but the CAAT box was destroyed; CAAT+, SF-1 nt -73 to -49 mutant in which the CAAT box was maintained but the E box was destroyed; C/EBP consensus binding site (27); CTF/NF-1 consensus binding site (26); CBF consensus binding site (28). Supershift assay with anti-CBF: SF-1 nt -73 to -49 probe in the absence and presence of anti-CBF polyclonal antibody; CBF consensus site probe in the absence and presence of anti-CBF antibody. The free probe did not remain on the gel due to the longer electrophoresis time required to achieve appropriate resolution.

 
The Functional Element at -30 to -24 Binds Sp1
As noted above, the GA-rich sequence that occupies nucleotides -30 to -24 of the SF-1 promoter could potentially bind transcription factors such as Ets (20), GAGA (47), and Sp1 (22). Gel mobility shift assays using Y1 nuclear extracts and an oligonucleotide spanning nt -40 to +12 reveals a single specific complex (Fig. 7Go, lane 1). The complex is effectively competed by its homologous sequence (lane 3) but not by an analogous mutant oligonucleotide in which nt -30 to -24 region had been replaced (lane 2), confirming that the complex was formed via interactions at nt -30 to -24. Gel shift assays using consensus competitors for Sp1, Ets, and GAGA demonstrate that this complex is effectively competed by the Sp1 site (lane 4) but not the Ets (lane 5) and GAGA sites (lane 6), suggesting that the complex is formed by an Sp1-like factor. To directly test whether Sp1 binds to the SF-1 promoter at this site, we performed a supershift assay with an anti-Sp1 antibody, which diminished formation of the complex (lane 7). The preimmune serum control had no effect upon the complex, and the specificity of the antiserum against Sp1 was demonstrated by its nonreactivity against an unrelated probe (data not shown).



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Figure 7. Gel Shift Analysis of the GA-Rich Element in the SF-1 Promoter

The DNA sequences of all oligonucleotides are shown in Table 1Go. A 32P-labeled probe corresponding to SF-1 nt -40 to +12 was incubated with Y1 nuclear extract in the absence (lane 1) or presence of a 100-fold molar excess of the indicated unlabeled competitor [SF-1 nt -40 to +12, mutated within nt -30 to -24 (lane 2); wild type SF-1 nt -40 to +12 (lane 3); consensus site for Sp1 (22) (lane 4); consensus site for Ets (20) (lane 5); consensus site for GAGA (47) (lane 6)], or in the presence of anti-Sp1 antibody. Anti-Sp1 antibody abolishes the complex yielded by the SF-1 nt -40 to +12 probe. A supershift was achieved using an Sp1 consensus oligonucleotide and anti-Sp1 antibody (lane 7). The free probe did not remain on the gel due to the longer electrophoresis time required to achieve appropriate resolution.

 
The Identified Elements Confer Differential Activity to a Heterologous Promoter
The E box, CAAT box, and Sp1 site are all required for SF-1 promoter activity (see Fig. 3Go). To characterize the functional role of each identified element, we tested their ability to activate the heterologous minimal PRL promoter, which contains only a TATA box as an identified promoter element. Transient transfection of Y1 cells demonstrates that the -90 to -14 region of the SF-1 promoter (which spans the E box, CAAT box, and Sp1 site) is sufficient for ~100-fold activation of this heterologous promoter (Fig. 8Go, construct 1). In this context, mutation of either the Sp1 site (construct 2) or both the Sp1 site and CAAT box (construct 3) does not appear to have a significant effect over promoter activity, indicating that they are not required in the presence of a TATA box. In contrast, mutation of the putative E box (construct 4) significantly reduced promoter activity. Furthermore, when a construct containing promoter E box element in the incorrect orientation 5' to the TATA-box was tested, full promoter activity was observed (data not shown). Therefore, on a TATA-containing promoter, the E box appears to serve as an enhancer, whereas the CAAT box and Sp1 site do not. Moreover, the wild type -90 to -14 fragment in this heterologous context was inactive in NIH-3T3 cells, further indicating that the E box serves as a steroidogenic cell-specific element on the SF-1 promoter (data not shown).



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Figure 8. Functional Analysis of the Identified Promoter Elements

Schematic representations of luciferase reporter constructs containing a TATA box from the minimal PRL promoter and various SF-1 mutant promoter fragments are shown. All constructs were transiently transfected into Y1 cells, and luciferase activities were measured. The graph shows luciferase activities averaged (±SD) from three separate experiments, relative to the minimal PRL promoter construct, and controlled by measurement of ß-gal activity as directed by the plasmid RSV-ß-gal.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SF-1 is a transcription factor that regulates steroidogenic enzyme expression and is a critical developmental regulator for the adrenal gland and gonads (11, 12, 13). Because SF-1 expression is restricted to the adrenal gland, gonads, and the neural and pituitary elements of the hypothalamic-pituitary-adrenal axis, the factors that regulate its temporal and spatial expression are crucial to the proper development and function of these organs. Our analysis of the SF-1 promoter has revealed several elements important for expression in steroidogenic cells. These include a bHLH transcription factor site at -83 (E box), a CAAT box protein-binding site at -68, and a GA-rich Sp1 site at -30.

The bHLH family of transcription factors includes both cell type-restricted and ubiquitously expressed members. This family of proteins includes master regulators of cell fate such as MyoD and myogenin, which have been shown to be instrumental for initiation of muscle cell differentiation (30). It is generally considered that the cell-restricted bHLH proteins heterodimerize with the ubiquitously expressed bHLH proteins and these dimers bind to E box elements (31, 32). The E box of the SF-1 promoter, which was also identified in a previous study (24), may determine cell-specific expression of SF-1 as the other factors, CBF and Sp1, are ubiquitously expressed. Moreover, the SF-1 promoter E box may bestow steroidogenic-specific expression as it alone can promote activation on the minimal PRL promoter in Y1 adrenocortical cells, but not in NIH-3T3 fibroblasts. While no steroidogenic cell-specific bHLH factors have yet been cloned, USF, which is a class B, E box-binding protein that is ubiquitously expressed, has been shown to bind to the E box within the SF-1 promoter (33) and may contribute to the regulation of the SF-1 gene in steroidogenic tissues. Because of its ubiquitous expression, however, USF is clearly not sufficient to direct tissue-specific expression of SF-1.

A second functional element identified in this study is recognized by the ubiquitous transcription factor, CBF. The CAAT box is a proximal promoter element that is found between nucleotides -80 and -60 in the promoters of many genes (17, 18). Although multiple factors, such as C/EBP, CTF/NF-1, and CBF, interact with the CAAT box, only CBF binds to the SF-1 CAAT element. CBF is a heteromeric factor that consists of three subunits, CBF-A, CBF-B, and CBF-C, and is highly conserved throughout evolution (21, 28, 34, 36). CBF has been identified as a critical factor in the regulation of many genes including albumin (35) and major histocompatibility complex class II genes (37). Although several lines of evidence suggest that CBF facilitates the formation of stable preinitation complexes, as CAAT elements are present in promoters with or without a TATA box, the role of CBF in transcription is unclear. It is possible that within the SF-1 promoter, CBF works in concert with Sp1 to direct and/or initiate site-specific initiation (38, 39). This is consistent with evidence gathered from other studies of TATA-less promoters, which show that Sp1 directs site-specific initiation. Indeed, our data reveal that Sp1 is not required for SF-1 promoter activity when a TATA element is present.

With regard to the ability of SF-1 to regulate its own promoter, our data differ from a previous report (25), which showed SF-1 binding to a divergent SF-1 site within the first intron of the gene, and 2- to 3-fold transactivation of the promoter through this element in CV-1 cells. While our results do not rule out SF-1’s ability to bind to this element, it would appear that this element plays little role in the regulation of SF-1 expression in steroidogenic tissues, as its incorporation into a promoter fragment offers no additional activity to a luciferase reporter in Y1 (SF-1 expressing) adrenocortical cells. Moreover, coexpression of SF-1 in CV-1 cells does not enhance the activity of a promoter fragment that includes this site. Therefore, whether SF-1 autoregulates the SF-1 gene remains unclear.

Our gel mobility shift assays using nuclear extracts from NIH-3T3 cells demonstrate that nuclear proteins from this cell line bind to the E box and CAAT box. However, in contrast to their function in steroidogenic cells, they are not sufficient to activate expression of the SF-1 gene in NIH-3T3 cells. There are four plausible reasons for this finding. First, the Y1 bHLH transcription factor(s) could be distinct from those found in nonsteroidogenic tissues (and yet yield similar migration on a mobility shift assay). Second, a steroidogenic cell-specific coactivating molecule may be required for transcriptional activity of the bHLH transcription factor but not for its ability to bind DNA. Third, a repressive molecule may be present in nonsteroidogenic tissues that prevents transcriptional activation through E boxes in certain contexts. Finally, steroidogenic cells may posttranslationally modify and thereby activate the bHLH transcription factor. Given the numerous bHLH transcription factors expressed in a tissue-specific fashion that bind as heterodimers, these possibilities are by no means mutually exclusive. The activities of bHLH transcription factors are often tissue-specific by virtue of the presence of refined interactive (activating or repressive) factors characteristic of that tissue (40, 41, 42, 43). Therefore, it is likely that tissue-specific expression of the SF-1 gene is guided by a bHLH transcription factor and associated tissue-specific coactivators and repressors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of SF-1 Genomic Sequences
Isolation of the mouse SF-1 genomic clone (strain 129Sv) has been reported (13). A genomic P1 clone of human SF-1 was obtained from Genome Systems, Inc. (St. Louis, MO). Both the mouse and human genomic clones were mapped, subcloned, and partially sequenced by the dideoxynucleotide method on a DNA sequencer (Applied Biosystems, Foster City, CA; model 373A). Sequence construction and nucleic acid homology searches were performed by the Lasergene sequence analysis program (DNAStar, Madison, WI).

Plasmids
For construction of the SF-1 promoter-luciferase reporter plasmids, appropriate restriction sites were used to insert fragments of the mouse SF-1 gene in a 5' to 3' orientation into the polylinker of the promoterless plasmid pLuc. The -1885, -712, -190, -90, and -12 -bp constructs were generated using genomic fragments spanning from the KpnI (nt -1885), XbaI (nt -712), PstI (nt -90), and PvuI (nt -12) sites, respectively, to the EcoRI site located at +140 (see Fig. 2Go). For site-directed mutagenesis of the SF-1 promoter, four 71-mer oligonucleotide sets comprising DNA corresponding to the -90 to -17 region with the indicated nucleotide substitution mutations were synthesized (see Fig. 3Go). The -90-Luc reporter constructs were cloned by replacement of the PstI (nt -90)/PvuI (nt -12) fragment of -90-Luc or -1885-Luc with mutant oligonucleotides. The construct -3100-Luc contains a 6.7-kb 5'-flanking fragment that was cloned as four smaller fragments from the mouse genomic clone (NotI-KpnI, nt -3100 to -1885; KpnI-XbaI, nt -1885 to -712; XbaI-EcoRI, nt -712 to +140; EcoRI-SacII, +140 to +3600) into pBluescript KS (Stratagene, La Jolla, CA) and subsequently the promoter-less pLuc. The expression vector for SF-1 (CMV-SF-1) was generated by sequential subcloning of the SF-1 cDNA (a gift from K. Parker) using linker-generated EcoRI sites to clone into the EcoRI site of pBluescript KS, and then the flanking KpnI and XbaI sites from the bluescript polylinker to clone into pCMVneo. To generate constructs for the heterologous promoter experiments, the individual SF-1 promoter fragments mutated as above were cloned into the BamHI site immediately upstream of the minimal PRL promoter that contains nucleotides -26 to +1, which directs expression of the luciferase reporter from the plasmid pPrl-Luc (a gift from Stuart Adler).

Cell Culture and Transient Transfections
Y1 adrenocortical cells were grown in 50% DMEM/50% Ham’s F-12 medium supplemented with 10% FBS and antibiotics. CV-1 monkey kidney and NIH-3T3 fibroblast cells were grown in DMEM supplemented with 10% FCS and antibiotics. DC3 ovarian granulosa cells (44) were grown in Iscoves’s modified DMEM supplemented with 20% FBS and antibiotics. All plasmid DNA samples tested were prepared over Qiagen columns (QIAGEN Inc., Chatsworth, CA), and at least three preparations of each plasmid were tested. Cells were plated at ~6 x 104 cells per 35-mm well and transfected 24 h later with 0.5 µg of reporter and a total of 2 µg plasmid DNA per well. Transient transfection of DC3 cells was performed using LipofectAMINE reagent (GIBCO BRL, Gaithersburg, MD) as described by the manufacturer. Transient transfection of NIH-3T3, CV-1, and Y1 cells was performed by the calcium phosphate coprecipitation method (45). Luciferase assays were performed 48 h after transfection and normalized to ß-galactosidase activity from the cotransfected plasmid RSV-ß-galactosidase.

Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared as described (46). Probes (refer to Table 1Go) were prepared by end-labeling 100 ng of the primary strand oligonucleotide by 20 µCi of [{gamma}-32P]ATP, using T4 polynucleotide kinase, and annealing to the complementary oligonucleotide. Five micrograms of nuclear extract were mixed with 1 µg of poly (deoxyinosinic-deoxycytidylic) acid in a binding buffer consisting of 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. After a 5-min preincubation, 1.0 ng of labeled probe was added, and the mixture was incubated for 20 min at 25 C. The binding reactions (50 µl total volume) were loaded onto 4% polyacrylamide gels and run at 150 V for 4 h in 0.5 x Tris-borate-EDTA at 4 C. The gels were dried and exposed to film or a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). For competition experiments, the competing unlabeled oligonucleotide was added at the indicated concentration and preincubated at 25 C for 5 min before addition of 32P-labeled oligonucleotide. For antibody supershift experiments, the reaction mixture was incubated with the 32P-labeled oligonucleotide for 5 min, after which 1 µl of antibody [anti-CBF (36), a gift from Benoit de Crombrugghe, or anti-Sp1 (Promega, Madison, WI)] was added and the mixture was incubated for an additional 15 min at 25 C.


    ACKNOWLEDGMENTS
 
We thank Shelly Audrain for excellent technical assistance, and Robert Heuckeroth and John Svaren for critical comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Jeffrey Milbrandt, M.D., Ph.D., Departments of Pathology and Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Box 8118, St. Louis, Missouri 63110.

This work was supported by Grant P01 CA49712-06A1 from the Nation Cancer Institute. J.M. is an Established Investigator of the American Heart Association.

1 The first three authors contributed equally to this study. Back

Received for publication August 16, 1996. Revision received October 25, 1996. Accepted for publication November 4, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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