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Molecular Endocrinology 12 (5): 714-726
Copyright © 1998 by The Endocrine Society

The Basic Helix-Loop-Helix, Leucine Zipper Transcription Factor, USF (Upstream Stimulatory Factor), Is a Key Regulator of SF-1 (Steroidogenic Factor-1) Gene Expression in Pituitary Gonadotrope and Steroidogenic Cells

Adrienne N. Harris and Pamela L. Mellon

Department of Reproductive Medicine (A.N.H., P.L.M.) Department of Neuroscience and the Center for Molecular Genetics (P.L.M.) University of California, San Diego La Jolla, California 92093-0674


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue-specific expression of the mammalian FTZ-F1 gene is essential for adrenal and gonadal development and sexual differentiation. The FTZ-F1 gene encodes an orphan nuclear receptor, termed SF-1 (steroidogenic factor-1) or Ad4BP, which is a primary transcriptional regulator of several hormone and steroidogenic enzyme genes that are critical for normal physiological function of the hypothalamic-pituitary-gonadal axis in reproduction. The objective of the current study is to understand the molecular mechanisms underlying transcriptional regulation of SF-1 gene expression in the pituitary. We have studied a series of deletion and point mutations in the SF-1 promoter region for transcriptional activity in {alpha}T3–1 and LßT2 (pituitary gonadotrope), CV-1, JEG-3, and Y1 (adrenocortical) cell lines. Our results indicate that maximal expression of the SF-1 promoter in all cell types requires an E box element at -82/-77. This E box sequence (CACGTG) is identical to the binding element for USF (upstream stimulatory factor), a member of the helix-loop-helix family of transcription factors. Studies of the SF-1 gene E box element using gel mobility shift and antibody supershift assays indicate that USF may be a key transcriptional regulator of SF-1 gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The maintenance of physiological homeostasis and sexual differentiation in mammals is predominantly controlled by the appropriate temporal and spatial production of specific steroid hormones. The biosynthesis of steroid hormones in specific tissues requires the concerted action of a related group of cytochrome P450 steroid hydroxylases (1), and considerable effort has been directed toward defining the molecular mechanisms that regulate expression of this family of biosynthetic enzymes. Functional analyses of the promoter regions of the steroid hydroxylase genes revealed a conserved element with homology to the estrogen receptor half-site that was required for maximal gene expression (2). As a result of these efforts, a developmentally regulated nuclear receptor, known as steroidogenic factor-1 (SF-1) (3) or Ad4BP (4, 5), was identified that interacts with this element to effect transcriptional regulation of specific cytochrome P450 enzyme expression in gonadal and adrenal tissues (2, 6).

SF-1 is one of two proteins encoded by the mammalian homologue of fushi tarazu factor-1 (FTZ-F1) (3, 7, 8), a Drosophila gene product that has been identified as a regulator of the fushi tarazu homeobox gene (9). Isolation and characterization of SF-1 cDNA clones (3, 7, 8, 10) revealed that the factor is a member of the steroid/thyroid hormone receptor superfamily (10), containing a zinc finger region and a ligand-binding/dimerization domain. It was recently demonstrated that endogenous oxysterols selectively enhance SF-1-mediated transcriptional activity, suggesting that SF-1 is a ligand-activated receptor (11).

SF-1 is expressed in a highly tissue-restricted manner during embryonic development (6, 12). SF-1 transcripts are first detected in the mouse on embryonic day 9 (e9) in the urogenital ridge and localized to the adrenal glands by e11. Interestingly, expression of SF-1 in the embryonic gonad is sexually dimorphic by e14.5, with high levels of SF-1 in testes and trace levels of detectable protein in ovaries. In the adult rodent, SF-1 is expressed in all the primary steroidogenic tissues, including the adrenal cortex, testicular Leydig cells, and the theca and granulosa cells and corpus luteum of the ovary (6, 12). Since SF-1 was thus strongly implicated in tissue-specific gene expression and sexual differentiation, studies were initiated to analyze the gene in intact mice. Targeted disruption of the mouse FTZ-F1 locus defined SF-1 as the essential transcript of the gene (13) and as responsible for the dramatic developmental abnormalities associated with FTZ-F1-null mice (13, 14, 15). Adrenal glands in gene-disrupted mice are absent from early stages in development, resulting in neonatal lethality, presumably due to adrenocortical insufficiency (13, 14, 15). In addition, FTZ-F1 null mice of both sexes lacked gonads but displayed internal female genitalia. Thus SF-1 plays a pivotal role in the formation and function of steroidogenic tissue in the developing mammal and may represent one of the target genes of SRY in the cascade of gene activation of sex determination genes (16, 17).

Importantly, control of reproductive function by the ventromedial hypothalamus and pituitary is also selectively disrupted in SF-1-knockout mice (15, 18). Of the five different endocrine cell types present in the anterior pituitary, SF-1 is selectively expressed in the gonadotrope (19, 20). In response to GnRH release from the hypothalamus, the gonadotrope secretes the gonadotropin hormones LH and FSH, resulting in increased steroid production in the target gonads. In SF-1-knockout mice, ventromedial hypothalamus control of GnRH secretion is impaired, which presumabably results in both decreased GnRH delivery to the gonadotropes and decreased LH/FSH secretion (15, 18). Recently, several targets of SF-1 transcriptional regulation through a consensus DNA-binding site for SF-1 [or GSE (gonadotrope-specific element) (20)] have been defined in the pituitary gonadotrope by analyzing the 5'-flanking sequences of gonadotrope-specific genes, including the GnRH receptor (21), the {alpha}-subunit (20), and the LH-ß-subunit of the glycoprotein hormones (22). Thus, SF-1 appears to play a global role at the molecular and physiological level in the integrated interaction of the hypothalamic-pituitary-gonadal axis in mammalian reproductive function (23).

Progress toward defining the molecular mechanisms that control cell type-specific expression of SF-1 in the pituitary gonadotrope has been enhanced by the isolation of the promoter region of the gene (24, 25, 26). In particular, recent reports have defined the functional importance of an E box motif in the proximal promoter of the rat (24), mouse (26), and human (26) SF-1 genes in tissue-specific expression, yet the factors that interact with this motif have not been characterized. Here we report that the E box motif in the proximal promoter of the rat SF-1 gene binds the transcription factor USF (upstream stimulatory factor), a member of the helix-loop-helix (HLH) family of transcription factors. In addition, we present evidence that nuclear proteins in the {alpha}T3–1 pituitary gonadotrope cell line exhibit different binding affinities for distinct E box motifs that are important for gonadotrope-specific gene regulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Promoter Activity of the Rat SF-1 Gene
To investigate the promoter activity of the SF-1 gene, 5'-flanking sequences were inserted upstream of the chloramphenicol acetyltransferase (CAT) reporter gene in recombinant plasmids and used in transient transfection assays. Previously, these SF-1 promoter plasmids had been studied in the Y1 adrenocortical cell line (24). Although SF-1 is also known to be expressed in the pituitary gonadotrope (19) and {alpha}T3–1 pituitary gonadotrope cell line (20), transcriptional regulation of the SF-1 gene has not been characterized in these cell types. In this study, we assessed transcriptional regulation of the SF-1 gene promoter in the {alpha}T3–1 and LßT2 pituitary gonadotrope cell lines.

Activity of the SF-1 gene directed by the proximal -4800 bp of the SF-1 promoter was tested by transient transfection in {alpha}T3–1 (mouse pituitary gonadotrope), CV-1 (monkey kidney), JEG-3 (human choriocarcinoma), and Y1 (mouse adrenocortical) cell lines (Fig. 1Go). CAT activity of the SF-1 promoter plasmids increased gradually when promoter sequences were deleted from -4800 to -265 (CV-1 and Y1) or -92 ({alpha}T3–1 and JEG-3). However, a dramatic reduction in CAT activity was observed when promoter sequences between -92 and -60 were deleted (82% decrease in {alpha}T3–1 cells, 83% in CV-1 cells, 90% in JEG-3 cells, and 75% in Y1 cells). Upstream sequences between -4800 and -265 are regulated differently in distinct cell types when normalized to CAT expression driven by the constitutively active thymidine kinase (TK) promoter. Sequences between -4800 and -265 appear to contain negative regulatory elements that are active in steroidogenic Y1 adrenocortical cells and also in {alpha}T3–1 pituitary gonadotropes and CV-1 cells. However, overall expression of the promoter is 2- to 4-fold higher in Y1 cells, a result that correlates well with previously published studies (24).



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  		Figure 1. Fig. 1. Effect of SF-1 5' Flanking Sequence on CAT Expression Transient transfection assays using plasmids carrying 5'-truncation mutations in the rat SF-1 promoter were performed in {alpha}T3–1 (pituitary gonadotrope), CV-1, JEG-3 (human choriocarcinoma), and Y1 (adrenocortical) cells. Numbers below the bars indicate base pairs included 5' from the transcription initiation site of the SF-1 gene inserted upstream of a CAT reporter gene in pSV00CAT (pSV00), a promoterless CAT expression vector (see Materials and Methods). CAT enzyme activity values were corrected for transfection efficiency by normalizing to a cotransfected CMV-ß-galactosidase expression vector. Normalized CAT values are expressed as a percentage of normalized TK-CAT activity in each cell type. All values represent the result of a minimum of three separate transfection experiments, with error bars representing the SEM.

 
The deletional analysis of the rat SF-1 gene promoter revealed the presence of functional regulatory sequences between -92 bp and -60 bp. Previous studies had established that a consensus E box motif (CACGTG) at -82/-77 was required for maximal expression of the SF-1 gene in steroidogenic cell types (24). In this study, we have established the importance of the E box for expression of SF-1 in the {alpha}T3–1 and LßT2 pituitary gonadotrope cell lines (Fig. 2Go). An SF-1 promoter-CAT reporter gene plasmid (-800) containing 800 bp of proximal 5'-flanking sequence was tested by transient transfection assays in pituitary gonadotrope ({alpha}T3–1, LßT2), steroidogenic (Y1), and nonsteroidogenic (JEG-3, CV-1) cell lines. CAT activity of -800 in each cell type was set at 100% (black bars) and compared with CAT activity derived from expression of the identical plasmid containing a specific nucleotide substitution mutation in the E box at -82/-77 (E-mut) generated by site-directed mutagenesis (24). This mutation converts the wild-type sequence from CACGTG to CTGTAG. The presence of this mutation has a direct functional effect on transcriptional activation of the SF-1 promoter and results in a decrease in CAT activity by 85% in {alpha}T3–1, LßT2, and CV-1 cells, 68% in JEG-3 cells, and 90% in Y1 cells (open bars). Results of these studies further establish the importance of the E box as a cis-regulatory element necessary for expression of the proximal promoter of the SF-1 gene and, in particular, represents the first transcriptional analysis of this element in the {alpha}T3–1 and LßT2 pituitary gonadotrope cell lines. Unlike Woodson et al. (26) we have not found that the SF-1 proximal E box imparts tissue specificity to SF-1 gene expression in any cell type tested.



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  		Figure 2. A Functional E box Is Required for Expression of the SF-1 Gene

{alpha}T3–1 and LßT2 (pituitary gonadotrope), JEG-3, CV-1, and Y1 cells were transfected with either the -800 (wild-type) or E-mut [-800 containing a 4-bp substitution mutation (underlined) in the E box at -82/-77] plasmids. Normalized CAT activity from the E box mutant plasmid (open bars) is expressed as a percentage of wild-type CAT activity of the -800 plasmid set to 100% (black bars) in the corresponding cell type. CAT enzyme activity values were corrected for transfection efficiency by normalizing to a cotransfected CMV-ß-galactosidase expression vector. All values represent the result of a minimum of three separate transfection experiments (± SEM). The sequence and location of the E box mutation relative to the transcription start site are indicated.

 
Mutation of the E Box, but Not the SF-1-Binding Site in the First Intron, Has a Significant Effect on Transcriptional Activity
Previous studies had established the importance of a consensus DNA recognition site for SF-1 [or GSE (20)] in the first intron as a possible mechanism of transcriptional autoregulation by SF-1 in Y1 cells (25). In the current study we have used a series of plasmids [previously described (25)] to assess the importance of this SF-1-binding site as an autoregulatory mechanism in the pituitary gonadotrope compared with steroidogenic and nonsteroidogenic cell types (Fig. 3Go). The -800Int plasmid (representing 100% of CAT activity) includes the 3.4-kb first intron of SF-1 in addition to -800 bp of 5'-flanking sequence linked upstream of the CAT reporter gene. Nucleotide substitution mutations were generated in the SF-1 binding site at +160/+167 in the first intron, converting the SF-1 site from GAAGGCCG to GAATATCG (S-mutant). Previous results indicated that this mutation in the SF-1 site significantly decreased transcriptional activation in Y1 cells (25). Surprisingly, in our studies, this mutation resulted in a moderate increase in transcriptional activity in all cell types tested, including Y1 cells. However, inclusion of the 3.4-kb first intron, which increases CAT activity by 400-2000% in these cells compared with the CAT activity of -800 (data not shown), cannot compensate for loss of the E box motif. CAT activity of the plasmid carrying the E box mutation (Ebox-mutant) is dramatically reduced to 23% in {alpha}T3–1, 20% in LßT2, 10% in JEG-3, 22% in CV-1, and 42% in Y1 cells of wild-type expression. CAT activity of the plasmid carrying both mutations (S+Ebox-mutant) is not significantly different from the Ebox-mutant alone, with activity reduced to 25% in {alpha}T3–1, 22% in LßT2, 10% in JEG-3, 26% in CV-1, and 65% in Y1 cells of wild-type expression. These data indicate that the E box motif in the proximal promoter is essential for full transcriptional activity of the SF-1 gene in a variety of cell types, while the SF-1-binding site is inconsequential.



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  		Figure 3. E Box, but Not SF-1-Binding Site, Mutations Decrease SF-1 Promoter Activity

Recombinant plasmids were constructed from the -800Int (wild-type) plasmid by nucleotide substitution mutations in either or both the E box at -82/-77 and the SF-1 binding site located in the first intron of the SF-1 gene (see Materials and Methods). -800Int includes the 3.4-kb first intron linked upstream of the CAT reporter gene in addition to -800 bp of 5' flanking sequence. Cells were transfected with either -800Int or mutant plasmids, and results are expressed in terms of normalized CAT activity of the mutant plasmid as a percentage of CAT activity of -800Int (set to 100%). CAT enzyme activity values were corrected for transfection efficiency by normalizing to a cotransfected thymidine kinase promoter-luciferase expression vector. All values represent the result of a minimum of three separate transfection experiments (± SEM). -800Int is represented schematically below the graph, with mutations in the E box and SF-1-binding sites indicated by crosses. The S mutant construct (open bars) contains a mutation in the SF-1-binding site at +160/+167 in the first intron converting GAAGGCCG to GAATATCG. The Ebox-mutant construct (black bars) contains a mutation in the E box at -82/-77 converting CACGTG to CTGTAG, and the S+Ebox-mutant construct (gray bars) contains both mutations. The hatched bar represents Exon I (+1 to +153), with the arrow indicating the transcription start site.

 
Nuclear Proteins Form Distinct Complexes on the E Box Element in the SF-1 Promoter
The effect on transcriptional activity of a site-specific mutation of the E box at -82 in the SF-1 promoter suggests the possibility that regulatory proteins interact with the E box to mediate full promoter activity. To examine this possibility, we performed electrophoretic mobility shift assays (EMSAs) with an oligonucleotide probe containing SF-1 gene sequences from -90 to -68, which includes the E box element at -82 (Fig. 4Go). The sequences of all double-stranded oligonucleotide probes and competitors are given in Materials and Methods and Table 1Go. The EMSA performed in the presence of {alpha}T3–1, CV-1, HeLa, JEG-3, and Y1 nuclear extracts revealed the formation of distinct complexes with the wild-type E box probe. The specificity of nuclear proteins for the E box is convincingly demonstrated by comparing the pattern of binding complexes formed with the E-mut probe to that obtained with the wild-type probe. The E-mut oligonucleotide probe contains a site-specific mutation in the E box at -82 (CACGTG to CTGTAG), identical in sequence to the mutation created in the promoter plasmid used in transient transfections (E-mut). Formation of the C1 and C2 complexes seen with the wild-type E-box probe (left panel) is completely eliminated in EMSAs employing the E-mut probe (right panel). The variable complex above C1 is also partially eliminated in specific extracts using the E-mut probe. The low-molecular weight complex observed below C2 with CV-1, HeLa, and JEG-3 nuclear extracts may represent non-E box-binding protein complexes formed elsewhere on the probe. An equivalent mass of nuclear protein (10 µg) was used in each EMSA; therefore the C1 and C2 complexes may represent ubiquitous proteins present in a wide variety of cell types. However, the C2 complex is more variable in intensity and suggests the possibility that a different cellular context may determine the expression pattern of E box-binding proteins.



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  		Figure 4. An E box Mutation Reduces Nuclear Factor Binding as Analyzed by EMSA

Wild-type SF-1 E box (E-box) and E box mutant (E-mut) 32P-labeled oligonucleotide probes were used in an EMSA containing nuclear extracts (10 µg total protein in each lane) from {alpha}T3–1, CV-1, HeLa, JEG-3, or Y1 cells. Specific complexes (C1 and C2) formed with the wild-type E box probe with all extracts are absent when an identical oligonucleotide containing an E box mutation (E-mut) is used as the labeled probe. -, No extract; Free, unbound oligonucleotide probe. The size of the oligonucleotide probes and sequence of the specific E box mutation are indicated above the corresponding lanes. The full-length sequence of the oligonucleotides is given in Materials and Methods and Table 1.

 

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  		Table 1. Sequence of Oligonucleotide Probes or Competitors Used in EMSA Reactions

 
SF-1 E Box-Binding Proteins Migrate with Identical Mobility to USF
USF is a ubiquitously expressed basic HLH-leucine zipper (bHLH-LZ) transcription factor that is involved in transcriptional activation of several promoters by forming functional heterodimers with ubiquitous and cell-specific partners (27, 28, 29). In this capacity, basal transcription is often facilitated by USF through a proximal E box motif characterized by a core CACGTG sequence (30). The E box motif in the SF-1 promoter is identical to the specific DNA sequence that is recognized by USF and required for transcriptional activation (30).

To address the identity of the factors that form the specific C1 and C2 complexes and to test the hypothesis that one or both of these complexes may contain USF, labeled SF-1 E box and USF probes were used in EMSAs performed with nuclear extracts from multiple cell types (Fig. 5Go). When the E box probe was incubated with {alpha}T3–1, CV-1, HeLa, JEG-3, or Y1 nuclear extracts, the specific C1 and C2 complexes were observed in all lanes (left panel). Interestingly, when the USF probe was incubated with the identical nuclear extracts, a very similar shift pattern emerged with the appearance of the prominent C1 and C2 complexes migrating with the same mobility as complexes formed on the E box probe (right panel). HeLa cell nuclear extracts were included as a positive control for USF binding (31). Due to the relatively long exposure time required to obtain the E-mut binding complex signal seen in Fig. 4Go (96 h exposure), the nonspecific binding complexes (below C2 and above C1) are not observed in Fig. 5Go (16 h exposure).



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  		Figure 5. Complexes with Identical Mobility to USF Are Formed with the E box Oligonucleotide Probe

SF-1 E box and USF consensus 32P-labeled oligonucleotide probes were used in an EMSA containing nuclear extracts (10 µg total protein in each lane) from {alpha}T3–1, CV-1, HeLa, JEG-3, or Y1 cells. HeLa cell nuclear extracts have been included as a positive control for USF binding. The specific C1 and C2 complexes formed with both the wild-type E box and USF probes are present in all cell types tested. -, No extract; Free, unbound oligonucleotide probe. The full-length sequence of oligonucleotides is given in Materials and Methods and Table 1.

 
USF exhibits a heterogeneous pattern of expression in which proteins with molecular masses of 43 kDa (USF-1) (31) and 44-kDa (USF-2) (29) are responsible for USF activity, yet the two different forms of USF show identical DNA-binding properties (29). Specific antibodies (29) against these widely expressed proteins were used in EMSA supershift experiments to further characterize the E box-binding complexes C1 and C2 (Fig. 6Go). Nuclear extracts from {alpha}T3–1, Y1, HeLa, CV-1, and JEG-3 cells were preincubated with 1 ng of specific rabbit polyclonal antibody raised against USF-1 or USF-2 before the addition of the labeled E box oligonucleotide probe. One nanogram of whole rabbit serum was included as a negative control for the specificity of antibody reactions. Inclusion of control nonspecific antibody [(-) lane] or whole mouse serum (IgG lane) has no effect on the formation of the prominent C1 and C2 complexes. However, the addition of specific {alpha}USF-1 antibody produces a supershifted complex and a reduction in C1 and C2 complex formation in all cell types. The addition of specific {alpha}USF-2 antibody has a weaker supershift effect in all cell types, particularly in {alpha}T3–1 and JEG-3 extracts. However, specific supershifted complexes are seen using the {alpha}USF-2 antibody with Y1, HeLa, and CV-1 nuclear extracts.



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  		Figure 6. Anti-USF-1 and USF-2 Antibodies Supershift the Specific Complexes Formed with the E box Oligonucleotide Probe

An SF-1 E box 32P-labeled oligonucleotide probe was used in an EMSA containing nuclear extracts (10 µg total protein in each lane) from {alpha}T3–1, CV-1, HeLa, JEG-3, or Y1 cells. Specific reactions were additionally incubated with either anti-USF-1 antibody ({alpha}USF-1), anti-USF-2 antibody ({alpha}USF-2), or whole mouse serum (IgG) as described in Materials and Methods. Formation of the specific C1 and C2 complexes are greatly reduced in all cell types in the presence of {alpha}USF-1 and slightly reduced with {alpha}USF-2 antibody. SSC, Supershifted complex; -, no extract or antibody; Free, unbound oligonucleotide probe.

 
Together, these data fit the hypothesis that the SF-1 promoter E box is preferentially recognized by USF proteins (or proteins antigenically related to USF). Previous studies have reported that USF-1 and USF-2 readily form hetero- and homodimers in solution and that these species represent the predominant DNA-binding complexes (32). Our data suggest the possibility that C1 represents a USF-1:USF-2 heterodimer-DNA complex composed of 43- and 44-kDa proteins that migrates with a relatively slow mobility. Thus, this complex is recognized by both antibodies, as we have observed. Since the C2 complex is also preferentially supershifted by {alpha}USF-1 antibodies, C2 may be composed of a USF-1:USF-1 homodimer complex of 43-kDa proteins that moves with a slightly faster mobility than C1. This pattern has been observed using different ratios of bacterially produced USF-1 and USF-2 proteins in EMSAs, wherein USF-1:USF-1>USF-1:USF-2>USF-2:USF-2 in terms of relative mobility (32). In addition, the protein charge and cellular context may also play a role in determining the ratio and binding affinity of USF-1 and USF-2 hetero- and homodimers for the SF-1 E box. The antigenic site required for recognition may be relatively less accessible to the {alpha}USF-2 antibody in the heterodimer, possibly leading to the appearance of a lower affinity of this antibody for the C1 complex. If HeLa cell nuclear proteins may be used as a standard from which to judge the formation of hetero- and homodimer complex formation, USF-2:USF-2 homodimers are a relatively minor component of an E box-binding complex (32), and such complexes have not been observed under our EMSA conditions. Based on this observation, we can conclude that the complex with higher electrophoretic mobility compared with C1 is probably not a USF-2:USF-2 complex, but may represent a complex with a relatively low affinity for the SF-1 E box compared with USF.

Pituitary Gonadotrope Nuclear Proteins Exhibit Different Binding Affinities for Distinct E Box Probes
A recent report provided evidence for E box-dependent transcriptional activation of the human {alpha}-subunit of the glycoprotein hormones in {alpha}T3–1 pituitary gonadotrope cells through two E box motifs ({alpha}EB1 and {alpha}EB2) located at -51 and -21, respectively, from the transcriptional start site (33). In this report it was determined by supershift EMSA analysis using a cross-reactive polyclonal rabbit anti-USF antibody that USF binds to {alpha}EB2, but not {alpha}EB1, E box sequences. The {alpha}-subunit is expressed by cells committed to the gonadotrope and thyrotrope cell lineage (34), and thus it was of interest to determine whether {alpha}EB1 or {alpha}EB2 could compete for SF-1 E box-binding proteins present in {alpha}T3–1 nuclear extracts.

To test this hypothesis, an SF-1 E box probe was used in an EMSA containing {alpha}T3–1 nuclear extracts with the inclusion of a 100-fold molar excess of unlabeled double-stranded competitor oligonucleotides comprising the E box, E-mut, USF, {alpha}EB1, or {alpha}EB2 sequences (Fig. 7AGo and Table 1). The specific C1 and C2 complexes are competed by an excess of both the wild-type SF-1 E box and USF oligonucleotides. However, {alpha}EB1 and {alpha}EB2 were poor competitors for SF-1 E box-binding proteins, demonstrating that the proteins forming the C1 and C2 complexes may not have the equivalent affinity for the {alpha}EB1 or {alpha}EB2 E box sequences. The sequence differences in the E box motifs between oligonucleotide probes and competitors are shown in Table 1.



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  		Figure 7. Nuclear Proteins from Pituitary Gonadotrope Cells Exhibit Different Binding Affinity for Three Different E Box Probes

A, An SF-1 E box 32P-labeled oligonucleotide probe was used in an EMSA containing {alpha}T3–1 nuclear extracts (10 µg total protein in each lane). Specific reactions were additionally incubated with a 100-fold molar excess of unlabeled double-stranded competitor oligonucleotides comprising the SF-1 E box (E-box); the SF-1 E box mutation (E-mut); the USF consensus element (USF); and the E boxes at -51/-45 ({alpha}EB1) or -21/-16 ({alpha}EB2) of the human {alpha}-glycoprotein subunit promoter (33). The specific C1 and C2 complexes are competed with an excess of both the wild-type SF-1 E box and USF oligonucleotides. B, An {alpha}EB1 or {alpha}EB2 32P-labeled oligonucleotide probe was used in an EMSA containing {alpha}T3–1 nuclear extracts (10 µg total protein in each lane). Specific reactions were additionally incubated with a 100-fold molar excess of unlabeled double-stranded competitor oligonucleotides as specified in panel A. The specific {alpha}C1 complex is competed from both probes with an excess of each oligonucleotide containing an intact E box. -xt, no extract or competitor; Free, unbound oligonucleotide probe. The sequence of oligonucleotide probes and competitors is given in Materials and Methods and Table 1.

 
The different affinity of the E box, {alpha}EB1, and {alpha}EB2 probes for E box-binding proteins is further highlighted by the results presented in Fig. 7BGo. EMSAs were performed in which specific reactions using the {alpha}EB1 or {alpha}EB2 probes and {alpha}T3–1 nuclear extracts were additionally incubated with a 100-fold molar excess of unlabeled double-stranded competitor oligonucleotides as specified in Fig. 7AGo. Two predominant shifted complexes, {alpha}C1 and {alpha}C2, were formed with both the {alpha}EB1 or {alpha}EB2 probes. The {alpha}EB1 probe appears to have a higher affinity for {alpha}T3–1 nuclear proteins (left panel), as the {alpha}EB2 autoradiograph shows a weaker signal than that of {alpha}EB1 (right panel). The {alpha}C1 complex migrates with equal mobility to the C1 complex on the SF-1 E box probe (data not shown), and it is likely that {alpha}C1 represents the same band previously described as C4 (33) that is competed here by both the {alpha}EB1 (CAGGTG) and E box and USF (CACGTG) oligonucleotides. However, the {alpha}C2 complex is competed by an excess of all oligonucleotides including E-mut, and so most likely does not represent a specific E box-binding complex. Finally, the C2 band possibly representing a USF-1:USF-1 homodimer complex is completely absent using either {alpha}EB1 or {alpha}EB2 probes. These results further highlight the qualitative and quantitative differences in binding patterns obtained with probes containing the CACGTG motif (E box and USF) or CAGGTG motif ({alpha}EB1 and {alpha}EB2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SF-1, also known as Ad4BP (5), was first identified as an orphan nuclear receptor that regulates cytochrome P450 steroid hydroxylase gene expression (2, 3, 5, 7, 10, 35, 36, 37, 38). An essential role for SF-1 in the development of the adrenal glands and gonads was established in mice by targeted disruption of the mammalian FTZ-F1 locus, which encodes the SF-1 and ELP transcripts (13, 14, 15, 18). These studies extended the role of SF-1 in the control of reproductive function by establishing that SF-1 knockout mice demonstrated defects at all levels of the hypothalamic-pituitary-gonadal axis.

In its role as a transcriptional regulator, SF-1 has been implicated in the selective regulation of key markers of the pituitary gonadotrope phenotype, including the human {alpha}-subunit (20), the LH ß-subunit in multiple species (Ref. 22 and references therein), and the GnRH receptor (21). We have focused primarily on the molecular mechanism for directing expression of the rat SF-1 gene to the gonadotropes of the anterior pituitary, and thus an analysis of the cis elements and trans-acting factors governing expression of SF-1 is critical to our understanding of the role of SF-1 in reproductive function. In this study, we have demonstrated that optimal promoter activity of the rat SF-1 gene in distinct cell types is primarily regulated by a proximal E box at -82. We have defined members of a specific subset of HLH transcription factors, USF-1 and USF-2, that may bind the E box in dimer form and may be responsible for transactivation of SF-1 in vivo. We have also demonstrated that nuclear proteins obtained from the {alpha}T3–1 pituitary gonadotrope cell line contain USF, and that these nuclear proteins exhibit distinct binding affinities for different E box elements.

Previous studies have provided evidence that the E box regulates expression of SF-1 in the Y1 and I-10 steroidogenic cell lines and not in the nonsteroidogenic CV-1 cell line (24). We have presented data demonstrating that regulation of SF-1 gene expression appears to occur primarily through the E box in all cell types that were transiently transfected, including the {alpha}T3–1 and LßT2 pituitary gonadotrope cell lines, and nonsteroidogenic CV-1 and JEG-3 cells. Our data differ from previously published reports (24, 25) with regard to expression of the SF-1 promoter in nonsteroidogenic cells and the ability of SF-1 to regulate its own promoter through a consensus DNA recognition site (SF-1 site). We may have obtained different results based on our cell culture and transfection technique (different media was used for Y1 cells). In addition, there appears to be variation in the characteristics of Y1 clones in different laboratories (K. Morohashi, personal communication). Cells transfected by the lipofection method (24, 25) may yield significantly different quantitative and qualitative results compared with the calcium phosphate method (39). The sensitivity of transcriptional assays may be enhanced by the calcium-phosphate method, allowing us either to transfect a larger amount of plasmid or lower the threshold of detection for CAT activity.

However, such results allow us to speculate on the mechanism by which the consensus E box may regulate SF-1 gene expression in a wide variety of cell types. We hypothesize that USF-1 and, to a lesser degree, USF-2 or a heterodimer of USF-1:USF-2 form complexes on the SF-1 proximal E box and are significantly involved in basal transcription of the gene. This hypothesis is supported by several recent observations of the function of USF proteins in transcriptional regulation and the structure of the SF-1 gene. The SF-1 gene lacks a recognizable TATA box, which is the hallmark of many eukaryotic gene promoters that use RNA polymerase II for transcription initiation (40). Although the mechanism of transcription initiation is less well understood in TATA-less promoters, it has been shown that TFII-I, a transcription initiation factor that activates core promoters through an initiator motif (Inr) can cooperatively interact on binding with USF at both the Inr and an upstream E box (41, 42). A potential Inr sequence (43) is located in the rat (24), mouse (26), and human (26) promoters at +1.

USF was first identified as a factor that bound to an upstream element in the adenovirus major late promoter that stimulated transcription (30, 41), and has subsequently been found to activate transcription of the promoters of a number of cellular genes (27, 28). USF is a ubiquitous nuclear protein that exists in two major biochemical forms with apparent molecular masses of 43-kDa (USF-1) (31) and 44-kDa (USF-2) (29). Sequence analysis of USF cDNA clones has demonstrated that USF belongs to the myc family of regulatory proteins characterized by a C-terminal basic region followed by a bHLH-LZ structure responsible for dimerization and DNA binding (44). bHLH-LZ proteins bind either as homodimers or heterodimers to a specific DNA element, which contains the core sequence CANNTG or E box (45). Dimerization between cell-specific [e.g. MyoD (46)] and ubiquitously expressed HLH proteins [e.g. E12/E47 (47)] is a critical step in controlling transcriptional activation and cell type specificity during differentiation of diverse tissues (46, 48, 49).

The ubiquitous expression pattern of nuclear factors that was observed to bind to the SF-1 E box and the specific architecture of the E box led us to examine USF as a potential regulator of SF-1 gene expression. USF binding is somewhat constrained at the first level by the central core nucleotides at position -1 and +1 (C-3A-2N-1N+1T+2G+3), preferring CG in these sequential positions (30, 50). In addition, binding discrimination can be conferred by specific nucleotides 5' to the E box core sequence, with USF exhibiting a pyrimidine selectivity at -4 and a purine selectivity at -5 (50). The rat (24), mouse (26), and human (26) SF-1 promoters contain a thymidine at -4 (relative to the E box). Based upon these data, we tested the binding specificity of pituitary nuclear factors for the USF E box from the SF-1 promoter compared with the sequence of E box elements ({alpha}EB1 and {alpha}EB2) that had been previously defined in the human {alpha}-subunit promoter using {alpha}T3–1 pituitary gonadotrope nuclear extracts (33). We observed that only oligonucleotides containing the USF binding sequence (CACGTG) could compete for the proteins bound to the SF-1 E box. However, oligonucleotides containing the sequence CACGTG or CAGGTG could compete for {alpha}EB1-binding proteins in the {alpha}C1 complex, a complex that contributed a relatively low percentage to overall probe binding. Previously, USF proteins had been characterized in {alpha}T3–1 nuclear extracts using the {alpha}EB2 probe in supershift analyses employing an anti-USF antibody that was cross-reactive between USF-1 and USF-2 (33). In this same report, functional studies demonstrated that overexpression of the Id protein compromised the activity of the {alpha}-subunit promoter, possibly through interference with USF binding at the {alpha}EB2 site. However, HLH proteins that contain a leucine zipper motif (e.g. USF-1 and USF-2) resist inactivation by Id (51), and so it is likely that the effect of Id in reducing {alpha}-subunit gene expression as described is indirect. Therefore, whether the {alpha}C1 complex contains specific USF proteins is inconclusive, although in our studies the {alpha}EB1 and {alpha}EB2 probes encourage binding by proteins comprising the {alpha}C1 complex that are specifically competed by the CACGTG E box.

Results from previous Southwestern and far-Western experiments demonstrated that factors sharing the conserved properties of bHLH proteins are present in pituitary nuclear cell extracts (52). Further, in gel mobility shift experiments using nuclear extracts from mature adult pituitary tissue, complexes distinct from those produced using the {alpha}T3–1 pituitary extracts are observed. These results may have significant implications for our understanding of developmental regulation of the pituitary gonadotrope lineage. We may use pituitary cell lines immortalized at discrete stages of development, which express differentiated markers of the pituitary gonadotrope cell (34), to determine the temporal and spatial expression pattern of HLH proteins that may be required for the differentiation process to occur. USF-1 and USF-2 proteins exhibit a heterogeneous pattern of expression in both human and mouse, resulting from differential splicing and alternative poly(A) site usage (32). Thus it is possible that the abundance of functional USF is regulated under physiological conditions by the predominance or ratio of encoded proteins in different cell types. Our studies represent a first step in the identification of a subset of HLH proteins that appear to be required for expression of a marker gene in the pituitary gonadotrope. Results from these studies may allow us to develop a model with which to understand the mechanism by which the ubiquitous USF bHLH-LZ proteins may interact with a tissue-specific partner to dictate expression of SF-1 to the appropriate tissues, as has been demonstrated in other systems utilizing HLH transcriptional determinants of gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
Construction of rat SF-1 gene promoter-CAT reporter gene plasmids has been previously described (24). Briefly, a 4.8-kb fragment containing the 5'-flanking region upstream from the EcoRI site located in the first exon of the SF-1 gene was ligated upstream of the CAT structural gene in pSV00CAT to produce Ad4CAT4.8K. The deletion mutant constructs including, and created from, Ad4CAT4.8K have been designated here as -4800, -2000, -1200, -800, -265, -92, and -60 (numbers indicate base pairs included 5' from the transcription initiation site) and correspond to Ad4CAT4.8K, Ad4CAT2.0K, Ad4CAT1.2K, Ad4CAT0.8K, Ad4CAT265, Ad4CAT92, and Ad4CAT60, respectively, as described previously (24). The E-mut plasmid (generated from Ad4CAT0.8K) contains a site-specific mutation in the SF-1 E box that converts the E box at -82/-77 from CACTGT to CTGTAG. E-mut corresponds to the Ad4CATM3 plasmid as previously described (24).

Construction of SF-1 gene promoter/intron reporter gene plasmids containing mutations in either or both the E box at -82 or the SF-1 binding site in the first intron has been previously described (25). The -800Int plasmid (corresponding to plasmid Ad4ECAT0.8K) contains the 4.4-kb DNA fragment located between the BamHI site at 0.8-kb upstream from the transcription initiation site and a SmaI site generated by site-directed mutagenesis in the second exon at 5 bp upstream from the initiation methionine. Mutations in either the E box (Ebox-mutant), SF-1 binding site (S-mutant), or both (S+Ebox-mutant) were generated from the wild-type -800Int plasmid by site-directed mutagenesis. These constructs correspond to Ad4ECAT0.8KM, Ad4ECAT0.8KA, and Ad4ECAT0.8KMA, respectively (25).

Cell Culture and Transfection
{alpha}T3–1 (53) and LßT2 (54) (mouse pituitary gonadotrope), CV-1 (monkey kidney), JEG-3 (human choriocarcinoma), and HeLa (human cervical carcinoma) cells were maintained in DMEM (GIBCO BRL) supplemented with 10% FBS (HyClone. Logan, UT), 4.5 mg glucose per ml, 100 U of penicillin per ml, and 0.1 mg of streptomycin per ml in a humidified atmosphere of 5% CO2. Y1 (mouse adrenocortical) cells were cultured in Ham’s F-10 medium (GIBCO BRL) supplemented with 15% horse serum (GIBCO BRL), 5% FBS, and additional supplements and atmosphere as indicated above.

Transient transfections were performed by the calcium phosphate precipitation method without glycerol shock (55). Cells were plated at a density of 40–60% confluency on 10-cm plates 18 h before transfection. Five micrograms of SF-1 gene promoter-CAT reporter expression plasmids were cotransfected with 5 µg of TK promoter-luciferase plasmid or ß-galactosidase under the control of the human cytomegalovirus promoter (CMV-ß-gal) as an internal transfection control. Additional plates were transfected with a TK-CAT reporter gene plasmid as a control for transfection in each different cell type. Cells were incubated with precipitates for 5 h in a humidified atmosphere of 5% CO2 and washed once each in PBS and culture medium, followed by a change of medium. Cells were harvested after 36 h by scraping into a buffer of 150 mM NaCl, 1 mM EDTA, and 40 mM Tris-Cl (pH 7.4) at 4 C. Extracts of harvested cells were made by brief centrifugation and resuspension of cell pellets in 100 µl of 250 mM Tris-Cl (pH 7.8) at 4 C followed by three cycles of freeze-thawing. Extracts were clarified by centrifugation for 5 min in an Eppendorf 5415C centrifuge. Supernatants were assayed for luciferase activity (56) with an AutoLumat 953 or MicroLumat 96P luminometer (EG&G Berthold, Bad Wildbad, Germany), ß-galactosidase activity (57), and CAT activity by the organic phase extraction method (58) with minor modifications (59). Results are reported as the mean CAT activity from at least three separate transfection experiments corrected for the activity of the cotransfected internal control. Error is reported as SEM.

EMSAs
Nuclear extracts from {alpha}T3–1, CV-1, JEG-3, Y1, and HeLa cells were prepared as previously described (60). Shift reactions were performed at a final volume of 15 µl in a solution containing 10 mM HEPES, pH 7.9, at 25 C, 10% (vol/vol) glycerol, 50 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, 0.2 mg BSA per ml, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1% Nonidet P-40, and 15 ng/µl poly(dI-dC). One to two microliters of nuclear extract were included at 5–10 µg/µl as determined relative to a BSA standard by the method of Bradford (61). Reaction mixtures with extract were preincubated for 10 min on ice followed by the addition of 20–50 fmol of radiolabeled, double-stranded oligonucleotide probe either with or without the addition of unlabeled competitor oligonucleotide at a 100-fold molar excess. Reaction mixtures were further incubated for 5 min at 25 C, and DNA-protein complexes were resolved by electrophoresis on 5% acrylamide-N,N'-bisacrylamide (30:1) gels at 9 V/cm. An autoradiogram of the gels was made by exposing dried gels to XAR-5 film (Kodak, Rochester, NY) using DuPont Cronex intensifying screens at -80 C. Antibody supershift assays were performed identically with the inclusion at the preincubation step of 1 ng of purified, reconstituted rabbit anti-USF-1 or anti-USF-2 antibody (a generous gift of Michele Sawadogo), or normal rabbit IgG (Vector Laboratories, Inc., Burlingame, CA).

The self-complementary oligonucleotide representing the SF-1 E box (E box) contains the sequence (5'-tTGCAGAGTCACGTGGGGGCAGAG-3') and comprises the nucleotide sequence from -90 to -68 of the rat SF-1 gene (24). The self-complementary E-mut oligonucleotide is identical to the E box oligonucleotide sequence, with the exception that CACGTG was converted to CTGTAT, creating a site-specific mutation from -81/-78 in the E box. The self-complementary oligonucleotide representing the consensus USF binding site (USF) contains the sequence 5'-tCTGAATTCCTGGTCACGTGACCGCAGCTGT-3' (62). The self-complementary oligonucleotides representing two different E boxes in the human {alpha}-subunit promoter (33) contain the sequences 5'-tGCTTAGATGCAGGTGGAAACACT-3' ({alpha}EB1) and 5'-tGTATAAAAGCAGGTGAGGACTTC-3' ({alpha}EB2). One thymidine nucleotide (t) was included at the 5'-end of each synthetic oligonucleotide to facilitate end-labeling reactions (57). Ten to 20 pmol of each double-stranded oligonucleotide were used in fill in labeling reactions with Klenow fragment and 100 µCi of [{alpha}32P]dATP (3000 Ci/mmol, DuPont NEN, Boston, MA) to generate oligonucleotide probes. Probe reactions were stopped with the addition of 5 mM EDTA, extracted with phenol-chloroform (1:1), and purified over a Probe-Quant G-50 microcolumn (Pharmacia Biotech, Piscataway, NJ). The probe concentration was adjusted to 20–50 nM in 10 mM Tris-Cl (pH 7.4), 1 mM EDTA, and 50 mM NaCl and stored at -20 C.


   ACKNOWLEDGMENTS
 
We thank Ken-Ichirou Morohashi and Masatoshi Nomura for generously providing the rat SF-1 promoter-CAT reporter plasmids. We also thank Michele Sawadogo for providing anti-USF-1 and USF-2 antibodies, and Sandra Holley and Flavia Pernasetti for critical review of the manuscript.


   FOOTNOTES
 
Address requests for reprints to: Pamela L. Mellon, Departments of Reproductive Medicine-0674, University of California, San Diego, School of Medicine, Cellular and Molecular Medicine Building, Room 2057, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail: pmellon{at}ucsd.edu

This work was supported by NRSA Grant DK-09468 from the NIDDK (to A.N.H.) and NIH Grants RO1DK-20377 and HD-12303 (to P.L.M.).

Received for publication September 10, 1997. Revision received December 12, 1997. Accepted for publication January 14, 1998.


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N. A. Ciccone, C. T. Lacza, M. Y. Hou, S. J. Gregory, K.-Y. Kam, S. Xu, and U. B. Kaiser
A Composite Element that Binds Basic Helix Loop Helix and Basic Leucine Zipper Transcription Factors Is Important for Gonadotropin-Releasing Hormone Regulation of the Follicle-Stimulating Hormone {beta} Gene
Mol. Endocrinol., August 1, 2008; 22(8): 1908 - 1923.
[Abstract] [Full Text] [PDF]