The Murine Dax-1 Promoter Is Stimulated by SF-1 (Steroidogenic Factor-1) and Inhibited by COUP-TF (Chicken Ovalbumin Upstream Promoter-Transcription Factor) via a Composite Nuclear Receptor-Regulatory Element

Richard N. Yu, Masafumi Ito and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, Illinois 60611


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Dax-1 gene encodes a protein that is structurally related to members of the orphan nuclear receptor superfamily. Dax-1 is coexpressed with another orphan nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal, gonads, hypothalamus, and pituitary gland. Mutations in Dax-1 cause adrenal hypoplasia congenita, a disorder that is characterized by adrenal insufficiency and hypogonadotropic hypogonadism. These developmental and endocrine abnormalities are similar to those caused by disruption of the murine Ftz-F1 gene (which encodes SF-1), suggesting that these nuclear receptors act along the same developmental cascade. Cloning of the murine Dax-1 gene revealed a candidate SF-1-binding site in the Dax-1 promoter. In transient expression assays in SF-1-deficient JEG-3 cells, SF-1 stimulated expression of the Dax-1 promoter. However, deletion or mutation of the consensus SF-1-binding site did not eliminate SF-1 stimulation. Further analyses revealed the presence of a cryptic SF-1 site that creates an imperfect direct repeat of the SF-1 element. When linked to the minimal thymidine kinase promoter, each of the isolated SF-1 sites was sufficient to mediate transcriptional regulation by SF-1. Mutation of both SF-1 sites eliminated SF-1 binding and stimulation of the Dax-1 promoter. Unexpectedly, mutation of either half of the composite SF-1 sites increased basal activity in JEG-3 cells, suggesting interaction of a repressor protein. Gel shift analyses of the composite response element revealed an additional complex that was not supershifted by SF-1 antibodies. This complex was eliminated by mutation of either half-site, and it was supershifted by antibodies against chicken ovalbumin upstream promoter-transcription factor (COUP-TF). We propose that Dax-1 is stimulated by SF-1, and that SF-1 and COUP-TF provide antagonistic pathways that converge upon a common regulatory site.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Dax-1 gene was identified based upon its association with adrenal hypoplasia congenita (AHC), an X-linked disorder that causes adrenal insufficiency and hypogonadotropic hypogonadism (1, 2). The Dax-1 locus on Xp21 has also been associated with dosage-sensitive sex reversal in males in which this region of the X-chromosome is duplicated (3). These features are reflected in its name, DAX-1 (dosage-sensitive sex-reversal-AHC critical region on the X-chromosome) (1).

The cloning of Dax-1 revealed that it encodes a variant member of the orphan nuclear receptor superfamily (1). The highest degree of homology with other nuclear receptors resides in the C-terminal portion of the DAX-1 protein. This putative ligand-binding domain is most homologous with that of chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and steroidogenic factor-1 (SF-1) (1, 4, 5). However, DAX-1 is unique by virtue of the amino-terminal domain that lacks the zinc-finger DNA-binding domain that is characteristic of other nuclear receptors. Instead, DAX-1 contains a unique N-terminal domain composed of a repeating 66- to 67-amino acid motif (1, 6). Recently, DAX-1 has been shown to bind to palindromic stem-loop sequences that are present in the promoters of some of its target genes (7), and it acts as a potent repressor of transcription (1, 8, 9).

DAX-1 is expressed in a tissue-specific manner that reflects sites of endocrine dysfunction in patients with AHC, including the adrenal cortex, testis, ovary, anterior pituitary gonadotropes, and ventral medial hypothalamus (10, 11, 12, 13). These sites of expression are also characteristic of tissues that express SF-1 (13, 14, 15, 16, 17). Moreover, disruption of the Ftz-f1 locus that encodes SF-1 results in a phenotype that partially resembles AHC (18, 19, 20, 21). Mice that are homozygous for the SF-1 gene knockout exhibit absent adrenal glands and gonads, as well as hypogonadotropic hypogonadism (15, 16, 20, 22). The male mice are sex-reversed, apparently reflecting impaired development of the primordial cells that give rise to the differentiated gonad (18). There may also be a defect in SF-1 regulation of the Müllerian-inhibiting substance (MIS) gene (15) and the steroidogenic enzyme genes (23, 24). The absence of male sex-differentiation in the SF-1 knockout mice is distinct from the effects of Dax-1 mutations, which allow normal development of the male phenotype in affected humans (25).

The similar phenotypic features caused by Ftz-f1 and Dax-1 mutations, and their similar spatial and developmental patterns of expression, have led to the suggestion that there is a functional relationship between these two factors (8, 11, 13, 26). Although little is known about the function of DAX-1, SF-1 is known to regulate an array of steroidogenic enzyme genes (24), as well as genes involved in sex differentiation (15) and gonadotropin regulation (15, 16, 27, 28, 29, 30). It has been suggested that DAX-1 might interact with SF-1 to either stimulate or antagonize its transcriptional properties (7, 8, 13, 24). Alternatively, it is possible that DAX-1 and SF-1 participate in a developmental cascade in which the product of one gene regulates expression of the other gene (13, 24, 26). An SF-1-binding site has been reported in the promoter of the human Dax-1 gene (31) and the mouse Dax-1 gene (13). In this report, we examined whether SF-1 regulates the murine Dax-1 gene. Unexpectedly, we found redundant SF-1-binding sites in the Dax-1 promoter and demonstrate that this duplicated region also creates a binding site for COUP-TF, which acts as a repressor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Murine Dax-1 Promoter Contains a Duplicated Binding Site for SF-1
Previous analyses of the human Dax-1 promoter indicated the presence of an SF-1-binding site (13, 31). The murine Dax-1 gene was isolated by screening a genomic library with the human Dax-1 cDNA. The 5'-flanking region (2.9 kb) of the murine Dax-1 promoter was sequenced, and the proximal region near the transcriptional start site is shown in Fig. 1Go. The indicated transcriptional start site was confirmed using ribonuclease (RNase) protection assays of mRNA from murine adrenocortical Y1 and murine anterior pituitary gonadotrope {alpha}T3 cells (data not shown), and it corresponds to the site reported by Ikeda et al. (13). Analysis of the Dax-1 promoter sequence revealed that a putative SF-1-binding site (-129 to -121, TCGAGGTCA) is also present in the murine gene.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 1. Structure of the Murine DAX-1 Promoter

The DNA sequence of the proximal region of the murine DAX-1 promoter is shown, and the nucleotides are numbered relative to the transcriptional start site (arrow), identified by RNase protection assays. The TATA-box is underlined and the translation initiation codon (ATG) is boxed with the encoded amino acids shown below the codons. Two overlapping elements resembling the SF-1 consensus sequence are indicated by horizontal arrows as site A (-129 to -121) and site B (-123 to -115).

 
Electrophoretic mobility shift assays (EMSAs) were performed to examine SF-1 binding to the murine Dax-1 promoter (Fig. 2Go). Using in vitro translated murine SF-1, a major complex was formed that was eliminated by the addition of an antibody to SF-1 (see below). A mutation (GG -> TT) was introduced into the putative SF-1 site (-129 to -121) to confirm that it binds SF-1 (Fig. 2AGo). This two-nucleotide mutation eliminates SF-1 binding to a consensus SF-1 site (data not shown). Unexpectedly, this mutation (134 m1a) had no apparent effect on SF-1 binding (Fig. 2BGo). This finding led us to consider whether an additional SF-1-binding site might reside within this region of the Dax-1 promoter. Inspection of the sequence suggested a possible cryptic SF-1 site (site B) adjacent to the consensus sequence (site A). Mutations were therefore also introduced into site B, or sites A and B. Like the mutation in site A (134 m1a), the mutation in site B alone (134 m1b) was not sufficient to eliminate SF-1 binding. However, a double mutation in sites A and B (134 m1ab) prevented SF-1 binding. An additional mutation (134 m3), which was predicted not to change nucleotides critical for SF-1 binding, had no effect on the formation of the SF-1 complex. These results are consistent with the ability of SF-1 to bind to either sites A or B in the composite element.



View larger version (36K):
[in this window]
[in a new window]
 
Figure 2. SF-1 Binds Specifically to the Murine DAX-1 Promoter

A, Oligonucleotide DNA sequences (sense strands) used in the EMSAs. Mutations introduced into the sequence are indicated below the wild-type oligonucleotide (134wt). B, EMSAs were performed using in vitro translated murine SF-1 (3 µl lysate). 32P-labeled probe (20 fmol) was incubated with 2 µl reticulocyte lysate and resolved on 4% polyacrylamide gels. The SF-1 band is indicated by an arrow. NP indicates the use of nonprogrammed reticulocyte lysate.

 
The binding characteristics of sites A and B were also examined using nuclear extracts prepared from {alpha}T3 cells and Y1 cells (Fig. 3Go). Both of these cell lines have been shown previously to contain abundant SF-1 protein (22, 32). Using a 25-bp sequence that contains both of the putative SF-1 binding sites (134wt; -134 to -110 bp), nuclear extracts from {alpha}T3 and Y1 cells resulted in three major protein/DNA complexes, A, B, and C (Fig. 3Go, A and B). An antibody directed against the DNA-binding domain of SF-1 specifically prevented the formation of complex A, indicating that this band corresponds to bound SF-1. The anti-SF-1 antibody did not alter complexes B or C, suggesting that they do not contain SF-1.



View larger version (85K):
[in this window]
[in a new window]
 
Figure 3. Competition for SF-1 Binding to Sites A and B in the Dax-1 Promoter

EMSAs were performed using nuclear extracts (5 µg) prepared from a murine {alpha}T3 pituitary gonadotrope cell line (panel A) and from a murine Y-1 adrenocortical cell line (panel B). The indicated 32P-labeled probes (20 fmol) were incubated with nuclear extracts and resolved on 4% polyacrylamide gels. Three distinct protein/DNA complexes were formed (A, B, and C) with the 134wt probe. The identity of SF-1 was determined by using a polyclonal rabbit antimurine SF-1 antibody (complex A).

 
Because site B might be configured as either a direct repeat, or as a palindrome, two sets of mutations were introduced into this site (134 m1b and 134 m1c, respectively) (see Fig. 2AGo). As found with in vitro translated SF-1, mutations of site A (134 m1a) or site B (134 m1b, 134 m1c) alone were not sufficient to eliminate SF-1 binding (complex A). The site A mutation (134 m1a) eliminated complexes B and C, whereas the site B mutations only partially decreased binding to the slower mobility complexes (complexes B and C). The double mutation of both sites A and B (134 m1ab) eliminated the binding of all three major complexes, including SF-1 (complex A). These results confirm that the murine Dax-1 promoter contains duplicated SF-1 sites and indicate that additional proteins also bind to this composite element.

Competition studies were performed to define further the characteristics of the complexes binding to the composite SF-1 sites (Fig. 4Go). Using extracts from Y1 cells, competition with a canonical SF-1 sequence (gatcTCAAGGTCAgatc) inhibited the binding of all three major complexes, although complex C was affected less than complexes A and B (Fig. 4AGo and data not shown). These competition studies also revealed the presence of minor bands at the positions of complexes A and B that are not inhibited by the canonical SF-1 site. As before, an antibody directed against SF-1 impaired the binding of complex A, but did not alter complexes B or C. An irrelevant control antibody directed against retinoid X receptor-{alpha} did not affect the SF-1 complex.



View larger version (64K):
[in this window]
[in a new window]
 
Figure 4. Competition for Binding to the Murine DAX-1 Composite Element

EMSAs were performed using nuclear extracts (5 µg) prepared from Y-1 cells and the wild-type -134 radiolabeled probe. In panel A, competition was performed using 100-fold or 500-fold excess of an unlabeled canonical SF-1 sequence (gatcTCAAGGTCAgatc). Antibodies include anti-retinoid X receptor-{alpha} and anti-SF-1. In panels B and C, the indicated competitor (100-fold excess) oligonucleotides were incubated in the presence of nuclear extract, and EMSAs were performed in the absence (panel B) or presence (panel C) of the anti-SF-1 antibody.

 
Competition studies were also performed using various mutants of the Dax-1 composite element (Fig. 4BGo). The site A mutation (134 m1a) retained competition for SF-1 (complex A), but failed to compete for complexes B and C, consistent with the ability of site B to bind SF-1 but not the other complexes. The single mutations of site B (134 m1b, 134 m1c) retained competition for each of the complexes, indicating that, like the canonical SF-1 sequence, site A is sufficient to bind all three complexes. Mutations in both sites A and B (134 m1ab) did not compete for any of the complexes. Similar competition studies were also performed in the presence of the anti-SF-1 antibody (Fig. 4CGo). The antibody did not affect complexes B or C, even in the presence of a selective competitor for SF-1 (134 m1a). Taken together, these results are consistent with SF-1 binding to both sites A and B. In addition, mutations in site A, but not site B, impair the binding of complexes B and C.

SF-1 Stimulates the Murine Dax-1 Promoter in SF-1-Deficient JEG-3 Cells
JEG-3 cells are a placental choriocarcinoma cell line that is deficient in SF-1 as assessed by RT-PCR and by Western blot analysis (8). The effect of SF-1 on promoter activity was examined in this cell line by cotransfecting various Dax-1 promoter deletion mutants in the absence or presence of an SF-1 expression vector (cytomegalovirus-driven SF-1). In the absence of cotransfected SF-1, there was little change in basal Dax-1 promoter activity until deletion from -114 to -84 bp, which resulted in about 2-fold stimulation (Fig. 5AGo). Further deletion from -84 to -50 bp markedly decreased promoter activity, suggesting the presence of a basal element in this region.



View larger version (33K):
[in this window]
[in a new window]
 
Figure 5. Transcriptional Activity of the Murine DAX-1 Promoter in the Absence or Presence of an SF-1 Expression Construct

A and B, Deletion series of murine DAX-1 promoter-luciferase reporter constructs transfected into JEG-3 cells with an empty expression construct (panel A) or with a CMV-driven, SF-1 expression construct (panel B). C and D, Mutant series of murine DAX-1 promoter-luciferase reporter constructs transfected into JEG-3 cells with an empty expression construct (panel C) or with a CMV-driven, SF-1 expression construct (panel D). Each reporter plasmid (0.5 µg) was transfected into JEG-3 cells with 100 ng of the empty or SF-1 expression constructs and cultured for 48 h. Basal activity is shown in ALU. Fold-stimulation by SF-1 is calculated for each construct as the ratio of promoter activity in the presence and absence of transfected SF-1. The error bars represent mean ± SD. Control refers to a promoterless luciferase plasmid.

 
Because the deletion mutations alter the level of basal activity, the effect of cotransfected SF-1 is presented as fold-stimulation relative to the basal activity of each construct (Fig. 5BGo). The full-length Dax-1 promoter (2938 bp) was stimulated 17-fold by cotransfection of SF-1. SF-1 had no effect on the promoterless plasmid or control viral promoters (data not shown). There was a progressive decline of SF-1 stimulation between -2938 and -1376 bp before returning to the maximal level with further deletions between -861 and -134 bp. Deletion of the SF-1-binding sites (sites A and B) between -134 and -114 bp resulted in an abrupt loss of SF-1-mediated transcription.

Point mutations of site A or site B, or in sites A and B, were used to correlate the functional regulation by SF-1 with its binding properties. These mutations in the native -134 Dax-1 promoter are identical to those introduced into the EMSA probes (Fig. 2AGo). Unexpectedly, the 134 m1a point mutation in site A caused a 4- to 5-fold increase in basal promoter activity relative to the 134wt reporter gene (Fig. 5CGo). This result raised the possibility that a repressor may bind to site A (this mutation eliminates complexes B and C). A less pronounced increase in basal activity was also observed with the 134 m1b mutation in site B, and the double mutant of sites A and B (134 m1ab) increased basal activity to a level similar to that of the site A mutant alone. SF-1 stimulation of the 134wt promoter was reduced from 17-fold to less than 5-fold by each of the individual SF-1-binding site mutations. These data indicate that each of the SF-1-binding sites are required for maximal induction of the Dax-1 promoter by SF-1.

A series of heterologous promoter constructs was also created to determine whether the isolated composite regulatory element is sufficient for SF-1-mediated transcriptional regulation (Fig. 6Go). The -134 to -110 sequence from the murine Dax-1 promoter was inserted upstream of a minimal thymidine kinase (TK) promoter (TK81-luc). Specific point mutations of site A or site B, or both sites A and B, were examined with the TK reporter constructs. The results with the TK promoter largely parallel the findings with the native Dax-1 promoter. Relative to the wild-type element, the 134 m1a (site A) mutation increased basal promoter activity, but there was less effect with the 134 m1b and the double mutant, 134 m1ab (Fig. 6AGo). As with the native promoter, mutation of either site A or site B, or sites A and B, reduced or eliminated stimulation by SF-1.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 6. SF-1-Regulatory Elements from the DAX-1 Promoter Are Functional When Linked to a Heterologous Promoter

Regulatory activity of the isolated composite response element of the murine DAX-1 promoter. Oligonucleotides containing the wild-type and mutant SF-1 response elements described in Fig. 2Go were fused to a TK minimal promoter luciferase reporter construct. These constructs were transfected into JEG-3 cells with an empty expression construct (panel A) or with a CMV-driven, SF-1 expression construct (panel B). Each reporter plasmid (1.0 µg) was transfected into JEG-3 cells with 100 ng of the empty or SF-1 expression construct. Luciferase activity (mean ± SD) was determined after 48 h. Basal activity is shown in ALU. Fold-stimulation by SF-1 is calculated for each construct as the ratio of promoter activity in the presence and absence of transfected SF-1.

 
Mutation of the Dax-1 Promoter SF-1 Sites Reduces Basal Activity in SF-1-Containing {alpha}T3 Cells and Y1 Cells
The functional role of the SF-1-binding sites was also analyzed in cell lines (pituitary gonadotrope {alpha}T3 cells and murine adrenocortical Y1 cells) that express SF-1 endogenously (22, 33). Both cell lines exhibited a high basal level of expression of the full-length 2.9-kb Dax-1 promoter construct, consistent with the presence of SF-1 in these cell lines (Fig. 7Go, A and C). Sequential deletion of the 5'-flanking region did not alter promoter activity substantially until deletion between -134 and -114 bp (includes sites A and B), which caused ~50% loss of activity in both {alpha}T3 and Y1 cells. Further deletion from -114 to -84 bp had little effect, but deletion to -50 bp reduced activity to background levels.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 7. Native Murine DAX-1 Promoter Transcriptional Activity in SF-1-Containing Cell Lines

Luciferase activity of the deletion series (panels A and C) and mutant series (panels B and D) of murine DAX-1 promoter luciferase reporter constructs. Each reporter plasmid (0.5 µg) was transfected into {alpha}T3 anterior pituitary cells (panels A and B) or into Y1 adrenal cells (panels C and D). Luciferase activity (mean ± SD) was measured after 48 h. Basal activity is shown in ALU.

 
When -134 bp promoter constructs containing specific point mutations of the composite regulatory element were transfected, a reduction of basal luciferase activity was observed upon mutation of either site A or site B, or both sites A and B (Fig. 7Go, B and D). The deletion and point mutation studies indicate that the SF-1 sites confer 30–50% of the basal activity of the Dax-1 promoter in cell lines that express high levels of SF-1.

The isolated SF-1 elements linked to the TK promoter confirmed the results of the native promoter studies in both {alpha}T3 and Y1 cells (Fig. 8Go). The site A -134/-110 m1a-TK construct caused 30–50% reduction of activity. The site B mutation (-134/-110 m1b), and the combined site A and site B mutations (-134/-110 m1ab), reduced activity close to that of the TK promoter alone.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 8. The Composite SF-1 Response Element is Sufficient for Murine DAX-1 Promoter Activity in {alpha}T3 and Y1 Cells

Oligonucleotides containing the wild-type and mutant SF-1 response elements described in Fig. 2Go were fused to a TK minimal promoter luciferase reporter construct. These constructs were transfected into {alpha}T3 (panel A) or into Y-1 cells (panel B). Each reporter plasmid (1.0 µg) was transfected into JEG-3 cells with 100 ng of the empty or SF-1 expression construct. Luciferase activity (mean ± SD) was determined after 48 h. Basal activity is shown in ALU.

 
The Composite SF-1-Binding Site Binds COUP-TF Family Members
As shown above, in addition to SF-1, the 134wt probe binds a complex (complex B) that has slower mobility and appears to contain several distinct bands. A series of antibodies were used in an attempt to identify additional proteins that might bind to SF-1-like sequences. Using {alpha}T3 nuclear extracts, antibodies directed against COUP-TF caused a supershift of most of the proteins in complex B (Fig. 9AGo). As shown before, anti-SF-1 antibody eliminated the binding of complex A. Antibodies against DAX-1, cAMP-response element binding protein (CREB), and phospho-CREB did not alter the binding pattern of the complexes. Inspection of the DNA sequence of the composite SF-1 site revealed the presence of an imperfect palindrome (-129/-115) that overlaps the SF-1 direct repeat (Fig. 9BGo). The palindromic element exhibits similarity to several sequences previously reported to bind and mediate COUP-TF activity (34, 35).



View larger version (40K):
[in this window]
[in a new window]
 
Figure 9. COUP-TF Binds to the Composite Regulatory Element

A, Demonstration of COUP-TF binding to the SF-1 regulatory composite element. EMSAs were performed using nuclear extracts (5 µg) prepared from a murine pituitary gonadotrope cell line, {alpha}T3. 32P-labeled 134wt probe (20 fmol) was incubated with the nuclear extracts as described in Fig. 2Go. Supershift analyses were performed using antibodies against DAX-1, SF-1, COUP-TF, CREB, and phospho-CREB proteins. B, Comparison of the composite element DNA sequence to known COUP-TF-binding sites. PAL, Palindromic version of the GGTCA sequence; mPOMC, mouse POMC element. C and D, EMSAs were performed using in vitro translated Ear3/COUP-TF1 (panel C) or ARP1/COUP-TF2 (panel D) proteins. Wild-type or mutant 32P-labeled probe (20 fmol) was incubated with in vitro translated products as described in Fig. 2Go. NP indicates the use of nonprogrammed reticulocyte lysate.

 
Murine COUP-TF1 and COUP-TF2 were in vitro translated and tested in EMSAs for binding to the composite element (Fig. 9Go, C and D). Using the 134wt probe, both COUP-TF1 and COUP-TF2 bound to the composite site, although greater binding was seen with COUP-TF2. As seen with the native extracts, mutation of site A eliminated COUP-TF binding, whereas mutations of sites B or C reduced COUP-TF binding. The double mutation of sites A and B also eliminated the binding of COUP-TF. These data indicate that site A is necessary for the binding of COUP-TF, but sequences in site B also serve to provide maximal binding of COUP-TF.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The murine Dax-1 promoter contains a duplicated binding site for SF-1. Both sites are capable of binding SF-1 and can mediate transcriptional activation. The combined sites (-134 to -114 region) function as a composite element that is capable of interacting with a number of different proteins, resulting in the formation of three major complexes. Supershift analyses have identified two of the these complexes as SF-1 (complex A) and COUP-TF (complex B). Complex C has not yet been characterized. Both half-sites are required for full SF-1-mediated activation. Disruption of these sites lowered the basal activity of the native Dax-1 promoter in SF-1-containing {alpha}T3 and Y1 cells, demonstrating the requirement for the intact -134/-114 element in maintaining nominal transcriptional activity.

Deletion of the -134/-114 region, or specific mutation of half-site A, increases basal promoter activity in JEG-3 cells, suggesting a relief of repression that requires the presence of site A. This site corresponds to the binding site for complex B, which contains COUP-TF. The inhibitory activity of COUP-TF may be mediated through a direct interaction with a novel class of ubiquitous proteins (36). These factors, N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoic acid receptor and thyroid hormone receptor) (37, 38), possess strong silencing activity. Thus, inhibition by COUP-TF may involve the recruitment of corepressors (36), as well as its ability to block SF-1 interactions with its target site. In the mouse Dax-1 promoter, site A, but not site B, is required for the formation of complex B. Based upon the mobility of complex B, it is likely that COUP-TF binds as a dimer, but further studies will be required to define the nature of the COUP-TF complexes.

In general, COUP-TF serves as a negative regulator of a wide array of genes (39). The two major forms of COUP-TF, I and II, are widely expressed, and their levels are high particularly during organogenesis and neurogenesis when they are believed to exert a repressive function on target genes (39, 40, 41). The spatial expression of COUP-TF overlaps that of DAX-1, although detailed comparisons of their developmental patterns of expression have not been performed. Targeted disruption of COUP-TFI and COUP-TFII result in perinatal (39, 42) and embryonic (39) lethality, respectively. In view of our studies suggesting a role for COUP-TF in the control of the Dax-1 promoter, it will be of interest to determine whether the expression of DAX-1 is altered in these animals.

Inhibition by COUP-TF can be effected through several mechanisms: direct repression, trans-repression, competition for binding to a shared DNA element, or competition for cofactors or heterodimeric partners (39). In the case of the Dax-1 promoter, COUP-TF appears to bind to the composite element with an affinity equal to or better than SF-1. Transfection studies to examine the direct effect of COUP-TF competition on SF-1-mediated transactivation of the Dax-1 promoter have been limited by the fact that transfected COUP-TF activates the proximal Dax-1 promoter and most other TATA-containing promoters tested under the experimental conditions employed in this study (data not shown). Thus, it is difficult to distinguish the repressive and activating effects of COUP-TF that occur through distinct promoter regions. Competition assays demonstrate that the binding of COUP-TF is specific and that it is not composed of a heterodimeric complex with SF-1. These results suggest that the relative levels of SF-1 and COUP-TF may determine which factors occupy the composite element, thereby determining the level of promoter activity.

SF-1 and DAX-1 colocalize to identical tissues (cells) within the hypothalamic-pituitary-gonadal and -adrenal axes (13). In terms of temporal expression, SF-1 usually precedes or is coexpressed with DAX-1 (13). In light of the phenotypes caused by the mouse Ftz-f1 gene disruption and in the human condition of X-linked AHC with hypogonadotropic hypogonadism, a relationship between these two factors seems highly likely. An interaction along this shared developmental cascade may occur through direct protein-protein interactions, through positive transcriptional regulation of one factor by the other, or through intermediary factors. SF-1 and DAX-1 have been shown to interact directly using in vitro protein interaction studies, but it is unclear whether such interactions occur in transfected cells or in vivo (8). DAX-1 has been shown to inhibit SF-1-mediated transcription (8), and it has also been shown to bind to hairpin loops that are present in the StAR and Dax-1 promoters and to inhibit transcription of these genes (7). A corepressor for DAX-1 has not been identified, but represents an alternative mechanism by which DAX-1 might alter SF-1-mediated effects since DAX-1 has been shown to contain a potent repressor domain (8, 9).

The possibility that Dax-1 might be transcriptionally regulated by SF-1 or vice versa has not been thoroughly examined. The finding that SF-1 expression precedes or coincides with expression of DAX-1 (11, 14) corresponds well with the identification of SF-1-binding sites in the mouse and human Dax-1 promoters and is consistent with the hypothesis that SF-1 may regulate Dax-1 promoter activity. On the other hand, DAX-1 expression was not eliminated in the SF-1 knockout mouse (13), indicating that SF-1 is not obligatory for the expression of Dax-1. This study also found that removal of the SF-1 site had little effect on the activity of the human Dax-1 promoter (13). In contrast to these results, we find that SF-1 clearly contributes to Dax-1 promoter activity. The SF-1 sites contributed about 50% to basal Dax-1 promoter activity in cell lines ({alpha}T3 and Y-1) that express SF-1 endogenously. However, the role of SF-1 was revealed more clearly in SF-1-deficient JEG-3 cells in which it caused 17-fold induction. In addition to the presence of endogenous SF-1, differences between cell lines may also reflect the amounts of COUP-TF, coactivators, and perhaps endogenous ligands for SF-1. Recently, it has been reported that SF-1 is activated by oxysterols, which are generated by the action of P450c27 (e.g. 25-hydroxycholesterol) (43). These, or other ligands, may be present in JEG-3 cells, which are highly steroidogenic (44). Mutation of both SF-1 sites clearly does not eliminate Dax-1 promoter activity, consistent with the idea that multiple factors are involved in the control of its expression. In view of this finding, it is possible that SF-1 modulates Dax-1 expression at certain times during development. This issue may be complicated further by the fact that DAX-1 may autoregulate its expression by acting to inhibit SF-1 (7, 8).

The duplicated SF-1 elements in the murine Dax-1 promoter are not completely conserved in the human gene. We have found that the human sequence also contains two SF-1-binding sites (data not shown). In the murine Dax-1 promoter, these sites act additively to potentiate SF-1-mediated transcriptional activation. It is unlikely that two SF-1 receptors bind to this region simultaneously because only monomeric complexes were observed in EMSA assays, and the duplicated SF-1 sites overlap partially. However, the availability of two sites for SF-1 binding may increase the likelihood that SF-1 can bind to and activate the murine promoter. Perhaps of greater significance, the presence of the second site creates a composite site that may facilitate the binding of COUP-TF. Further studies will be required to assess whether this pathway for regulation of the Dax-1 promoter has been evolutionarily conserved. There is reason to suspect that the interaction between SF-1 and COUP-TF may also occur for other genes that are targets for SF-1. In the case of the bovine Cyp17 gene (encoding P450 steroid 17{alpha}-hydroxylase), SF-1 and COUP-TF have been shown to bind to repeated sequences (AAGTCA and AGGTCA) that are spaced by six nucleotides in the repCRS2 element (34, 45). As with the Dax-1 promoter, SF-1 stimulates this element, and COUP-TF acts as an inhibitor. Thus, it is possible that SF-1 and COUP-TF represent antagonistic pathways at least for a subset of target genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of Murine Dax-1 Gene
Dax-1 genomic DNA clones were isolated from a 129Sv/J murine genomic {lambda} DNA library (Stratagene, La Jolla, CA). The library was screened using full-length human Dax-1 cDNA (8) that was radiolabeled using a random nonamer-labeling kit (Stratagene). After purification of DNA from positive clones using a {lambda} DNA purification kit (Promega, Madison, WI), genomic fragments were excised using NotI and subcloned into the NotI site of pGEM 5Zf(+) (Promega). DNA sequencing was performed using an ABI Prism 377 DNA Sequencer (Perkin-Elmer, Foster City, CA).

Reporter Plasmid Construction
A 2938-bp KpnI–NcoI mDAX promoter DNA fragment was isolated from pGEM 5Z-mDX2–1 and subcloned into the KpnI–NcoI polylinker site of the luciferase reporter construct pGL3 Basic (Promega). This construct, pGL3B-mDX(-2938), was used to generate sequential 5'-deletions of the DAX-1 promoter by exonuclease III/mung bean nuclease digestion (Stratagene). The DNA sequences of the resulting deletion constructs were confirmed by DNA sequencing. Site-directed mutagenesis and deletion of more proximal DAX-1 promoter fragments were performed by PCR amplification using Deep Vent polymerase (New England Biolabs, Beverly, MA) and synthetic oligonucleotide primers (GIBCO/BRL, Bethesda, MD). Primer pairs consisted of a 5'-sense DAX-1 promoter primer and a 3'-antisense luciferase gene primer (LUCseq, 5'-GAATGGCGCCGGGCCTTTCTT-3'). The following DAX-1 promoter primers were used for site-directed mutagenesis (sense sequence, XhoI restriction enzyme site in lowercase; DAX promoter in uppercase):

mDX134wt, 5'-gatcctcgagAGCTTTCGAGGTCATGGCCA-3';

mDX134m1a, 5'-gatcctcgagAGCTTTCGATTTCATGGCCA-3';

mDX134m1b, 5'-gatcctcgagAGCTTTCGAGGTCATTTCCACA-3';

mDX134m1ab, 5'-gatcctcgagAGCTTTCGATTTCATTTCCACA-3';

mDX134m3, 5'-gatcctcgagAGCTTTCGAGGCCATTTCCACA-3'

mDX114wt, 5'-gatcctcgagCACACATTCAAGCACAAAGG-3';

mDX84wt, 5'-gatcctcgagTCTGCGCCCTTGTCCAAGAG-3';

mDX50wt, 5'-gatcctcgagGCTTGCGTGCGCATTCAGTA-3'.

Amplification products were digested with XhoI and NcoI and subcloned into the XhoI–NcoI polylinker site of pGL3 Basic. All DAX-1 promoter reporter constructs were confirmed by DNA sequencing. The pTK81-mDAX heterologous constructs were prepared using the following double-stranded oligonucleotides that correspond to the DAX-1 composite element primers (sense strand:

81/mDX134wt, 5'-tcgagAGCTTTCGAGGTCATGGCCACACACactagta-3';

81/mDX134m1a, 5'-tcgagAGCTTTCGATTTCATGGCCACACACactagta-3';

81/mDX134m1b, 5'-tcgagAGCTTTCGAGGTCATTTCCACACACactagta-3';

81/mDX134m1ab, 5'-tcgagAGCTTTCGATTTCATTTCCACACACactagta-3'.

Each annealed primer pair was subcloned into the polylinker site of pTK81 (46, 47), immediately upstream of the thymidine kinase minimal promoter.

Cell Culture, Transfections, and Luciferase Assays
Murine pituitary gonadotrope {alpha}T3 cells (48) and human placental JEG-3 cells (American Type Culture Collection, HTB-36) were grown in DMEM supplemented with 10% FBS in a 5% CO2 atmosphere at 37 C. Murine adrenocortical Y1 cells (American Type Culture Collection, CCL-79) were grown in Ham’s F10 medium supplemented with 15% horse serum and 2.5% FBS. Cells were transfected by the calcium phosphate method as previously described (49). Luciferase assays (50) were performed 48 h after transfection and are reported in arbitrary light units (ALU). Basal activity is expressed in ALU, and fold-stimulation by SF-1 is expressed for each construct as the ratio of promoter activity in the presence and absence of transfected SF-1 expression vector. Results are the mean ± SD of triplicate transfections.

EMSAs
Nuclear extracts were isolated from the indicated cell lines as previously described (51). Protein concentrations were determined using the Bradford assay system (Bio-Rad, Hercules, CA). The following oligonucleotides (sense strand) were used for EMSAs:

mDX134wt, 5'-AGCTTTCGAGGTCATGGCCAC-3';

mDX134m1a, 5'-AGCTTTCGATTTCATGGCCAC-3';

mDX134m1b, 5'-AGCTTTCGAGGTCATTTCCAC-3';

mDX134m1ab, 5'-AGCTTTCGATTTCATTTCCAC-3'.

The oligonucleotide pairs were annealed and labeled with [{alpha}-32P]dCTP using Klenow DNA polymerase. Nuclear extracts (5 µg) were incubated with 20 fmol radiolabeled, double-stranded oligonucleotides for 30 min at room temperature in a volume of 20 µl. Protein-DNA complexes were resolved on 4% nondenaturing, polyacrylamide gels using 0.5x Tris-borate-EDTA (TBE) buffer.

In vitro transcription and translation were performed with the TnT reticulocyte lysate system (Promega) as recommended by the manufacturer. T3 or T7 RNA polymerase was used for the transcription of SF-1 (8), COUP-TF1 (52), and COUP-TF2 (53, 54). In vitro translated products (2 µl) were incubated with 20 fmol radiolabeled, double-stranded oligonucleotides for 30 min at room temperature in a final volume of 20 µl. The DNA and protein complexes were resolved as described above. Antibodies against COUP-TF were provided by M. J. Tsai (Baylor College of Medicine, Houston, TX). DAX-1 antibodies were raised in rabbits using a peptide fragment (LTEHIRMMQREYQIR; Research Genetics, Huntsville, AL). SF-1, CREB, and phospho-CREB antibodies were obtained from Upstate Biotechnology (Lake Placid, NY).


    ACKNOWLEDGMENTS
 
We are grateful to M. J. Tsai for kindly providing COUP-TF antibodies and expression vectors.

This work was performed as part of the National Cooperative Program for Infertility Research (NIH Grant U54-HD-29164); R.N.Y. was the receipient of NIH training grant (T32 DK-07169).


    FOOTNOTES
 
Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Tarry Building 15–709, Chicago, Illinois 60611. E-mail: ljameson{at}nwu.edu

Received for publication July 3, 1997. Revision received January 21, 1998. Accepted for publication March 9, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, Lalli E, Moser C, Walker AP, McCabe ER, Meitinger T, Monaco AP, Sassone-Corsi P, Camerino G 1994 An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635–641[CrossRef][Medline]
  2. Muscatelli F, Strom TM, Walker AP, Zanaria E, Recan D, Meindl A, Bardoni B, Guioli S, Zehetner G, Rabl W, Schwarz H-P, Kaplan J, Camerino G, Meitinger T, Monaco AP 1994 Mutations in the DAX-1 gene give rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature 372:672–676[CrossRef][Medline]
  3. Bardoni B, Zanaria E, Guioli S, Floridia G, Worley KC, Tonini G, Ferrante E, Chiumello G, McCabe ER, Fraccaro M, Zuffardi O, Camerino G 1994 A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal. Nat Genet 7:497–501[Medline]
  4. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[Medline]
  5. Enmark E, Gustafsson JA 1996 Orphan nuclear receptors–the first eight years. Mol Endocrinol 10:1293–1307[Medline]
  6. Burris TP, Guo W, McCabe ER 1996 The gene responsible for adrenal hypoplasia congenita, DAX-1, encodes a nuclear hormone receptor that defines a new class within the superfamily. Recent Prog Horm Res 51:241–259[Medline]
  7. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311–315[CrossRef][Medline]
  8. Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17:1476–1483[Abstract]
  9. Lalli E, Bardoni B, Zazopoulos E, Wurtz J-M, Strom TM, Moras D, Sassone-Corsi P 1997 A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita. Mol Endocrinol 11:1950–1960[Abstract/Free Full Text]
  10. Guo W, Burris TP, McCabe ER 1995 Expression of DAX-1, the gene responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism, in the hypothalamic-pituitary-adrenal/gonadal axis. Biochem Mol Med 56:8–13[CrossRef][Medline]
  11. Swain A, Zanaria E, Hacker A, Lovell-Badge R, Camerino G 1996 Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function. Nat Genet 12:404–409[Medline]
  12. Majdic G, Saunders PT 1996 Differential patterns of expression of DAX-1 and steroidogenic factor-1 (SF-1) in the fetal rat testis. Endocrinology 137:3586–3589[Abstract]
  13. Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL 1996 Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development. Mol Endocrinol 10:1261–1272[Abstract]
  14. Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654–662[Abstract]
  15. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312[Abstract]
  16. Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear factor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 9:478–486[Abstract]
  17. Morohashi K, Iida H, Nomura M, Hatano O, Honda S, Tsukiyama T, Niwa O, Hara T, Takakusu A, Shibata Y, Omura T 1994 Functional difference between Ad4BP and ELP, and their distributions in steroidogenic tissues. Mol Endocrinol 8:643–653[Abstract]
  18. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[Medline]
  19. Luo X, Ikeda Y, Schlosser DA, Parker KL 1995 Steroidogenic factor 1 is the essential transcript of the mouse Ftz-F1 gene. Mol Endocrinol 9:1233–1239[Abstract]
  20. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, Tourtellotte LM, Simburger K, Milbrandt J 1995 Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA 92:10939–10943[Abstract]
  21. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Morohashi K-I, Li E 1995 Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dynam 204:22–29[Medline]
  22. Barnhart KM, Mellon PL 1994 The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone alpha-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878–885[Abstract]
  23. Morohashi KI, Omura T 1996 Ad4BP/SF-1, a transcription factor essential for the transcription of steroidogenic cytochrome P450 genes and for the establishment of the reproductive function. FASEB J 10:1569–77[Abstract/Free Full Text]
  24. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr Rev 18:361–377[Abstract/Free Full Text]
  25. Kletter GB, Gorski JL, Kelch RP 1991 Congenital adrenal hypoplasia and isolated gonadotropin deficiency. Trends Endocrinol Metab 2:123–128
  26. Ryner LC, Swain A 1995 Sex in the ’90s. Cell 81:483–93[Medline]
  27. Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone beta subunit promoter in gonadotropes of transgenic mice. J Biol Chem 271:10782–10785[Abstract/Free Full Text]
  28. Albarracin CT, Kaiser UB, Chin WW 1994 Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene. Endocrinology 135:2300–6[Abstract]
  29. Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of luteinizing hormone beta gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. J Biol Chem 271:6645–6650[Abstract/Free Full Text]
  30. Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina G, Milbrandt J 1996 Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273:1219–1221[Abstract]
  31. Burris TP, Guo W, Le T, McCabe ER 1995 Identification of a putative steroidogenic factor-1 response element in the DAX-1 promoter. Biochem Biophys Res Commun 214:576–581[CrossRef][Medline]
  32. Ikeda Y 1996 SF-1: a key regulator of development and function in the mammalian reproductive system. Acta Paediatr Jpn 38:412–419[Medline]
  33. Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor 1, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fush tarazu-factor 1. Mol Endocrinol 6:1249–1258[Abstract]
  34. Bakke M, Lund J 1995 Mutually exclusive interactions of two nuclear orphan receptors determine activity of a cyclic adenosine 3',5'-monophosphate-responsive sequence in the bovine CYP17 gene. Mol Endocrinol 9:327–339[Abstract]
  35. Bakke M, Lund J 1995 Transcriptional regulation of the bovine CYP17 gene: two nuclear orphan receptors determine activity of cAMP-responsive sequence 2. Endocr Res 21:509–516[Medline]
  36. Shibata H, Nawaz Z, Tsai SY, O’Malley BW, Tsai MJ 1997 Gene silencing by chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is mediated by transcriptional corepressors, nuclear receptor-corepressor (N-CoR) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT). Mol Endocrinol 11:714–724[Abstract/Free Full Text]
  37. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  38. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  39. Tsai SY, Tsai M-J 1997 Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): coming of age. Endocr Rev 18:229–240[Abstract/Free Full Text]
  40. Pereira GA, Qiu Y, Tsai M-J, Tsai SY 1994 COUP-TF: expression during mouse embryogenesis. J Steroid Biochem Mol Biol 53:503–508[CrossRef]
  41. Jonk LJ, de Jonge ME, Pals CE, Wissink S, Vervaart JM, Schoorlemmer J, Kruijer W 1994 Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors. Mech Dev 47:81–97[CrossRef][Medline]
  42. Qiu Y, Pereira FA, DeMayo FJ, Lydon JP, Tsai SY, Tsai M-J 1997 Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization. Genes Dev 11:1925–1937[Abstract/Free Full Text]
  43. Lala DS, Syka PM, Lazarchik SB, Mangelsdorf DJ, Parker KL, Heyman RA 1997 Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols. Proc Natl Acad Sci USA 94:4895–4900[Abstract/Free Full Text]
  44. Kohler PO, Bridson WE, Hammond JM, Weintraub B, Kirschner MA, Van Thiel DH 1971 Clonal lines of human choriocarcinoma cells in culture. Acta Endocrinol (Copenh) [Suppl] 153:137–153[Medline]
  45. Zhang P, Mellon SH 1997 Multiple orphan nuclear receptors converge to regulate rat P450c17 gene transcription: novel mechanisms for orphan nuclear receptor action. Mol Endocrinol 11:891–904[Abstract/Free Full Text]
  46. Nordeen SK 1988 Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6:454–457[Medline]
  47. Wood WM, Kao MY, Gordon DF, Ridgway EC 1989 Thyroid hormone regulates the mouse thyrotropin ß-subunit gene promoter in transfected primary thyrotropes. J Biol Chem 264:14840–14847[Abstract/Free Full Text]
  48. Windle JJ, Weiner RI, Mellon PL 1990 Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol 4:597–603[Abstract]
  49. Graham FL, van der Eb AJ 1973 Transformation of rat cells by DNA of human adenovirus 5. Virology 52:456–487[Medline]
  50. deWet JR, Wood KV, DeLuca M, Helinski DR 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737[Medline]
  51. Shapiro DJ, Sharp PA, Wahli WW, Keller MJ 1988 A high-efficiency HeLa cell nuclear transcription extract. DNA 7:47–55[Medline]
  52. Wang LH, Tsai SY, Cook RG, Beattie WG, Tsai MJ, O’Malley BW 1989 COUP transcription factor is a member of the steroid receptor superfamily. Nature 340:163–166[CrossRef][Medline]
  53. Wang LH, Ing NH, Tsai SY, O’Malley BW, Tsai MJ 1991 The COUP-TFs compose a family of functionally related transcription factors. Gene Expr 1:207–216[Medline]
  54. Mietus-Snyder M, Sladek FM, Ginsburg GS, Kuo CF, Ladias JA, Darnell Jr JE, Karathanasis SK 1992 Antagonism between apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and hepatocyte nuclear factor 4 modulates apolipoprotein CIII gene expression in liver and intestinal cells. Mol Cell Biol 12:1708–1718[Abstract]