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Molecular Endocrinology, doi:10.1210/me.2003-0176
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Molecular Endocrinology 18 (6): 1428-1437
Copyright © 2004 by The Endocrine Society

Generation of Two Distinct Functional Isoforms of Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita-Critical Region on the X Chromosome Gene 1 (DAX-1) by Alternative Splicing

Anwar Hossain, Chun Li and Grady F. Saunders

Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-4009

Address all correspondence and requests for reprints to: Grady F. Saunders, Department of Biochemistry and Molecular Biology, Unit 117, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030-4009. E-mail: gsaunders{at}mdanderson.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita-critical region on the X chromosome gene 1; NR0B1) is an orphan nuclear receptor that plays an important role in the development and functioning of the adrenal gland and hypothalamic-pituitary gonadal axis. The DAX-1 protein acts as a transcriptional repressor of genes involved in the steroidogenic pathway. We have identified a novel isoform encoded by the known exon 1 of DAX-1 and a previously unrecognized exon (exon 2{alpha}) that lies within intron 1 of DAX-1. This novel transcript, which we designated DAX-1{alpha}, is terminated at exon 2{alpha}; the last 70 amino acids of the C-terminal repressor domain encoded by exon 2 are absent. DAX-1{alpha} encodes a protein of 401 amino acids; the first 389 amino acids are encoded by exon 1 and the last 12 are encoded by exon 2{alpha}. Using conventional RT-PCR and real-time RT-PCR analyses, we found that DAX-1{alpha} is abundantly expressed in the adrenal gland, brain, kidney, ovary, and testis. We also found that DAX-1{alpha} can bind to steroidogenic factor 1 and to DNA but is unable to repress steroidogenic factor 1-mediated transcriptional activation of the reporter gene and acts as an antagonist of DAX-1 under certain conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ADRENAL HYPOPLASIA CONGENITA (AHC) is a rare inherited disorder of the adrenal cortex commonly manifested as an early-onset adrenal insufficiency syndrome. Two forms of AHC have been recognized: an X-linked form and a rare autosomal-recessive, or sporadic, form (1). If untreated, AHC is potentially life threatening, because it can cause mineralocorticoid deficiency. A long-term follow-up study of affected individuals surviving to adulthood revealed that AHC is commonly associated with hypogonadotropic hypogonadism (HHG), a condition that manifests as a failure of sexual maturation at the expected time of puberty. HHG is caused by a combination of hypothalamic failure in GnRH release and a pituitary defect in gonadotropin production (2). Investigators who cloned DAX-1 (dosage-sensitive sex reversal-AHC-critical region on the X-chromosome gene 1; NR0B1) found that loss-of-function mutations in the gene caused X-chromosome-linked AHC and HHG (3, 4). Duplication of this region has been found to cause dosage-sensitive sex reversal in male humans, a condition for which DAX-1 appears to be responsible (5).

DAX-1 consists of two exons, one composed of 1168 bp and the other composed of 245 bp, separated by a 3385-bp intron. It encodes a protein that consists of 470 amino acids (3). An unusual orphan member of the nuclear hormone receptor superfamily (3, 4), DAX-1 displays a novel DNA-binding domain that lacks the characteristic zinc-finger motif that is highly conserved in other nuclear receptors (3, 4). The amino terminus of DAX-1 consists of three and one half repeats of an alanine- and glycine-rich amino acid motif (65–67 amino acids) that has been proposed to likely serve as its DNA-binding domain (3, 6, 7). DAX-1 is a transcriptional repressor of several genes involved in steroid hormone metabolism (6, 8, 9, 10) and has also been shown to act as a corepressor of the estrogen receptors {alpha} and ß (11). A bipartite transcriptional repression domain lies in the C terminus of the DAX-1 protein (12, 13). Transcriptional repression by DAX-1 is thought to be mediated by its interaction with the corepressors N-CoR (nuclear receptor corepressor) (14) and Alien (15). Importantly, all DAX-1 mutations identified to date in patients with AHC share the ability to alter the sequence of the protein C-terminal domain and impair the transcriptional repression activity of DAX-1 (3, 4, 12, 13, 14, 15, 16, 17). In addition, DAX-1 is a nucleocytoplasmic shuttling protein associated with ribonucleoprotein structures in the nucleus and polyribosomes in the cytoplasm (18, 19). These findings suggest that DAX-1 plays an additional regulatory function in the posttranscriptional processes.

DAX-1 also plays a role in reproductive functioning and phenotypic sex determination. The DAX-1 gene lies inside a critical region on the X chromosome, the duplication of which causes dosage-sensitive sex reversal in males (5). Furthermore, overexpression of DAX-1 in certain mouse strains causes phenotypic sex reversal (20). Because of its expression pattern and ability to repress the expression of genes that mediate male development, DAX-1 was initially thought to play an important role in ovarian development; this hypothesis was challenged, however, by findings in knockout mice lacking Dax-1 (21): female Dax-1 knockout mice exhibited completely normal ovarian development and reproductive functioning, whereas male Dax-1 knockout mice exhibited impaired spermatogenesis.

In this study, we identify, for the first time, the new, shorter isoform of human DAX-1 (DAX-1{alpha}) that results from alternative splicing and compare the properties of this new isoform with those of the longer isoform, DAX-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation and Characterization of 5'- and 3'-Rapid Amplification of cDNA Ends (RACE) cDNA Clones of Human DAX-1
To obtain additional exons for human DAX-1, the RACE procedure was carried out with nested primers and an adaptor-ligated cDNA library prepared from human testis tissue. The RACE products were subcloned and sequenced. Because the clones obtained from the 5'-RACE products matched the known cDNA of DAX-1, we concluded that no new exons were located upstream of the known exon 1. Two sequences, which diverged exactly at the junction of exon 1 and exon 2, were obtained for the 3'-RACE cDNA clones (Fig. 1Go, A and B). In addition to the 3'-RACE sequence that exactly matched the previously identified exon 2, a second set of clones revealed the presence of a shorter splice variant (Fig. 1CGo); we designated this alternate form DAX-1{alpha} and herein refer to the longer form as DAX-1. These DAX-1{alpha} clones had an additional 191 nucleotides that did not correspond to the exon 2 sequences but perfectly matched those of intron 1 of DAX-1. We found a poly-A tail at the end of all the clones analyzed. These findings indicated that a previously unknown exon was present in intron 1 of DAX-1 and that the new transcript, DAX-1{alpha}, ended at this exon. DAX-1{alpha} is 1.6 kb long and encodes a shorter protein consisting of 401 amino acids and lacking amino acids 390–470 of the longer isoform, DAX-1 (Fig. 1CGo). As shown in Fig. 1AGo, DAX-1{alpha} lacks the amino acids previously identified as being essential for the transcriptional repression encoded by exon 2 (12).




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  		Fig. 1. Cloning of Human DAX-1{alpha} Isoform

A, Schematic representation of the DAX-1 gene and transcripts. The DAX-1{alpha} isoform results from a cryptic exon within intron 1. B, Exon-intron boundary of DAX-1. Splicing donor and acceptor sites are shown in boldface type, exonic sequences are shown in uppercase, and truncated intronic sequences are shown in lowercase. C, Complete cDNA sequence of DAX-1{alpha} and its amino acid sequences. The open reading frame is shown in uppercase, and the 5'- and 3'-UTR sequences are shown in lowercase. New exon 1{alpha} sequences are shown in italics.

 
Tissue-Specific Expression Pattern of DAX-1 and DAX-1{alpha} mRNA
To compare the tissue distribution of DAX-1{alpha} with that of the longer isoform, DAX-1, we first attempted a Northern blot analysis. However, the exon 1{alpha}-specific probe was small and thus worked poorly in the Northern blot. In addition, the mRNA sizes for both isoforms were similar, so a common probe could not be used to differentiate the two isoforms on the Northern blot. Instead, we employed RT-PCR, using primers specific for each isoform. PCR was performed on the first-strand cDNA derived from various human tissues using primers specific for DAX-1 and DAX-1{alpha} (Table 1Go) that generated a 237-bp product for DAX-1 and a 237-bp product for DAX-1{alpha}. The PCR amplifications from 14 different human tissues showed that the 237-bp fragment representing DAX-1 was present in the adrenal gland, brain, kidney (fetal), ovary, pancreas, and testis and absent or very low in the bone marrow, breast, kidney (adult), heart, liver, lung, spleen, and thymus (Fig. 2Go). The 237-bp fragment of DAX-1{alpha} was less tissue specific. Expression of DAX-1{alpha} was highest in the RNA of the adrenal gland, brain, breast, kidney (fetal), ovary, pancreas, testis, and thymus, but it was also readily detectable in all the other tissues examined.


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  		Table 1. Sequence of Primers Used in RACE and RT-PCR

 


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  		Fig. 2. Tissue Distributions of DAX-1 and DAX-1{alpha} Transcripts

First-strand cDNA was synthesized from various human tissues and subjected to RT-PCR using exon-specific primers. The resulting PCR products were separated using a 1.5% agarose gel. GAPDH primers were used as an internal control. The upper band represents DAX-1{alpha} (237 bp), the middle band represents DAX-1 (237 bp), the lower band represents GAPDH.

 
Quantitation of DAX-1 and DAX-1{alpha} Transcript by Real-Time RT-PCR
Having obtained the mRNA distribution profiles for the DAX-1 and DAX-1{alpha} splice variants using conventional RT-PCR, we next assessed the relative levels of each variant using real-time RT-PCR. The adrenal gland, brain, fetal kidney, ovary, pancreas, and testis expressed the highest total DAX-1{alpha} mRNA, as determined by the copy number, whereas the other tissues expressed it at levels that were medium to nonexistent (Fig. 3Go). The copy number of the DAX-1 transcript was highest in testis tissue and either was not detectable or was expressed in very low concentrations in all the other tissues. As found with conventional RT-PCR (Fig. 2Go), DAX-1{alpha} expression was higher than that for DAX-1, except in the testis (Fig. 3Go), in which DAX-1 expression was higher.



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  		Fig. 3. Quantification of DAX-1 and DAX-1{alpha} Expression by Real-Time RT-PCR

The primers and cDNAs used were the same as for the conventional RT-PCR analysis described in Fig. 2Go. The expression levels are given as the ratio of the target gene to the control gene (GAPDH) to correct for variations in the starting amount of mRNA. The ratios were calculated on the basis of 1000 copies of GAPDH.

 
Expression of DAX-1{alpha} Protein
We also evaluated the expression of the endogenous DAX-1{alpha} protein in human tissues. Tissue extracts from the testis were immunoblotted with a DAX-1 antibody directed against the N terminus of DAX-1. Tissue extracts from the liver, in which DAX-1 had not been detected by RT-PCR and DAX-1{alpha} expression was also very low, were used as a negative control, and breast tissue extracts, in which DAX-1{alpha} was detectable by RT-PCR only, were also used. The DAX-1 antibody specifically recognized two bands in the extracts from the testis at the expected size (Fig. 4Go). Thus, both isoforms of the DAX-1 protein were expressed in the human tissues. Because the quality of commercially available anti-DAX-1 antibody was poor, a lower dilution, it recognized many nonspecific bands in a Western blot. We overcame some of these problems by using an ultrasensitive detection reagent, which allowed us to use the antibody at a higher dilution, thus increasing the specificity.



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  		Fig. 4. Endogenous Expression of DAX-1{alpha} Protein

Human tissue extracts were subjected to 4–20% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with an anti-DAX-1 N-terminal-specific antibody. The bands were visualized using a Femto-Western blotting detection kit. Tissue types are indicated at the top of each lane; M, positions of molecular mass weight markers.

 
Functional Analysis of the New Isoform DAX-1{alpha}
To analyze the function of DAX-1{alpha}, we used transient transfection experiments to asses its ability to repress steroidogenic factor 1 (SF-1)-mediated activation of the steroidogenic acute regulatory (StAR) promoter. We used expression vectors coding for wild-type human DAX-1 as positive controls. As shown in Fig. 5AGo, this promoter was robustly activated by an SF-1 expression construct. Cotransfection of increasing amounts of wild-type DAX-1 greatly reduced the reporter gene activation by SF-1. In contrast, the construct expressing DAX-1{alpha} did not substantially repress activation at the highest dose of DNA.



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  		Fig. 5. Inability of DAX-1{alpha} to Repress SF-1-Mediated Activation of the Reporter Gene

HeLa cells were transfected with (A) the StAR promoter- and (B) the CYP17 promoter-driven luciferase reporter constructs and the indicated amounts of SF-1 in the presence or absence of either DAX-1 and DAX-1{alpha} expression constructs or the empty expression vector pcDNA3 (control). To control the transfection efficiency 50 ng of the CMV promoter-driven ß-galactosidase gene, CMV-ßgal, was cotransfected with each sample. Luciferase activity was measured after 48 h of transfection. All results are expressed as the mean ± SD of the results of at least three experiments. C, Equal expression of DAX-1 and DAX-1{alpha} in transfected HeLa cells. Nuclear lysates were separated using SDS-PAGE and immunoblotted with an anti-DAX-1 antibody. The immunoblots were developed using an ECL-chemiluminescence kit (Amersham Pharmacia Biotech).

 
We also determined the ability of DAX-1{alpha} to repress SF-1-mediated activation of the cytochrome P450 17-{alpha}-hydroxylase (CYP17) promoter. The results were identical to those obtained with the StAR promoter (Fig. 5BGo). Cotransfection of SF-1 increased reporter gene activity by several times that of the empty vector. As expected, coexpression of wild-type DAX-1 repressed this activation in a dose-dependent manner. However, similar doses of DAX-1{alpha} did not considerably reduce the SF-1-mediated activation of the CYP17 promoter (Fig. 5BGo), although the two proteins were expressed at almost equal levels in the transfected cells (Fig. 5CGo). We concluded from these results that DAX-1{alpha} does not have repressor activity, which is a known function of DAX-1.

Antagonistic Action of DAX-1{alpha} over DAX-1
Because DAX-1{alpha} did not show any repressive activity, we hypothesized that DAX-1{alpha} competed with DAX-1 for binding with SF-1 and might relieve DAX-1-mediated repressor activity. When we cotransfected SF-1, DAX-1, and DAX-1{alpha} with the StAR promoter driven-luciferase reporter gene, luciferase activity increased with increasing dosages of DAX-1{alpha} (Fig. 6AGo). Similar results were obtained with the CYP17 promoter-driven luciferase reporter gene activity (data not shown). At a lower dose of SF-1, DAX-1{alpha} gave an additive effect in reporter gene activity (Fig. 6BGo). SF-1 alone saturated the reporter gene activity and masked the effects of DAX-1{alpha} at a higher dose. We also saw some squelching effects when we cotransfected higher doses of both SF-1 and DAX-1{alpha} (Figs. 5Go, A and B, and 6A). These results suggested that DAX-1{alpha} acts as an antagonist of DAX-1 under specific conditions.



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  		Fig. 6. Antagonistic Effects of DAX-1{alpha} over DAX-1 in SF-1-Mediated Activation of the StAR Promoter

HeLa cells were transfected with the StAR promoter-driven luciferase reporter constructs and the indicated amounts of SF-1 (200 ng) in the presence or absence of DAX-1 (40 ng) and an increasing amount of DAX-1{alpha} expression constructs (panel A). B, The lower concentration of SF-1 (25 ng) was used in the presence of an increasing amount of DAX-1{alpha}, as indicated in the figure. To control the transfection efficiency 50 ng of the CMV promoter-driven ß-galactosidase plasmids, CMV-ßgal, were cotransfected with each sample. Luciferase activity was measured after 48 h of transfection. All results are expressed as the mean ± SD of the results of at least three experiments.

 
Similar DNA Binding Abilities of DAX-1{alpha} and DAX-1
Using its N-terminal DNA-binding domain, DAX-1 can bind in the hairpin structure of its own promoter and in the StAR promoter. Because the N-terminal DNA-binding domain of DAX-1{alpha} is the same as that of DAX-1, we investigated whether DAX-1{alpha} could bind to StAR DNA sequences. In vitro-translated DAX-1{alpha} specifically bound to the StAR promoter sequences in a fashion similar to DAX-1 (Fig. 7Go).



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  		Fig. 7. DNA Binding of DAX-1 and DAX-1{alpha} to DNA

DNA binding assays were performed with the radiolabeled DAX-1 binding site (–61 to –27) in the StAR promoter and incubated with in vitro-translated DAX-1 and DAX-1{alpha}. A 100x cold probe was used as the competitor in DAX-1 reaction mixture.

 
Interaction of DAX-1 and DAX-1{alpha} with SF-1 and Alien in Vitro
To clarify why DAX-1{alpha} did not repress the promoter activity mediated by SF-1, we examined the interaction between DAX-1{alpha} and SF-1. To determine whether DAX-1{alpha} interacted directly with SF-1, we performed coimmunoprecipitation assays using whole-cell extracts of DAX-1{alpha}- and SF-1-cotransfected cells. For comparison, we also examined the interaction of SF-1 with DAX-1. As shown in Fig. 8AGo, SF-1 was coimmunoprecipitated with both DAX-1 and DAX-1{alpha} using the anti-DAX-1 antibody. These results indicate that DAX-1{alpha}, like DAX-1, can interact directly with SF-1.



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  		Fig. 8. Interactions between DAX-1{alpha} and SF-1, DAX-1{alpha}, and Alien

A, FLAG-tagged SF-1 and DAX-1 (lane 1) or DAX-1{alpha} (lanes 2 and 3) were cotransfected in COS-1 cells and coimmunoprecipitated with a rabbit anti-DAX-1 (N-terminal specific) antibody and immunoblotted with a rabbit anti-SF-1 antibody. Normal rabbit IgG was used as a negative control. B, In vitro-translated GST-Alien was mixed with in vitro translated 35S-labeled DAX-1 (lane 1) and DAX-1{alpha} (lane 3) and performed the GST-pull-down assays. Lanes 2 and 4 are GST-only negative control.

 
We also tested the interaction between DAX-1 and DAX-1{alpha} with Alien. DAX-1 interacted with Alien as expected; however, DAX-1{alpha} was unable to interact with Alien as judged by the in vitro-translated glutathione-S-transferase (GST) pull-down assays (Fig. 8BGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we cloned a new isoform of DAX-1 that results from alternative splicing and named the new, shorter variant, DAX-1{alpha}. To our knowledge, this report is the first to describe an alternatively spliced variant of DAX-1. In the novel isoform, DAX-1{alpha}, the last 80 amino acids of the C terminus, which are encoded by exon 2 of DAX-1, are missing, including part of the repressor domain of DAX-1.

The level of expression and the tissue distribution of this newly identified isoform are very different from those of the longer DAX-1. We analyzed RNA from 14 different human tissues to measure the expression of DAX-1 and DAX-1{alpha} and found that these two isoforms were expressed at various levels in several of the tissues. We have observed that the real-time RT-PCR is less sensitive than the conventional RT-PCR. We also identified new domains of DAX-1 expression, such as fetal kidney, and detected DAX-1 and DAX-1{alpha} proteins in whole-cell extracts from human testis. Thus, both isoforms of DAX-1 are expressed endogenously in human tissue. Because of the poor quality of the antibody used, we could not perform the quantitative analysis at the protein level.

An unexpected phenomenon observed in this study was that DAX-1{alpha} was unable to repress the reporter transcription mediated by SF-1. The repression domain was present within the C-terminal portion of DAX-1. DAX-1-mediated repression of SF-1-induced transcription of the reporter gene occurred by direct interaction with SF-1 and subsequently by recruitment of two corepressors, N-CoR and Alien. DAX-1{alpha}, like DAX-1, was able to interact directly with SF-1 but was unable to repress the SF-1-mediated activation of the StAR and the CYP17 promoters. The C terminus of DAX-1, which is essential for biological functioning (21), is the site of most of the missense mutations in DAX-1 found in patients with AHC (22). It has been shown in vitro that the C-terminal amino acids of DAX-1 are important for recruiting N-CoR (14) and Alien (15). DAX-1{alpha}, however, cannot interact with Alien, as judged by coimmunoprecipitation (Fig. 8BGo). DAX-1{alpha} can bind with SF-1, but it is unable to recruit these corepressors and thus cannot repress SF-1-mediated activation of the promoters.

In this study, we identified a novel role of DAX-1{alpha} using an in vitro system, that DAX-1{alpha} plays a dominant positive role over DAX-1 under certain conditions. It was not clear, however, how DAX-1{alpha} synergized with SF-1 in activating the reporter gene. It is possible that DAX-1{alpha} simply competes with DAX-1 for binding with SF-1. Another possibility is that the 12 amino acids encoded by exon 1{alpha} create a novel protein-interacting domain that helps recruit coactivators and activates target genes that are supposed to be repressed by DAX-1. Further studies are required to explain this question. In this study, DAX-1{alpha} was overexpressed in most tissues two to three times more than DAX-1, except in testis tissue. The DAX-1{alpha} action should have been predominant over DAX-1, because the mRNA of DAX-1{alpha} was more highly expressed than that of DAX-1 in most of the tissue types examined. Spatial and temporal expression levels of these two isoforms may determine the outcome of DAX-1-mediated steroidogenesis during the development and normal physiological functioning of the adrenal gland and hypothalamic gonadal axis. No ligand has yet been identified for DAX-1; it will be interesting to learn how these two isoforms act in the presence of a ligand.

DAX-1 contains a novel DNA-binding domain lacking the classical zinc-finger motif that is highly conserved in other nuclear hormone receptors (3). The amino terminus of DAX-1 contains three and one half repeats of an alanine- and glycine-rich amino acid motif that serves as the DAX-1 DNA-binding domain and binds to the DNA hairpin structures (6). In this study, DAX-1 and DAX-1{alpha} showed similar DNA binding activities in vitro, indicating that the differences between the isoforms did not disrupt the structure of the DNA-binding domain. Although some reports have shown that DNA binding to DAX-1 is essential for DAX-1 repressor activity (6), other studies have shown that it is the direct interaction between DAX-1 and SF-1 that is most important for the repressor activity (12, 13). DAX-1 also acts as a direct repressor of transcription activated by estrogen receptors through direct interaction (11). Our finding that the C-terminal amino acids are important for the repressor activity of DAX-1 is consistent with the results of other studies (12, 13, 23).

In a mouse model for AHC that was developed in a previous study by targeting the DAX-1 locus (21), exon 2 of DAX-1 was removed using the Cre-lox system. However, the phenotypes of these mice did not exactly mimic the AHC and HHG phenotypes seen in humans (21). The next step in determining the function of DAX-1{alpha} will be to clone mouse Dax-1{alpha} and generate Dax-1{alpha}-specific mutant mice to evaluate the exact role of this isoform during development.

In summary, we have described, for the first time, alternative splicing for the human DAX-1 gene and the resulting isoforms, which are expressed differentially in various healthy human tissues. Whereas both isoforms can bind to SF-1 and DNA, these two isoforms function differentially: DAX-1 is a potent repressor of SF-1-mediated transcriptional activation of a reporter gene, and DAX-1{alpha}, which is unable to repress the target promoter, acts as an antagonist of DAX-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
5'-RACE and PCR Cloning
To identify previously unknown exons upstream and downstream of the known exon 1 of DAX-1, we employed the RACE procedure using the Marathon-Ready human testis-specific cDNA kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s recommendations. First-round PCR was performed with exon 1-specific sense (DAXRACE-F1) and antisense (DAXRACE-R1) primers and the sense anchor primer AP1 or the antisense anchor primer AP1, supplied by CLONTECH. Nested PCR was performed with the plus primer AP2 and either the new sense primer (DAXRACE-F1.1) or the new antisense primer (DAXRACE-R1.1). Several bands were obtained and cloned into a TOPO vector (Invitrogen, Carlsbad, CA) and sequenced. The sequencing results were compared with the known DAX-1 cDNA sequences.

RT-PCR Analysis
RNA was purchased from commercial sources (Ambion, Inc., Austin, TX; and CLONTECH). Single-stranded cDNA was prepared using the ThermoScript RT-PCR System (Invitrogen, Carlsbad, CA). PCRs were performed using the Expand High-Fidelity PCR system (Roche Diagnostics, Indianapolis, IN) with primers DAX1RT-F and DAX1RT-R for DAX-1 and primers DAX1{alpha}RT-F and DAX1{alpha}RT-R for the DAX-1{alpha} isoform. The PCR products were separated on 1.5% agarose gels. Each band was subcloned and confirmed by sequencing from both directions.

Real-Time RT-PCR
The primers and cDNA were used for optimization and quantitation in real-time RT-PCR. To ensure that both the gene of interest and the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified at the same efficiency, several dilutions of cDNA (1, 5, and 10 ng) were amplified. Optimization was performed similarly using different concentrations of MgCl2. PCR was performed using a Light-Cycler system (Roche Diagnostics) in a total reaction mixture of 10 µl containing 5 ng cDNA, 1x Light-Cycler Hotstart DNA Master SYBER Green 1, 4 mmol/liter MgCl2, and 1 µmol/liter of each primer. After denaturation at 95 C for 10 min, 40 cycles were performed at 95 C for 5 sec, 55 C for 5 sec, and 72 C for 15 sec. The data were normalized using the ratio of the target cDNA concentration to GAPDH to correct for differences in RNA quantity among the samples.

Plasmids
pcDNA3DAX-1 and pcDNA3DAX-1{alpha} constructs were made using PCR amplification from human testis RNA. The resulting PCR products were subcloned into a modified pcDNA3 vector (24) harboring the 5'-untranslated region of the herpes simplex virus-thymidine kinase gene.

The StAR and CYP17 promoter sequences that drove transcription of the luciferase gene were amplified using PCR from human genomic DNA and cloned in front of the luciferase gene in the vector pGL3basic (Promega Corp., Madison, WI). These constructs were designated StARPLuc and CYP17Pluc, respectively. All constructs were confirmed by sequencing from both directions.

Cell Culture and Transfection
HeLa and COS-1 cells were grown at 37 C in DMEM-F12 medium supplemented with 10% fetal calf serum in an atmosphere containing 5% CO2. The cells were seeded at a density of 50,000–70,000 cells per well in 24-well plates 16–18 h before transfection. The cells were cotransfected with expression and reporter plasmids as indicated in the figure legends. The plasmid cytomegalovirus (CMV)-ßgal was cotransfected as an internal control to normalize for differences in transfection efficiency. The transfections were performed with the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations, and the cells were harvested after 40–48 h. Luciferase activity was measured with a luciferase assay kit (Tropix, Inc., Bedford, MA) and a Lumat LB9507 luminometer (EG&G Berthold, Bad Wildbad, Germany). ß-Galactosidase was measured with the Galacto-Light Plus kit (Tropix, Inc.).

Gel Shift Assay
DNA-binding assays were performed with possible hairpin structures (–61 to –27) in the StAR promoter (12). The initiation site was defined as +1. Gel shift reactions were performed in a total volume of 20 µl at 37 C. A radiolabeled probe was prepared with [{gamma}-32P]ATP, 10 pmol of the labeled probe, and 5 µl of the in vitro-translated protein were used for each reaction. To assay competition with wild-type probe, a 100-fold molar excess of the unlabeled probe was added to the reaction mixture 15 min before the addition of the labeled probe. Samples were resolved in a 6% polyacrylamide gel cast in Tris-borate-EDTA buffer containing 10 mM magnesium acetate at 4 C for 3 h. The gels were dried and analyzed using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Western Blotting
Tissue extracts (50 µg) (GenoTech) were separated using a 4–20% SDS-PAGE gel and immunoblotted with a specific antibody. The primary and secondary antibodies used were rabbit anti-DAX-1 at a 1:2 x 104 dilution and goat antirabbit IgG conjugated with peroxidase at a dilution of 1:105 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Western blots were developed by SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Chemical Co., Rockford, IL).

Coimmunoprecipitation Assay
FLAG-tagged SF-1 and DAX-1 or DAX-1{alpha} were cotransfected in the COS-1 cells and immunoprecipitated with rabbit anti-DAX-1 (Santa Cruz Biotechnology) or rabbit control IgG (Santa Cruz Biotechnology). Immunocomplexes were collected using Protein A/G sepharose (Pierce), separated using a 4–20% SDS-PAGE gel, and immunoblotted with rabbit anti-SF-1 antibodies (Upstate Biotechnology, Inc., Lake Placid, NY).


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants CA 34936 and CA 16672.

Abbreviations: AHC, Adrenal hypoplasia congenital; CMV, cytomegalovirus; CYP17, cytochrome P450 17-{alpha}-hydroxylase; DAX-1, dosage-sensitive sex reversal-AHC critical region on X chromosome gene 1; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; GST, glutathione-S-transferase; HHG, hypogonadotropic hypogonadism; N-CoR, nuclear receptor corepressor; RACE, rapid amplification of cDNA ends; StAR, steroidogenic acute regulatory protein; SF-1, steroidogenic factor 1.

Received for publication May 13, 2003. Accepted for publication March 16, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   DAX1  |  SF-1
Coregulators:   Alien  |  NCOR



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