Dax-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1) Gene Transcription Is Regulated by Wnt4 in the Female Developing Gonad

Hirofumi Mizusaki, Ken Kawabe1, Tokuo Mukai2, Etsuko Ariyoshi, Megumi Kasahara, Hidefumi Yoshioka, Amanda Swain and Ken-ichirou Morohashi

Department of Developmental Biology (H.M., K.K., T.M., K.-I.M.), National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan; Department of Molecular Biomechanics (H.M., K.-I.M.), School of Life Science, The Graduate University for Advanced Studies, Myodaiji-cho, Okazaki 444-8585, Japan; Core Research for Evolutional Science and Technology (K.K., H.Y., K.-I.M.), Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi 332-0012, Japan; Department of Natural Sciences (E.A., M.K., H.Y.), Hyogo University of Teacher Education, Yashiro-cho, Hyogo 673-1494, Japan; and Section of Gene Function and Regulation (A.S.), Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, United Kingdom

Address all correspondence and requests for reprints to: Ken-ichirou Morohashi, Professor, Ph.D., Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan.


    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 acting as a suppressor of Ad4 binding protein/steroidogenic factor 1 [Ad4BP/SF-1 (NR5A1)] and as an anti-Sry factor in the process of gonadal sex differentiation. The roles of these nuclear receptors in the differentiation of the gonads and the adrenal cortex have been established through studies of the mutant phenotype in both mice and humans. However, the mechanisms underlying transcriptional regulation of these genes remain largely unknown. Here, we examined the relationship between Dax-1 gene transcription and the Wnt4 pathway. Reporter gene analysis revealed that Dax-1 gene transcription was activated by ß-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with Ad4BP/SF-1. Interaction between ß-catenin and Ad4BP/SF-1 was observed using yeast two-hybrid and in vitro pull-down assays. The region of Ad4BP/SF-1 essential for this interaction consists of an acidic amino acid cluster, which resides in the first helix of the ligand-binding domain. Mutation of the amino acid cluster impaired transcriptional activation of Dax-1 as well as interaction of Ad4BP/SF-1 with ß-catenin. These results were supported by in vivo observations using Wnt4 gene-disrupted mice, in which Dax-1 gene expression was decreased significantly in sexually differentiating female gonads. We thus conclude that Wnt4 signaling mediates the increased expression of Dax-1 as the ovary becomes sexually differentiated.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DAX-1 [DOSAGE-SENSITIVE SEX reversal (DSS)-adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] has been positionally cloned from the DSS and AHC critical region located at Xp21 (1, 2, 3). Structurally, DAX-1/Dax-1 [NR0B1 (4)] is related to the nuclear receptor superfamily (2, 5). However, its N-terminal region lacks the Zn-finger DNA-binding domain conserved in nuclear receptors and instead is composed of three repeats each containing an LXXLL motif (6, 7).

Dax-1 is expressed in the steroidogenic tissues such as the adrenal cortex and the gonads, and its expression mostly overlaps with that of Ad4BP/SF-1 [Ad4 binding protein (8)/steroidogenic factor 1 (9)] (NR5A1) (4). This coexpression strongly suggested a functional correlation between the two factors (10, 11, 12). In fact, mutations in Ad4BP/SF-1 (13, 14, 15, 16) and in DAX-1 (2, 3, 17, 18, 19, 20) have been shown to affect the structure and function of the steroidogenic tissues in both mice and humans. Moreover, Dax-1 has been reported to repress a class of nuclear receptors including Ad4BP/SF-1 through interaction with the LXXLL motifs (2, 6, 7, 21, 22, 23). Together, these findings support the view that the two nuclear receptors share functional correlation as the constituents in the gonadal and adrenocortical differentiation pathways.

As another correlation between the two genes, it is interesting to note that the Dax-1 gene was identified as the target gene of Ad4BP/SF-1 (11, 24, 25, 26, 27). Indeed, Dax-1 gene transcription is mediated by Ad4BP/SF-1 through multiple Ad4 sites on the gene promoter. This regulation of Dax-1 gene transcription was confirmed by an in vivo observation that Dax-1 expression was absent or severely impaired in Ad4BP/SF-1 gene-disrupted mice (11).

It is well accepted that gonadal sexes in mammals are determined primarily by the presence or absence of Sry, the sex determining gene on the Y chromosome (28). In addition to Sry, several autosomal genes such as Sox9 (29, 30, 31), M33 (32), Wt-1 (33), Wnt4 (34), Fgf9 (35), Dhh (36), and Gata4 (37) are known to be involved in gonadal sex determination. When these genes were disrupted or overexpressed in mice, the resultant animals displayed sex reversal and delayed sex differentiation of the gonads. Two genes on the X chromosome, Dax-1 (38) and Arx (39), were also shown to be critical for proper gonadal sex differentiation. The expression level of DAX-1 was assumed to affect sex determination and lead to the DSS phenotype. This assumption was verified by a transgenic study in which male gonads expressing a larger amount of Dax-1 than wild type showed delayed and incomplete testicular differentiation in mice carrying a weak Sry allele (38). Thus, it was concluded that Dax-1 antagonizes Sry function.

Wnt4 is another gene whose activity antagonizes Sry: gene-disrupted mice displayed abnormal expression of both Leydig and Sertoli cell marker genes, which are normally not expressed in wild type fetal and neonatal ovaries, respectively (34). Moreover, it was shown that two XY patients with a duplication of a part of 1p containing the WNT4 gene displayed XY sex reversal (40, 41). These findings, together with the DSS phenotype of Dax-1 overexpression, led to the hypothesis that both Dax-1 and Wnt4 are critical for female sex determination. In support of this view, Jordan et al. (42) showed that Wnt4 induced Dax-1 expression in cultured cells. However, the functional correlation between the two genes during the process of female sex determination remained unclear.

In the present study, we provide evidence that Dax-1 gene transcription is activated by ß-catenin, a key molecule acting synergistically with Ad4BP/SF-1 as a Wnt signal transducer, and that a direct interaction between the two proteins is critical for this activation. These findings were further supported by in vivo studies using Wnt4 gene-disrupted mice.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synergistic Transcriptional Activation of the Dax-1 Gene by Ad4BP/SF-1 and ß-Catenin
As reported previously (11, 24, 25, 26, 27), Dax-1 gene transcription is activated by Ad4BP/SF-1, which binds to multiple Ad4 sites on the 5'-upstream region. We examined whether Dax-1 gene transcription is activated by ß-catenin by using the reporter plasmid Dax 2.5K-Luc, which carries 2.5 kb of the upstream region of the mouse Dax-1 gene. As expected, expression of Ad4BP/SF-1 resulted in activation of Dax-1 gene transcription, as indicated by the activity of the luciferase reporter. Interestingly, an increase in fold activation of Dax-1 gene transcription was observed after the addition of ß-catenin (Fig. 1AGo). However, ß-catenin was unable to activate Dax-1 gene transcription in the absence of Ad4BP/SF-1, whereas in the presence of Ad4BP/SF-1 it activates transcription in a dose-dependent manner (Fig. 1BGo). These results show that Ad4BP/SF-1 and ß-catenin act in synergy, rather than additively, to activate Dax-1 gene transcription.



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Figure 1. Synergistic Activation of Dax-1 Gene Transcription by ß-Catenin and Ad4BP/SF-1

A, A luciferase reporter gene construct, Dax2.5K-Luc (200 ng), was transfected into human embryonic kidney 293 cells with increasing amounts of the bovine Ad4BP/SF-1 expression vector (0, 100, 250, 500 ng) in the presence (+) or absence (-) of the ß-catenin expression vector (100 ng). B, Dax2.5K-Luc (200 ng) was transfected with increasing amounts of the ß-catenin expression vector (0, 20, 50, 100 ng) in the presence (+) or absence (-) of the bovine Ad4BP/SF-1 expression vector (500 ng). The total amount of transfected plasmids was adjusted to 2 µg with empty vectors. Luciferase activity was measured 36 h after transfection. Results are the mean ± SD of triplicate transfections.

 
Identification of the Lymphoid Enhancer Binding Factor (LEF)/T Cell-Specific Factor (TCF) Binding Site Responsible for the Synergistic Activation of Dax-1 Gene Transcription
Because it is known that ß-catenin activates transcription of target genes through heterodimerization with LEF/TCF, an HMG-box-containing transcription factor, we assumed that the binding sequence of LEF/TCF would be found in the Dax-1 promoter region. Searching for the LEF/TCF binding site revealed five candidate sequences (LEF-A to LEF-E, indicated as open ovals in Fig. 2A-aGo) residing within the 2.5-kb region upstream of Dax-1. The binding activities of the candidate sequences were analyzed by an EMSA using the oligonucleotides listed in Fig. 2A-bGo. When the oligonucleotides were incubated with an in vitro synthesized Flag-tagged LEF-1, a signal was seen at the same location as TCR{alpha} [authentic LEF/TCF binding site of the T cell receptor {alpha} (43)]. However, the binding strength varied between the different regions; stronger signals were detected with LEF-A and LEF-D than with the others. As expected, these signals disappeared or were supershifted after the addition of an anti-Flag antibody. Similarly, bacterially synthesized ß-catenin also gave supershifted signals, indicating heterodimer formation on these candidate sequences (Fig. 2A-cGo).



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Figure 2. Localization of the Regions Responsible for Synergistic Activation of the Dax-1 Promoter

A, Binding of LEF-1/ß-catenin complexes to the Dax-1 promoter. The Dax-1 gene upstream region is schematically represented in panel a. LEF-A, LEF-B, LEF-C, LEF-D, and LEF-E are candidate LEF-1/ß-catenin complex binding sites in the Dax-1 promoter. b, Comparison of the nucleotide sequence of the candidates with the consensus binding site and the representative LEF/TCF site found in human TCR{alpha} (T cell receptor {alpha}) gene enhancer. Mismatches with the consensus sequence are underlined. Ad4–1, Ad4–2a, Ad4–2b, and Ad4–3 indicate Ad4BP/SF-1 binding sites previously identified (11 ). EMSAs with oligonucleotides containing LEF-A, LEF-B, LEF-C, LEF-D, and LEF-E were performed using in vitro translated Flag-LEF-1. The LEF-1 binding site in the TCR{alpha} enhancer (TCR{alpha}-WT) and the disrupted form (M) were used as the positive and negative controls, respectively. GST-ß-catenin and anti-Flag M2 antibody were added to the reaction mixtures as indicated. The positions of the free probes, the dominant DNA-LEF-1 complexes, and the supershifted complexes are indicated at the left of panel c. B, Localization of regions responsible for synergistic transcriptional activation between ß-catenin and Ad4BP/SF-1. The deletion series of the Dax-1 reporter constructs, Dax2.5K-Luc, Dax1.5K-Luc, Dax1.0K-Luc, Dax600-Luc, Dax540-Luc, Dax265-Luc, Dax117-Luc, and Dax40-Luc, shown at the left, was constructed as described in Materials and Methods. Solid and open ovals indicate the location of Ad4BP/SF-1 binding sites and the candidates LEF/TCF binding sites, respectively. These deletion constructs were transfected into human embryonic kidney 293 cells in the presence (+) or absence (-) of the expression vectors for bovine Ad4BP/SF-1 (500 ng) and ß-catenin (100 ng). The total amount of transfected plasmids was adjusted to 2 µg with empty vectors. Luciferase activity was measured 36 h after transfection. Results are the mean ± SD of triplicate transfections.

 
To localize the regions responsible for the activation of transcription by Ad4BP/SF-1 and ß-catenin, transcriptional activity was examined using a series of deletion constructs (Fig. 2BGo). Deletion of the upstream region from 2.5 kb (Dax-2.5K-Luc) to 265 bp (Dax265-Luc) had no effect on the synergistic activation by Ad4BP/SF-1 together with ß-catenin or on activation mediated by Ad4BP/SF-1 alone. In contrast, both synergistic activation and activation by Ad4BP/SF-1 alone were decreased considerably by deletion of the 265 to 117 bp. Thus, these results suggest that LEF-D, one of the strong binding sites, is predominantly implicated in the activation of Dax-1 gene transcription by the complex of Ad4BP/SF-1 and ß-catenin.

The function of the LEF/TCF binding sites was confirmed by the following experiments: the two LEF/TCF binding sites, LEF-D and LEF-E, lying within 265 bp of the gene, were disrupted to give the construct LEF-DEM, which was tested using a reporter gene assay. As shown in Fig. 3Go, disruption of the LEF/TCF sites resulted in a significant decrease in Dax-1 gene transcription when Ad4BP/SF-1 and ß-catenin are acting together. Although in the Dax265-Luc wild-type construct relative luciferase activity is 100%, in the LEF-DEM mutant, activity is approximately 60%. The mutation had no effect on transcription mediated by Ad4BP/SF-1 or ß-catenin alone, but significantly affected the synergy of the two proteins acting as a complex. This is apparent in the increase in activity seen when ß-catenin is added to Ad4BP/SF-1: with the wild-type construct, a 60% increase in relative activity is seen, whereas in the mutant, only a 15% increase is seen. Thus, synergistic activation by Ad4BP/SF-1 and ß-catenin together is, in the LEF-DEM mutant, decreased to approximately one fourth of the wild-type construct. As reported previously (11), the mutant Ad4–2ab3M, which carries simultaneous mutations of three Ad4 sites (Ad4–2a, Ad4–2b, and Ad4–3), completely abolished Ad4BP/SF-1-mediated transcriptional activity. As expected, ß-catenin failed to activate transcription of Ad4–2ab3M even when LEF-D and LEF-E were intact. A fourth construct, LEF-DEM Ad4–2ab3M, which carries mutations at all the binding sites for LEF/TCF and Ad4BP/SF-1, also gave negligible transcriptional activity.



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Figure 3. Different Roles of the Binding Sites for LEF/TCF and Ad4BP/SF-1 in Synergistic Transcriptional Activation

Three luciferase reporter gene plasmids, LEF-DEM, Ad4–2ab3M, and LEF-DEM/Ad4–2ab3M, were constructed by introducing mutations into Dax265-Luc. LEF-DEM and Ad4–2ab3M have mutations at the LEF/TCF binding sites and the Ad4BP/SF-1 binding sites, respectively. LEF-DEM/Ad4–2ab3M carries mutations at all the binding sites for LEF/TCF and Ad4BP/SF-1. These constructs were transfected into human embryonic kidney 293 cells in the presence (+) or absence (-) of the expression vectors for bovine Ad4BP/SF-1 (500 ng) and ß-catenin (100 ng). The total amount of transfected plasmids was adjusted to 2 µg with empty vectors. Luciferase activities were measured 36 h after transfection. Results are the mean ± SD of triplicate transfections.

 
Localization of Amino Acids Critical for Interaction between Ad4BP/SF-1 and ß-Catenin
The activation of Dax-1 gene transcription by Ad4BP/SF-1 in synergy with ß-catenin suggested an interaction between the two factors. ß-Catenin was isolated in a yeast two-hybrid screening using a library prepared from mouse fetal gonads and a part of Ad4BP/SF-1 (Ad4BP/SF-1[129–263]) as a bait plasmid. To localize the interaction domain of ß-catenin, we examined the interaction of Ad4BP/SF-1 with the deletion constructs shown in the left panel of Fig. 4AGo. In the assay used, appearance of yeast colonies resistant to 3 mM 3-aminotriazole (3-AT) indicates the presence of an interaction between Ad4BP/SF-1 and ß-catenin. As shown in the right panel of Fig. 4AGo, interaction with Ad4BP/SF-1 was observed with ß-catenin[312–781], ß-catenin[474–781], and ß-catenin[312–696], but was not observed with ß-catenin[624–781] and ß-catenin[312–575]. These results indicate that the amino acids corresponding to armadillo repeats 9, 10, 11, and 12 are sufficient for the interaction between Ad4BP/SF-1 and ß-catenin.



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Figure 4. Interaction between Ad4BP/SF-1 and ß-Catenin Examined in Yeast Two-Hybrid Studies

A, Localization of a region in ß-catenin interacting with Ad4BP/SF-1. Wild-type ß-catenin and the truncated ß-catenins used in this assay are indicated schematically on the left. The numbers at either side of the constructs indicate amino acid residues, whereas the numbers (1–12) in the structure of the full-length ß-catenin indicate armadillo repeats. Yeast expression vectors for GAL4 AD fused with the ß-catenin derivatives and pGAD424, an empty expression vector for GAL4 AD, were transformed together with an expression vector for GAL4 DBD-Ad4BP/SF-1[129–263]. An expression vector for GAL4 DBD-LEF-1[1–109] and GAL4 DBD were used as controls. Transformed cells were plated on SD-Leu-Trp plates and SD-Leu-Try-His plates, and incubated at 30 C. B, Localization of a region in Ad4BP/SF-1 interacting with ß-catenin. The structure of Ad4BP/SF-1 is indicated schematically. A Zn-finger DNA binding domain (Zn) and a LBD are indicated, while helix 1 in the LBD is indicated by a closed box. Truncated mutants of Ad4BP/SF-1 used in this assay are schematically indicated on the left. Yeast expression vectors for GAL4 DBD fused with Ad4BP/SF-1 derivatives and pGBT9, an empty expression vector for GAL4 DBD, were transformed together with the expression vector for GAL4 AD-ß-catenin[312–781] or pGAD424. Transformed cells were plated on SD-Leu-Trp plates and SD-Leu-Try-His plates and incubated at 30 C. C, Identification of amino acids in Ad4BP/SF-1 critical for interaction with ß-catenin. GAL4 DBD-Ad4BP/SF-1[129–263] and its mutant forms were used in this assay. The mutant forms carry four consecutive amino acid substitutions to alanine (underlined). In the case of 129–263[220–4A], four amino acids from the 220th onward were substituted by alanines. Yeast expression vectors for GAL4 DBD fused with the Ad4BP/SF-1 derivatives and pGBT9 were transformed with the expression vector for GAL4 AD-ß-catenin[312–781] or pGAD424. Liquid ß-galactosidase assays were performed using three independent colonies obtained from each transformation. Results are the mean ± SD values of three independent experiments. Fold activities over empty pGAD424 are indicated on the right. D, Effect of point mutations at the 234 to 239 amino acids of Ad4BP/SF-1 on interaction with ß-catenin. GAL4 DBD-Ad4BP/SF-1[129–263] and its mutant forms were used in this assay. To examine the contribution of single amino acids from 234 to 239 in Ad4BP/SF-1, they were substituted to alanine. Yeast expression vectors for GAL4 DBD fused with the Ad4BP/SF-1 derivatives and pGBT9 were transformed together with the expression vector for GAL4 AD-ß-catenin[312–781] or pGAD424. Liquid ß-galactosidase assays were performed using three independent colonies obtained from each transformation. Results are the mean ± SD of three independent experiments. Fold activities over empty pGAD424 are indicated on the right. DBD, DNA binding domain; AD, activation domain.

 
The amino acids in Ad4BP/SF-1 essential for interaction with ß-catenin were examined with the constructs listed in the left panel of Fig. 4BGo. Interaction with ß-catenin was observed with Ad4BP/SF-1[129–263] but not with Ad4BP/SF-1[1–129] or with Ad4BP/SF-1[259–462] (Fig. 4BGo). Subsequently, the interaction was analyzed with three deletion constructs, Ad4BP/SF-1[129–253], Ad4BP/SF-1[129–243], and Ad4BP/SF-1[129–225]. An interaction was still observed even when the amino acid residues from 253 to 244 were deleted, but it completely disappeared with deletion of residues 243–226. Structurally, this region, shown here to be responsible for the interaction, corresponds to helix 1 in the ligand-binding domain (LBD).

Because the above observation suggested that the amino acids responsible for the interaction are located in the 226–243 region, we prepared a series of constructs in which four consecutive amino acids were substituted by alanine. As negative controls, the amino acid residues from 220–223 and from 245–248 were substituted by alanine. To quantitatively evaluate the alteration of the binding strength, we analyzed the ß-galactosidase activity resulting from interaction between the prey and bait molecules. ß-Galactosidase activity with 129–263(220–4A) and 129–263(245–4A) was not decreased when compared with the wild type, 129–263(Wild Type). Likewise, neither 129–263(225–4A) nor 129–263(240–4A) showed substantial decrease of the activity, whereas 129–263(230–4A) showed only a slight decrease. By contrast, the activity of 129–263(235–4A) was reduced significantly, strongly suggesting that amino acids 235–238 are predominantly implicated in the interaction. Interestingly, this region consists of a cluster of acidic amino acids. To determine whether any one amino acid is responsible for the interaction, peptides carrying a single amino acid substitution from 234 to 239 were analyzed. As indicated in Fig. 4DGo, no substantial decrease of ß-galactosidase activity was observed, probably indicating that at least three acidic amino acids are enough for the interaction.

Direct Interaction between Ad4BP/SF-1 and ß-Catenin Is Essential for Synergistic Transcriptional Activation
The interaction between Ad4BP/SF-1 and ß-catenin was investigated directly in vitro using glutathione-S-transferase (GST) pull-down assays. GST fusion proteins with residues 129–263 of Ad4BP/SF-1 [GST-(WT)] and its mutant form [GST-(235–4A)] were synthesized in Escherichia coli and incubated with 35S-labeled ß-catenin[312–781] in a binding buffer containing 100 or 250 mM NaCl. As shown in Fig. 5AGo, a certain amount of ß-catenin was recovered by GST-(WT), whereas a markedly smaller amount was recovered by GST-(235–4A), and this decreased recovery was observed with both buffers. These in vitro results indicate that the interaction with ß-catenin requires amino acid residues 235–238 of Ad4BP/SF-1 and are consistent with our earlier observations in the yeast two-hybrid experiments.



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Figure 5. Requirement of Direct Interaction between Ad4BP/SF-1 and ß-Catenin for Synergistic Transcriptional Activation

A, Direct interaction between Ad4BP/SF-1 and ß-catenin is shown by an in vitro binding assay. GST fusion proteins containing the 129th-263rd amino acid of mouse Ad4BP/SF-1 [GST-(WT)] and its mutant form [GST-(235–4A)] were incubated with 35S-labeled ß-catenin[312–781] in the binding buffer containing 250 mM NaCl or 100 mM NaCl. The bound proteins were analyzed by SDS-PAGE followed by autoradiography. Five percent of the total input of 35S-labeled ß-catenin[312–781] is indicated as a control. B, Effect of amino acid substitution (235–4A) on Ad4BP/SF-1-mediated transcription. Increasing amounts (200, 600, and 1800 ng) of the wild-type and mutant forms (235–4A) of mouse Ad4BP/SF-1 were transfected with Dax265-Luc (200 ng). The total amount of the transfected plasmid was adjusted to 2 µg with empty vectors. C, Effect of amino acid substitution (235–4A) in Ad4BP/SF-1 on the synergistic activity. Dax265-Luc (200 ng) was transfected with increasing amounts of ß-catenin (0, 100, 200 ng) in the presence (+) or absence (-) of mouse Ad4BP/SF-1 expression vector (1800 ng). The total amount of transfected plasmid was adjusted to 2.2 µg with empty vectors; 36 h after transfection, luciferase assays were performed. Results are the mean ± SD of triplicate transfections.

 
We wished to investigate whether the physical interaction between Ad4BP/SF-1 and ß-catenin is also necessary in the activation of Dax-1 gene transcription, and thus used the mutant form of Ad4BP/SF-1, Ad4BP/SF-1(235–4A). Because there is a possibility that this amino acid substitution abolishes the intrinsic transcriptional activity of the protein, we examined first whether this mutant form retains this activity. Although the transcriptional activity of Ad4BP/SF-1(235–4A) was reduced when compared with wild type, the mutant form showed a dose-dependent activation of transcription, indicating that this mutation does not completely abolish the transcriptional activity of Ad4BP/SF-1 (Fig. 5BGo). Nevertheless, in complex with ß-catenin, the mutant form (235–4A) of Ad4BP/SF-1 gave minimal activity when compared with wild type, thus demonstrating that the interaction between the two proteins is necessary for efficient activation of Dax-1 transcription.

Dax-1 Gene Expression Is Affected by Wnt4 Gene Disruption
All the results described above strongly suggest that a Wnt signal activates Dax-1 gene transcription. Because it was reported that Wnt4 is expressed in the developing urogenital system of mouse fetuses (34), we used whole-mount in situ hybridization to investigate in detail the expression of Wnt4 at the sexually differentiating stages of gonads at embryonic days 11.5 and 12.5 (E11.5 and E12.5). As shown in Fig. 6Go, Wnt4 expression is detected in the female genital ridge (G) of E11.5. In the male genital ridge, Wnt4 expression is barely detectable except in the rostral and caudal poles, whereas the signal is clearly detected at the rostral side of the mesonephros (M) where the nephric tubules are differentiating. At E12.5, expression in the male accumulates in the same mesonephric region of the rostral side, whereas that in the female is decreased in the gonad distal to the mesonephros but is maintained in the gonad proximal to the mesonephros. Expression appeared to be increased in the mesonephros of the female.



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Figure 6. Down-Regulation of Dax-1 Expression in Wnt4-Knockout (Wnt4-KO) Mice

Whole-mount in situ hybridization using Wnt4 probe was performed with gonads of both sexes (XY and XX) from E11.5 and E12.5 wild-type mouse embryos. Dax-1 gene expression was analyzed by in situ hybridization with the gonads of both sexes prepared from Wnt4 gene-disrupted [Wnt4 (-/-)] and heterozygous [Wnt4 (+/-)] mouse embryos. Gonads (G) and mesonephroi (M) are indicated by arrows.

 
The fetal gonads of Wnt4 gene-disrupted mice were subjected to in situ hybridization with a Dax-1 probe. The expression of Dax-1 in the heterozygous mice [Wnt4(+/-)] is higher in the female than in the male gonads both at E11.5 and E12.5, as was shown with the fetal gonads of the wild type (12, 44). When comparing the Dax-1 expression between the gene-disrupted [Wnt4(-/-)] and heterozygous [Wnt4(+/-)] mouse gonads, the in situ signal is significantly decreased in the female gonads of the Wnt4(-/-) both at E11.5 and E12.5. Such a clear decrease was not observed in the male gonads.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Wnt4 Signal Is Upstream of the Dax-1 Gene
Müllerian inhibiting substance (Mis) and steroidogenic 3ß-hydroxysteroid dehydrogenase (Hsd) genes are expressed in Sertoli and Leydig cells, respectively, in the male, but not in the female, fetal and neonatal gonads (34, 45, 46). Normally, these genes start to be expressed postnatally in the female gonads. Therefore, these genes have been frequently used as markers for testis differentiation. Interestingly, it was reported that expression of these testicular marker genes started earlier in the female gonads of Wnt4 gene-disrupted mice than in those of the wild type. Based on this phenotype, it was strongly suggested that Wnt4 suppresses Mis and 3ß-Hsd gene transcription in the fetal and neonatal ovary.

It has been established that Wnt regulates a variety of cellular events via canonical or noncanonical pathways (47, 48, 49). As one of the major components of the former pathway, the function and regulation of ß-catenin has received some attention (50). Under stimulus from certain types of Wnt, ß-catenin stabilized through suppressed phosphorylation translocates into the nucleus to heterodimerize with the LEF/TCF transcription factor containing an HMG box. Thereafter, the complex up-regulates transcription of target genes. However, as described above, Wnt4 is possibly acting as a suppressive signal for Mis and 3ß-Hsd gene transcription in the fetal ovary, which is clearly inconsistent with the activating events occurring downstream of the well known canonical pathway. To give a reasonable explanation for this inconsistency, it was hypothesized that Wnt4 activates a putative suppressor gene, and thereafter this gene product suppresses the transcription of the Mis and 3ß-Hsd genes in the fetal ovary. Because Dax-1 suppresses the transcription mediated by Ad4BP/SF-1, which is known as a transcriptional activator of the Mis and 3ß-Hsd genes, we further hypothesized that Dax-1 acts as the suppressor downstream of the Wnt4 signal. This hypothesis seemed to be supported by the phenotypes of human patients caused by gene duplication of DAX-1 and WNT4. As reported by Elejalde et al. (40) and Jordan et al. (42), WNT4 gene duplication leads to gonadal dysgenesis in XY patients, which is similar to the phenotype of DSS patients carrying DAX-1 gene duplication. Thus, considering the similar phenotypes of the two diseases, it was reasonable to suppose that the enhanced WNT4 signal caused by the gene duplication induces the DAX-1 gene transcription up to a certain level equivalent to that induced by DAX-1 gene duplication.

To examine the hypothesis described above, we investigated whether ß-catenin activates Dax-1 gene transcription. Because Wnt4 was shown to activate the canonical pathway by two studies with embryonic stem cells (51) and thymocytes (52), we investigated the effects of ß-catenin. As expected, it was clearly shown that transcription is activated by the action of ß-catenin through the LEF/TCF binding sites on the Dax-1 gene promoter. In support of our observation, Jordan et al. (42) showed that Wnt4 up-regulated transcription of the endogenous Dax-1 gene in cultured cells. These observations from our in vitro studies were further confirmed by an in vivo study using Wnt4 gene-disrupted mice. Absence of Wnt4 from the sexually differentiating gonads led to down-regulation of Dax-1 gene expression in the female. Taken together, as the molecular mechanism underlying female sex determination, we propose that Wnt4 suppresses the expression of the testicular marker gene through up-regulation of Dax-1. The present study revealed for the first time the interactions between Wnt4 and Dax-1 at the molecular level.

As described previously (12, 44, 53, 54), both Dax-1 and Ad4BP/SF-1 show sexually dimorphic expression. Namely, the expression of Dax-1 in the female is more abundant than in the male, whereas the expression of Ad4BP/SF-1 in the female is less abundant than in the male. However, this reciprocal relationship seems paradoxical, if we consider that Ad4BP/SF-1 acts as a positive regulator of the Dax-1 gene transcription (11, 24, 25, 26, 27). Therefore, additional factors were assumed to regulate Dax-1 gene transcription. In this study, we present the interpretation that the Wnt4 signal, which is abundant in the female, compensates for the lower expression of Ad4BP/SF-1 and leads to higher expression of Dax-1.

ß-Catenin and Ad4BP/SF-1 Interact Directly to Give a Synergistic Transcriptional Activation of Dax-1
Previous studies indicated that Dax-1 gene transcription is activated by Ad4BP/SF-1 through multiple binding sites (11, 24, 25, 26, 27). In the present study, it was shown that a ß-catenin-LEF/TCF complex is implicated in Dax-1 gene transcription, and in particular this complex activates transcription synergistically with Ad4BP/SF-1. This activation was clearly shown to be due to a direct interaction between Ad4BP/SF-1 and ß-catenin.

A number of transcription factors and cofactors have been known to interact with the armadillo repeats in ß-catenin. In this study, it was shown that armadillo repeats 9–12 are critical for interaction with Ad4BP/SF-1, whereas LEF/TCF interacts specifically with armadillo repeats 3–8 (55). Although we could not detect any interaction between Ad4BP/SF-1 and LEF/TCF (data not shown), it is likely that the three factors form a ternary complex using ß-catenin as the tether between Ad4BP/SF-1 and LEF/TCF. This hypothesis awaits further investigation.

Amino Acids in Ad4BP/SF-1 Critical for Interaction with ß-Catenin
It has been reported that the ß-catenin interacting regions identified in cadherin, adenomatous polyposis coli, and LEF/TCF family members all contain acidic amino acids (56). The present study revealed that Ad4BP/SF-1 also contains an acidic amino acid cluster in the ß-catenin interacting region. A similar sequence with a cluster of acidic amino acids was reported to be critical for the interaction of ICAT (inhibitor of ß-catenin and TCF-4) with ß-catenin (57). Although the ß-catenin-interacting amino acids of many other proteins are still unknown, it seems reasonable to assume that, as proposed previously (56, 58), an acidic region interacts with the basic groove of ß-catenin. Our observation strongly supports this.

Members of the nuclear receptor superfamily, retinoic acid receptor-{alpha} (RAR{alpha}; NR1B1) and androgen receptor (AR; NR3C4), were reported to show synergistic transcriptional activation with ß-catenin (59, 60, 61). However, as shown in Fig. 7Go, alignment of helix 1 revealed that the acidic amino acid cluster is not found in the helices of AR and RAR{alpha}, indicating that these nuclear receptors are interacting with regions other than helix 1. By contrast, amino acid clusters containing at least three acidic residues are conserved in Ad4BP/SF-1 in all vertebrate species. However, we showed here that where only a single amino acid is substituted, the mutant shows essentially no difference in interaction. Accordingly, it is possible to assume that Ad4BP/SF-1 in all vertebrate species has the potential to interact with ß-catenin, and therefore that the synergistic activation between Ad4BP/SF-1 and the Wnt signal is a mechanism found widely in vertebrates. Moreover, we aligned LRH-1 (NR5A2), which is the nuclear receptor showing highest homology with Ad4BP/SF-1. Interestingly, liver receptor homolog-1 (LRH-1) also has the conserved acidic amino acid cluster, raising the possibility that LRH-1 shows synergistic activation with the Wnt signal.



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Figure 7. Sequence Alignment of Helix 1 in the LBD of Nuclear Receptors

The amino acid sequence of the helix 1 in the LBD of mouse Ad4BP/SF-1 is compared with those of human AR, human RAR{alpha}, Ad4BP/SF-1 orthologs from the vertebrate species (wallaby, chick, turtle, and frog), and mouse LRH. AR and RAR{alpha} are nuclear receptors previously shown to interact with ß-catenin. The amino acids of mouse Ad4BP/SF-1 required for interaction with ß-catenin and the corresponding residues in the other nuclear receptors are shown with larger letters. Asterisks indicate acidic amino acids, specifically aspartic acid (D) or glutamic acid (E). Numbers on the right indicate the amino acid residues of each receptor.

 
An increasing number of proteins have been reported to interact with Ad4BP/SF-1. Some of them are known to be implicated in gonadal differentiation and gonadal sex differentiation from observations in transgenic and gene-disrupted studies as well as studies of human diseases. In the present study, we resolved the questions arising from the phenotype of Wnt4 gene-disrupted mice, through an investigation of the interactions of the key proteins, and finally revealed a probable link between the Wnt4 pathway and Dax-1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Two and five-tenths kilobases, 1.5 kb, 1.0 kb, 600 bp, 540 bp, 265 bp, 117 bp, and 40 bp upstream from the transcription initiation site of the mouse Dax-1 gene were used to prepare a series of the Dax-1 gene reporter constructs, Dax2.5K-Luc, Dax1.5K-Luc, Dax1.0K-Luc, Dax600-Luc, Dax540-Luc, Dax265-Luc, Dax117-Luc, and Dax40-Luc, respectively. pGL3-Basic was the original plasmid for the luciferase reporter gene constructs (Promega Corp., Madison, WI). To disrupt the LEF/TCF binding sites, LEF-D (5'-CCTTTGAG-3') at position -140/-133 and LEF-E (5'-CACAAAGG-3') at position -102/-95 in Dax265-Luc were replaced by 5'-AATTTCAG-3' and 5'-CACCCCGG-3', respectively, using PCR-based site-directed mutagenesis. For expression of ß-catenin in mammalian cells, MMßcha (62) was used. MMßcha encodes a carboxy-terminal 3x hemagglutinin-tagged ß-catenin in which the 33rd, 37th, and 45th serine residues, and the 41st threonine residue are substituted by alanine to produce a form resistant to ubiquitin-dependent proteolytic digestion (63). Bovine (8) and mouse (9) cDNAs for Ad4BP/SF-1 were used for expression in mammalian cells as noted in the figure legends. Amino acid residues of mouse Ad4BP/SF-1 at 235–238 were substituted by four alanines to produce Ad4BP/SF-1[235–4A].

Cell Culture and Transfection
Human embryonic kidney 293 cells were grown in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) and 1x penicillin-streptomycin-glutamine (Invitrogen, Carlsbad, CA) at 10% CO2 and 37 C. Cells were seeded at 7 x 105 per 60-mm dish 24 h before the relevant combination of DNAs was transfected using lipofectamine reagent (Invitrogen) according to the manufacturer’s protocol. pCMV-SPORT-ß-gal (50 ng; Invitrogen) was used as an internal control to normalize transfection efficiency. The total amount of DNA used in the assays is described in the figure legends. Cells were harvested 36 h after transfection and subjected to a luciferase assay as described previously (64). All experiments were performed in triplicate.

EMSAs
EMSAs were basically performed as described previously (65). pCS2-hLEF-1 (66) was used for in vitro synthesis of amino-terminal Flag-tagged human LEF-1, using a coupled transcription and translation kit (Promega Corp.). GST-ß-catenin (62) was expressed in E. coli (DH5{alpha}) and purified by affinity chromatography on glutathione-sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden). Anti-Flag M2 monoclonal antibody (Sigma) was used for supershift analysis. The following double-stranded oligonucleotides were used as probes: TCR{alpha}WT [LEF-1 binding site in the human TCR{alpha} enhancer (43)]: 5'-GAGGGCACCCTTTGAAGCTCTCCC-3' and 5'-GGGGAGAGCTTCAAAGGGTGCCCT-3'; TCR{alpha}M [mutated LEF-1 binding site in the human TCR{alpha} enhancer (43)]: 5'-GAGGGCACAATTTCAAGCTCTCCC-3' and 5'-GGGGAGAGCTTGAAATTGTGCCCT-3'; LEF-A (a putative LEF-1 binding site on the mouse Dax-1 upstream at -2604/-2596): 5'-GCCTAAGAGACTTTGATACAGGTT-3' and 5'-GAACCTGTATCAAAGTCTCTTAGG-3'; LEF-B (a putative LEF-1 binding site on the mouse Dax-1 upstream at -1460/-1452): 5'-GATATCTATATCAATCTTCCAAGA-3' and 5'-GTCTTGGAAGATTGATATAGATAT-3'; LEF-C (a putative LEF-1 binding site on the mouse Dax-1 upstream at -991/-984): 5'-GCTGTAAGAGCAAAGGCTTGCACT-3' and 5'-GAGTGCAAGCCTTTGCTCTTACAG-3'; LEF-D (a putative LEF-1 binding site on the mouse Dax-1 upstream at -140/-133): 5'-GGTTTGCCCCTTTGAGCTTTCGAG-3' and 5'-GCTCGAAAGCTCAAAGGGGCAAAC-3'; LEF-E (a putative LEF-1 binding site on the mouse Dax-1 upstream at -102/-95): 5'-GCATTCAAGCACAAAGGCGCGTCC-3' and 5'-GGGACGCGCCTTTGTGCTTGAATG-3'.

Yeast Two-Hybrid Assay
In the yeast two-hybrid study, pGBT9 and pGAD424 (CLONTECH Laboratories, Inc., Palo Alto, CA) were used for expression of fusion proteins with the GAL4 DNA-binding domain and the GAL4 activation domain, respectively. pGAD424-ß-catenin[312–781] was isolated in a yeast two-hybrid screening. Other pGAD424-ß-catenin constructs, pGAD424-ß-catenin[1–575], pGAD424-ß-catenin[474–781], pGAD424-ß-catenin[624–781], pGAD424-ß-catenin[312–696], and pGAD424-ß-catenin[312–575] were prepared by inserting fragments corresponding to the respective portions of ß-catenin into pGAD424. pGBT9-LEF-1[1–109] was constructed by inserting the EcoRI-XhoI fragment from pCS2-hLEF-1 into pGBT9. The DNA fragments from mouse Ad4BP/SF-1 (67) were generated by PCR and inserted into pGBT9 to produce Ad4BP/SF-1[1–129], Ad4BP/SF-1[129–263], Ad4BP/SF-1[259–462], Ad4BP/SF-1[129–253], Ad4BP/SF-1[129–243], and Ad4BP/SF-1[129–225]. Mutant forms of pGBT-Ad4BP/SF-1[129–263], Ad4BP/SF-1[129–263(220–4A)], Ad4BP/SF-1[129–263(225–4A)], Ad4BP/SF-1[129–263(230–4A)], Ad4BP/SF-1[129–263(235–4A)], Ad4BP/SF-1[129–263(240–4A)], and Ad4BP/SF-1[129–263(245–4A)], were constructed to give four consecutive amino acid substitutions to alanine at the indicated positions. Similarly, Ad4BP/SF-1[129–263(L234A)], Ad4BP/SF-1[129–263(E235A)], Ad4BP/SF-1[129–263(P236A)], Ad4BP/SF-1[129–263(E237A)], Ad4BP/SF-1[129–263(E238A)], and Ad4BP/SF-1[129–263(D239A)] were constructed to carry a single amino acid substitution to alanine at the indicated position. Yeast two-hybrid experiments were basically performed according to the recommended protocol (CLONTECH Laboratories, Inc.). The bait plasmid was transformed into a yeast strain PJ69–4A (68). A cDNA library prepared from fetal gonads of mouse embryos of both sexes (E11.5–E13.5) was used for screening. Transformants were selected on Sabouraud dextrose (SD) medium lacking leucine, tryptophan, and histidine. Interactions were determined by cell growth on the selection medium or by a liquid ß-galactosidase assay as described (69). ß-Galactosidase activities were measured using the Galacto-light Plus kit (Tropix Inc., Bedford, MA).

In Vitro Binding Assay
Expression vectors for GST-Ad4BP/SF-1[129–263aa] (GST-WT) and its mutant form [GST-(235–4A)] were constructed using pGEX-4T-1 (Amersham Pharmacia Biotech, Uppsala, Sweden). The GST fusion proteins were expressed in E. coli (BL21) and bound to glutathione-sepharose 4B beads (Amersham Pharmacia Biotech). An expression vector for ß-catenin [312–781aa] was constructed in pCMX containing a T7 promoter (70). ß-Catenin was synthesized in vitro in the presence of 35S-methionine using a TNT-coupled reticulocyte lysate system (Promega Corp.). Equal amounts of GST-Ad4BP/SF-1 fusion proteins coupled to glutathione-sepharose beads were incubated with 35S-labeled ß-catenin at 4 C for 2 h in a binding buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM or 250 mM NaCl, 1 mM dithiothreitol, 0.1% Triton X-100, and 1x complete protease inhibitor cocktail (Roche, Indianapolis, IN). Beads were carefully washed four times with the binding buffer, after which the bound proteins were analyzed by SDS-PAGE followed by autoradiography.

Whole-Mount In Situ Hybridization
Digoxygenin-labeled antisense riboprobes from Wnt4 (kindly provided by Dr. Andrew McMahon) and Dax-1 were generated using T7 RNA polymerase (Promega Corp.). Whole-mount in situ hybridization was performed as described previously (11, 38).


    ACKNOWLEDGMENTS
 
We thank Drs. Keith Parker and Ohtsura Niwa for the mouse SF-1 plasmids, Dr. Hiroshi Shibuya for the human LEF-1 plasmid, Drs. Tetsu Akiyama and Satoru Ishihara for the ß-catenin plasmids, Dr. Andrew McMahon for the Wnt4 plasmid, Dr. Philip James for yeast strain PJ69-4A, and Dr. Kazuhiko Umesono for pCMX. We also thank Drs. Yoshinori Ohsumi, Hiroshi Shibuya, and Masuo Goto for their suggestions on the yeast two-hybrid experiments.


    FOOTNOTES
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. This study was also supported by the Cell Science Research Foundation.

1 Present address: Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Back

2 Present address: Department of Pediatrics, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan. Back

Abbreviations: Ad4BP/SF-1, Ad4 binding protein/steroidogenic factor 1; AR, androgen receptor; Dax-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; DSS, dosage-sensitive sex reversal; E, embryonic day; GST, glutathione-S-transferase; Hsd, hydroxysteroid dehydrogenase; LBD, ligand-binding domain; LRH-1, liver receptor homolog-1; LEF, lymphoid enhancer binding factor; Mis, Mullerian inhibiting substance; RAR, retinoic acid receptor; SD, Sabouraud dextrose; TCF, T cell-specific factor; TCR{alpha}, T cell receptor {alpha}.

Received for publication November 1, 2002. Accepted for publication January 13, 2003.


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