DAX1 Function Research Articles

A TRIM66/DAX1/Dux axis suppresses the totipotent 2-cell-like state in murine embryonic stem cells.

2-cell-like cells (2CLCs)-which comprise only ∼1% of murine embryonic stem cells (mESCs)-resemble blastomeres of 2-cell-stage embryos and are used to investigate zygotic genome activation (ZGA). Here, we discovered that TRIM66 and DAX1 function together as negative regulators of the 2C-like state in mESCs. Chimeric assays confirmed that mESCs lacking TRIM66 or DAX1 function have bidirectional embryonic and extraembryonic differentiation potential. TRIM66 functions by recruiting the co-repressor DAX1 to the Dux promoter, and TRIM66's repressive effect on Dux is dependent on DAX1. A solved crystal structural shows that TRIM66's PHD finger recognizes H3K4-K9me3, and mutational evidence confirmed that TRIM66's PHD finger is essential for its repression of Dux. Thus, beyond expanding the scope of known 2CLC regulators, our study demonstrates that interventions disrupting TRIM66 or DAX1 function in mESCs yield 2CLCs with expanded bidirectional differentiation potential, opening doors for the practical application of these totipotent-like cells.

Nonsense variant of NR0B1 causes hormone disorders associated with congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a rare X-linked recessive inherited disease that is considered a major cause of steroidogenesis disorder and is associated with variants or complete deletion of the NR0B1 gene. The DAX-1 protein (encoded by NR0B1) is a vertebrate-specific orphan nuclear receptor and is also a transcriptional factor for adrenal and reproductive development. CAH usually causes adrenal insufficiency in infancy and early childhood, leading to hypogonadotropic hypogonadism in adulthood; however, few adult cases have been reported to date. In this study, we examined a Chinese family with one adult patient with CAH, and identified a putative variant of NR0B1 gene via next-generation sequencing (NGS), which was confirmed with Sanger sequencing. A novel nonsense variant (c.265C>T) was identified in the NR0B1 gene, which caused the premature termination of DAX-1 at residue 89 (p.G89*). Furthermore, mutant NR0B1 gene displayed a partial DAX-1 function, which may explain the late pathogenesis in our case. Additionally, qPCR revealed the abnormal expression of four important genes identified from ChIP-seq, which were associated with energy homeostasis and steroidogenesis, and were influenced by the DAX-1 mutant. In addition, hormone disorders can be caused by DAX-1 mutant and partially recovered by siRNA of PPARGC1A. Herein, we identified a novel nonsense variant (c.265C>T) of NR0B1 in a 24-year-old Chinese male who was suffering from CAH. This mutant DAX-1 protein was found to have disordered energy homeostasis and steroidogenesis based on in vitro studies, which was clinically consistent with the patient’s phenotypic features.

Pluripotency-State-Dependent Role of Dax1 in Embryonic Stem Cells Self-Renewal.

Dax1(also known as Nr0b1) is regarded as an important component of the transcription factor network in mouse embryonic stem cells (ESCs). However, the role and the molecular mechanism of Dax1 in the maintenance of different pluripotency states are poorly understood. Here, we constructed a stable Dax1 knockout (KO) cell line using the CRISPR/Cas9 system to analyze the precise function of Dax1. We reported that 2i/LIF-ESCs had significantly lower Dax1 expression than LIF/serum-ESCs. Dax1KO ES cell lines could be established in 2i/LIF and their pluripotency was confirmed. In contrast, Dax1-null ESCs could not be continuously passaged in LIF/serum due to severe differentiation and apoptosis. In LIF/serum, the activities of the Core module and Myc module were significantly reduced, while the PRC2 module was activated after Dax1KO. The expression of most proapoptotic genes and lineage-commitment genes were drastically increased, while the downregulated expression of antiapoptotic genes and many pluripotency genes was observed. Our research on the pluripotent state-dependent role of Dax1 provides clues to understand the molecular regulation mechanism at different stages of early embryonic development.

Live Birth in Sex-Reversed XY Mice Lacking the Nuclear Receptor Dax1

The nuclear hormone receptor Dax1 functions during development as a testes-determining gene. However, the phenotype of male mice lacking Dax1 is strain-dependent due to the background-specific abundance of male-determining Sry gene-transcripts. We hypothesised that inter-individual variation in Sry mRNA-abundance would result in a spectrum of phenotypes even within-strain. We found that while all XY C57BL/6J mice lacking Dax1 presented as phenotypic females, there was a marked inter-individual variability in measures of fertility. Indeed, we report rare occasions where sex-reversed mice had measures of fertility comparable to those in control females. On two occasions, these sex-reversed XY mice were able to give birth to live offspring following mating to stud-males. As such, this work documents within-strain variability in phenotypes of XY mice lacking Dax1, and reports for the first time a complete sex-reversal capable of achieving live birth in these mice.

Abstract 2533: Regulation of proliferation and metastasis gene expression by DAX-1 (NR0B1) in human breast cancer cells

Abstract The orphan nuclear receptor DAX-1 (Dosage Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, critical region on the X chromosome, gene 1) is known to serve an important role early on in mammalian sex determination as well as in the regulation of expression of steroid hormones. While it is unclear whether DAX-1 functions independently, DAX-1 has been demonstrated to act as a repressor of many nuclear hormone receptors, including ER, AR, PR, LRH-1 and SF-1. DAX-1 plays a role in select adult tissues, including gonadal tissue. However, it has also been shown to play a role in several types of cancer, though its specific function is not well understood. For example, in some types of cancer DAX-1 expression is upregulated, while in other cancers it is highly downregulated or completely absent. In an effort to better understand DAX-1 function both in normal and disease states, we examined the actions of DAX-1 in MCF7 breast cancer cells, which do not express the DAX-1 gene. We probed three different PCR arrays (breast cancer genes, proliferation genes and metastatic genes) to determine which genes were influenced by DAX-1 expression. With the introduction of DAX-1 in ERα positive breast cancer cells (MCF-7), we find that DAX-1 inhibits cell proliferation and blocks estradiol activation of key cell cycle regulatory genes, such as cyclin D1, as well as important metastatic genes. We show that DAX-1 binds directly to ERα and the ERα-DAX-1 complex binds to the cyclin D1 promoter. The outcome of DAX-1 expression in MCF7 cells is cell cycle arrest through the repression of key cell cycle regulatory genes. These results demonstrate that DAX-1 functions as a corepressor of ERα in MCF7 breast cancer cells by preventing the activation of growth-promoting genes and decreasing cell proliferation in response to estrogens. These findings could have significant implications for future drug development strategies specifically aimed at ER-interacting proteins such as DAX-1. Citation Format: Christina Tzagarakis-Foster. Regulation of proliferation and metastasis gene expression by DAX-1 (NR0B1) in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2533.

Timing of adrenal regression controlled by synergistic interaction between Sf1 SUMOylation and Dax1.

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1, Ad4bp) is crucial for formation, development and function of steroidogenic tissues. A fetal adrenal enhancer (FAdE) in the Sf1 gene was previously identified to direct Sf1 expression exclusively in the fetal adrenal cortex and is bound by both Sf1 and Dax1. Here, we have examined the function of Sf1 SUMOylation and its interaction with Dax1 on FAdE function. A diffused prolonged pattern of FAdE expression and delayed regression of the postnatal fetal cortex (X-zone) were detected in both the SUMOylation-deficient-Sf12KR/2KR and Dax1 knockout mouse lines, with FAdE expression/activity retained in the postnatal 20αHSD-positive postnatal X-zone cells. In vitro studies indicated that Sf1 SUMOylation, although not directly influencing DNA binding, actually increased binding of Dax1 to Sf1 to further enhance transcriptional repression of FAdE. Taken together, these studies define a crucial repressor function of Sf1 SUMOylation and Dax1 in the physiological cessation of FAdE-mediated Sf1 expression and the resultant regression of the postnatal fetal cortex (X-zone).

Dax1 and Nanog act in parallel to stabilize mouse embryonic stem cells and induced pluripotency.

Nanog expression is heterogeneous and dynamic in embryonic stem cells (ESCs). However, the mechanism for stabilizing pluripotency during the transitions between Nanoghigh and Nanoglow states is not well understood. Here we report that Dax1 acts in parallel with Nanog to regulate mouse ESC (mESCs) identity. Dax1 stable knockdown mESCs are predisposed towards differentiation but do not lose pluripotency, whereas Dax1 overexpression supports LIF-independent self-renewal. Although partially complementary, Dax1 and Nanog function independently and cannot replace one another. They are both required for full reprogramming to induce pluripotency. Importantly, Dax1 is indispensable for self-renewal of Nanoglow mESCs. Moreover, we report that Dax1 prevents extra-embryonic endoderm (ExEn) commitment by directly repressing Gata6 transcription. Dax1 may also mediate inhibition of trophectoderm differentiation independent or as a downstream effector of Oct4. These findings establish a basal role of Dax1 in maintaining pluripotency during the state transition of mESCs and somatic cell reprogramming.

A novel DAX1/NR0B1 mutation in a patient with adrenal hypoplasia congenita and hypogonadotropic hypogonadism

We report a case of adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) due to a novel DAX1 mutation. A 19-month-old boy with hyperpigmentation and failure to thrive came to our service for investigation. Three brothers of the patient had died due to adrenal failure, and a maternal cousin had adrenal insufficiency. Adrenoleukodystrophy was excluded. MRI showed normal pituitary and hypothalamus. Plasma hormone evaluation revealed high ACTH (up to 2,790 pg/mL), and low levels of androstenedione, DHEA-S, 11-deoxycortisol, and cortisol. At 14 years of age the patient was still prepubescent, his weight was 43.6 kg (SDS: -0.87) and his height was 161 cm (SDS: -0.36), with normal body proportions. In the GnRH test, basal and maximum values of LH and FSH were respectively 0.6/2.1 and < 1.0/< 1.0 U/L. Molecular investigation identified a novel mutation that consists of a deletion of codon 372 (AAC; asparagine) in exon 1 of DAX1. This mutation was not found in a study of 200 alleles from normal individuals. Prediction site analysis indicated that this alteration, located in the DAX1 ligand-binding domain, may damage DAX1 protein. We hypothesize that the novel (p.Asp372del) DAX1 mutation might be able to cause a disruption of DAX1 function, and is probably involved in the development of AHC and HH in this patient.

The Orphan Nuclear Receptor DAX-1 Functions as a Potent Corepressor of the Constitutive Androstane Receptor (NR1I3)

Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function.

Damage in brain development by morpholino knockdown of zebrafish dax1

DAX1 is an orphan nuclear receptor and involved in development of steroidogenic organs, which activates transcription of genes involved in steroidogenesis. In this study, we analyzed the function of the zebrafish dax1 during early development of central nervous systems to appear unidentified aspects of DAX1 and decrease confusions concerned with functions of DAX1 in early development of vertebrates. By whole-mount in situ hybridization of embryo at the 32 h post fertilization (hpf), expression of zebrafish dax1 was detected around the forebrain, midbrain, hindbrain, and the extending tail tip. Embryos injected with zebrafish dax1 morpholino antisense nucleotide (MO) exhibited delayed development. When the developmental stage of wild type embryos was at Prim-15 (32 hpf), zebrafish dax1MO injected embryos were at Prim-5 (24 hpf). Concurrently with developmental delay, the MO injected embryos showed high mortality. At 48 hpf, the MO injected embryos exhibited abnormal development in the central nervous systems. The enlarged tectum and the protruded rhombomeres were observed. Moreover, development of central nervous systems, especially midbrain-hindbrain boundary, became narrower. At 5 day post fertilization, the MO injected embryos formed edemas around head, pericardial sac and abdomen. Collectively, our results indicated that the zebrafish dax1 is important for brain development.

2094 ADENOVIRUS-MEDIATED DAX1GENE TRANSFER ENHANCES THE FUNCTION OF SERTOLI CELLS VIA THE SCF/C-KIT PATHWAY

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20122094 ADENOVIRUS-MEDIATED DAX1GENE TRANSFER ENHANCES THE FUNCTION OF SERTOLI CELLS VIA THE SCF/C-KIT PATHWAY Satoshi Kurokawa, Yoshiyuki Kojima, Kentaro Mizuno, Akihiro Nakane, Takahiro Yasui, Noriyasu Kawai, Keiichi Tozawa, Tetsuji Maruyama, Yutaro Hayashi, and Kenjiro Kohri Satoshi KurokawaSatoshi Kurokawa Nagoya, Japan More articles by this author , Yoshiyuki KojimaYoshiyuki Kojima Nagoya, Japan More articles by this author , Kentaro MizunoKentaro Mizuno Nagoya, Japan More articles by this author , Akihiro NakaneAkihiro Nakane Nagoya, Japan More articles by this author , Takahiro YasuiTakahiro Yasui Nagoya, Japan More articles by this author , Noriyasu KawaiNoriyasu Kawai Nagoya, Japan More articles by this author , Keiichi TozawaKeiichi Tozawa Nagoya, Japan More articles by this author , Tetsuji MaruyamaTetsuji Maruyama Nagoya, Japan More articles by this author , Yutaro HayashiYutaro Hayashi Nagoya, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2261AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES DAX1, an essential transcription factor in sex differentiation, is expressed in the cells of the hypothalamic-pituitary-testicular axis. DAX1 plays important roles in both steroidogenesis in Leydig cells and spermatogenesis in Sertoli cells in the testes. DAX1 expression is synchronized with the spermatogenic cycle, especially in Sertoli cells. However, DAX1 function in Sertoli cells is unclear. We therefore analyzed the function of DAX1 in Sertoli cells using the adenoviral gene transfer technique. METHODS 1) In vitro study: An adenovirus vector containing full-length mouse Dax1 cDNA was constructed from the human adenovirus serotype 5. This vector was tested on the mouse Sertoli cell line, TM4, to assess the efficiency of gene transfer at various multiplicities of infections (MOIs): 1, 10, and 100. TM4 cells were harvested and mRNA was extracted 48 hours after gene transfer. The expression level of 16 Sertoli cell-specific genes was examined by quantitative RT-PCR. 2) In vivo study: Sl/Sld and W/Wv mice with male infertility were used. Ten-week-old male mice were sacrificed and their testes were harvested. Dax1 expression in the testes was analyzed by quantitative RT-PCR and western blotting. RESULTS 1) More than 70% of TM4 cells showed gene transfer with the adenovirus vector at a MOI of 100. Among the Sertoli cell-specific genes, Scf, Sox9, and Star were expressed at significantly higher levels after Dax1 gene transfer. Scf showed the largest expression level increase (3.1 fold). 2) Dax1 mRNA expression was significantly higher in the testes of Sl/Sld and W/Wv mice than in normal mice. Strong DAX1 staining was detected in the nuclei of Sertoli cells. CONCLUSIONS Strong Scf expression was observed after Dax1 gene transfer, indicating that the nuclear receptor DAX1 controls SCF expression in Sertoli cells. The Sl/Sld mouse has mutant SCF and the W/Wv mouse has mutant C-KIT, which is a SCF receptor. DAX1 expression was accelerated in the testes of these mice, which may be caused by a feedback system based on the functional loss of SCF. SCF, which is produced in the Sertoli cells of the testis, binds to C-KIT on the spermatogonia and plays an important role in the differentiation and proliferation of germ cells. DAX1 may activate Sertoli cells to stimulate spermatogenesis via a SCF/C-KIT signaling pathway. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e844 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Satoshi Kurokawa Nagoya, Japan More articles by this author Yoshiyuki Kojima Nagoya, Japan More articles by this author Kentaro Mizuno Nagoya, Japan More articles by this author Akihiro Nakane Nagoya, Japan More articles by this author Takahiro Yasui Nagoya, Japan More articles by this author Noriyasu Kawai Nagoya, Japan More articles by this author Keiichi Tozawa Nagoya, Japan More articles by this author Tetsuji Maruyama Nagoya, Japan More articles by this author Yutaro Hayashi Nagoya, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

2011 ROLE OF TRANSCRIPTION FACTOR DAX-1 AS A NEW FUNCTIONAL MARKER OF UNDIFFERENTIATED SERTOLI CELLS IN AZOOSPERMIA PATIENTS

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20112011 ROLE OF TRANSCRIPTION FACTOR DAX-1 AS A NEW FUNCTIONAL MARKER OF UNDIFFERENTIATED SERTOLI CELLS IN AZOOSPERMIA PATIENTS Yoshiyuki Kojima, Yutaro Hayashi, Kentaro Mizuno, Yukihiro Umemoto, Shoichi Sasaki, and Kenjiro Kohri Yoshiyuki KojimaYoshiyuki Kojima Nagoya, Japan More articles by this author , Yutaro HayashiYutaro Hayashi Nagoya, Japan More articles by this author , Kentaro MizunoKentaro Mizuno Nagoya, Japan More articles by this author , Yukihiro UmemotoYukihiro Umemoto Nagoya, Japan More articles by this author , Shoichi SasakiShoichi Sasaki Nagoya, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2239AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES DAX-1 is an orphan member of the nuclear hormone receptor superfamily and acts as a transcriptional repressor. It plays a critical role in gonadal function and steroidogenesis. The characterization of Dax-1-deficient male mice suggested that Dax1 plays an important role in the maintenance of spermatogenesis. In our study, we examined the expression levels of DAX-1 in several azoospermiac patients with Sertoli cell-only testes and discuss the possibility of DAX-1 as a functional marker of undifferentiated Sertoli cells in azoospermia patients. METHODS All patients were diagnosed as Sertoli cell-only by histological observation. Ten idiopathic azoospermia patients with Sertoli cell-only syndrome, five azoospermia patients with cryptorchidism, and four azoospermia patients with hypogonadropic hypogonadism were enrolled. Five obstructive azoospermia patients with normal spermatogenesis were included as additional testicular samples. We performed immunohistochemical study and quantitiative RT-PCR using testicular biopsy samples to examine the DAX-1 expression, and analyzed the role of DAX-1 in Sertoli cells. In addition, transcriptional activities of DAX-1 by FSH stimulation were examined with TM4 derived from mouse testicular Sertoli cells. RESULTS Immunohistochemical study showed that DAX-1 was strongly expressed in the nuclei of Sertoli cells in patients with cryptorchidism, hypogonadropic hypogonadism or normal spermatogenesis. On the other hand, it was faintly expressed in the nuclei of Sertoli cell in patients with idiopathic azoospermia. The average expression levels of DAX-1 mRNA in idiopathic azoospermia patients with Sertoli cell-only syndrome (0.13±0.08) were lower than those with cryptorchidism (1.38±0.37), hypogonadropic hypogonadism (0.98±0.34) or normal spermatogenesis (1.30±1.41). Identical amounts of DAX1 were detected regardless of FSH stimulation in TM4 cells, and DAX1 was not activated significantly by FSH with TM4. CONCLUSIONS These results suggest that the function of DAX-1 in Sertoli cells may be required for normal spermatogenesis and fertility. Idiopathic azoospermia may result in abnormal Sertoli cell function, which disrupts the normal progression of spermatogenesis. Transcription of the DAX-1 gene may be negatively regulated by factors other than FSH, especially in Sertoli cells in Sertoli cell-only syndrome. Given the essential role of DAX-1 in Sertoli cells with normal spermatogenesis, its defect could reflect a new functional marker of undifferentiated Sertoli cells. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e804-e805 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshiyuki Kojima Nagoya, Japan More articles by this author Yutaro Hayashi Nagoya, Japan More articles by this author Kentaro Mizuno Nagoya, Japan More articles by this author Yukihiro Umemoto Nagoya, Japan More articles by this author Shoichi Sasaki Nagoya, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Dax-1 Knockdown in Mouse Embryonic Stem Cells Induces Loss of Pluripotency and Multilineage Differentiation

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here, we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore, Dax-1 knockdown induced upregulation of multilineage differentiation markers, and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis, we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly, the great majority of these genes are upregulated, showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells, as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis, respectively.

The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1.

The Dax-1 protein is an enigmatic nuclear receptor that lacks an expected DNA binding domain, yet functions as a potent corepressor of nuclear receptors. Here we report the structure of Dax-1 bound to one of its targets, liver receptor homolog 1 (LRH-1). Unexpectedly, Dax-1 binds to LRH-1 using a new module, a repressor helix built from a family conserved sequence motif, PCFXXLP. Mutations in this repressor helix that are linked with human endocrine disorders dissociate the complex and attenuate Dax-1 function. The structure of the Dax-1:LRH-1 complex provides the molecular mechanism for the function of Dax-1 as a potent transcriptional repressor.

DAX1, a direct target of EWS/FLI1 oncoprotein, is a principal regulator of cell-cycle progression in Ewing's tumor cells

The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT-PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.

An Amino-Terminal DAX1 (NROB1) Missense Mutation Associated with Isolated Mineralocorticoid Deficiency

Mutations in DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1; NR0B1) cause X-linked adrenal hypoplasia congenita, a disease characterized by primary adrenal failure, testicular dysgenesis, and gonadotropin deficiency. Most DAX1 mutations are deletions, nonsense, or frameshift mutations that markedly impair its transcriptional activity. Missense mutations have been restricted to the carboxy-terminal domain and are associated with more variable clinical phenotypes. The objective was to identify novel clinical phenotypes associated with DAX1 missense mutations. We investigated the genetic basis of isolated mineralocorticoid deficiency in a patient who carries a unique missense mutation (W105C) in the amino-terminal region of DAX1. The W105C DAX1 mutation in the proband was present in three asymptomatic hemizygous males, but it was not detected in the general population. Using in vitro studies of DAX1 expression and function in transfected cells, we demonstrate that the mutant DAX1 protein exhibits mild loss of function, whether studied for genes it represses or for genes it activates. Structure-function studies suggest that the W105C and other mutations in the aminoterminus are compensated by the presence of repeated LXXLL motifs that mediate DAX1 interactions with other proteins. We describe the first missense mutation in the aminoterminus of DAX1 and conclude that mutations in this region may be partially compensated by redundant functional domains. Mild DAX1 mutations may be a cause of isolated mineralocorticoid deficiency.

Zebrafish dax1 Is Required for Development of the Interrenal Organ, the Adrenal Cortex Equivalent

Mutations in the human nuclear receptor, DAX1, cause X-linked adrenal hypoplasia congenita (AHC). We report the isolation and characterization of a DAX1 homolog, dax1, in zebrafish. The dax1 cDNA encodes a protein of 264 amino acids, including the conserved carboxy-terminal ligand binding-like motif; but the amino-terminal region lacks the unusual repeats of the DNA binding-like domain in mammals. Genomic sequence analysis indicates that the dax1 gene structure is conserved also. Whole-mount in situ hybridization revealed the onset of dax1 expression in the developing hypothalamus at approximately 26 h post fertilization (hpf). Later, at about 28 hpf, a novel expression domain for dax1 appeared in the trunk. This bilateral dax1-expressing structure was located immediately above the yolk sac, between the otic vesicle and the pronephros. Interestingly, weak and transient expression of dax1 was observed in the interrenal glands (adrenal cortical equivalents) at approximately 31 hpf. This gene was also expressed in the liver after 3 dpf in the zebrafish larvae. Disruption of dax1 function by morpholino oligonucleotides (MO) down-regulated expression of steroidogenic genes, cyp11a and star, and led to severe phenotypes similar to ff1b (SF1) MO-injected embryos. Injection of dax1 MO did not affect ff1b expression, whereas ff1b MO abolished dax1 expression in the interrenal organ. Based on these results, we propose that dax1 is the mammalian DAX1 ortholog, functions downstream of ff1b in the regulatory cascades, and is required for normal development and function of the zebrafish interrenal organ.

Thirteen novel mutations in theNR0B1(DAX1) gene as cause of adrenal hypoplasia congenita

X-linked adrenal hypoplasia congenita (AHC) is a rare developmental disorder associated with primary adrenal insufficiency and combined primary and secondary male hypogonadism. It is caused by deletions or mutations of the NR0B1 (DAX1) gene encoding DAX1, an atypical orphan member of the nuclear receptor superfamily. The continuous molecular genetic analysis of male patients with primary adrenal insufficiency revealed 13 novel mutations within the coding region of the NR0B1 gene which are predicted to inactivate the DAX1 function. These were three nonsense mutations (c.312C>A, p.Cys104X, c.670C>T, p.Gln224X; and c.873G>A, p.Trp291X), five duplications (c.269_270dup, c.421_422dup, c.895_896dup, c.989dup, c.999_1000dup), and five deletions (c.483del, c.745_746del, c.734_740del, c.1092del, and c.1346del). All of the mutations resulted in a premature stop codon destroying the ligand binding domain of the predictive DAX1 protein.

Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development

Mutations of orphan nuclear receptors SF1 and DAX1 each cause adrenal insufficiency and gonadal dysgenesis in humans, although the pathological features are distinct. Because Dax1 antagonizes Sf1-mediated transcription in vitro, we hypothesized that Dax1 deficiency would compensate for allelic loss of Sf1. In studies of the developing testis, expression of the fetal Leydig cell markers Cyp17 and Cyp11a1 was reduced in heterozygous Sf1-deficient mice at E13.5, consistent with dose-dependent effects of Sf1. In Sf1/Dax1 (Sf1 heterozygous and Dax1-deleted) double mutant gonads, the expression of these genes was unexpectedly reduced further, indicating that loss of Dax1 did not compensate for reduced Sf1 activity. The Sertoli cell product Dhh was reduced in Sf1 heterozygotes at E11.5, and it was undetectable in Sf1/Dax1 double mutants, indicating that Sf1 and Dax1 function cooperatively to induce Dhh expression. Similarly, Amh expression was reduced in both Sf1 and Dax1 single mutants at E11.5, and it was not rescued by the Sf1/Dax1 double mutant. By contrast, Sox9 was expressed in single and in double mutants, suggesting that various Sertoli cell genes are differentially sensitive to Sf1 and Dax1 function. Reduced expression of Dhh and Amh was transient, and was largely restored by E12.5. Similarly, there was recovery of fetal Leydig cell markers by E14.5, indicating that loss of Sf1/Dax1 delays but does not preclude fetal Leydig cell development. Thus, although Sf1 and Dax1 function as transcriptional antagonists for many target genes in vitro, they act independently or cooperatively in vivo during male gonadal development.

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients.

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.