molecular mechanisms of dax1 action -...

Molecular Genetics and Metabolism 83 (2004) 60–73

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Molecular mechanisms of DAX1 action

Anita K. Iyera, Edward R.B. McCabea,b,c,d,e,¤

a Department of Human Genetics, David GeVen School of Medicine at UCLA, Los Angeles, CA, USAb Department of Pediatrics, David GeVen School of Medicine at UCLA, Los Angeles, CA, USA

c UCLA Molecular Biology Institute, Los Angeles, CA, USAd Mattel Children’s Hospital at UCLA, Los Angeles, CA, USA

e UCLA Center for Society, The Individual and Genetics, Los Angeles, CA, USA

Received 25 May 2004; received in revised form 12 July 2004; accepted 13 July 2004

Abstract

DAX1 (dosage sensitive sex reversal (DSS), adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1)encoded by the gene NR0B1, is an unusual orphan nuclear receptor that when mutated causes AHC with associated hypogonadotro-pic hypogonadism (HH), and when duplicated causes DSS. DAX1 expression has been shown in all regions of the hypothalamic–pituitary–adrenal–gonadal (HPAG) axis during development and in adult tissues, suggesting a critical role for DAX1 in the normaldevelopment and function of this axis. Steroidogenic factor 1 (SF1, NR5A1) knockout mice show similar developmental defects asAHC and HH patients, but paradoxically, DAX1 is a negative coregulator of SF1 transactivation. The function of DAX1 as anantagonist of SF1 in gonadal development is consistent with the fact that in humans, duplication of the region of the X chromosomecontaining DAX1 causes a similar phenotype as mutations in SF1. However, how disruption of DAX1 leads to adrenal, hypotha-lamic, and pituitary developmental defects similar to SF1 disruption remains to be clariWed. The exact mechanism of DAX1 action ineach of these tissues during adulthood and critical stages of development are not fully understood. Recent evidence suggests abroader functional role for DAX1 as a negative coregulator of estrogen receptor (ER, NR3A1-2), liver receptor homologue-1 (LRH-1, NR5A2), androgen receptor (AR, NR3C4), and progesterone receptor (PR, NR3C3), each by distinct repression mechanisms.DAX1 may have pleiotropic roles in addition to its function as a negative regulator of steroidogenesis during the development andadult function of the HPAG axis. 2004 Elsevier Inc. All rights reserved.

Keywords: DAX1; NR0B1; Adrenal hypoplasia congenita; SF1; AR; ER; PR; LRH-1; Hypothalamic–pituitary–adrenal–gonadal axis; Nuclearreceptor

Introduction

Adrenal hypoplasia congenita (AHC) is an inheriteddisorder characterized by underdevelopment of the adre-nal cortex [1]. It has an estimated frequency of 1:12,500live births and presents in two histological forms: theminiature adult and the cytomegalic forms. The adrenalglands of patients with the miniature adult form have a

* Corresponding author. Fax: +1 310 206 4584.E-mail address: [email protected] (E.R.B. McCabe).

1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ymgme.2004.07.018

permanent zone that has normal structural zonation butis smaller than normal, with a minimal or absent fetal cor-tex. This form is generally associated with abnormal cen-tral nervous system and pituitary development andfunction, and is either sporadic or inherited in an autoso-mal recessive manner. In the cytomegalic form of AHC,associated with NR0B1 mutations, the permanent zone ofthe cortex is absent or nearly absent, and the residualadrenal cortical tissue is structurally disorganized withlarge vacuolated cells that most closely resemble those inthe fetal adrenal cortex, resulting in an adrenal cortex thatlacks normal postnatal zonation and is dysfunctional.

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A.K. Iyer, E.R.B. McCabe / Molecular Genetics and Metabolism 83 (2004) 60–73 61

This form primarily aVects males as it is inherited in anX-linked manner.

Patients with X-linked cytomegalic AHC present withadrenal insuYciency early in childhood, and exhibit salt-wasting, hypotension, hyperpigmentation, hyponatre-mia, hyperkalemia, hypoglycemia, decreased glucocorti-coid and aldosterone production, and increased levels ofadrenocorticotropic hormone (ACTH) [1]. This diseaseis lethal without glucocorticoid and mineralocorticoidreplacement therapy. Some patients with X-linked AHCwho survive beyond childhood develop hypogonadotro-pic hypogonadism (HH), in which a mixed hypothalamicand pituitary defect in the secretion of gonadotropinsprevents normal puberty and requires treatment withtestosterone for sexual maturity [1,2].

X-linked AHC was originally mapped to Xp21 andthe NR0B1 gene was subsequently identiWed by posi-tional cloning as the causative agent of AHC, with muta-tions or deletions in the NR0B1 gene identiWed in AHCpatients [3–5]. AHC can also present as part of an Xp21contiguous gene syndrome along with phenotypes ofglycerol kinase (GK) deWciency, Duchenne musculardystrophy (DMD), and mental retardation due to alarge deletion encompassing NR0B1 and the neighbor-ing GK and DMD loci [1]. AHC patients with defects inthe NR0B1 gene also develop HH, thus identifying themutant DAX1 as the causative agent of both disorders[3–5]. DAX1 has also been shown to be involved in sexdetermination and gonadal development [6,7]. Male tofemale sex reversal in XY individuals with an intact SRYgene was mapped to a 160 kb region of Xp21, whichincludes NR0B1 [6]. Duplication of NR0B1, the genethat encodes DAX1, in these sex reversed patients makesNR0B1 a very strong candidate for the dosage sensitivesex reversal gene (DSS).

NROBI genomic organization and DAX1 protein domainstructure

NR0B1 has a very simple genomic structure with twoexons separated by a single intron [3,5]. Exon 1 is1168 base pairs (bp) in length, Exon 2 is 245 bp, and theintron is 3385 bp [8]. The 1413 nucleotide cDNAencodes a 470 amino acid protein. Hossain et al. [9] haverecently identiWed an alternatively spliced isoform des-ignated as DAX1� that speciWes a protein of 401 aminoacids encoded by Exon 1 and a previously unidentiWedExon 2�. This isoform was shown to be expressed in abroad range of tissues, but elucidation of the signiW-cance and functional role of DAX1� will require furtherinvestigation.

DAX1 has been classiWed as an orphan member ofthe nuclear receptor superfamily [10–12]. Superfamilymembers have a characteristic domain structure consist-ing of subregions A–E (Fig. 1A). The A/B region is the

most evolutionarily divergent and varies in size amongfamily members. It is considered a modulator domainand may contain a hormone independent transactivationdomain (Activation Function 1 or AF-1). The C region istypically the most highly conserved and represents aDNA-binding domain (DBD) containing two zinc Wngersthat allow the receptor to recognize and bind hormoneresponse elements in the promoters of target genes. The Cregion also contains receptor dimerization interfaces. TheD region serves as a hinge between the DBD and theligand-binding domain (LBD) and has been shown toserve as a docking site for corepressors. The E region isthe second most highly conserved LBD, and mediatesligand binding, dimerization, and nuclear localization. Itconsists of 12 helices with an AF-2 hormone-dependenttransactivation domain in helix 12 that undergoes alloste-ric conformational changes in response to ligand binding.

The DAX1 domain structure is rather unusual (Fig.1B) [11,12]. The carboxy-terminal domain (CTD) ishomologous to the LBD of other nuclear receptors andalso contains an AF-2 transactivation domain, butDAX1 lacks the conventional DBD (Region C), modu-lator domain (Region A/B), and hinge region (RegionD). Instead, the DAX1 amino-terminal domain (NTD)has a novel structure consisting of 3.5 alanine/glycinerich repeats of a 65–70 amino acid motif that has noknown homology to any other proteins, with the excep-tion of the related nuclear receptor superfamily member,small heterodimer partner (SHP), encoded by NR0B2[1]. The repeats show 33–70% identity to each other, andalso contain cysteine residues in conserved positions thatcould potentially form zinc Wngers [1]. The C-terminaldomain of DAX1 has strongest amino acid similarity tothe LBD of the testis receptor, COUP-TF, and retinoidX receptor (RXR) [12,13]. However, the similarities withother receptors are unable to provide informationregarding a putative ligand, and to date, no ligand hasbeen identiWed for DAX1. DAX1 is structurally mostsimilar to SHP, in the sense that SHP also lacks the typi-cal nuclear receptor DBD, but has an N-terminaldomain similar, but shorter than DAX1 that containsone 65–70 amino acid repeat, and contains a C-terminalregion homologous to nuclear receptor LBDs [14].

Fig. 1. Comparison of functional domain structure of members of thenuclear receptor superfamily (A) with DAX1 (B).

62 A.K. Iyer, E.R.B. McCabe / Molecular Genetics and Metabolism 83 (2004) 60–73

Molecular mechanisms of DAX1 action

The complex endocrine phenotype caused by DAX1defects is consistent with its pattern of expressionthroughout the hypothalamic–pituitary–adrenal–gonadal (HPAG) axis. DAX1 expression has beenshown in the developing adrenal cortex, gonad, anteriorpituitary, and hypothalamus, and also in adult adrenalcortex, Sertoli and Leydig cells in the testis, theca, granu-losa, and interstitial cells in the ovary, anterior pituitarygonadotropes, and the ventromedial nucleus of thehypothalamus [15–18]. Such a pattern of expression sug-gests that DAX1 is involved in the development andfunction of the HPAG axis.

The DAX1/SF1 functional paradox

Interestingly, the tissue expression proWle of DAX1in development and in the adult organism is similar tothat of SF1/NR5A1, an orphan nuclear receptor that isan essential regulator of HPAG axis diVerentiation andsteroid hormone biosynthesis [19]. The phenotype ofSF1-disrupted mice is similar to the phenotype of AHCpatients. These mice have aplasia of the gonads andadrenals, and also have pituitary and hypothalamicdefects [19]. This observation led to the idea that thesetwo receptors could be involved in a common develop-mental pathway, with the concerted eVort of SF1 andDAX1 necessary for normal development of the HPAGaxis. DAX1 and SF1 were shown to have a colocalizedtissue proWle during embryonic development and alsoin adult tissues [16,17], suggesting a functional interac-tion between DAX1 and SF1, perhaps in a transcrip-tional cascade or through cooperative transcriptionalregulation.

Classifying DAX1 as a member of the nuclear recep-tor superfamily suggested that it may have a bona Wdereceptor function to activate target genes involved inHPAG axis development and function [12,20]. Paradoxi-cally, DAX1 has been shown to function primarily as atranscriptional repressor. It has been proposed thatDAX1 inhibits expression of steroidogenic acute regula-tory protein (StAR) by binding to DNA hairpin struc-tures in the StAR promoter [21]. Most notably, however,DAX1 was shown to act as an inhibitor of SF1-mediatedtranscriptional transactivation [20,22]. SF1 functions asa transcriptional activator of many genes involved in ste-roid hormone biosynthesis in the HPAG axis, andDAX1 appears to act by complexing with and inhibitingthe activator function of SF1 [23]. The function ofDAX1 as an antagonist of SF1 in gonadal developmentis consistent with the fact that duplication of the NR0B1gene causes a similar XY sex reversal phenotype asmutations in SF1, with DAX1 repressing, or SF1 unableto activate, genes involved in sex determination. Therequirement of SF1 for proper development of the hypo-

thalamus, pituitary, and adrenal glands is consistentwith its role as an activator, in which the loss of SF1function leads to decreased transcription of critical tar-get genes. On the other hand, how disruption of DAX1,the antagonist of SF1 function, can lead to adrenal,hypothalamic, and pituitary developmental defectsremains a functional conundrum.

DAX1 expression colocalizes with that of SF1[16,17]; however, the colocalization, as shown by doubleimmunoXuorescence studies, is not absolute [17]. DAX1and SF1 show distinct expression patterns duringgonadal development. DAX1 positive, SF1 negativecells were found in the embryonic mouse testis, postna-tal ovary, and most notably in the developing pituitaryand hypothalamus, which suggests a function for DAX1independent of SF1. The expression of DAX1� in aneven broader range of tissues [9] suggests far greaterpleiotropy.

Immunohistochemical studies in developing andadult tissues have shown DAX1 to have primarily anuclear localization [17,24]. However, studies of DAX1in cultured cell lines and in ES cells show that DAX1 ispresent in both the nucleus and the cytoplasm [25–29].Kawajiri et al. [29] also showed nucleocytoplasmic local-ization in vivo with immunohistochemical analyses ofDAX1 within the developing pituitary in Rathke’spouch, a region where SF1 is not expressed. In addition,Lalli et al. [26] showed an association of DAX1 with pol-ysomes and RNA in the cytoplasm, suggesting a regula-tory role at the posttranscriptional level, though thephysiological consequence of such an association is notclear. This group also demonstrated shuttling of DAX1between the nucleus and cytoplasm. These observationssuggest that DAX1 may function both in the nucleusand the cytoplasm independent of SF1 and possiblyindependent of transcriptional silencing.

DAX1 may have pleiotropic roles with diVerentmolecular functions in the context of developing com-pared with adult tissues, or may have distinct mecha-nisms of action in speciWc cell types and tissues duringdevelopment. How disruption of DAX1 leads to AHCand HH appears to be more complex than previouslyconsidered, and likely involves molecular functionsbeyond its role as a repressor of SF1 action. DAX1 con-tains an AF-2 transactivation domain that is characteris-tic of nuclear receptors, and therefore it is possible thatcertain physiological conditions and/or cellular develop-mental environments could convert this repressor intoan activator. Recent studies provide evidence for a widerfunctional role for DAX1 as a transcriptional repressorof other nuclear receptors expressed in the HPAGaxis. DAX1 can also repress the action of the androgen(AR; NR3C4), estrogen (ER; NR3A1-2) and progester-one receptors (PR; NR3C3), and also liver receptorhomologue-1 (LRH-1; NR5A2) [22,28,30,31]. What isknown about molecular mechanisms of DAX1-mediated


repression of SF1 and its involvement with these othernuclear receptors are described below.

DAX1-mediated repression of SF1 action

DAX1 is a negative regulator of SF1-mediated trans-activation of many genes in the steroid biosyntheticpathway [23]. In addition, DAX1 inhibits the transcrip-tional synergy of SF1 with other associated coregulatorsthroughout the HPAG axis. DAX1 antagonizes thecooperation of SF1 with Wilms Tumor 1 (WT1) andwith GATA-4 in regulation of Müllerian inhibiting sub-stance (MIS) in the gonad [32,33], SF1 with GATA-6 intranscriptional activation of genes in androgen biosyn-thesis in the adrenal [34], SF1 with Egr-1 in the tran-scription of LH� in pituitary gonadotropes [35], and SF1with CREB in the transcription of gonadotropin-induc-ible ovarian transcription factor 1 (GIOT1) in ovariangranulosa cells [36]. DAX1 also negatively regulates SF1and SREBP-1a-mediated transcription of the HDL-Rgene [37].

Mechanisms of repressionTranscriptional silencing of SF1 is thought to involve

direct protein–protein interactions between DAX1 andDNA-bound SF1 via the DAX1 N-terminal domain,with the subsequent recruitment of corepressors to thepromoters of target genes via a DAX1 C-terminal tran-scriptional silencing domain [20,38] (Fig. 2A). It has beensuggested that DAX1 inhibition of SF1 requires multipleSF1-binding sites in the promoters of target genes [39].Transcriptional coactivators typically bind the AF-2domain of activated nuclear receptors through a

Fig. 2. Mechanisms of DAX1-mediated repression of SF1, ER, andLRH-1. (A) DAX1 binds the AF-2 domain of the nuclear receptors viaits LXXLL motifs and recruits corepressor proteins to target gene pro-moters. (B) EVects of intracellular levels of DAX1 and SF1 on tran-scriptional repression. Increased SF1 levels relative to DAX1 favortranscriptional activation (left), and increased levels of DAX1 relativeto SF1 favor transcriptional repression (right).

conserved LXXLL motif called the NR box. DAX1 con-tains three LXXLL motifs, and has been shown to bindthe AF-2 domain of SF1 via these motifs, making DAX1an LXXLL containing-corepressor [22,30]. Mutations ordeletions of the DAX1 LXXLL motifs impair its repres-sor activity against SF1 [22]. SF1 has been shown tointeract with a number of transcriptional coactivators,including CBP/p300, GRIP1, and SRC-1 [40]. SRC-1typically interacts through its LXXLL motifs and hasbeen shown to interact with the AF-2 domain of SF-1[41]. It is possible that DAX1 and the transcriptionalcoactivators compete for a similar binding site on SF1,with DAX1 displacing the coactivators from SF1 in cer-tain conditions to promote transcriptional repression.This coactivator competition mechanism of repressionhas been shown for SHP, the orphan nuclear receptorthat has strongest structural similarity to DAX1 and isalso an LXXLL-containing corepressor [42]. SHP hasbeen shown to interact with the AF-2 domains of its tar-get receptors, including HNF-4�, RXR, and ER, and hasbeen shown to compete with coactivators for binding tothe AF-2 [42,43], which makes this a likely mechanismfor DAX1-mediated repression.

The transcriptional silencing domain of DAX1 hasbeen mapped to a bipartite region in the C-terminalLBD-like (LBD-L) domain which includes two groupsof residues, one at each end of the LBD-L [20,38]. Inter-estingly, all documented AHC missense mutations alsomap to this region [44], and it has been shown that AHCmutations abolish transcriptional silencing activity, sug-gesting that a lack of silencing could be involved in thepathogenesis of AHC. The mechanism of DAX1-medi-ated transcriptional repression involving corepressorrecruitment was prompted by a squelching experimentwhere cotransfection of excess DAX1 was able to relieveDAX1-mediated repression, suggesting that this reliefwas due to the titration of cellular corepressors by theexcess DAX1 [38]. DAX1 was subsequently shown tointeract with the corepressors NCo-R and Alien, but notSMRT [45,46] through portions of the C-terminaldomain that corresponded with transcriptional silencing.However, the interaction between DAX1 and NCo-Rappears to be very weak [27,45], which indicates that theinteraction could involve protein domains diVerent fromthose used in these studies, or could be stabilized byunknown proteins or conditions in speciWc cell typeswhere DAX1 action could be taking place. A recentstudy by Eckey et al. [47] demonstrates a direct interac-tion between Alien and mixed lineage kinase 2 (MLK2)and also shows an enhancement of DAX1-mediatedrepression in the presence of MLK2, suggesting that theinteraction of Alien and MLK2 possibly stabilizes acorepressor complex or recruits additional complexesfor eYcient repression. In addition, DAX1 could notfully relieve its own transcriptional silencing [38], sug-gesting corepressor-independent mechanisms of


transcriptional repression. It is also possible that DAX1could interact with other novel unidentiWed corepressorssimilar to those that interact with other nuclear recep-tors that function as corepressors, such as testis receptorand COUP-TF.

Though DAX1 and SHP share structural similarities,mechanisms of SHP-mediated transcriptional repressionappear to be diVerent from DAX1. Similar to DAX1,transcriptional silencing by SHP not only involves coac-tivator competition mediated by the LXXLL motifs, butalso a C-terminal repression domain. The precise mecha-nism of silencing via this domain is unclear but isthought to involve the recruitment of unidentiWed core-pressors, as SHP has been shown not to interact withNCo-R [48], but has been shown to interact with thecofactor EID-1, which antagonizes the function of CBP/p300 coactivators [49]. DAX1 and SHP both contain a25 and 12 amino acid insertion, respectively, betweenhelix 6 and helix 7 of the LBD-L/transcriptional silenc-ing domain that is not present in other members of thesuperfamily [13,38,50]. Recent studies by Park et al. [50]elucidated the role of this insertion in DAX1 and SHP-mediated transcriptional repression. This insertionappears to have an important role in repression by SHP,but not DAX1, as deletion of this insertion impaired theability of SHP to repress its target orphan receptors, butdid not impair the ability of DAX1 to repress SF1. Theinsertion was necessary for the interaction of SHP withEID-1, as it is thought to provide structural spacing foreYcient interaction. Interestingly, DAX1 did not inter-act with EID-1, providing further evidence for a distinctrepression mechanism for SHP, and also lending cre-dence to the concept of DAX1 recruiting corepressorsinvolved in histone deacetylase function. Though theinsertion did not impair the ability of DAX1 to repressSF1, DAX1 could repress other nuclear receptorsthrough additional mechanisms that may rely on thisinsertion.

Gene dosage and subcellular localizationTranscriptional regulation of SF1 target genes,

including those in steroidogenesis both during develop-ment and in the adult organism appear to be, in part,regulated by intracellular levels of SF1 and DAX1, withthe ratio of these two factors determining whether thetarget genes are activated or repressed (Fig. 2B). If moreSF1 is present in a cell than DAX1, SF1 molecules willoutnumber the SF1–DAX1 complexes and the targetgenes will be activated. Conversely, if more DAX1 ispresent, the SF1–DAX1 complexes will outnumber theSF1 molecules and the target genes will not be activatedand therefore will be repressed. This mechanism alsoalters the balance of coactivators and corepressors thatare recruited to the promoter. This concept applies totarget genes that require the synergy of SF1 with anassociated cofactor, such as WT-1 and GATA-4 in the

transcription of MIS [32,33]. DAX1 does not inhibittranscription of or interact with WT-1 and GATA-4alone, but does inhibit synergy with SF1. It is not clearwhether DAX1 forms a complex with SF1 and thecofactors, or whether DAX1 competes with the cofac-tors for binding to SF1, though evidence from Nachtigalet al. [32] seem to suggest the former.

This type of regulation emphasizes the critical natureof gene dosage in development. For example, reductionof WT1 in Denys Drash syndrome, or increased DAX1levels in DSS seem to explain the observed antagonizedmale development. Both of these situations shift the bal-ance of cofactors to favor the DAX1–SF1 interactionand thus transcriptional repression. An increase inDAX1 expression and a downregulation of SF1 expres-sion via the MAPK pathway has been suggested toexplain the decreased steroidogenesis in response to pro-longed stimulation of ovarian granulosa cells withgonadotropins [51]. In the zona glomerulosa in the adre-nal gland, aldosterone biosynthesis is promoted byangiotensin II in part by downregulating DAX1 expres-sion, and by stimulation of the cAMP signal to mimic anACTH signal that results in downregulation of DAX1expression and upregulation of SF1 expression [52].

The subcellular localizations of DAX1 in cultured celllines and in ES cells have been shown by many groups tobe nuclear and cytoplasmic [25–29], with evidence thatDAX1 can shuttle between the compartments [26].Coexpression of DAX1 and SF1 in cultured cells resultsin DAX1 shifting completely to the nucleus [29]. A simi-lar phenomenon has been observed for SHP andHNF4� [53]. This nuclear localization with SF1 appearsto require direct interaction with SF1 as the LXXLLmotifs in isolation were able to promote nuclear locali-zation in the presence of SF1, and mutation of the thesemotifs impaired this process. This phenomenon alsoappears to require the AF-2 domain of DAX1, as muta-tion of these residues also lowered the frequency ofnuclear localization in the presence of SF1, implicatingthe AF-2 in nuclear localization processes. This observa-tion appears to indicate that in order for DAX1 to carryout its function as a repressor of SF1, it needs to localizeto the nucleus. The mechanism of nuclear import is notclear. DAX1 may shuttle between the cytoplasm andnucleus as previously described [26], and may be retainedin the nucleus by SF1. Alternatively, DAX1 may bindSF1 in the cytoplasm followed by translocation into thenucleus.

Once in the nucleus, DAX1 can repress SF1 asdescribed above. A recent confocal imaging study inKGN granulosa cells describes how the protein kinase A(PKA) pathway stimulates SF1 transactivation andinvolves DAX1 [54]. In the absence of stimulation withforskolin, the nuclear distribution of SF1 is very diVuse,but in the presence of stimulation, SF1 is rearranged intodistinct foci, which are indicative of sites of transcrip-


tionally active nuclear receptors. In the presence of stim-ulation, these foci also colocalize with transcriptionalcoactivators GCN5 and TRRAP suggestive of a func-tional interaction of SF1 and the recruitment of a coacti-vator complex. DAX1 and SF1, when coexpressed in theabsence of stimulation, have a speckled/dotted distribu-tion with no diVuse background, and colocalize com-pletely in the nucleus. This is consistent with the resultsof Kawajiri et al. [29]. When stimulated, the speckled/dotted distribution remains, but DAX1 and SF1 appearto separate from each other. The activation of the PKApathway may weaken the DAX1–SF1 interaction. Fluo-rescence recovery after photobleaching (FRAP) studiesshow that DAX1 immobilizes SF1 and anchors it to agiven site in the nucleus. These results suggest thatDAX1 may be capable of interacting with the nuclearmatrix. This immobilization is relieved in the presence ofa PKA signal. This phenomenon has been observed forER in the presence of antagonist [55]. The simultaneousdistribution between DAX1, SF1, and the GCN5/TRRAP complex was not studied. These results arelikely not an artifact of overexpression as transientlytransfected cells that expressed amounts of protein closeto physiological levels were selected for imaging. Theseobservations suggest that coexpression of DAX1 andSF1 causes DAX1 to localize fully to the nucleus whereit can repress SF1 transactivation through direct proteininteractions and the recruitment of corepressors and alsoby immobilizing SF1. Activation of pathways that stim-ulate SF1 action cause DAX1 and SF1 to dissociate,promoting SF1 interactions with coactivators, and acti-vation of SF1 target genes.

DAX1-mediated repression of ER and LRH-1 action

DAX1 has been shown to repress transcriptionaltransactivation of ER and LRH-1 [22,30,56]. Thisrepression is thought to occur through a similar mecha-nism as the repression of SF1 action (Fig. 2A). TheLXXLL motifs of DAX1 have been shown to interactwith the AF-2 domain of ER [22,30], and these motifsappear to be required for interaction with LRH-1 [22].DAX1 interacts with both ER� and ER�. The interac-tion with ER� is ligand dependent, whereas the interac-tion with ER� appears to be ligand independent. DAX1does not interfere with dimerization or DNA binding ofER, but forms a ternary complex on ER response ele-ments. DAX1 is expressed in many estrogen target tis-sues, with a suggested coexpression in testis and ovaryduring development, making the ER a likely physiologi-cal target in the developing and adult reproductive sys-tem [30]. It is possible that DAX1 is involved in thecontrol of the physiological response to estrogen in theadult organism, or may repress ER target genes duringcritical times and cell types in development. SpeciWc tar-get genes have not yet been identiWed, and further

studies of colocalization of DAX1 and ER expressionduring development and adult reproductive processesneed to be conducted in order to understand fully thephysiological role of this interaction.

LRH-1, in addition to expression in pancreas, liver,and intestine and its function in bile acid metabolism, isalso expressed in the ovary, testis, and adrenal. LRH-1 isa monomeric orphan nuclear receptor that can recognizethe same response elements as SF1 and can substitutefor SF1 in the activation of steroidogenic enzymes, sug-gesting a function in the regulation of steroidogenesis[57]. LRH-1 has been shown to upregulate expression ofaromatase and 3�-hydroxysteroid dehydrogenase type II(3�HSD2) in the ovary [56,58]. SF1 and LRH-1 areexpressed in diVerent sites in the ovary at diVerent timesduring the menstrual cycle and during pregnancy [58].SpeciWcally, during the conversion of a mature ovarianfollicle to the corpus luteum following the LH surge,there is a shift to progesterone biosynthesis. LRH-1 isthought to be involved in this regulation of steroidogen-esis as SF1 expression is downregulated in the corpusluteum [56,58]. DAX1 and LRH-1 both appear to beexpressed in granulosa cells [58,59] and DAX1 wasshown to inhibit LRH-1-mediated transcription of3�HSD2 in granulosa cells [56]. It is possible that DAX1can repress other LRH-1 target genes in the ovary. TheDAX1 interaction with LRH-1 appears to be noticeablystronger than the interaction with SF1 as seen in an invitro binding assay and also a mammalian two-hybridassay [22]. These results may be indicative of Wne tuningof a regulatory mechanism to allow for a critical amountof repression with regard to SF1 to prevent gain of func-tion and loss of function phenotypes as observed inAHC and DSS. Perhaps the SF1/DAX1 interaction istransient in the context of physiological and develop-mental function, occurring for very speciWc lengths oftime that could alter an overall physiological responseshould the interaction be stronger and more stable. SF1may be in a conformation to discourage a strong interac-tion compared to LRH-1. It is also possible that thereare cell-speciWc coregulators in cell types within whichSF1 and DAX1 would exert their actions to stabilize theinteraction.

Since DAX1 appears to interact with ER and LRH-1through the LXXLL motifs and repress ER and LRH-1by similar mechanisms, it can be expected that coexpres-sion of DAX1 with either ER or LRH-1 will causeDAX1 to localize to the nucleus. This has been shown forER� [29] and thus seems highly likely for LRH-1. It isalso possible that DAX1 could immobilize these recep-tors in the nucleus, but this remains to be determined.

Moore et al. [60] have recently shown an interactionbetween an LXXLL motif of DAX1 and the thyroidreceptor � (TR�) LBD through an in vitro bindingassay. The functional implications of this physical inter-action require further investigation, but it is possible


that DAX1 could repress TR�, and could do so via arepression mechanism similar to that of SF1, ER, andLRH-1.

DAX1-mediated repression of AR and PR action

DAX1 has been shown to interact with and functionas a negative coregulator of AR [28,31,61] and PR [31],suggesting a role for DAX1 in the normal developmentand adult function of the reproductive system, but themechanism of DAX1-mediated repression of thesereceptors appears to be diVerent from that of SF1, ER,and LRH-1 (Figs. 3A and B).

Unliganded AR is present in the cytoplasm, but istransported to the nucleus upon ligand binding in orderto carry out its function. Holter et al. [28] showed thatDAX1 inhibits AR transactivation by interfering withthe dimerization and interdomain communication of theAR, and further showed through transient transfectionsand intracellular localization studies that cytoplasmicDAX1 tethered AR in the cytoplasm in the presence ofligand, preventing its translocation into the nucleus. Thistethering occurs independently of ligand and is betweenthe AR LBD and the N-terminus of DAX1. NuclearDAX1 colocalized with AR in the nucleus in the pres-ence of ligand, suggesting not only that the liganded-ARcan enter the nucleus properly, but that DAX1 may alsopossess a mechanism for repressing AR in the nucleus.

Agoulnik et al. [31] further investigated DAX1 repres-sion of AR and provided evidence of a nuclear mecha-nism of transcriptional repression. DAX1 was shown torepress AR activity in the presence of an agonist and anantagonist. An in vivo interaction of endogenous DAX1with AR was also conWrmed in an agonist-treated pros-tate cancer cell line, suggesting a physiologically relevant

Fig. 3. Other mechanisms of DAX1-mediated repression. (A) NuclearDAX1 inhibits AR- and PR-mediated transactivation through inter-fering with functional dimerization of the nuclear receptors. (B) Cyto-plasmic DAX1 inhibits AR transactivation by preventing itstranslocation into the nucleus. (C) DAX1 may inhibit transcription bybinding to hairpin elements in the promoters of target genes.

interaction. The repression of AR by DAX1 does notappear to involve histone deacetylases, as there was norelief of repression in the presence of a histone deacety-lase inhibitor. DAX1-mediated silencing was reversed bythe addition of excess coactivator. DAX1 does not inter-fere with AR interaction with SRC-1, rather it interfereswith the dimerization and interdomain communicationof AR, similar to the results of Holter et al. [28]. DAX1appears to interfere with the formation of functionalcoactivator complexes by binding to sites diVerent thancoactivators like SRC-1 while also preventing the forma-tion of transcriptionally active AR dimers. Repression ofAR by DAX1 involves both the N- and C-terminaldomains of DAX1, consistent with the previous observa-tion that the DAX1–AR interaction involves bothhalves of DAX1 [28].

Whereas Holter et al. [28] showed that cytoplasmictethering involves the AR LBD, Agoulnik et al. [31]show that DAX1 is capable of repressing a truncatedAR lacking the LBD. Perhaps this provides evidence fortwo distinct mechanisms by which DAX1 can repressAR depending on its localization. Despite the lack ofcytoplasmic tethering in the truncated AR lacking theLBD, DAX1 can still repress AR via the AR N-terminusin the nucleus [31]. DAX1 does not interfere with bind-ing of AR to DNA, which seems to conXict with the con-cept of DAX1 tethering AR in the cytoplasm and thuspreventing its binding to DNA. However, the localiza-tion of DAX1 varies depending on culture conditions,and the number of cells in a given population with cyto-plasmic versus nuclear DAX1 varies. DAX1 is present inboth the nucleus and the cytoplasm regardless of thepresence of AR, so it is feasible for both cytoplasmic andnuclear mechanisms to be valid. While these results seemto suggest that DAX1 may possess two mechanisms ofrepressing AR, depending on whether DAX1 is presentin the cytoplasm or in the nucleus, it is also possible thatthe cytoplasmic tethering mechanism is an artifact ofoverexpression. It has been suggested that DAX1 mayinteract with AR through the LXXLL motifs for cyto-plasmic retention [28]; however this logic conXicts withthe observation that an interaction through the LXXLLmotifs with SF1 and ER causes translocation of DAX1and the nuclear receptor into the nucleus. The speciWcresidues involved in the DAX1–AR interaction have notbeen studied. These results indicate a repression mecha-nism distinct from that of SF1 and ER.

Agoulnik et al. [31] also studied DAX1 repression ofthe PR. DAX1 repressed agonist-, but not antagonist-bound PR activity. DAX1 interacted with PR-A andPR-B in vivo in an agonist-treated breast cancer cell line.DAX1 did not interfere with the interaction of PR andSRC-1, indicating that DAX1 binds to diVerent sites onPR than SRC-1. DAX1 did, however, interfere with PRhomodimerization. A functional interaction takes placebetween the PR LBD and the N-terminus of DAX1,


though the C-terminus may also be involved as fulllength DAX1 was a more potent repressor of PR. It ispossible that this interaction may involve the LXXLLmotifs of DAX1 and the AF-2 of PR, but this seemsunlikely in light of the lack of competition for binding ofPR to SRC-1. DAX1 did not interfere with the bindingof PR to DNA. These results suggest a mechanism diVer-ent from that of SF1 and ER, but also slightly diVerentfrom AR with regard to protein domains involved.DAX1 may interfere with the speciWc mechanisms oftranscriptional activation of a given receptor.

DiVerences in mechanisms of DAX1-mediated repression

DAX1 has been shown to repress transactivation ofother nuclear receptors by both nuclear and cytoplasmicmechanisms. For example, DAX1 functions as a tran-scriptional repressor of SF1 by heterodimerization withSF1 in the nucleus, and thereby interfering with the pro-tein–protein interactions between SF1 and the heterodi-meric partners with which SF1 cooperates intranscriptional activation [32–37] (Fig. 2). As anotherexample, however, DAX1-mediated repression of ARaction appears to involve cytoplasmic and nuclear mech-anisms (Figs. 3A and B). DAX1 has been observed totether AR in the cytoplasm, even in the presence ofligand, and to prevent nuclear translocation of AR [28].DAX1 also appears to repress AR in the nucleus [28,31].

Transcriptional repression by DAX1 displays speci-Wcity for certain nuclear receptors. DAX1 does notrepress the action of HNF4�, ROR�, VDR, and p53[22,31]. Thus, molecular mechanisms of DAX1 actionappear to be restricted in terms of target receptor

speciWcity, but DAX1 repression of ER, LRH-1, AR,PR, and possibly TR�, suggests that DAX1 may havebroader functional roles in HPAG development andadult function than previously considered (Fig. 4).

It is informative to speculate on the origin and natureof DAX1 repression. DAX1 serves as a transcriptionalsilencer for a broad range of nuclear receptors and yetDAX1 is restricted in its target speciWcity. DAX1 evi-dences nuclear or cytoplasmic mechanisms for repres-sion of its target receptors’ transactivation functions,and for some targets DAX1 may function, perhaps bydistinct and independent mechanisms, in both cellularcompartments. Therefore, the breadth of receptors withwhich DAX1 interacts could be a consequence of diversemolecular mechanisms acquired in the course of its evo-lution [62]. Alternatively, these mechanistic diVerencescould reXect variations in the experimental designsemployed by various investigators. Supra-physiologicallevels of DAX1 expression, for example in transienttransfection assays [31], could lead to spurious interac-tions with nuclear receptors that would not be targetedunder normal conditions or would interact through adiVerent mechanism in the presence of physiologicalconcentrations of DAX1 protein.

Role of DAX1 in HPAG axis development and adultfunction

Though DAX1 expression has been shown in allregions of the HPAG axis during development and inadult tissues, and coregulator and posttranscriptionalfunctions have been shown in vitro, the exact mechanisms

Fig. 4. Role of DAX1 and mechanisms of action. DAX1 may have pleiotropic roles in early embryonic development, bone cell development, andHPAG axis development and adult function, and may repress the action of various nuclear receptors depending on cellular and physiological con-text. Solid lines show deWnitive stimulatory and inhibitory relationships. Dotted lines indicate relationships where tissue coexpression has beenshown, but functional activity remains to be determined. See text for details.


of DAX1 action in each of these tissues, and how disrup-tions of these mechanisms cause AHC and HH are notfully understood. The following sections outline possibleroles for DAX1 in HPAG axis development and func-tion. Combined evidence from in vitro and in vivo mod-els suggest that DAX1 may have pleiotropic roles, withcomplex and distinct functions in development and adultfunction throughout the HPAG axis (Fig. 4).

DAX1 as an inhibitor of steroidogenesis

The observation of DAX1 functioning as a negativecoregulator of SF1 transactivation suggested that DAX1may have a role as an inhibitor of steroidogenesis in theadrenal gland and the gonads [20]. DAX1 blocks overallsteroid production in forskolin-stimulated Y-1 adreno-cortical cells at multiple levels of the steroid biosyntheticpathway without an eVect on the cAMP signaling path-way [63]. DAX1 has been shown to inhibit SF1-mediatedtransactivation of many steroidogenic enzymes [23].

GonadsThe transcriptional inhibition of speciWc steroido-

genic enzymes by DAX1 seems to depend on the sex ofthe organism, the organ, and physiological context.DAX1 was shown to inhibit transcription of LRH-1-mediated transcription of 3�HSD2 in a granulosa cellline, implicating DAX1 in the regulation of ovarian ste-roidogenesis during the menstrual cycle [56]. In the Ley-dig cells of DAX1-deWcient mice, aromatase isspeciWcally upregulated at the mRNA and protein level,while StAR, P450 side chain cleavage enzyme (P450scc),17�-hydroxylase (Cyp17), 3�HSD2, and 17�HSD3 werenot [64]. These results suggested that increased estrogenproduction could explain the infertility in these mice. Inaddition, aromatase expression was not upregulated inthe ovaries of female DAX1-deWcient mice [64]. Theobservation of the lack of DAX1 repression for othersteroidogenic enzymes appears to conXict with observa-tions in cultured cell lines that DAX1 inhibits StAR and3�HSD expression [21,56,63]. These diVerences couldrepresent bona Wde physiological tissue speciWcity, orthey could also be artifacts of overexpression of DAX1in transfected cultured cell lines or due to the possiblepresence of a hypomorphic allele in the DAX1-deWcientmice [65].

Adrenal glandDAX1 has been shown to repress SF1-mediated tran-

scription of genes involved in adrenal androgen biosyn-thesis (StAR, P450scc, and Cyp17) in cultured cells [34].DAX1 has also been shown to repress aldosterone bio-synthesis in bovine adrenal zona glomerulosa cells thatcan be relieved by angiotensin II [52]. DAX1-deWcientmice appear to have increased levels of 21-hydroxylase(Cyp21) and the ACTH receptor in the adrenal glands in

response to stress compared with wildtype [66]. DAX1represses SF1-mediated transcription of Cyp17 in ahuman adrenocortical cell line [39]. These results portrayDAX1 as a negative regulator of steroidogenesis in theadrenal gland.

Disruption of normal DAX1 function in the adrenal glandand pathogenesis of AHC

Patients with the cytomegalic form of AHC presentwith adrenal insuYciency due to low levels of steroidproduction [1]. Although there is a great deal of evidencefor DAX1 functioning as a negative regulator of steroi-dogenesis, this appears to be inconsistent with thepatient phenotype. These AHC patients have small, non-functioning adrenal glands, and thus the adrenal insuY-ciency is due to a developmental defect rather than adefect in postnatal function. These observations indicatea critical role for DAX1 in the normal development ofthe adrenal cortex.

DAX1 is expressed throughout adrenal cortical devel-opment, but its function in adrenal development ispoorly understood. The phenotype of DAX1-deWcientmice [65] was quite diVerent from that of AHC patients.These mice had fully developed adrenal glands withfunctional zonation and normal serum corticosteronelevels, but the X zone (roughly equivalent to the humanfetal zone) failed to regress at the normal time ofpuberty. These observations suggest that DAX1 isinvolved in fetal adrenal degeneration but is not neces-sary for formation of the deWnitive zone or for steroido-genesis. The diVerences between AHC patients and themice may be due to species diVerences or the disruptedDAX1 gene acting as a hypomorphic allele.

DAX1 may be involved in adrenocortical growth andmay interact with SF1 in an antagonistic manner duringdevelopment. This is supported by the fact that SF1haploinsuYcient mice display adrenal glands that aresmaller than wildtype, while compound DAX1-deWcient,SF1 haploinsuYcient mice do not display the growthdefect [67]. DAX1-deWcient mice contain a unique steroi-dogenically active cell layer contiguous with the X zonethat is rescued by SF1 haploinsuYciency, suggesting thatDAX1 is involved in the regression of this particular celllayer [68]. The signiWcance of steroid production duringdevelopment is unclear [23], but it is possible that DAX1could inhibit steroid production at critical periods indevelopment. The possibility exists that DAX1 may alsoplay an unknown function in the context of develop-ment, possibly as a transcriptional activator, or throughmodulating the actions of other nuclear receptors, thatcould explain the AHC phenotype.

The function of DAX1 as a negative regulator of ste-roidogenesis may have a more signiWcant role in theadult adrenal. Adrenal steroid production is tightly regu-lated, as steroids are secreted on demand in response to


physiological stimuli with secretion regulated at the levelof biosynthesis. DAX1 is likely involved in the control ofadrenal steroid production, possibly repressing steroido-genic genes when steroid synthesis is not required, keep-ing steroid production in control when synthesis isrequired, and also regulating the transcription of speciWcbiosynthetic genes depending on the steroid beingsecreted. DAX1 may be involved in negatively regulatingthe physiological response to stress. DAX1-deWcientmice have increased corticosterone production and alsoincreased adrenal responsiveness following restraintstress and also after treatment with exogenous ACTH[66].

DAX1 mutations found in AHC patients result inreduced transcriptional silencing activity which couldcontribute to AHC pathogenesis [20,38,45]. It was previ-ously thought that these mutations caused a direct tran-scriptional eVect with a reduced capacity for DAX1mutants to interact with its target nuclear receptors andcorepressor proteins [45,46]. However, it has been shownthat these DAX1 mutants localize predominantly to thecytoplasm presumably due to protein misfolding [27,69].This observation explains the impaired repressor activityof these mutants, since mutant DAX1 is thus unable toenter the nucleus to interact with nuclear receptors andrecruit corepressors for full repressor function.

Disruption of normal DAX1 function and pathogenesis ofsex determination/diVerentiation abnormalities

Sex determinationSince patients with a duplication of a 160 kb region of

Xp21 that contains DAX1 are XY sex reversed females,DAX1 has been thought to function as an anti-testis orpro-ovarian factor during gonadal development [6].Overexpression of DAX1 in mice in the presence of aweakened SRY allele caused XY sex reversal, suggestingthat DAX1 is acting as an “anti-testis” gene that antago-nizes SRY and male development [70]. DAX1 has beenshown to repress transcription of MIS by antagonizingtranscriptional synergy of SF1 with WT1 or GATA-4[32,33]. MIS repression prevents regression of the Mülle-rian ducts. These observations are consistent with therepressor function of DAX1 and also its putative role inDSS.

Testicular development and functionStudies using the DAX1-deWcient mouse model pro-

vide evidence that DAX1 is necessary for proper testicu-lar development and function, suggesting a role beyondthat of simply an “anti-testis” factor. The DAX1-deW-cient mice are hypogonadal and infertile, and have testic-ular and spermatogenic defects with loss of germ cellsand degeneration of the seminiferous epithelium, con-trary to the hypothalamic or pituitary defect seen in HHpatients [65]. Meeks et al. [71] recently showed that

DAX1-deWcient XY mice in the presence of the weak-ened SRY allele develop as phenotypic females, suggest-ing that DAX1 is required for testis determination,though humans with mutations in DAX1 develop asmales. Subsequent studies have shown that DAX1 isinvolved in testis cord organization during development[72]. Sertoli and Leydig cell-speciWc expression of DAX1in the DAX1-deWcient mice appear to rescue fertility andsperm production, suggesting a role of DAX1 in sper-matogenesis. However, expression of DAX1 in each ofthese cell types individually was not suYcient to rescuethe testicular pathology, suggesting that DAX1 functionin both Sertoli and Leydig cells, in addition to othersomatic cell types is necessary for proper testiculardevelopment [73,74]. It has been proposed that embry-onic Leydig cell development involves cooperativeactions of DAX1 and SF1, as DAX1-deWcient/SF1haploinsuYcient mice show no improvement in testicu-lar pathology [75]. DAX1 is thought to repress aroma-tase expression, and thus estrogen production in thetestis, as estrogen levels were close to normal in thetransgenic rescue mice [73,74], making aromatase aphysiological target for DAX1. Increased estrogen pro-duction in the infertile DAX1-deWcient mice suggestsincreased ER action, indicating that DAX1 repression ofER action may have functional signiWcance in the testisand adult reproductive function. LRH-1 expression hasalso been detected in Leydig cells, and aromatase expres-sion was stimulated by LRH-1 in a Leydig cell line [76].It is possible that DAX1 could repress LRH-1 action inthe testis. MIS expression is also present in adult testes[77], and DAX1 may be involved in regulating its expres-sion during puberty and adulthood. The demonstrationthat expression of DAX1 in rat Sertoli cells is hormon-ally regulated by FSH and also during postnatal devel-opment further suggest a role for DAX1 inspermatogenic cell development [78]. The testicular roleof DAX1 in spermatogenesis is consistent with theobservation that the infertility in some AHC/HHpatients is due to spermatogenic defects in additiondefects in gonadotropin secretion. These patients showazoospermia with resistance to gonadotropin treatment[79].

Ovarian development and functionThe function of DAX1 in ovarian development is not

clear. DAX1 expression is detected in the developingovary at various stages of embryonic development [17],but homozygous DAX1-deWcient female mice, except fora slight ovarian follicular defect, were otherwise normaland fertile, implying that DAX1 is not required for ovar-ian development [65].

DAX1 may have a role in the adult ovary as a regula-tor of steroidogenesis during the menstrual cycle. DAX1expression varies among follicles [17,24], and thereforeDAX1 likely plays a role in regulating gene expression in


various ovarian cell types during speciWc stages of follic-ular development. Expression is detected strongly in thegranulosa cells with more varied expression in the stro-mal and thecal cells from the primordial follicular stageto the corpus luteum. Since the cyclic changes in sex ste-roid production in the ovary are due to spatiotemporallocalization of key enzymes involved in steroid biosyn-thesis, DAX1 could be involved in the regulation ofthese enzymes in the various cell types and follicularstages as an inhibitor of SF1- or LRH-1-mediated tran-scription [59]. It has been postulated that DAX1 couldinhibit SF1-mediated transcription of steroidogenicenzymes in earlier stages of follicular development [59].DAX1 has also been shown to inhibit LRH-1-mediatedtranscription of 3�HSD2, which likely takes place in thecorpus luteum [56], and also to inhibit transcription ofaromatase in a granulosa cell line [80]. The observationthat DAX1-deWcient female mice have normal levels ofovarian aromatase [64] may reXect the transient natureof induction of aromatase expression during folliculardevelopment, or that aromatase may not be a physio-logic target of DAX1 in the ovary.

Disruption of DAX1 function in the hypothalamus andpituitary glands and pathogenesis of HH

The role of DAX1 in the hypothalamus and pituitaryglands has not been studied extensively, although HHappears to be a mixed defect of hypothalamic and pitui-tary function [2]. AHC patients also develop HH, andthey have low levels of gonadotropins. However, DAX1has been shown to inhibit SF1-mediated transcription ofLH� [35], and a lack of DAX1 would presumably causean upregulation of gonadotropins. The HH is thus likelydue to a developmental defect of the hypothalamus andpituitary glands, suggesting a role for DAX1 in properdevelopment of these organs. In contrast to AHC/HHpatients, DAX1-deWcient mice show pituitary glandsidentical in size to wildtype and show no deWciencies ingonadotropin production, possibly reXecting speciesdiVerences or residual DAX1 function in these mice [65].DAX1 could also have a role in adult pituitary gonado-tropes as a negative regulator of gonadotropin secretion.However, the mechanisms of DAX1 action in the hypo-thalamus and pituitary during development and adultfunction remain unknown.

Other functional roles for DAX1

Early embryonic developmentDAX1 expression has been detected in totipotent

murine embryonic stem (ES) cells and also in preimplan-tation embryos with reduced expression upon diVerenti-ation into individual germ layer fates [25]. Thisobservation of expression in undiVerentiated cells sug-gests a role for DAX1 in development much earlier than

previously considered, prior to the development of thesteroidogenic axis. DAX1 likely has a critical role inearly embryonic development since complete deletion ofDAX1 in ES cells is lethal [65]. These results suggest afunction for DAX1 independent of steroidogenesis, butthe exact mechanism of DAX1 action in early embryonicdevelopment remains to be determined.

Bone cell developmentDAX1 expression was found to increase with osteo-

blast cell diVerentiation in a transcriptional proWlingstudy [81]. As estrogen and the ER are known to beinvolved in bone cell development and homeostasis, thepresence of DAX1 in bone cells suggests a physiologicalrole for DAX1-mediated repression of ER. DAX1involvement in bone cell development could provide anexplanation for skeletal abnormalities and pathologicfractures observed in two brothers with complex glycerolkinase deWciency [82].

DAX1 and cancer

DAX1 has been proposed to be involved in the devel-opment of cancers of a variety of tissues, which includeadrenal and pituitary adenomas, breast and ovarian car-cinoma, and prostate cancer. High levels of DAX1expression are associated with a non-functional pheno-type in adrenal adenomas [83]. In addition, low levels ofDAX1 are detected in cortisol-producing tumors caus-ing Cushing Syndrome, and high levels of DAX1 aredetected in deoxycorticosterone-producing adenomas,suggesting that DAX1 is involved in the regulation ofsteroidogenesis of adrenal tumors [84]. DAX1 expres-sion has been detected in non-functioning gonadotropicpituitary adenomas along with SF1 in some instances[85,86], and has also been demonstrated in a breast can-cer cell line and human breast carcinomas [31,87]. DAX1immunoreactivity in ovarian carcinoma is thought to beassociated with poor clinical prognosis [88]. DAX1expression is strongly reduced in benign prostate hyper-plasia compared to normal prostate, suggesting that alack of repression of AR can account for the elevatedAR activity in these tumors [28,31].

Conclusions

DAX1 was previously considered to be only a repres-sor of SF1-mediated transcription of genes involved insteroidogenesis and male development. However, theconundrum of how disruption of both these orphannuclear receptors leads to similar phenotypes suggestsadditional complexity in DAX1 function. There isincreasing evidence for a role of DAX1 independentfrom SF1 as a repressor of ER, AR, PR, and LRH-1action. These functional interactions may provide


insight into mechanisms of DAX1 action during develop-ment. DAX1 may have a pleiotropic role as a tissue orcell-speciWc transcriptional coregulator of ER, AR, PR,LRH-1, and SF1 target genes throughout HPAG axisdevelopment (Fig. 4), many of which remain to be identi-Wed. The possibility exists that DAX1 may have a yetundiscovered function. Many questions remain unan-swered, but recent advances into possible roles for DAX1in HPAG axis development and function conWrm thecomplexity in the molecular mechanisms of DAX1 action.

Acknowledgments

This research was supported by USPHS NationalResearch Service Award GM07104 (A.K.I.) and R01HD39322 (E.R.B.M.C.).

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