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Human Molecular Genetics, 2003, Vol. 12, No. 9 1063-1072
DOI: 10.1093/hmg/ddg108
© 2003 Oxford University Press

Structure–function analysis reveals the molecular determinants of the impaired biological function of DAX-1 mutants in AHC patients

Sylvia G. Lehmann, Jean-Marie Wurtz, Jean-Paul Renaud, Paolo Sassone-Corsi and Enzo Lalli*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, BP 163, 67404 Illkirch, Strasbourg, France

Received January 23, 2003; Accepted February 21, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the DAX-1 (NR0B1) gene cause the X-linked form of adrenal hypoplasia congenita (AHC), which is constantly found associated with hypogonadotropic hypogonadism (HHG). DAX-1 encodes an atypical orphan member of the nuclear hormone receptor superfamily. DAX-1 acts at multiple levels to repress the expression of genes involved in steroid hormone metabolism through a potent transcriptional repression domain present in its C-terminus, which is similar to the nuclear receptors' ligand binding domain. All DAX-1 mutations causing AHC/HHG alter the protein C-terminal domain, impairing its nuclear localization and, consequently, its transcriptional repression activity. Here we show that DAX-1 AHC mutants have a misfolded conformation, which correlates with their cytoplasmic retention. Extensive structure–function analysis reveals that the chemical nature of amino acid residues at positions interested by AHC mutations and critical determinants in helix 12 affect DAX-1 nuclear localization and transcriptional silencing. Surprisingly, mutations in a conserved putative corepressor binding surface have a negative effect upon DAX-1 transcriptional repression only when they also affect protein expression levels. These data suggest that a folding defect underlies the impaired function of DAX-1 missense mutants found in AHC/HHG patients and that interactions with transcriptional cofactors different from known corepressors mediate DAX-1 silencing properties.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In primates the fetal zone occupies most of the adrenal cortex during fetal life, being present starting from weeks 8–10 of gestation and regressing within the first 3 months after birth. Anatomic and functional zonation of the adrenal cortex into mineralocorticoid-producing glomerulosa and glucocorticoid-producing fasciculata-reticularis then ensues. These zones together form the permanent zone of the adrenal cortex. Adrenal hypoplasia congenita (AHC) is an inherited disorder of the adrenal cortex usually, but not always, manifesting clinically as an acute form of adrenal insufficiency in the infant (1). The OMIM database reports two genetically and histologically distinct forms of AHC (www.ncbi.nlm.nih.gov/htbin-post/omim/dispmim?300200). In the X-linked, cytomegalic form of AHC the disorganized adrenal cortex shows poor differentiation of the three cortical zones. Scattered clumps of large, eosinophilic and vacuolated cells similar to fetal zone cells are the only remaining adrenal cortical tissue. A constant feature of the X-linked form of AHC is the association with hypogonadotropic hypogonadism (HHG), a syndrome caused by a lack of pituitary FSH and LH production. In the other form of AHC both the fetal and permanent cortex are absent. This has been defined as the ‘miniature adult’ type of AHC because the small adrenal cortex consists almost exclusively of a very reduced permanent cortex. This type of AHC occurs either sporadically or as an autosomal-recessive condition and may occur alone or accompanied by anomalies of the brain and pituitary gland, as in anencephaly. If left untreated, AHC is a lethal disorder essentially because of dehydration (water loss) and electrolyte imbalance due to mineralocorticoid deficit.

Mutations in the DAX-1 (NR0B1) gene, situated in Xp21, are the cause of the X-linked form of AHC (2,3). In addition, DAX-1 lies inside the critical region whose duplications produce dosage-sensitive sex reversal in XY individuals (4). DAX-1 encodes an atypical member of the nuclear hormone receptor family (2). Its C-terminal domain has high similarity with the ligand-binding domain of other members of this family. On the basis of this similarity, it has been possible to build a model of the DAX-1 C-terminal domain (5,6). However, no ligand has been identified for DAX-1 up to date, and for this reason it is classified in the group of the orphan receptors. Conversely, the DAX-1 N-terminal domain is highly divergent from other nuclear hormonereceptors, being composed of three repetitions of an about 70-amino acid motif which has no homologues in the human genome (5). DAX-1 expression is restricted to the adrenal cortex and to other tissues producing steroid hormones (testis, ovary) and involved in reproductive functions (pituitary gonadotropes, hypothalamic VMH) (7,8). This expression pattern explains the clinical characteristics of the disorder caused by DAX-1 mutations and suggests that this gene also plays a direct role in gonadal functions. Divergence in the biological function of the DAX-1 gene in humans compared to rodents is suggested by the absence of AHC and HHG in mice null for its Ahch homologue (9).

The DAX-1 gene product acts at multiple levels to repress the expression of genes involved in steroid hormone metabolism through a potent transcriptional repression domain present in its C-terminus (5,10–13). DAX-1 can be recruited to gene promoters by direct binding of stem-loop DNA sequences (10) and by interaction with transcriptional activators belonging to the nuclear receptor family (13) and exerts silencing through interaction with corepressors (5). Recent data suggest that DAX-1 plays additional roles in post-transcriptional regulations, being associated to actively translating polyribosomes as a complex with mRNA (14). Importantly, all DAX-1 mutations identified in patients with AHC alter the protein C-terminus and invariably impair its transcriptional repression activity (5,13). We have recently shown that an altered nuclear localization of DAX-1 AHC mutants accounts for their loss of transcriptional silencing. The activities of the natural DAX-1 nuclear localization signal (NLS), present in the protein N-terminus, or of a heterologous NLS are both overridden by AHC mutations (15).

Here we have performed a detailed structure–function analysis of the DAX-1 protein in order to:

  • understand the mechanism through which AHC mutations alter its subcellular localization. We show that DAX-1 AHC mutants have a misfolded conformation, as revealed by limited proteolysis;
  • investigate the effect that residues of different chemical nature localized at protein sites affected by AHC mutations have upon DAX-1 subcellular localization and transcriptional repression;
  • identify DAX-1 surfaces involved in transcriptional repression by systematic mutagenesis of protein residues predicted to be exposed in our model.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Limited proteolysis of DAX-1 AHC mutants reveals their misfolded conformation
Folded proteins have been proven to have a different exposure to proteases than unfolded or misfolded proteins. Therefore limited tryptic proteolysis has been widely used to study protein folding (16). In the case of ligand-bound nuclear receptors, ligand induces a tightly folded conformation in the holoreceptor which renders it resistant to protease digestion (17). Crystal structures of liganded RAR{gamma} and TR{alpha} have confirmed that the ligand-binding domain undergoes a conformational change in the holo form, mainly involving the repositioning of the C-terminal H12 helix towards the domain core to close the ligand-binding pocket (18,19). The repositioning of the H12 helix is essential for the formation of the surface interacting with coactivators.

We have used the same method of limited digestion with trypsin to get clues about the structure of DAX-1 AHC mutants compared with the wild-type protein. When varying trypsin concentrations are used to digest [35S]-labelled wild-type DAX-1, the full-length protein is partially resistant to digestion up to a concentration of 15 µg/ml. In addition, a doublet of about 15 kDa proteolytic fragments is produced, which is highly protease-resistant (Fig. 1, lanes 2–5). Conversely, trypsin digestion of DAX-1 R267P, {Delta}V269, N440I and 1–461 AHC mutants completely degrades the mutant proteins at a protease concentration as low as 10 µg/ml and no trypsin-resistant fragments are produced at higher protease concentrations (Fig. 1). These data show that DAX-1 AHC mutants have an altered folding, compared with the wild-type protein. The folding defect correlates with retention of mutant proteins into the cytoplasm, which we have previously described (15).



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  		Figure 1. Limited trypsin digestion of [35S]-labelled in vitro translated wild-type DAX-1 (lanes 1–6) and R267P (lanes 7–12), {Delta}V269 (lanes 13–18), N440I (lanes 19–24), 1–461 (lanes 25–30) mutants. Inputs are shown in lanes 1, 7, 13, 19 and 25, respectively. Proteins were digested with 5, 10, 15, 20 and 25 µg/ml trypsin for 10 min at 25°C and analysed by SDS–PAGE and fluorography.

 
Effects of amino acid substitutions at positions affected by AHC mutations
A three-dimensional model of the structure of the DAX-1 C-terminal domain (5) is shown in Figure 2. The positions of residues subject to mutagenesis are indicated and grouped into three differently colour-coded classes: positions affected by AHC mutations (green); residues in a putative corepressor binding surface (magenta); and H12 helix residues (yellow).



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  		Figure 2. DAX-1 mutations positions on a three-dimensional homology model. The DAX-1 C-terminal domain is in grey with helices drawn as tubes and beta sheets as arrows. The mutated residues are shown with a sphere around their beta carbon. AHC, putative corepressor-binding and helix 12 mutants are shown in green, magenta and yellow, respectively. Views (B) and (C) are rotated by 180° and 90°, respectively, compared with view (A). The homology model was generated as previously described (5) and the figure has been drawn with SETOR (40).

 
We introduced by site-directed mutagenesis different amino acid residues at specific positions in the DAX-1 protein where AHC mutations have been identified, with the purpose of studying how the chemical nature of the residue affects subcellular localization, transcriptional repression activity and protein levels (Fig. 3 and Table 1). It is well understood, in fact, that mutation of conserved residues in the protein core destabilizes its fold, whereas mutation of conserved exposed residues may affect the protein functions. The positions investigated lie in different locations in the DAX-1 C-terminal domain, according to our model (Fig. 2). All these mutations affect protein expression levels by less than 1.5-fold (Fig. 4).



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  		Figure 3. (A) Histogram showing repression activities of DAX-1 mutants on the StAR promoter expressed as a percentage of the repression activity of wild-type DAX-1, set as equal to 100. AHC mutants are shown with black histograms. SEM is indicated. (B) For each protein the percentage of nuclear localization is indicated with black histograms, nucleo-cytoplasmic localization in grey and cytoplasmic localization in white. At least 200 transfected cells were analysed for each sample. (C) Left, an example of a typical cytoplasmic distribution of a DAX-1 mutant protein (W291C). Right, nuclear staining with the Hoechst 33342 dye.

 

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  		Table 1. DAX-1 proteins harbouring amino acid substitutions at positions affected by AHC mutations. Residues are assigned to specific positions in alpha helices 1–12 of the DAX-1 C-terminal domain according to our homology model with the RXR{alpha} and RAR{gamma} ligand binding domains (5). The accessibility index expresses the accessibility of a given residue to solvent as a fraction of its accessibility when it is flanked by glycines in the sequence Gly-Gly-X-Gly-Gly, where the residue is fully exposed (accessibility index=1). An accessibility index<0.200 indicates that the residue is buried. Accessibility indices and exposed surface areas were calculated with the DSSP program (41)
 


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  		Figure 4. Immunoblot showing expression of the various DAX-1 proteins with amino acid substitutions at positions affected by AHC mutations. Lactate dehydrogenase (LDH) levels in the same samples are also shown. DAX-1 mutants levels were quantified by densitometry (histogram) and normalized against the amount of endogenous LDH, with the expression of wild-type DAX-1 set equal to 100. AHC mutants are shown with black histograms.

 
The {Delta}V269 AHC mutation is an in-frame deletion affecting a residue (V269) in the H3 helix, which is directly involved in maintaining the structure of the hydrophobic core of the putative ligand-binding domain (20). Interestingly, this valine residue is conserved in all DAX-1 homologues cloned in different species, while most other nuclear receptors have an alanine residue at the corresponding position (20). The DAX-1 {Delta}V269 mutant has a predominantly cytoplasmic localization and impaired transcriptional repression activity (Fig. 3). We replaced V269 with alanine, leucine, isoleucine or arginine. The three first amino acid residues have hydrophobic lateral chains of different size, while arginine is positively charged at physiological pH, due to its guanidinium group, but can also mediate hydrophobic interactions via its three carbon atom-lateral chain. V269A, V269L and V269I mutants have the same predominantly nuclear localization and transcriptional repression activity as wild-type DAX-1, also being expressed at approximately the same levels as the wild-type protein (Figs 3 and 4). Conversely, the V269R mutation produces the shift of localization of DAX-1 to the cytoplasm and loss of transcriptional repression activity, mimicking the effect of the AHC mutation at this position. These data show that other hydrophobic residues may substitute for V269 in mediating critical interactions stabilizing the core of the DAX-1 C-terminal domain and that introduction of a charged bulky residue destabilize the protein fold.

The W291C AHC mutation affects a buried residue predicted to be situated in the N-terminal portion of the H5 helix. This tryptophan is conserved in the subfamily of nuclear receptors related to RXR and in steroid receptors, while it is substituted by residues of different chemical nature in other nuclear receptors, and notably by a cysteine in human RAR{gamma} and TRß1. When we replaced W291 with the charged amino acid arginine, we could observe that W291R DAX-1 behaves as the W291C mutant, being predominantly cytoplasmic and impaired in its transcriptional repression activity (Fig. 3).

The K382N AHC mutation is particularly interesting since it affects a residue in the H8 helix that in our model is predicted to play a key role in core domain stabilization by forming a salt bridge with the deeply buried E298 in the H5 helix (5). Introduction of a polar asparagine at the place of the positively charged lysine disrupts the salt bridge. As expected, substitution of K382 with a negatively charged glutamate has the same effect as the AHC mutation, shifting protein localization to the cytoplasm and impairing its transcriptional activity (Fig. 3).

R425 at the beginning of H10 contacts E377 in H8. The position of this salt bridge is conserved among nuclear receptors. Remarkably, both of these residues are interested by AHC mutations (R425G; E377K) (12). The introduction of a negatively charged glutamate at position 425 mimicks the effect of the AHC mutations, disrupting the charge pair and affecting protein localization and transcriptional repression (Fig. 3).

The I439S mutation was found in a patient with adult-onset adrenal failure and incomplete HHG. This mutation produces only a partial impairment of nuclear localization of the DAX-1 protein and incomplete loss of its transcriptional repression activity (21). I439 is located at the end of the H10 helix and is conserved as a hydrophobic residue throughout the nuclear receptor family, making contacts with residues in H7 (Q359, A360 and C363). The weakly hydrophilic character of serine probably only partially perturbs the hydrophobic cluster in which I439 is involved. We chose then to replace this amino acid with alanine or leucine, which are residues of increasing hydrophobic character, and monitor the effect of these substitutions upon subcellular localization and transcriptional repression. A clear correlation exists between the size of the hydrophobic lateral chains and the extent of nuclear localization and transcriptional repression impairment (Fig. 3).

Transcriptional repression by DAX-1 is insensitive to mutagenesis of a putative corepressor binding surface
A largely overlapping molecular surface is used by nuclear hormone receptors for interaction with both coactivators and corepressors. In both cases this surface comprises residues positioned in helices H3 and H4 but, unlike what happens with coactivators, the corepressor interaction surface does not include the exposed surface of helix 12 and extends to residues which are located underneath the position occupied by the H12 helix in the presence of ligand. This corepressor binding surface includes a cluster of hydrophobic residues surrounded by polar and charged residues (22–25). These residues are conserved in DAX-1 and in most other nuclear receptors. In addition, recent studies concerning human TRß have evidenced that two additional surfaces may be involved in corepressor interaction, one lying alongside helix 1 and the other above helix 11 (site 2 and site 3 in 25). We then set up to mutagenize DAX-1 surface residues located at positions corresponding to residues in TRß and PPAR{alpha} whose mutations affect interaction with corepressors (Fig. 2 and Table 2), in order to evaluate the relative importance that different molecular surfaces have for DAX-1 transcriptional repression, subcellular localization and protein levels in transfected HeLa cells. Similarly to previous studies, DAX-1 charged surface residues were mutated into alanine, while other residues were mutated into charged residues (arginine or lysine). R288 was mutagenized both into alanine and into a glutamate.


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  		Table 2. DAX-1 proteins analysed harbouring amino acid substitutions at surface residues. Residues are assigned to specific positions in alpha helices 1–12 of the DAX-1 C-terminal domain according to our homology model with the RXR{alpha} and RAR{gamma} ligand binding domains (5). The accessibility index expresses the accessibility of a given residue to solvent as a fraction of its accessibility when it is flanked by glycines in the sequence Gly-Gly-X-Gly-Gly, where the residue is fully exposed (accessibility index=1). An accessibility index >0.200 indicates that the residue is exposed. Accessibility indices and exposed surface areas were calculated with the DSSP program (41) and refer to residues present in the wild-type DAX-1 sequence
 
The results clearly show that mutations of the DAX-1 residues homologous to the ones directly involved in corepressor interaction for PPAR{alpha} have a negative effect upon transcriptional repression only when mutations also affect protein expression (Figs 5 and 6). This is the case of the L266R, R267A and R288E mutations. Mutations L263R, K270A, L284R and R288A also have a negative impact upon protein levels without affecting repression by DAX-1 significantly. Conversely, mutation of each of the TR residues K288 (homologous to DAX-1 K270), I302 (homologous to DAX-1 L284) and K306 (homologous to DAX-1 R288) dramatically impairs both corepressor and coactivator interaction with consequent impairment of ligand-independent repression and ligand-dependent transactivation, respectively (25,26). In this series, the only DAX-1 mutation reducing transcriptional repression by about 50%, while keeping a predominantly nuclear localization and expression similar to the wild-type protein, interested S374. This residue is situated in the loop between H7 and H8, in the same position as D366 in TRß, whose mutation into arginine strongly affects corepressor interaction, even if it belongs to a different surface than the one directly contacting the corepressor's receptor interacting motifs (site 2 in 25). Interestingly, also mouse and rat dax-1 have an aspartate at this position, and a polar residue (serine or threonine) or an aspartate is present at the corresponding position in most other nuclear receptors, including steroid receptors. However, mutation of DAX-1 residues homologous to TRß W219 and Y406 (P217 and H414, respectively, in DAX-1), which belong to the same molecular surface as D366 and whose mutation also strongly affects TR–corepressor interaction (25), has no effect on its transcriptional properties, subcellular localization and expression levels. Moreover, mutation of DAX-1 R437 into alanine also had no impact on these parameters, while mutation of the homologous residue in TRß (R429), strongly diminishes corepressor interaction (25) (Fig. 5).



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  		Figure 5. (A) Histogram showing repression activities of DAX-1 surface mutants on the StAR promoter expressed as a percentage of the repression activity of wild-type DAX-1, set as equal to 100. SEM is indicated. (B) For each protein the percentage of nuclear localization is indicated with black histograms, nucleo-cytoplasmic localization in grey and cytoplasmic localization in white. At least 200 transfected cells were analysed for each sample.

 


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  		Figure 6. Immunoblot showing expression of the various DAX-1 proteins with amino acid substitutions at surface residues. Lactate dehydrogenase (LDH) levels in the same samples are also shown. DAX-1 mutants levels were quantified by densitometry (histogram) and normalized against the amount of endogenous LDH, with the expression of wild-type DAX-1 set equal to 100.

 
DAX-1 helix 12 contains critical determinants for nuclear localization and transcriptional repression
Nuclear receptors' H12 helix constitutes an essential part of their ligand-dependent transactivation domain (AF-2), the dynamic one that is assembled upon agonist binding. Structural studies have indeed shown that the H12 helix repositions upon ligand binding, occupying part of the hydrophobic groove bound by corepressors in the absence of ligand. In many cases a conserved glutamate in the H12 helix is re-oriented together with a conserved lysine residue present at the end of helix 3 to form a charge clamp stabilizing the interaction of coactivator LXXLL motifs with the receptor (27). H12 helix residues required for ligand-dependent transactivation are conserved in DAX-1, even if this acts as a transcriptional repressor in all contexts investigated so far and lacks a known ligand. Our previous studies have shown that the H12 helix plays a critical role in directing DAX-1 nuclear localization. In fact, truncation of the four C-terminal amino acids is sufficient to induce a significant shift of the protein to the cytoplasm. Cytoplasmic localization and loss of transcriptional repression activity are virtually complete when the last nine residues are deleted (15). Moreover, an AHC mutation has been found in the H12 helix (L466R) that nearly completely shifts the protein to the cytoplasm, impairing silencing (15). For these reasons we decided to perform a detailed mutagenesis of residues lying in DAX-1 H12 helix and monitor mutations' effect upon transcriptional repression, subcellular localization and expression of the protein (Fig. 8). The position and nature of the mutants we have investigated is reported in Figures 2 and 7. No mutation had an important effect upon protein levels in transfected HeLa cells, except for M465A/L466A (Fig. 9).



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  		Figure 8. (A) Histogram showing repression activities of DAX-1 H12 mutants on the StAR promoter expressed as a percentage of the repression activity of wild-type DAX-1, set as equal to 100. AHC mutants are shown with black histograms. SEM is indicated. (B) For each protein the percentage of nuclear localization is indicated with black histograms, nucleo-cytoplasmic localization in grey and cytoplasmic localization in white. At least 200 transfected cells were analysed for each sample.

 


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  		Figure 7. Amino acid substitutions introduced into the DAX-1 H12 helix. The L466R AHC mutation is shown in bold.

 


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  		Figure 9. Immunoblot showing expression of the various DAX-1 proteins with amino acid substitutions at positions in the H12 helix. Lactate dehydrogenase (LDH) levels in the same samples are also shown. DAX-1 mutants levels were quantified by densitometry (histogram) and normalized against the amount of endogenous LDH, with the expression of wild-type DAX-1 set equal to 100. AHC mutants are shown with black histograms.

 
As we have also reported before, truncation of DAX-1 at residues 451, 456 and 461 induces a prevalent cytoplasmic localization, with the truncation at aa. 466 having a milder effect (Fig. 8). We have also confirmed that the AHC L466R mutation shifts protein localization to the cytoplasm, while the M465A/L466A double mutation induces a less important change in subcellular localization. All these mutants have a more or less severe impairment of transcriptional repression activity correlated with the extent of their loss of nuclear localization, which, in the case of the M465A/L466A mutant, is also determined in part by its reduced protein levels.

Remarkably, the M461A/M462A double mutant keeps a predominantly nuclear localization and expression levels similar to the wild-type protein, but its repression activity is impaired of about 50% (Fig. 8). Conversely, M461R, L463A, L463R, E464R, R288E/E464R and C467A mutants have normal transcriptional repression activity, subcellular localization and expression levels (Figs 8 and 9).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The most common DAX-1 mutations found in AHC/HHG patients are frameshifts and nonsense mutations. Missense mutations have been found in about 20% of patients (28). The common feature of known DAX-1 mutations causing AHC/HHG is that they are all predicted to alter the protein C-terminus, which has high similarity with the nuclear hormone receptor ligand-binding domain. Even if the DAX-1 C-terminal domain contains a predicted hydrophobic pocket and all structural features required to function as a ligand-dependent transcriptional activator, including conserved residues in the H12 helix, no ligand has been identified for DAX-1 and the protein acts as a powerful transcriptional repressor in all contexts investigated so far (5,9,13). For other ligand-activated nuclear hormone receptors, like thyroid hormone receptor beta or androgen receptor, many examples are known of clinical syndromes due to mutations affecting ligand binding (29,30). One argument suggesting that DAX-1 may indeed lack a physiological ligand is that all missense mutations identified map in a structural subdomain of the protein C-terminal portion and none of them localizes to positions that may contact a putative ligand, as shown by homology modelling (5,6).

The transcriptional silencing activity of DAX-1 has been mapped to its C-terminal domain and in all DAX-1 AHC mutants this activity is consistently impaired (5,13). We have recently reported that transcriptional repression impairment is dependent upon an altered nuclear localization caused by DAX-1 AHC mutation, even if the protein nuclear localization signal (NLS), which lies in the protein N-terminal domain, is intact (15). Here we have shown that DAX-1 AHC mutants are significantly more sensitive to limited proteolysis than the wild-type protein. This finding indicates that the consequence of AHC mutation is to induce a misfolded state of the protein. Nuclear translocation of the misfolded DAX-1 proteins in AHC patients may be impaired consequently to stable interaction with anchoring sites in the cytoplasm. Interestingly, a global misfolding of DAX-1 AHC mutants may also explain their reduced RNA-binding capacity, which we have shown to be dependent upon both the N- and the C-terminal domains of the protein (14). Consequently, in addition to impairment of transcriptional repression, another important consequence of DAX-1 AHC mutations may be to affect the association of the protein to polyribosomes and its probable function in post-transcriptional regulations.

Protein misfolding is increasingly being recognized as a common cause of several human congenital and acquired diseases, with cystic fibrosis, nephrogenic diabetes insipidus, pulmonary emphysema, Alzheimer's and scrapie being only a few examples (31). Recently, therapeutic strategies have started to target misfolding to restore protein function in cystic fibrosis and other diseases (32,33). These approaches might also be worth considering in the cases of AHC due to missense mutations in DAX-1.

Our structure–function analysis shows that, in general, substitution of DAX-1 residues affected by AHC mutations with residues of similar chemical nature does not affect protein function and localization, while substitution of a hydrophobic residue with a charged one (V269R, W291R) or of a charged residue with one of reverse charge (K382E, R425E) has the same effect as AHC mutations. Particularly interesting is the case of amino acid substitutions at the I439 position. This residue was found mutated into a serine in a patient with adult-onset adrenal failure and incomplete HHG. This mutation only partially shifts the protein to the cytoplasm and impairs transcriptional repression (15). The extent of transcriptional repression and nuclear localization impairment is inversely proportional to the size of the hydrophobic lateral chain, with the I439A mutant being more severely affected than I439L. These results show that the conservation of the chemical nature of certain residues that make critical contacts stabilizing the DAX-1 C-terminal domain is required for normal protein localization and function.

Another aim of our work was to investigate the effect of the mutagenesis of a conserved putative corepressor interaction surface upon DAX-1 transcriptional repression. The conservation of key residues forming this molecular surface and its partial overlap with the coactivator binding surface has led to the hypothesis that all nuclear receptors share common surfaces for interaction with transcriptional cofactors (34). In particular, corepressors NCoR and SMRT contain an extended amphipathic helix containing the LXXI/HIXXXI/L motif, which represents their receptor-interaction surface (CoRNR box) (22,23,35). This extended helix can make contacts with a molecular surface comprising part of nuclear receptor helices H3 and H5 only in the absence of ligand or in the presence of antagonists. In fact only in these cases the position of the H12 helix does not make a steric clash with part of the corepressors' extended amphipathic helix. Ligand binding causes repositioning of the H12 helix in its holo position, with the consequent release of corepressors and recruitment of coactivators containing LXXLL motifs (NR boxes). Our results show that mutation of DAX-1 residues homologous to the ones that in other nuclear receptors mediate interaction with NCoR/SMRT corepressors does not impair its transcriptional repressor activity. These data lend further support to the hypothesis that interactions with transcriptional cofactors different from NCoR/SMRT mediate DAX-1 silencing properties (15). In addition, we have shown that mutations of some of the residues (L266R, R267A and R288E) forming the putative corepressor interaction surface destabilize the DAX-1 protein. Levels of mutant proteins were not measured in a previous study analysing the corepressor interaction surface in TRß (25) and it cannot be excluded that some of the effects reported to be consequent to mutations in specific TRß residues may depend upon protein destabilization.

The only two cases where we observed a reduction of DAX-1 transcriptional repression by about 50%, while keeping a predominantly nuclear localization and wild-type-like expression levels, were the S374R (loop H7–H8) mutation and the M461A/M462A (H12) double mutation. In the case of S374, the homologous residue in TRß (D366) belongs to a surface whose mutations also affect corepressor interaction (site 2 in 25). However, mutation of the DAX-1 residues P217 and H414, predicted to lie in the same molecular surface as S374, has no effect upon its transcriptional properties, expression levels and subcellular localization. Conversely, mutation of the homologous residues in TRß (W219 and Y406) strongly affects corepressor interaction (25). This suggests that either the S374R mutation has a global effect upon the structure of the entire DAX-1 C-terminal domain, weakening corepressor recruitment, or that only a subset of residues lying in the proximity of S374 participates in interaction with a corepressor. Further studies are required to discriminate between these two possibilities. The other residues (M461A/M462A) whose mutations affect transcriptional repression by DAX-1 lie at the beginning of the H12 helix. These residues lie inside a conserved {Phi}{Phi}XE{Phi}{Phi} motif, which is essential for ligand-dependent transcriptional activation in other nuclear receptors (36–38). The critical mutation affecting repression in DAX-1 is probably M462A, since the other single mutant M461R behaves like the wild-type protein. These data show that the function and integrity of the DAX-1 H12 helix is critical both for its nuclear localization and for its transcriptional silencing function. In fact, truncations of the H12 helix and the L466R AHC mutation cause a shift of protein localization to the cytoplasm, while the M461A/M462A double mutation impairs transcriptional repression by DAX-1. The relevance of the H12 helix for transcriptional silencing by a member of the nuclear receptor family is unusual, but it has been recently shown that the interaction of the orphan receptor ROR{alpha} with the corepressor Hr is also mediated through its H12 helix (39). However, the amino acid sequence of the DAX-1 H12 helix considerably diverges from the ROR{alpha} H12 helix, suggesting that corepressors different from Hr mediate DAX-1 transcriptional silencing activity. The information produced by our analysis will be very useful in the characterization of interactions between DAX-1 and specific transcriptional cofactors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Model building
The model of the DAX-1 C-terminal domain was built by sequence alignment with the non-liganded human RXR{alpha} (35% sequence similarity) and liganded human RAR{gamma} (29% sequence similarity), and has previously been described in detail (5).

DNA clones
Mutations were introduced into the pSV.DAX-1 expression vector (15) using the QuickChange mutagenesis system (Stratagene, CA, USA). All clones were verified by sequencing. The SF-1 (pSG.SF-1) and ß-galactosidase (pCH110) expression vectors have been described before (15).

Limited protease digestion
[35S]-labelled wild-type and R267P, {Delta}V269, N440I, 1–461 DAX-1 proteins were produced by coupled in vitro transcription–translation using the TnT system (Promega, France). Two microlitres of in vitro translated proteins were subjected to digestion with 5, 10, 15, 20 and 25 µg/ml trypsin, respectively, for 10 min at 25°C (17). Digestion was blocked by addition of denaturing loading buffer and boiling. Samples were analysed by 10% SDS–PAGE and fluorography.

Cell culture and transfections
HeLa cells were cultured in DMEM containing 2.5% calf serum, 2.5% fetal calf serum and gentamycin. Thirty-six hours after transfection cells were seeded into six-well plates (3x105 cells/well). The calcium phosphate method was used to transfect duplicate wells with 1 µg of StAR promoter–luciferase reporter, 0.5 µg of the SF-1 expression vector, 0.5 µg of wild-type or mutant DAX-1 expression vector and 0.4 µg of the ß-galactosidase expression vector. Twenty-four hours after transfection cells were lysed and luciferase activity measured with a Berthold Microlumat LB96P luminometer (Berthold, France). Luciferase activities were normalized against ß-galactosidase activities for each sample. At least three distinct experiments performed in duplicate were analysed for each sample.

Immunofluorescence
Immunofluorescence was performed as previously described (15). Briefly, transfected HeLa cells growing in chamber slides (Nunc, France) were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and incubated with the 2E5 monoclonal antibody, which recognizes an epitope in the DAX-1 N-terminal domain (8), to detect wild-type and mutant DAX-1 proteins. After washing with PBS containing 0.1% Triton X-100, the primary antibody was revealed by incubation with an Alexa594-labelled anti-mouse secondary antibody (Molecular Probes, The Netherlands). DNA was counterstained by Hoechst 33342 dye. Assessment of the subcellular distribution (nuclear, cytoplasmic, nucleo-cytoplasmic) of wild-type and mutant DAX-1 proteins was performed following the criteria described in (15). Data were computed from at least 200 transfected cells for each sample.

Immunoblot
Immunoblot was performed as previously described (15), using 10 µg per sample of total cell extract from transfected HeLa cells. The 2E5 monoclonal antibody was used to detect the wild-type and mutant DAX-1 proteins. Endogenous lactate dehydrogenase protein was revealed by a specific monoclonal antibody (Sigma, France). Signal intensities in each lane were quantified by densitometry with the NIH Image software and normalized against lactate dehydrogenase levels.


   ACKNOWLEDGEMENTS
 
We thank B. Bardoni for discussions, E. Heitz for technical assistance and the IGBMC cell culture, oligonucleotide–peptide synthesis, microscopy and sequencing facilities. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale and Association pour la Recherche sur le Cancer, Centre Hospitalier Universitaire Régional, Human Frontiers Science Program and Organon (Akzo/Nobel).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue L. Fries, BP 10142, 67404 Illkirch Cedex, France. Tel: +33 388653406; Fax: +33 388653246; Email: ninino{at}igbmc.u-strasbg.fr Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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