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A Transcriptional Silencing Domain in DAX-1 Whose Mutation Causes Adrenal Hypoplasia Congenita Enzo Lalli, Barbara Bardoni, Emmanuel Zazopoulos, Jean-Marie Wurtz, Tim M. Strom, Dino Moras, and Paolo Sassone-Corsi Institut de Génétique et de Biologie Moléculaire et Cellulaire (E.L., B.B., E.Z., J-M.W., D.M., P. S-C.) 67404 Illkirch-Strasbourg, France Biologia Generale e Genetica Medica (B.B.) Università di Pavia 27100 Pavia, Italy Abteilung für Pädiatrische Genetik (T.M.S.) Kinderpoliklinik der Ludwig Maximilians Universität 80336, München, Germany The DAX-1 gene encodes an unusual member of the nuclear hormone receptor superfamily. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. We have shown that DAX-1 binds to hairpin secondary structures and blocks steroidogenesis in adrenal cells via transcriptional repression of the steroidogenic acute regulatory protein (StAR) promoter. Here we have investigated the molecular mechanism of DAX-1-mediated repression. We show that the DAX-1 C terminus contains a potent transcriptional silencing activity, which can be transferred to a heterologous DNA-binding domain. Deletion analysis and modeling of DAX-1 structure identify two cooperating domains required for the silencing function, one located within helix H3 and the other within H12. The silencing function is cell- and promoter-specific. Strikingly, two point mutations (R267P and DV269) found in adrenal hypoplasia patients impair silencing. These findings suggest that transcriptional silencing by DAX-1 plays a critical role in the pathogenesis of adrenal hypoplasia congenita. (Molecular Endocrinology 11: 1950–1960, 1997) INTRODUCTION The human DAX-1 gene was cloned from the DSS region localized in Xp21, whose double dosage causes male-to-female sex reversal in individuals with a 46,XY karyotype (1). Mutations in the DAX-1 gene 0888-8809/97/$3.00/0 Molecular Endocrinology Copyright © 1997 by The Endocrine Society have been shown to be responsible for both adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG) (2–5). The permanent zone of the adrenal cortex is absent in AHC patients, while abnormally large fetal adrenal cells persist, resulting in structural disorganization of the adrenal gland and low serum levels of glucocorticoids, mineralocorticoids, and androgens. Individuals with AHC fail to respond to ACTH treatment and require steroid hormone replacement therapy for survival. HHG is a deficiency of production of pituitary gonadotropins, FSH and LH, and is manifested at the time of puberty. Males with HHG fail to undergo puberty unless treated with testosterone (2–5). The DAX-1 protein has an unusual structure. The N-terminal portion can be divided into three repeats of a 65- to 67-amino acid (aa) motif and a fourth incomplete repeat. Extensive search has revealed no sequence similarity between this domain and other known protein sequences. The DAX-1 C terminus shares significant homology to the ligand-binding domain (LBD) of some members of the nuclear hormone receptor superfamily (2). Recently we have shown that DAX-1 binds to DNA hairpin structures and blocks steroidogenesis in adrenal cells by inhibiting the expression of the steroidogenic acute regulatory protein (StAR) (6). Here we show that the DAX-1 C-terminal domain is endowed with transcriptional silencing activity. This property is restricted to a subset of the members of the nuclear hormone receptor superfamily: the thyroid hormone receptor (TR) and the related oncogene product v-erbA, retinoic acid receptor (RAR), and the chicken ovalbumin upstream promoter transcription factor (COUP-TF) (7, 8). Silencing domains in nuclear receptors are located in the C terminus of the protein (cor1950 Bipartite Silencing Domain in DAX-1 responding to the LBD). They function in the absence of ligand and have a modular nature, since they can be transferred to a heterologous DNA-binding domain. It has recently been shown that nuclear receptor ligandindependent silencing activity is mediated by the recruitment of corepressor factors termed TRACs (TRand RAR-associated corepressors) (9–11). Here we present a structural model of the DAX-1silencing domain, based on the homology with the ligand-binding domain of apo-RXRa and holo-RARg (12, 13). The DAX-1-silencing domain is bipartite in its nature, since the integrity of the a-helical modules H12 and H3 is required for its function. We show that two different single amino acid mutations, responsible for the AHC phenotype, abolish DAX-1-silencing activity. Our findings indicate that a direct relationship exists between the loss of DAX-1 transcriptional repression and AHC. Importantly, all DAX-1 mutations found in AHC patients have the common feature to alter its C terminus. RESULTS Structure Modeling of the DAX-1 C Terminus The three-dimensional structure of the LBD of apo-RXRa, which has about 35% similarity with DAX-1 C terminus, has recently been solved (12). Based on apo-RXRa and holo-RARg (13) structures, which have defined the existence of a common fold for the LBD of nuclear receptors (14, 15), and on the alignments of the sequences of human DAX-1 (2) and mouse Dax-1 (16), we have been able to identify in DAX-1 C terminus the domains corresponding to a-helices 1–12, which represent the hallmark of the nuclear receptor LBD structure (H1–12; Fig. 1). Surprisingly, we have found that helix H1 encompasses the region previously defined as the last and incomplete repeat. In the light of this finding, we have reconsidered our view of the DAX-1 structure. Indeed, the three highly conserved repeats in the N terminus are followed by a short stretch of five residues linking the third repeat to H1. The structure of the human DAX-1 C terminus, based on the model defined for apo-RXRa and holo-RARg, is presented in Fig. 2. Structural analysis of various LBDs has shown that H1 is an integral part of the hormonebinding domain (15), contacting H3, H5, and H8. The residues involved in H1 contacts follow two alternative patterns: [(I,L)lx(A,I)Exx] in RXRs and steroids receptors and [hxcAHxxT] in the RAR/TR subgroup (c, charged; l, long side-chain; h, hydrophobic; x, any amino-acid residue). In both cases, residues contained in the pattern anchor H1 to the LBD core (15). The first hydrophobic residue (V193 in hRARg; I235 in hRXRa) is buried and forms key contacts together with the adjacent histidine (H197 in hRARg) or glutamate (E239 in hRXRa). This last glutamate forms a buried salt bridge with R371 in H8 of hRXRa. 1951 In hDAX-1, the residue corresponding to hRXRa R371 (S332 in hRARg) is a lysine (K382). This suggests the presence of a RXR-like pattern for DAX-1 in H1. The pattern identified in DAX-1 is [(V,T)Sx(N,D)Qxx], and the deeply buried glutamate forming the salt bridge with the lysine in H8 is predicted to be located in H5 (E298). This glutamate residue corresponds to a serine in hRXRa and to an arginine in hRARg, which both point to the H197 and E239 in H1 of hRXRa and hRARg, respectively. The residue preceding this histidine and glutamate in the H1 pattern is an alanine, which is in close contact with an arginine at the end of H3 (R246 as in hRARg). This arginine is highly conserved among many members of the nuclear receptor superfamily. In DAX-1, this residue is replaced by a tyrosine (Y271 in hDAX-1), and either an asparagine or an aspartate (N221 in hDAX-1) are substituted for the alanine in the H1 pattern. A remarkable characteristic of the DAX-1 LBD is the presence of an unusually long insertion (26 amino acids) between H6 and H7 (Figs. 1 and 2), which lies in the proximity of the predicted ligand-binding pocket. This insertion represents an outstanding feature of DAX-1, being absent in hRARg, in hRXRa, and in most other members of the nuclear receptor superfamily. The conservation of this insertion in both human and mouse sequences suggests that it can play a relevant role for DAX-1 function. Since structural predictions of loops whose length is greater than about 10 residues are most likely to be inaccurate, we have chosen to replace it in Fig. 2 by a residue stretch similar in length to the hRARg loop 6–7. Another remarkable feature of DAX-1 is a conserved amphipathic a-helix motif in H12, whose integrity has been shown to be essential for the function of the ligand-dependent AF-2 activation domain of other nuclear receptors (17, 18). Intriguingly, all mutations in AHC patients have the common feature to alter DAX-1 C terminus. Most of these mutations are deletions, nonsense and frameshift mutations in the coding sequence (2–5). Two AHC patients present single amino acid changes (3). Both mutations reside in the N-terminal portion of the putative LBD; in one case arginine 267 is replaced by proline (3), while in the other case a 3-bp deletion suppresses valine 269, leaving the remainder of the sequence in frame (3). Structure prediction localizes the sites of these mutations in H3, inside (V269) or immediately adjacent (R267) to the conserved hydrophobic residues belonging to the core of the nuclear receptor structure (Fig. 2). The C Terminus of DAX-1 Possesses Transcriptional Silencing Activity We have recently shown that DAX-1 represses StAR promoter activity in Y-1 mouse adrenal cells (6). Repression is dependent on the presence of a hairpin secondary structure, which functions as the DAX-1- MOL ENDO · 1997 1952 Vol 11 No. 13 Fig. 1. Alignment of the Human and Mouse DAX-1 (hDAX-1 and mDAX-1, Respectively) Sequences with the hRXRa and Human RARg (RARg) Sequences Bipartite Silencing Domain in DAX-1 binding site, in the StAR promoter (located between positions 261 and 227 in the human StAR promoter), and results in a complete block of steroid production in Y-1 cells stably transfected with human DAX-1 (6). To assess the impact that the R267P and DV269 mutations have on DAX-1 transcriptional properties, we have studied the effect of DAX-1 proteins harboring these mutations on StAR promoter activity in Y-1 cells. While wild type DAX-1 efficiently represses both basal and forskolin-stimulated StAR promoter expression, the presence of either mutation in DAX-1 results in impairment of the repression effect (Fig. 3a). This cannot be accounted for by differences in DNA binding, since both mutant proteins bind with comparable affinity as wild type DAX-1 to the StAR promoter hairpin structure (Fig. 3b). The mutated proteins are expressed at levels comparable to the wild type DAX-1 in transfected cells (Fig. 3c). Ligand-independent transcriptional repression within the nuclear hormone receptor superfamily is restricted to TR (and its variants), RAR and COUP-TF (7, 8). This activity resides in the LBD and can be transferred to a heterologous DNA-binding domain. This prompted us to investigate whether DAX-1 C terminus is provided with a transferable silencing domain and how the R267P and DV269 mutations may affect this property. A Bipartite Silencing Domain in DAX-1 To identify the domains essential for transcriptional silencing in DAX-1, a series of deletions of its C terminus (aa 207–470) was produced and fused in-frame to the yeast GAL4 DNA-binding domain (aa 1–147). In addition, GAL4/DAX-1 fusion constructs containing either the R267P or the DV269 mutation were generated, as well as constructs where R267 and V269 were replaced by an alanine residue (Fig. 4). The effect of these mutated DAX-1 proteins on transcription driven by two different basal promoters [herpes simplex virus (HSV) thymidine kinase (tk) and rabbit b-globin promoters] was measured in two cell lines, mouse L tk2 fibroblasts and Y-1 adrenal tumor cells. All GAL4/DAX-1 fusion proteins are expressed at similar levels in transfected cells and bind to the cognate 17 mer sequence with comparable affinities (data not shown). The fusion construct G4D 207–470, which comprises H1-H12 of the DAX-1 C-terminus, is a potent silencer (generating about 15-fold repression) of the b-globin promoter, both in L tk2 and Y-1 cells (Fig. 5). Conversely, the tk promoter is silenced less efficiently 1953 (;10-fold) by G4D 207–470 in L tk2 cells and poorly (;3-fold) in Y-1 cells (Fig. 5). Removal of the most C-terminal 19 amino acids from G4D 207–470, encompassing the last portion of the predicted H11, H12, and the short loop between them, results in complete abrogation of silencing of both promoters in both cell types. Further deletion of sequences encompassing H10 to H7 has no effect on this loss of function (Fig. 5). When N-terminal deletions of G4D 207–470 were examined, we observed that deletion of aa 207–244, corresponding to the predicted H1 and to part of the loop between H1 and H3, significantly reduces silencing of the b-globin promoter in Y-1, but not in L tk2 cells. Considering the poor repression activity of G4D 207–470 on the tk promoter in Y-1 cells, it is difficult to evaluate the effect of G4D 245–470 in this context. Conversely, no loss of silencing activity of the tk promoter by G4D 245–470 was detected in L tk2 cells. G4D 272–470 and mutants with deletions spanning further in the N terminus display loss of silencing (Fig. 5). Introduction of the R267P and DV269 mutations into G4D 245–470 results in complete loss of silencing of both the tk and the b-globin promoters in L tk2 cells and of the b-globin promoter in Y-1 cells. It was not possible to assess the effect that these mutations have on silencing of the tk promoter in Y-1 cells since, as already mentioned, G4D 245–470 has negligible effect on the activity of this promoter in this cell type. We have also generated additional mutations at positions 267 and 269. While alanine substitution of R267 has no effect on silencing activity, mutation of V269 into alanine results in complete loss of silencing (Fig. 5). Recruitment of Corepressors The ligand-independent silencing effect of TR and RAR has been shown to be mediated by the interaction of their LBDs with corepressor molecules termed TRACs (9–11). Cotransfection of expression vectors encoding either TR and RAR causes the recovery of basal promoter expression silenced by unliganded GAL4/TR and GAL4/RAR (19). This phenomenon is believed to be produced by titration of cellular corepressors. When full-length DAX-1 is cotransfected together with G4D 245–470, attenuation of tk promoter silencing is observed (Fig. 6, a and b). This result implies that the mechanism of transcriptional repression by DAX-1 involves interaction with a corepressor molecule or a Conserved residues between hDAX-1 and mDAX-1 are highlighted in blue, while residues conserved in all four sequences are highlighted in yellow. The numbering of hDAX-1 and hRARg is given above and below the respective sequences. Below the alignment, the three DAX-1 N-terminal repeats are colored in red, green, and violet, respectively. Secondary structure elements are indicated as they are present in the liganded hRARg crystal structure (13). The residues lining the ligand-binding pocket of hRARg bound to all-trans-retinoic acid (13) are indicated with green dots. The ALSCRIPT (35) software was used to draw the multiple alignment. MOL ENDO · 1997 1954 Vol 11 No. 13 Fig. 2. 3D Model of the Human DAX-1 C Terminus, Based on the Liganded Form of the hRARg Hormone-Binding Domain Crystal Structure (13) Secondary structure elements shown to be essential for transcriptional silencing are shown in yellow. Key contacting residues between helices H1, H3, H5, and H8 are shown as blue spheres. The two hDAX-1 mutants discussed in the text, R267P and DV269, are highlighted as red spheres. The long 26-residue insertion between H6 and H7, which is impossible to model, has been replaced in the figure by a residue stretch similar in length to the hRARg loop 6–7, and colored in violet. The SETOR (36) software was used to draw the 3D model. component of the basal transcriptional machinery. Significantly, R267P and DV269 DAX-1 mutants are unable to relieve silencing by G4D 245–470 (Fig. 6b), suggesting that the impaired repression activity of these mutants can be accounted for by a less efficient interaction with corepressor molecules than the DAX-1 wild type protein. Coexpression of RARa does not relieve silencing by G4D 245–470 (Fig. 6, a and b). This result suggests that transcriptional repression caused by the DAX-1 C terminus may involve interaction with different molecules than the TRAC corepressors that have been described to interact with TR and RAR in the absence of ligand. Bipartite Silencing Domain in DAX-1 1955 DISCUSSION The unusual structure of the DAX-1 protein is suggestive of complex regulatory functions. We have recently shown that DAX-1 binds to hairpin DNA structures and suppresses steroidogenesis in adrenal cells via repression of StAR expression (6). Here we identify transcriptional silencing as another important regulatory property of DAX-1. Based on the structural model of DAX-1 C terminus, we show that two domains are necessary for this activity: a N-terminal domain, which minimally requires H3, and a C-terminal domain, including the final portion of H11, H12 and the intermediary short loop. Fusion of H1 sequences to the N-terminal silencing domain has a cell- and promoterspecific effect in reinforcing transcriptional repression. Single amino acid mutations R267P and DV269, located in the predicted H3, abolish DAX-1-silencing function, either in the context of the natural protein (Fig. 3) or when the C terminus is fused to a heterologous DNA-binding domain (Fig. 4). Remarkably, substitution of R267 with alanine has no effect, while alanine replacement of V269 causes loss of silencing (Fig. 4). While deletion or mutation of V269 is likely to affect the stability of the conserved hydrophobic core (12–15), R267 is predicted to be exposed on H3 surface (Fig. 2). Here we show that residues of different nature (charged vs. hydrophobic) can occupy this position without producing loss of transcriptional silencing, indicating that the effect of the R267P mutation is probably due to distortion in the spatial arrangement of the final portion of H3. Unliganded RAR and TR, as well as the orphan receptor COUP-TF, act as transcriptional repressors (7, 8); the domain responsible for their silencing function resides in the C terminus, corresponding to the LBD (7, 8). v-erbA and kindred S TR are variants of TR harboring mutations in their C terminus that impair hormone binding but that do not affect silencing ac- Fig. 3. Point Mutations in DAX-1 Which Abrogate Repression of StAR Promoter a, Top panel: Schematic representation of the DAX-1 protein. The three N-terminal repeats are indicated with arrows. The C terminus is indicated in black. The position of two point mutations found in two AHC patients is indicated. Lower panel: Repression of StAR promoter activity by DAX-1, R267P DAX-1, and DV269 DAX-1. Y-1 cells were transfected with a plasmid carrying the luciferase gene under the control of a 1.3-kb StAR promoter fragment (pGL1.3 kb StAR; 1 mg). StAR promoter activity is shown either in basal conditions or after stimulation with 10 mg/ml forskolin for 16 h. Histograms show cells cotransfected with the empty expression vector pSG5 (1 mg), pSG.DAX-1 (1 mg), pSG.R267P DAX-1 (1 mg), and pSG.DV269 DAX-1 (1 mg). StAR promoter activity in the presence of cotransfected pSG5 is set as equal to 1. Values were normalized using a cotransfected SV40-b-galactosidase expression plasmid and represent the average (6SEM) of three experiments, each one performed in duplicate. b, Electrophoretic mobility shift assay to test binding of GST.DAX-1, GST.R267P DAX-1, and GST.DV269 DAX-1 fusion proteins to an oligonucleotide encompassing StAR promoter hairpin 267/ 221 (6). Lane 1, No protein. Lane 2, GST (0.9 mg). Lanes 3–5, GST.DAX-1 (0.3, 0.6, and 0.9 mg, respectively). Lanes 6–8, GST.R267P DAX-1 (0.3, 0.6, and 0.9 mg, respectively). Lanes 9–11, GST.DV269 DAX-1 (0.3, 0.6, and 0.9 mg, respectively). Protein-DNA complexes were analyzed in a 8% acrylamide gel in 0.25 3 Tris-borate-EDTA. c, Western blot analysis showing equivalent expression of DAX-1 and of the mutant R267P and DV269 proteins. Cells were transfected with 10 mg of the expression vectors pSG5 (lane 1), pSG.DAX-1 (lane 2), pSG.R267P DAX-1 (lane 3), and pSG.DV269 DAX-1 (lane 4), respectively. The anti-DAX-1 2F4 monoclonal antibody (6) was used for DAX-1 protein detection in total cell lysates. MOL ENDO · 1997 1956 Fig. 4. GAL4/DAX-1 Fusion Constructs Used in Transient Transfection Assays The yeast GAL4 DNA-binding domain (aa 1–147) was fused in-frame with DAX-1 sequences, as indicated in the figure. tivity (7, 20). Constitutive silencing by v-erbA and kindred S TR is believed to be important in the pathogenesis of erythroid transformation and generalized thyroid hormone resistance, respectively (21, 20). Remarkably, all mutations in DAX-1 causing AHC/HHG reported to date have, as a common feature, the production of C-terminally modified proteins (2–5). Based on our deletion analysis, the result is invariably the impairment of transcriptional silencing by DAX-1. This represents a novel example of loss of transcriptional repression by a member of the nuclear receptor superfamily associated with a pathological situation. Our analysis shows that DAX-1 contains a transferable silencing domain that is able to repress the activity of various promoters, when appropriately tethered in their vicinity. This finding may be relevant to the understanding of the pathogenetic mechanism of AHC/HHG. Indeed, it is likely that DAX-1 modulates the expression of a set of genes involved in adrenal gland development. Some of these genes may be distinct from those whose expression is characteristic of the differentiated steroidogenic function of the adrenal cortex (i.e. StAR). Our data show that promoters containing a diverse array of elements supporting basal transcription can be a target for regulation by DAX-1. Several studies in Drosophila demonstrated the importance of transcriptional repressors in regulating developmental cascades. For example, even- Vol 11 No. 13 skipped is a homeodomain protein genetically defined as a repressor of segmentation-controlling genes (22), and the ecdysone-induced orphan receptor E75B regulates metamorphosis by repressing the function of another orphan receptor, DHR3 (23). DAX-1 provides one of the rare known examples of mammalian transcriptional repressors whose loss of function is associated with a congenital disease. Another case is represented by the WT1 tumor suppressor gene. A point mutation in a WT1 allele still present in a patient affected by WAGR (Wilms’ tumor, aniridia, genitourinary malformations and mental retardation) syndrome has been described (24), which converts glycine 201 into aspartic acid. The consequence is the transformation of the product encoded by the mutated WT1 allele from a transcriptional repressor into an activator (24). The variable degree of silencing by the DAX-1 C terminus, depending on the promoter and cell type, represents functional evidence that additional factors are needed to mediate repression by DAX-1. These might belong to the family of corepressor factors (TRACs) that are able to form a complex with unliganded TR/RXR and RAR/RXR heterodimers (9–11). Corepressors are released when specific ligands bind to the receptors, allowing recruitment of coactivators (11). Multiple modes of interaction of unliganded nuclear receptors with TRAC corepressors exist (9, 10, 25). DAX-1, however, lacks sequence similarity with motifs that have been shown to be required for interaction of TR/RAR (9, 10) and RevErb (25) with corepressors. In addition, repression by G4D 245–470 can be relieved by cotransfected DAX-1, but not RARa (Fig. 6), suggesting that distinct factors are required to mediate silencing by DAX-1. The abundance of these mediating factors might possibly vary according to the cell type. The particular promoter structure might also influence the efficiency of their recruitment, depending on the specific set of transcription factors bound to the promoter. This could explain the difference in silencing efficacy of the tk promoter by G4D 207–470 in L tk2 as compared with Y-1 cells. In addition, our data suggest that H1 sequences, which are absent in G4D 245–470, can significantly increase the availability of mediating factors to the DAX-1-silencing domain when they are either present in limiting amounts or inefficiently recruited to the promoter. Recent results indicate a direct and specific in vitro interaction between the transactivator SF-1 and DAX-1 (26). While analogous in vitro results have been obtained in our laboratory, we have not been able, by using several experimental approaches, to demonstrate an in vivo interaction between SF-1 and DAX-1 (Ref. 6 and our unpublished results). On the other hand, we have shown that DAX-1-mediated repression of both the dax-1 and the StAR promoters is dependent on specific binding of DAX-1 to DNA hairpin structures (6). It is conceivable that SF-1 and DAX-1 association may be possible in vitro under some experimental conditions that do not exist in physiological situations. It is also possible that tissue- Bipartite Silencing Domain in DAX-1 1957 Fig. 5. Transcriptional Repression Activity of the Various GAL4/DAX-1 Fusion Constructs Shown in Fig. 4 Construct activity was tested on two different basal promoters (HSV tk and rabbit b-globin), in L tk2 and Y-1 cells. Fold repression is calculated compared with promoter activity in the presence of cotransfected empty expression vector pSG5 (1 mg). In each case, 1 mg reporter plasmid and 1 mg GAL4/DAX-1 fusion plasmid were used, as indicated. Values were normalized using a cotransfected SV40-b-galactosidase expression plasmid. The mean value (6SEM) of three different experiments is reported. specific bridging factors may exist that could facilitate SF-1 and DAX-1 association. In conclusion, one mechanism by which DAX-1 exerts transcriptional repression is the recruitment of a powerful silencing domain to target promoters via binding to hairpin DNA structures (6). Due to the presence and conservation in the DAX-1 C terminus of a potential AF-2 domain, it is still possible that a ligand may induce a switch in DAX-1 function from a repressor to an activator. MATERIALS AND METHODS Sequence Alignment and Model Building These were performed as described (15), using the ClustalW 1.5 (27) and Modeller (28) packages. The model presented relies on the comparison of the nonliganded human RXRa (hRXRa) and liganded human RARg (hRARg) crystal structures, combined with sequence analysis. In this respect DAX-1 exhibits about 20% and 16% sequence identity and 35% and 29% sequence similarity with hRXRa and hRARg, respectively. In DAX-1 the conserved regions (from helix H3 to H5 and from helices H7 to H11) are clearly identified, and, in addition, DAX-1 also has a conserved C-terminal amphipathic a-helical domain (helix H12). We could clearly identify the conserved regions among nuclear receptors, which constitute the anchoring points on which the model relies. The conserved regions identified suggest that the fold is conserved and that a good starting model can be obtained, except for the loop 6–7 region. The loop 6–7 in DAX-1 is a rather long loop (30 amino acids) that cannot be modeled reliably. The importance of the loop 6–7 for DAX-1 structure and function requires further investigation. In the absence of any three-dimensional (3D) experimental data, we preferred not to include the loop in the model, as it brings no further information concerning the mutants discussed. Structural modeling was performed according to the sequence alignment shown in Fig. 1 and taking the liganded hRARg crystal structure (13) as a landmark. To obtain the final model, we first minimized the structure obtained from Modeller with the CHARMM package (MSI Inc., San Diego, CA). The minimization is conducted in two steps, each consisting of 1000 steps of the Powell algorithm. The Ca atoms were first restrained by a harmonic potential of 30 kcal/Å2 and then released to give the final structure. The united atom force field was used. The final structure was then analyzed with PROCHECK (29), which shows that more than 90% of the residues in the Ramachandran plot are in the most favored regions and that main-chain and side-chain parameter statistics are inside the range of or better than the statistics derived from crystal structures solved at a resolution of 2Å (data not shown). The quality/ validity of the 3D model can also be assessed by how well the DAX-1 sequence fits the native fold of the hRARg crystal structure. The program PROSAII (version 3.0) (30) gives a Z-score for Cb potentials of 24.2, which is in the MOL ENDO · 1997 1958 Vol 11 No. 13 Fig. 6. Cellular Factors Mediate Transcriptional Silencing by DAX-1 a, Coexpression of DAX-1 reduces silencing of the tk promoter by G4D 245–470. L tk2 cells were transfected with 2x17mer-tk-CAT reporter plasmid (1 mg) alone (lane 1) or in combination with GAL4 DNA-binding domain (aa 1–147) (200 ng; lane 2) or G4D 245–470 (200 ng; lanes 3–11) expression plasmids. Increasing quantities (5 and 10 mg) of the empty expression vector pSG5 (lanes 4 and 5), pSG.DAX-1 (lanes 6 and 7), and pSG.RARa in the absence (lanes 8 and 9) or in the presence (lanes 10 and 11) of 1026 M retinoic acid were added. b, Coexpression of DAX-1 R267P and DV269 mutants does not significantly relieve silencing of the tk promoter by G4D 245–470. L tk2 cells were transfected with 2x17mer-tk-CAT reporter plasmid (1 mg) and G4D 245–470 (200 ng). The effects of cotransfection with pSG5, pSG.DAX-1, pSG.R267P DAX-1, pSG.DV269 DAX-1, and pSG.RARa (10 mg each) are shown. The results are expressed as fold repression of basal CAT activity of cells transfected with GAL4 DNA-binding domain (aa 1–147) expression plasmid (200 ng). The mean value (6SEM of at least three different experiments is reported. range observed for crystal structures of the same size (range from 24 to 29; hRARg Z-score equal to 28.4 and hRXRa Z-score equal to 26.9). Plasmids Construction of the pSG5-based human DAX-1 expression vector (pSG.DAX-1) has been described (2). The DAX-1coding sequence (from position 3 to 1197 with respect to the translation start site) was PCR-amplified from genomic DNA of patients 2115 (DV269) and 2687 (R267P) (3). To insert each mutation into the wild type-coding sequence, generating pSG.DV269 DAX-1 and pSG.R267P DAX-1, the amplified DNA was excised with BspEI-PvuII and cloned into BspEIPvuII-digested pSG.DAX-1. The vector pG4MpolyII (31) was used to generate fusion constructs between the sequence encoding for the yeast GAL4 (aa 1–147) DNA-binding domain and various portions of the DAX-1 C terminus. DAX-1 sequences were PCR-amplified from plasmid pSG.DAX-1 using the appropriate primers and cloned into the KpnI-BamHI sites of pG4MpolyII. pG4D R267P and pG4D DV269 were constructed by PCR amplification of the sequence encoding for aa 245–470 from pSG.R267P DAX-1 and pSG.DV269 DAX-1, respectively, and subsequent insertion into the KpnI-BamHI sites of pG4MpolyII. PCR mutagenesis was used to introduce the mutations R267A and V269A into pG4D 207–470. Each plasmid was verified by sequencing. DAX-1-, R267P DAX-1-, and DV269 DAX-1-coding sequences were PCR amplified from pSG.DAX-1, pSG.R267P DAX-1, and pSG.DV269 DAX-1, respectively, and cloned into pGEX 4T-3 (Pharmacia, Piscataway, NJ), for glutathione-Stransferase (GST) fusion protein expression. Each plasmid was verified by sequencing. pGL1.3 kb StAR (32), 2x17mer-tk-chloramphenicol acetyltransferase (CAT) (33) (which has two GAL4 sites cloned upstream the 2105/151 HSV tk promoter), and 5x17merglobin-luc (which has five GAL4 sites cloned upstream from the 2109/110 rabbit b-globin promoter) were used as reporter plasmids in transient transfection assays. DAX-1 Protein Expression in Escherichia coli and Electrophoretic Mobility Shift Assay These were performed according to the described methods (6, 25). In the electrophoretic mobility shift assay, the labeled oligonucleotide 59-TTGCACAGTGAGTGATGGCGTTTTTATC-TCCTGATGATGATGCACAGCCTTCAGCGGGGGACATTTAAGACGCAGAA -39, encompassing StAR promoter hairpin 267/221 (6) was used as a probe. Protein Analysis Western blotting was performed as described in Ref. 6, using the anti-DAX-1 monoclonal antibody 2F4 raised in our laboratory for DAX-1 protein detection (6). Transient Transfection Assays Y-1 mouse adrenal cells were transfected by the calcium phosphate method, as described previously (2, 6). L tk2 mouse fibroblast cells were transfected by the diethylaminoethyl-dextran method (34). CAT and luciferase assays were performed as described (6, 31). Acknowledgments We thank D. M. Stocco, T. Meitinger, and G. Camerino for discussions; J. F. Strauss III for the gift of the plasmid pGL1.3 kb StAR; and E. Heitz, S. Vicaire, M. Acker, F. Ruffenach, and C. Werlé for technical assistance. Received August 7, 1997, Revision received September 22, 1997. Accepted September 25, 1997. Address requests for reprints to: Dr. P. Sassone-Corsi, Institut de Genetique et de Biologie Molecular et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, C.U. Strasbourg, France. Bipartite Silencing Domain in DAX-1 E. L. was supported by a Telethon Italy Fellowship. B. B. was supported by an EMBO short term fellowship. 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