direct interaction of sry-related protein sox9 and steroidogenic

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/98/$04.0010

Nov. 1998, p. 6653–6665 Vol. 18, No. 11

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Direct Interaction of SRY-Related Protein SOX9 andSteroidogenic Factor 1 Regulates Transcription of the

Human Anti-Mullerian Hormone GenePASCAL DE SANTA BARBARA,1 NATHALIE BONNEAUD,1 BRIGITTE BOIZET,1

MARION DESCLOZEAUX,1 BRIGITTE MONIOT,1 PETER SUDBECK,2

GERD SCHERER,2 FRANCIS POULAT,1 AND PHILIPPE BERTA1*

Centre de Recherche de Biochime Macromoleculaire, CNRS UPR1142, 34293 Montpellier cedex 5, France,1 andInstitute of Human Genetics, University of Freiburg, D-79106 Freiburg im Breisgau, Germany2

Received 28 May 1998/Returned for modification 7 July 1998/Accepted 20 July 1998

For proper male sexual differentiation, anti-Mullerian hormone (AMH) must be tightly regulated duringembryonic development to promote regression of the Mullerian duct. However, the molecular mechanismsspecifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for thesteroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximalpromoter has recently been characterized and demonstrated as being essential for AMH gene activation.However, the requirement for a specific promoter environment for SF-1 activation as well as the presence ofconserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorialprotein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding sitewithin the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closelyassociated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealedthat SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studiesindicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminalregion, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved inthe expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and inpart because of their ability to interact with each other. Our work thus identifies SOX9 as an interactionpartner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

In mammals, male sex determination starts by activation ofthe testis-determining factor gene, SRY, within cells of thesupporting cell precursor lineage (15, 44). When produced,SRY protein will then trigger differentiation of these embry-onic cells into Sertoli cells. After differentiation, the Sertolicells will export the male determining signal via the productionof a member of the transforming growth factor b family, theanti-Mullerian hormone (AMH), also known as Mullerian in-hibitory substance (for a review, see reference 25). AMH pro-duction promotes the regression of Mullerian ducts, the anla-gen of the female reproductive organs, and thus appears to becritical for establishing the male phenotype. Molecular studieshave been performed by several groups to reconstruct thepathway of primary male sex determination initiated by SRYand terminated by AMH secretion by Sertoli cells. Since AMHexpression will follow Sertoli cell determination initiated bySRY, one attractive hypothesis was the direct control of AMHexpression via the SRY gene product, a high-mobility group(HMG) box containing transcription factor (19–21). However,the time lag between SRY expression and AMH expression (18)and the absence of any transactivation domain in the humanSRY protein (8, 9) have led many investigators to refute thishypothesis (13, 14, 43). It has been suggested that other genesin the cascade located downstream of SRY have to fulfil thisrole. The recent description of additional transcription factorsinvolved in the sex-determining pathway and the compilation

of conserved DNA-binding sites located in the AMH promotersequences from diverse mammalian species have opened newtracks for investigating the control of AMH expression duringmale embryogenesis.

An important initial finding came from deletion analysis ofthe AMH promoter region that led to the identification of a180-bp segment required for correct AMH expression in pri-mary Sertoli cells (43). Characterization and analysis of thisregion in humans, bovines, mice, and rats (43) indicate that itcontains at least two highly conserved sequence elements plusa characteristic TATA box (see Fig. 1A). The proximal ele-ment that includes a single estrogen receptor half-site, AGGTCA, is known to interact with a protein designated steroido-genic factor 1 (SF-1), the mammalian homologue of theDrosophila orphan nuclear receptor fushi tarazu factor 1 (FTZ-F1), a factor that regulates expression of the fushi tarazu ho-meobox gene during early development (23, 28, 33). The func-tional importance of this conserved SF-1-binding site wassupported by several observations such as its high conservationamong species, its binding in vitro to purified SF-1 protein, acoincident expression profile between SF-1 and AMH, and theability of a DNA fragment containing this site to drive thetranscription of a reporter gene in a Sertoli cell-specific man-ner as demonstrated in transgenic animals (13, 43). However,the SF-1 binding site failed to activate gene expression fromthe AMH promoter in heterologous cells such as HeLa cells(43). This activation was shown to require removal of theligand-binding domain of the SF-1 protein, suggesting that aSertoli cell-specific ligand or cofactor must be necessary forSF-1 to fulfil its transcriptional activity.

While the existence of a SOX-binding element in the 180-bp

* Corresponding author. Present address: Human Molecular Genet-ics Group, Institut de Genetique Humaine, 141 rue de la cardonille,34396 Montpellier cedex 5, France. Phone: (33) 499619955. Fax: (33)499619901. E-mail: [email protected].

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minimal promoter led many investigators to postulate the tes-tis-determining factor SRY as a candidate to control AMHexpression, rather contradictory results and hypotheses havebeen produced so far (18, 20, 42, 43, 50). Among the additionalsex-determining genes described in recent years that wereshown to be expressed concomitant with or shortly after SRY,the SRY-related gene SOX9 is the most attractive candidate tocontribute to this control. SOX9 was initially identified by po-sitional cloning as associated with the skeletal malformationsyndrome campomelic dysplasia, in which two-thirds of XYindividuals show sex reversal (10, 47). The recent detailedanalysis of mouse Sox9 expression during gonadal develop-ment coincident with Sertoli cell differentiation and its upregu-lation preceding the onset of AMH expression in mice andchickens are the first arguments to support such a hypothesis(3, 26, 32). Furthermore, unlike human SRY, SOX9 was shownto act as a transcriptional activator during chondrocyte differ-entiation (2, 30) and to display a high level of protein conser-vation across vertebrate evolution.

We now address the possibility that the conserved SOX-binding sequence present within the 180-bp proximal promoterregion of the human AMH gene is a binding site for SOX9. Wedemonstrate that SOX9 interacts with this sequence and in-creases the expression of an AMH promoter/reporter geneconstruct. We provide evidence for direct protein-protein in-teraction between SOX9 and SF-1 and for additive activationof the human AMH promoter by these proteins. We alsodemonstrate that potentiation of SF-1 activity by SOX9 re-quires both the SOX9 transactivation domain and the SF-1ligand-binding domain. We conclude that SOX9 and SF-1 haveto function in a cooperative manner for the proper control ofAMH gene expression during early male gonadal develop-ment.

MATERIALS AND METHODS

Cloning and plasmid constructions. Human SF-1 cDNA was obtained byscreening a lZAPII human embryonic cDNA library (24) with a human SF-1-derived genomic 153-bp fragment. This fragment was the result of the reversetranscription-PCR amplification of human embryonic total RNA with the help ofprimers (forward, 59-GGTGTCCGGCTACCACTACGG-39; reverse, 59-CCAACGCCGACAAGGGACAGC-39) designed from the human SF-1 partialgenomic sequence deposited in GenBank under accession no. U32592. Theamplification product was next sequenced to verify its identity to the SF-1sequence. The SF-1-derived probe was labeled with [a-32P]dCTP by randompriming (Megaprime; Amersham) and then used for the screening. SF-1 cDNA-containing phagemids were excised in vivo from the lZAPII vector by coinfec-tion of Escherichia coli XL1-blue cells with VCSM13 helper phage (Stratagene)as specified by the supplier and then fully sequenced. This sequence is depositedin GenBank (accession no. U32591). Human SOX9 cDNA was cloned inpcDNA3 vector (Invitrogen).

Plasmid constructions used in this study are described below. Further detailsas well as plasmid maps are available upon request.

(i) Yeast expression plasmids. Full-length or partial SF-1 open reading frames,including the sequences encoding amino acids 1 to 461, 1 to 226, and 223 to 461,were obtained by PCR amplification with the appropriate oligonucleotides con-taining upstream BamHI sites and downstream SalI sites. PCR products werenext cloned into pUC18 (SureClone ligation kit; Pharmacia) and checked bysequencing. The different fragments were then subcloned into the BamHI andSalI sites of pGBT11 as a fusion with the GAL4 DNA-binding domain.pGADGH-SOX9 constructs were obtained by inserting the human SOX9 openreading frame or the SOX9 fragment, spanning positions 1 to 304, from thepcDNA3 construct into the BamHI site of pGADGH (Clontech) as a fusion withthe GAL4 activating-domain DNA sequence. Again, the authenticity of theconstructs and ligation junctions was checked by sequencing.

(ii) Bacterium expression vectors. Both SF-1 and SOX9 proteins were bacte-rially expressed as glutathione S-transferase (GST) fusion proteins after PCRamplification of the corresponding cDNA and cloning into the BamHI andEcoRI sites of the pGEX-4T3 expression vector (Pharmacia). The authenticity ofthe constructs was checked by sequencing.

(iii) Mammalian reporter and expression plasmids. To construct the reportergene plasmid, pEMBL8-AMH (16) was used as a template to amplify AMHpromoter DNA from positions 110 to 2154 by PCR. The two primers containthe recognition site for either SalI or SacI. After SalI-SacI digestion, one copy of

the 164-bp PCR product was inserted in the SalI-SacI sites of the pEMBL-CATvector (Stratagene) upstream of the chloramphenicol acetyltransferase (CAT)coding sequence. This construct is referred to as p154CAT. The same strategywas used to produce the p123CAT construct. Mutagenesis of the SOX-bindingsite to produce p154MUTSOXCAT was performed by using the QuickChangesite-directed mutagenesis kit (Stratagene) with the help of the SOX-MUT oli-gonucleotide (see below). Full-length SF-1 cDNA was cloned as a BamHI-EcoRIfragment into the pcDNA3 vector, which directs transcription from the cytomeg-alovirus promoter. pcDNA-SOX9 and pcDNA-SOX9 1–304 were described pre-viously (46).

(iv) Plasmid constructs for in vitro translation. pcDNA-SOX9 HMG wasdescribed previously (31). The SOX9 1–118 deletion mutant was obtained byBssHII digestion of pcDNA-SOX9. For the other two deletions (SOX9 1–208and SOX9 1–95), pcDNA-SOX9 was digested with BamHI and the releasedinsert was cloned in pBluescript vector. This construct was finally digested witheither PstI or HincII and ligated to create SOX9 1–208 and SOX9 1–95 mutants,respectively.

Synthesis of proteins in vitro and preparation of nuclear extracts. SOX9protein, SOX9 mutants, and SF-1 protein were synthesized by in vitro transcrip-tion-translation with the expression vectors described above and with the TNTsystem (Promega). Nuclear extracts from NT2/D1 cells were prepared as de-scribed previously (41).

Production and purification of bacterially expressed pGEX-SOX9 and pGEX–SF-1 fusion proteins. After being cloned in the pGEX-4T3 expression vector, thetwo recombinant proteins were produced in bacterial strain BL21(DE3) afterinduction with 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After 2 h ofinduction at 30°C, cells were collected by centrifugation, resuspended in lysisbuffer (150 mM NaCl, 1 mM dithiothreitol [DTT], 5 mM EDTA, 25% sucrose,50 mM Tris [pH 7.5]) supplemented with bovine DNase I (Pharmacia) and 0.5mM Pefabloc-SC-AEBSF (Interchim), and sonicated for 5 min at 4°C. Bacterialdebris were removed by centrifugation at 25,000 rpm for 20 min. Lysate wasloaded onto glutathione-Sepharose beads and washed three times with buffer I (5mM EDTA, 250 mM NaCl, 50 mM Tris [pH 7.6]) and three times with buffer II(5 mM EDTA, 120 mM NaCl, 50 mM Tris [pH 7.6]). The purified proteins werechecked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and used directly for in vitro binding assay studies or eluted fromthe matrix with buffer II plus 10 mM reduced glutathione after a 30-min incu-bation at 4°C.

Production, purification, and characterization of SF-1 and SOX9 antibodies.Polyclonal human SF-1-specific rabbit serum and rat serum were raised againstthe GST fusion protein containing amino acids 121 to 232 from the human SF-1protein. The fusion protein was overexpressed in BL21 (DE3) cells and purifiedon a glutathione-Sepharose column as described above. Male New ZealandWhite rabbits and rats were injected with purified protein mixed with completeFreund’s adjuvant (Sigma) and bled 10 days after each injection. For purification,only the rabbit polyclonal antiserum was loaded onto GST beads overnight andaffinity purified by blotting overnight onto the SF-1 peptide coupled to anImmobilon membrane (Millipore). After saturation with polyvinylpyrrolidone(Sigma), SF-1 antibody was eluted with 0.2 M glycine (pH 2.5) and dialyzedagainst 1 M Tris base (pH 7.5). The antibody was finally aliquoted and stored at280°C.

SF-1 antibody specificity was checked by immunostaining of SF-1-transfectedCOS-7 cells and also of empty COS-7 cells as control. In each case, nuclearextract proteins were transferred to nitrocellulose and immunostained as de-scribed previously (41). In the transfected cells, only an expected 53-kDa proteinwas detected (data not shown).

Polyclonal human SOX9-specific rat serum was raised against the bacteriallyexpressed SOX9 TA domain (amino acids 408 to 504) fused to GST. SOX9antibody specificity was checked as described above by using COS-7 cells ex-pressing full-length SOX9 protein and immunostaining.

DNA-binding assays. Protein binding to AMH promoter DNA probes wasassessed by the electrophoretic mobility shift assay (EMSA). For these experi-ments, double-stranded oligonucleotides were labeled by a fill-in reaction in thepresence of [a-32P]dCTP and DNA polymerase I Klenow fragment for 1 h at37°C. The labeled probe was purified on a 5% nondenaturing acrylamide gel andeluted overnight in 0.8 M ammonium acetate–5 mM EDTA–0.1% SDS buffer at50°C. For the binding reaction, 5 to 10 mg of nuclear extract or recombinantprotein was mixed with 32P-labeled probe (10,000 cpm) and 2 mg of poly(dI-dC)for SF-1 protein or 2 mg of poly(dG-dC) for SOX9 protein in a 20-ml final volumeof binding buffer (20 mM HEPES [pH 7.9], 20% glycerol, 0.1 M KCl, 0.2 mMEDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT). For competitionor supershift experiments, unlabeled competitor oligonucleotide or antibodysolution was incubated for 15 min at room temperature before the probe wasadded. The DNA-protein complexes were resolved by electrophoresis on a 5%polyacrylamide gel in Tris-borate-EDTA buffer at 4°C and visualized by autora-diography after being fixed with 10% methanol–10% acetic acid and dried.

The DNA probes used in these assays were complementary double-strandedDNA oligonucleotides including the SOX binding site of the AMH proximalpromoter (SOX-BS), a mutated version of this site (SOX-MUT), or the SF-1-binding site from the same promoter (SF-1-BS). The nucleotide sequences of thetop strands of the oligonucleotides are as follows: SF-1-BS, 59-GGCACTGTCCCCCAAGGTCGC-39; SF-1-MUT, 59-GGCACTGTCCCCCAATTTCGC-39;

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SOX-BS, 59-GGACAGAAAGGGCTCTTTGAGAAGGCCA-39; and SOX-MUT, 59-GGACAGAAAGGGCTCTGGGAGAAGGCCA-39. The mutated nu-cleotides are in boldface type.

Cell culture and transfection assays. The human NT2/D1 cells (N-Tera 2,clone D1, a human pluripotent embryonic carcinoma cell line [ATCC CRL1973]) were obtained from the American Type Culture Collection (Biovaley,France). NT2/D1 and COS-7 cells were cultured in Dulbecco’s modified Eagle’smedium (Imperial Laboratories, Flobio, France) containing 10% (vol/vol) fetalcalf serum (Life Technologies), penicillin/streptomycin, and 2 mM glutaminewith a partial pressure of CO2 of 5% at 37°C in humidified air. Plasmids used fortransfections were purified with the maxiprep reagent system (Qiagen). COS-7 orNT2/D1 cells at 60% to 80% confluence were washed twice with serum-freemedium before undergoing cotransfection with 1 mg of reporter plasmid, 200 ngof pCMV–b-galactosidase plasmid (Stratagene) used as an internal control fortransfection efficiency, and different SF-1 and SOX9 expression plasmids with 7ml of Lipofectamine (Life Technologies) in 200 ml of serum-free medium. Aftera 6-h incubation, the medium was replaced with 2 ml of medium supplementedwith 10% serum and the cells were harvested after 48 h of culture. CAT assayswere performed on cell extracts with [3H]acetyl coenzyme A (200 mCi/mmol;Amersham, Little Chalfont, United Kingdom) by a nonchromatographic methodas described by Nielsen et al. (37). Promoter activities were expressed as CATactivity units per b-galactosidase unit, and each value represents the mean of theresults from four separate wells. Error bars represent the standard errors.

Protein interaction assays. For protein association experiments on glutathio-ne-Sepharose beads, GST–SF-1 fusion proteins were overexpressed in BL21bacteria and purified as described above. Immobilized SF-1 proteins were thenincubated with the different 35S-labeled SOX9-derived proteins obtained by invitro translation and the diverse SOX9 constructs in pcDNA3 and pBluescriptvectors. Incubations were carried out in 300 ml of TBST buffer (10 mM Tris-HCl[pH 7.5], 130 mM NaCl, 0.5% Tween 20)–0.2% bovine serum albumin [BSA]–ethidium bromide (50 mg/ml) at room temperature for 30 min. The Sepharosebeads were washed three times with 1 ml of TBST buffer. Bound proteins wereeluted by the addition of 53 Laemmli buffer, boiled, and visualized after SDS-PAGE analysis and autoradiography.

Yeast two-hybrid interaction assays. After the different pGADGH-SOX9 con-structs (containing the leucine-selective marker) were obtained, each was trans-formed into the Mata Y187 yeast strain by standard procedures. On the otherhand, the Mata Hf7c yeast strain was transformed with the different pGBT11–SF-1 constructs (containing the tryptophan-selective marker). These two yeaststrains both harbor HIS3 and b-galactosidase reporter genes under the control ofGAL4-binding sites. Diploids were obtained by mating and were selected onDO-W-L medium without tryptophan and leucine, as reported previously (11).Interaction assays were done for three independent transformants. Histidineassays were conducted on Y187-H7fc diploid strains expressing the designatedconstructs on DO-W-L-H medium without tryptophan, leucine, and histidine.Quantitative b-galactosidase assays were conducted on the same diploids as thehistidine assays, and mean values are given in b-galactosidase units.

In vivo coimmunoprecipitation. Detection of SF-1/SOX9 complexes was ana-lyzed in vivo in the NT2/D1 cell line. After a 4-h labeling with 100 mCi of[35S]methionine (Amersham; specific activity, .800 Ci/mmol), the cells werewashed with phosphate-buffered saline (PBS), collected, lysed for 30 min at 4°Cin 1 ml of TBST or TLB (20 mM Tris-HCl [pH 7.6], 140 mM NaCl, 2 mM EDTA,1% Triton X-100, 25 mM b-glycerophosphate, 10% glycerol, 2 mM sodiumpyrophosphate) buffer supplemented with Complete protease inhibitor cocktail(Boehringer Mannheim) with or without 50 mg of ethidium bromide per ml, andvortexed. All subsequent steps were carried out on ice. Cell debris were removed,and the resulting lysate was precleared with protein A-Sepharose beads (Phar-macia). Typically, for each immunoprecipitation, 100 ml of cleared lysate wasincubated with anti-SF-1 antibody conjugated to protein A-Sepharose beads at4°C in a total volume of 1 ml for 1 h with continuous rocking. The beads werepelleted and washed five times in the lysis buffer, and the resultant proteins werediluted in 53 Laemmli buffer and subjected either to SDS-PAGE and autora-diography or to Western blot analysis with the SOX9 antibody (diluted 1/400)revealed with an ECL kit (Amersham).

DNase I footprinting assay. A double-stranded DNA fragment correspondingto the 164-bp region from the AMH promoter was used for DNase I footprintinganalysis. Briefly, a pUC18-AMH 164-bp construct was linearized by digestionwith BamHI and labeled with [a-32P]dCTP. The 164-bp fragment was thenreleased by EcoRI. The labeled fragment was gel purified and eluted at 50°C in0.8 M ammonium acetate–5 mM EDTA–0.1% SDS buffer. For each footprintingreaction, 104 cpm of the probe was added to various amounts of purified SF-1and SOX9 proteins in 50 ml of mixture containing 10 mM HEPES (pH 7.8), 40mM KCl, 3 mM MgCl2, 0.5 mM DTT, 5% glycerol, and 100 ng of poly(dI-dC) forSF-1 or 100 ng of poly(dG-dC) for SOX9 and the mixture was incubated at roomtemperature for 30 min. DNase I digestion was performed by adding 1 U ofenzyme diluted in the corresponding buffer (Boehringer-Mannheim) and incu-bating the mixture for 1 min at 25°C. The reaction was terminated by addingphenol-chloroform. The digested fragments were recovered by ethanol precipi-tation, resuspended in 3 ml of stop buffer (0.1% xylene cyanol, 0.1% bromophe-nol blue, 10 mM EDTA, 95% formamide), incubated for 2 min at 90°C, andresolved by electrophoresis on a 6% polyacrylamide–urea gel. A DNA ladder wasmade by dimethyl sulfide treatment for 5 min of 2 ml of labeled probe and

piperidine treatment at 90°C for 30 min. This ladder was used to assign nucle-otide positions in the gel.

Immunofluorescence staining of tissue culture cells. Cells were preincubatedin PBS buffer–1% BSA for 30 min at 37°C and then probed with the appropriateantibody, anti-SF-1, anti-SOX9, or anti-AMH diluted 1/100 in PBS/BSA. Thecells were washed in PBS, and the primary antibodies were visualized withbiotinylated anti-rabbit antibodies (dilution, 1/200) and Texas red-conjugatedstreptavidin or with Fluorolink Cy2-conjugated anti-rat labeled goat antibodies(dilution, 1/200). In each case, incubations were performed for 30 min under thesame conditions described for the primary antibodies. For colocalization analy-sis, incubation with both appropriate primary antibodies was performed in thesame incubation buffer and the same antibody dilutions were used with thesecondary antibodies. Cell nuclei were visualized with Hoechst 33286. The cellswere washed again and mounted in FluorSave reagent (Calbiochem). Imageswere collected and processed on a Bio-Rad confocal microscope or on a ZeissAxiophot.

RESULTS

A consensus and functional SOX-binding site resides withinthe proximal AMH promoter. Previous in vitro studies of theAMH promoter indicated that not more than 180 bp is re-quired for proper AMH expression in Sertoli cells (43). Di-verse observations including the presence of a conserved SF-1site (Fig. 1A), mutation analysis, or transgenic experimentshave revealed the requirement for this site in AMH genecontrol (13, 43). However, several results have also pointed outthe necessity for a specific Sertoli-related cell environment foractivation, indicating that an uncharacterized Sertoli cell factorcontributes to this activation (43). We now confirm this re-quirement by using the COS-7 cell line as the transfectioncontrol (Fig. 1B). Pursuing progressive deletions of the mini-mal human AMH promoter, we show that the nucleotide se-quence between 2154 and 2123 of the AMH promoter isimportant for promoter function, as demonstrated by transfec-tion assays in the NT2/D1 cell line (Fig. 1B). The NT2/D1 cellline still constitutes one of the rare cell models positive forSRY expression (4, 41). Inspection of the nucleotide sequenceconfirmed previous analysis by revealing a 7-of-8-bp identity(59-CCTTGAG, referred to as the SOX site in this study) to aknown binding site that represents a potential site for SRY andother related members of the HMG-box class DNA-bindingfactors such as SOX, TCF-1, or LEF-1 protein (12, 22, 30, 36).Finally, using direct mutagenesis, we also show the necessityfor the SOX site in such an additive activation (Fig. 1B).

Purified human SOX9 and SF-1 proteins bind to the AMHpromoter in vitro. In vitro DNase I footprint analysis showedthat purified GST-SOX9 protein could protect the 2154 to2135 sequence from the AMH proximal promoter, the se-quence spanning the SOX site (Fig. 2A). As a control, by usingthe same 164-bp labeled oligonucleotide, the same experimen-tal conditions, and the purified GST-SF-1 fusion protein, wealso confirmed the protection of the 297 to 282 DNA se-quence that includes the SF-1 binding site previously described(43) (Fig. 2B). A labeled 28-bp oligonucleotide probe includ-ing the SOX-binding site was next checked in EMSA experi-ments with GST-SOX9 protein. As shown in Fig. 2C, a singleDNA protein complex was evident. Complex formation wasblocked by using the same unlabeled oligonucleotide as thecompetitor. When a double mutation known to abolish SOXprotein binding was introduced into the oligonucleotide (SOX-MUT), the competition was abolished. These results indicatethat SOX9 binds directly to the DNA in the region of theAMH promoter. On the other hand, similar EMSA experi-ments with purified GST–SF-1 and the corresponding SF-1oligonucleotide-binding site designed from the AMH proximalpromoter confirm SF-1 protein interaction (Fig. 2D).

Transactivation of AMH expression by SOX9 and by SF-1 inCOS-7 cells. The functional relevance of SOX9 factor binding

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to the SOX site located in the AMH proximal promoter andthe possibility of its cooperation with the SF-1 nuclear receptorin activating the AMH promoter were examined. Cotransfec-tions of COS-7 cells with a reporter plasmid which contains the164-bp AMH DNA fragment adjacent to the CAT gene andtermed p154CAT (see Materials and Methods), together withincreasing SF-1 expression plasmid concentrations but with afixed SOX9 plasmid concentration, were performed (Fig. 3A).The use of 10 ng of pcDNA3-SOX9 along with 200 ng ofpcDNA3-SF-1 provides the best activation signal (more thanfivefold compared with the empty vector) and was used in thefollowing experiments (Fig. 3A). As a control, under theseexperimental conditions the specificity of this activation wasthen tested with a truncated form of SOX9 (deletion of aminoacids 305 to 509), a form described as being able to bind theDNA target but unable to activate transcription (30). As shownin Fig. 3B, the SOX9 C-terminal transactivation domain wasrequired for this activation. Similarly, no activation was ob-

served when the effector plasmid expressing SRY, a previouscandidate factor for contributing to AMH gene activation, wasused (Fig. 3B). While this result confirmed recent data ob-tained with an artificial promoter (8), it also showed that SRYcannot substitute for SOX9 in the AMH-positive control. If theSOX9 activation domain appears necessary in the activationeffect, its DNA-binding capacity is also required, as demon-strated when using a mutated version of the AMH proximalpromoter (Fig. 3C).

SOX9 and SF-1 interact directly with each other as shownby two-hybrid analysis and by in vitro assays. The results justpresented and the observation of rather closely spaced SOX-and SF-1-binding sites within the proximal AMH promoterregion (Fig. 1B), along with the ability of SOX proteins to bendDNA (12), raised the possibility that the two factors interactwith each other, as recently reported for another system in-volving Sox2 and Oct-3 (1, 52).

To obtain evidence for such an interaction, we first used the

FIG. 1. Deletional analysis of the AMH proximal promoter region. (A) Sequence comparison among human, mouse, rat, and bovine AMH proximal promoterregions. The characteristic TATA box, the SF-1-binding site (SF-1-BS), the SOX-binding site (SOX-BS), and a putative GATA site (GATA) that are conserved amongthe different sequences are indicated. Numbers below the sequences correspond to the human gene. (B) Intrinsic activity of the human AMH proximal promoter inNT2/D1 cells. Various human AMH promoter-CAT reporter gene constructs were transfected in NT2/D1 and COS-7 cells. CAT reporter activities were quantitatedafter cotransfection of 1 mg of constructs containing either the bp 2154 (p154CAT), the bp 2123 human AMH promoter (p123CAT), or the bp 2154 AMH promotermutated on the SOX-binding site (p154MUTSOXCAT) and 0.2 mg of pCMV–b-galactosidase. The values shown represent the means of four transfection experiments.Increases in activation are shown relative to pEMBL empty vector. Standard deviations are indicated by bars.


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FIG. 2. DNase I footprint analysis of the AMH proximal promoter by using SOX9 and SF-1 recombinant proteins. (A) SOX9 DNase I footprint. The upper strandof the 164-bp AMH promoter was 32P labeled; 104 cpm of the probe was incubated in the presence of 1 mg of either free GST or GST-SOX9. In each case, the reactionmix was resolved on a 6% polyacrylamide sequencing gel. The lane labeled Ladder designates a G1A Maxam-Gilbert sequence ladder obtained with the probe. Theprotected regions are indicated by a box. (B) SF-1 DNase I footprint. The same radiolabeled probe was incubated with 1 mg of either free GST or GST–SF-1. The lanelabeled Ladder represents the G1A Maxam-Gilbert sequence ladder. The protected regions are indicated by a box. (C) Affinity binding of GST-SOX9 to theSOX-binding site (SOX-BS) in the AMH proximal promoter probe. The double-stranded SOX-binding-site oligonucleotide was end labeled and incubated with 10 ngof purified GST-SOX9 protein in the absence (lane 2) or presence (lanes 4 and 5) of a 50-fold molar excess of unlabeled competitors. Free GST was used as the control(lane 3). (D) Affinity binding of GST–SF-1 recombinant protein to the SF-1-binding site (SF-1-BS) in the AMH promoter. The double-stranded SF-1-binding-siteoligonucleotide was end labeled and incubated with 10 ng of purified GST-SOX9 protein in the absence (lane 2) or presence (lanes 4 and 5) of a 50-fold molar excessof unlabeled competitors. Free GST was used as control (lane 3).


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yeast two-hybrid assay and a mating protocol (11). For this, theregion encompassing the SF-1 open reading frame was fused tothe GAL4 DNA binding domain in the pGBT11 vector andmated with the SOX9 open reading frame fused to the GAL4activation domain in the pGADGH vector. The yeast diploidtransformants were able to grow on selective medium lackinghistidine and were also b-galactosidase positive (Fig. 4B andC). This result indicates an interaction between the two pro-teins. We subsequently tested potential interactions betweenfull-length SOX9 and different regions of SF-1, as depicted inFig. 4A. We found that the carboxy-terminal region of SF-1(amino acids 225 to 461) was sufficient for this interaction.Correspondingly, SOX9 did not interact with the N-terminalportion of SF-1 (amino acids 1 to 226). It is worth noting thatidentical results were obtained when using only the N-terminalpart of SOX9 (amino acids 1 to 304) (Fig. 4B). To quantify thestrength of the interactions between the different constructs ofSF-1 and SOX9 proteins, b-galactosidase activity was mea-sured from the transformant lysates (Fig. 4C). These resultsconfirm that the strongest interaction occurs between the N-terminal region of SOX9 and the C-terminal region of SF-1,including its ligand-binding domain. Although these resultsdemonstrate that SOX9 and SF-1 interact, we cannot excludethe contribution of a posttranslational modification or the in-volvement of a third partner present in the yeast strain andcontributing to the interaction.

To clarify this point and to better delineate the SOX inter-action region, in vitro binding experiments were also per-formed. For this, SF-1 was expressed as a bacterial GST fusionprotein, immobilized on glutathione-Sepharose resin, and sub-jected to in vitro binding assays by incubation with in vitro-translated radiolabeled SOX9. SOX9 was produced either asfull-length protein or as deleted versions (depicted schemati-cally in Fig. 5A). After extensive washing of the resin andanalysis by SDS-PAGE, approximately 10% of the input[35S]methionine-labeled SOX9 remained bound on the GST–SF-1 beads (Fig. 5B). These coprecipitation assays show that aGST–SF-1 fusion protein binds specifically to SOX9 in vitroand that the SOX9 interaction domain maps to amino acids104 to 118, a domain located in the first one-third of the SOX9HMG box (Fig. 5B). As controls, neither the SOX9-derivedproteins bound to GST-Sepharose alone nor the full-lengthSOX9 protein interacted with an unrelated nuclear receptorsuch a c-erbA (Fig. 5B). Similar binding was observed afterintroduction of ethidium bromide, confirming a DNA-inde-pendent protein association mechanism (27). Taken together,these results demonstrate a direct protein-protein interactionbetween SOX9 and SF-1. Finally, this direct protein-proteininteraction was confirmed by using band shift experiments in-cluding GST–SF-1 purified protein, in vitro-translated SOX9protein, and the AMH proximal promoter-derived SF-1 bind-ing site (Fig. 5C). At the same time, this data demonstrates the

FIG. 3. Cooperation between SOX9 and SF-1 proteins in the activation of the AMH minimal promoter in COS-7 cells. (A) Cotransfection assay in COS-7 cells ofa reporter plasmid with the CAT gene under the control of an AMH minimal promoter (p154CAT), a constant amount of pcDNA3-SOX9 (10 ng), and increasingamounts of the pcDNA3–SF-1 construct. (B) The specificity of SOX9 activity was tested with a deleted version of pcDNA3-SOX9 (10 ng) or with pcDNA3-SRY (10ng). (C) Comparison of the SOX9–SF-1 activity on the wild-type AMH proximal promoter (p154CAT) with that on the same promoter mutated on the SOX-bindingsite p154MUTSOX. The CAT activity of the reporter plasmid alone was set as 1. All values represent the means of three separate transfection experiments (6 standarderrors).


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inability of SOX9 protein to interact with the SF-1 DNA-binding site used in this study.

SOX9 and SF-1 interact in vivo. We then examined whetherSOX9 and SF-1 form a protein complex in vivo. The ability ofSOX9 to interact directly with SF-1 independently of theSOX9 DNA-binding activity was first tested by EMSA in thepresence of NT2/D1 cell nuclear extracts and the labeled SF-1binding-site oligonucleotide whose sequence was derived fromthe AMH proximal promoter. Nuclear extracts of NT2/D1 cellsgave rise to a major protein-DNA complex (Fig. 6, lane 1), aDNA binding that was inhibited by an excess of unlabeled SF-1oligonucleotide (lane 2) but not by a mutated SF-1 oligonu-cleotide (lane 10). Evidence for the identity of the retardedmajor band was obtained in supershift experiments. Preincu-bation of nuclear extracts in the presence of SOX9 or SF-1

antisera produced a supershift band in both cases (lanes 4 and6). By contrast, addition of SRY antiserum did not modify themigration of the complex (lane 7), in agreement with previousresults (43).

To complete our investigations of the SOX9–SF-1 interac-tion, we performed coimmunoprecipitation experiments withextracts prepared from [35S]methionine-labeled NT2/D1 cells.Immunoprecipitation was carried out in two steps. First, radio-labeled NT2/D1 cells extracts were immunoprecipitated withSOX9- or SF-1-specific antiserum or with the respective pre-immune serum. As shown in Fig. 7A and B, the two antiseraprecipitated the corresponding proteins with the expected mo-lecular weights, but the preimmune sera did not. In a secondstep, after immunoprecipitation of NT2/D1 cell extracts withSF-1-specific antiserum, immunoprecipitates were collected on

FIG. 4. SOX9 interacts with the carboxy-terminal region of the SF-1 protein. The SOX9–SF-1 interaction was scored by a yeast two-hybrid assay (see Materials andMethods). (A) Schematic diagrams of SF-1 wild type, deletion constructs, and functional domains of the human SF-1 protein. (B) Qualitative histidine assays. A positiveinteraction results in the ability of the Y187-Hf7c diploid expressing the designated constructs to grow (1) or not grow (2) on a medium depleted of tryptophan, leucine,and histidine. Assays were done for three independent diploids. All the SF-1 constructs were tested against the empty pGADGH vector as a negative control, and theSOX9-derived constructs were tested against the empty pGBT11 vector. (C) Quantitative b-galactosidase assays for these interactions were conducted on the samediploids as those used in the histidine assays. Mean values are given in relative b-galactosidase units.


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protein A-Sepharose beads and then fractionated by SDS-PAGE. After blotting onto nitrocellulose and probing with therat SOX9 antibody, a 65-kDa protein with the size expected forSOX9 was detected (Fig. 7C, lane 2), demonstrating the co-immunoprecipitation of SOX9 with SF-1. The 65-kDa proteinwas not detected when SF-1 preimmune serum was used in theprecipitation step (lane 1). As a control, when the unrelatedbut immunoprecipitating rabbit SIP-1 antibody (40) was used

instead of the SF-1 antiserum, no SOX9 protein was detected(lane 3), underlining the specificity of the SOX9 antiserum.Moreover, any potential cross-reactivity of the SF-1 antibodywith SOX9 was ruled out by the unsuccessful immunostainingof in vitro-translated SOX9 protein after blotting (data notshown). The specificity of the interaction between SOX9 andSF-1 was confirmed by the use of different detergent conditionsand the introduction of ethidium bromide into the coimmuno-precipitation assay (Fig. 7D). The reverse experiment (SOX9immunoprecipitation followed by SF-1 Western blotting) wasunsuccessful because of the similarity between the molecularweights of SF-1 and immunoglobulin light chain.

SOX9 and SF-1 colocalize in nuclei of NT2/D1 cells. We nextanalyzed the subcellular localization of the SOX9 and SF-1proteins by performing indirect-immunofluorescence labelingexperiments. The use of purified rabbit anti-SF-1 and rat anti-SOX9 antibodies allowed double-labeling experiments on theNT2/D1 cells. Both antibodies revealed a nuclear, punctuatelocalization for the two proteins, an expression throughout the

FIG. 5. SOX9 and SF-1 interact in vitro. (A) The diagram shows the differentSOX9 deletion mutants which were used to investigate the physical interactionbetween SOX9 and SF-1. In each case, the respective percentage of proteinbound to the GST–SF-1 phase was determined by phosphorimager analysis. (B)The SOX9 polypeptides depicted in panel A were translated in vitro in thepresence of [35S]methionine and analyzed for binding to either GST, GST–SF-1or GST–c-erbA fusion proteins bound to glutathione-Sepharose. The GST pull-down assay was performed as described in Materials and Methods. The 10%input (left lane) and bound proteins (the other lanes) were separated by SDS-PAGE analysis and then autoradiographed. (C) Binding of the GST–SF-1 fusionprotein to the SF-1-binding-site (SF-1-BS) probe (lane 2) is shifted after prein-cubation with in vitro-translated TNT-SOX9 protein as shown by EMSA (lane4). The specificity was assessed by the use of either TNT or TNT plus GST alone(lanes 1 and 3). Lane 5 shows the absence of TNT-SOX9 protein binding to theSF-1-binding-site probe in the presence of 2 mg of poly(dI-dC). WT, wild type.


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cell culture (Fig. 8A and B). Cytoplasmic labeling of the AMHprotein was observed in the same cells (Fig. 8C). We furtheranalyzed the possibility of subcellular colocalization of SOX9with SF-1. A good, if incomplete, colocalization between thetwo proteins was observed after examination by confocal laser-scanning microscopy (Fig. 8F). These results strengthen thenotion that SOX9 and SF-1 interact in vivo, possibly as part ofa multimeric protein complex.

DISCUSSION

In the developing gonad, when the sex-determining factorSry is present, the supporting cell precursors differentiatealong the Sertoli cell pathway. The first known product of theembryonic Sertoli cell is AMH. Initiation, maintenance, anddown-regulation of AMH expression by Sertoli cells still re-main a matter of controversy. If AMH has to be tightly regu-lated during gestation in order to avoid Mullerian duct persis-tence (17), it also requires a sexually dimorphic regulationbecause of its necessary absence in females during embryogen-esis.

The aim of the work described here was to investigate thefunctional relationship between cis-acting conserved elementslocated in the AMH proximal promoter in order to contributeto our understanding of the transcription factors controllingAMH promoter activity and leading to its spatiotemporal reg-ulation during embryogenesis. Sequence analysis showed con-served binding sites for different transcription factors withinthe proximal and minimal 180-bp AMH promoter (13, 43). Inthis study, the functional importance of the nuclear receptor

SF-1 binding site located in the AMH promoter and previouslyrevealed by studies with primary Sertoli cells (43) was con-firmed. However, these results also shed light on the require-ment for an appropriate developmental context or cell envi-ronment for SF-1 activation of AMH, suggesting that a ligandor a cofactor (or both) for SF-1 is needed (43). As mentionedin this and other reports (18, 43, 50), apart from the nowwell-described nuclear receptor SF-1 site, a nearly perfectmatch with the consensus SOX-binding site appears conservedin the promoter of distantly related species. Performing pro-gressive 59 deletions of the AMH proximal promoter revealedthat a deletion of the sequence from 2154 to 2123 whichremoves this SOX-binding site caused a strong decrease in thebasal activity of the AMH proximal promoter after transfectioninto the “Sertoli-like” human NT2/D1 cell line. This result wasconfirmed after mutation of the candidate SOX site. Func-tional in vitro analysis of this site by either DNase I footprint-ing or in vitro DNA-binding experiments showed that thisputative site was able to specifically bind bacterially expressedSOX9 protein. The choice of this particular SOX protein wasdictated by several arguments, including its conservationamong vertebrates and its high level of expression closely cor-relating with Sertoli cell differentiation and subsequent AMHproduction during testicular development in mice and chickens(32) as well as in humans (unpublished data). Furthermore, thesex reversal phenotype observed in campomelic dysplasia pa-tients carrying an SOX9 mutant is consistent with failure ofSertoli cell differentiation and subsequent absence of AMHproduction (31). Finally, of the two SOX proteins, SRY andSOX9, that have been identified as being expressed in the male

FIG. 6. SOX9 and SF-1 form a common protein-DNA complex. Nuclear extracts were prepared from NT2/D1 cells. In an EMSA reaction, a-32P-labeledSF-1-binding-site (SF-1-BS) oligonucleotide and 2 mg of nuclear extract were incubated together. The specificity of the retarded bands was controlled by the use ofeither an excess of cold SF-1-binding-site probe (lane 2) or a mutated form of this oligonucleotide (lane 10). Supershift experiments were performed after preincubationof either 1 ml of SF-1 antibody (SF-1-Ab.) (lane 4) or 1 ml of SOX9 antibody (SOX9-Ab.) (lane 6). The corresponding preimmune serum (SF-1-Pre-imm. andSOX9-Pre-imm.) (lanes 3 and 5) or 1 ml of a specific anti-SRY antibody (SRY-Ab.) (lane 7) was used as the control. The specificity of the observed shifts with thedifferent antibodies was assessed by the use of the corresponding antibody only (lanes 11 and 12). Gel retardation of the TNT–SF-1 protein after binding to theSF-1-binding-site probe was used as a control (lane 8).


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sex-determining pathway, only SOX9 has been shown to act asa potent transcriptional activator in humans via a transactiva-tion domain mapped to its C terminus (30, 46). As suggested byAmbrosetti et al. (1), the requirement for DNA-sequence-specific HMG domain transcription factors such as Sox2 (1,52), Sox5 (7), and TCF-1 (48) in a particular promoter envi-

ronment suggests that they are unable to act autonomouslybut, instead, must collaborate with other transcription factors.Under our experimental conditions with the COS-7 cell line,transient-cotransfection experiments with the SOX9 expres-sion plasmid showed that SOX9 can activate AMH promoteractivity to an extent similar to that for SF-1 (data not shown)even when using a single copy of the AMH proximal promoter.Thus, the kidney-derived cell line COS-7 appears to provide aconvenient AMH promoter environment to test SF-1 or SOX9function. Furthermore, these transactivation experiments alsoconfirmed the strict requirement for the C-terminal domain ofSOX9, implying that AMH promoter activation by SOX9 is notsimply the result of the bending capacity of its HMG domain.In contrast, despite a 71% amino acid similarity to SOX9 inthe HMG domain (10), the SOX transcription factor familyfounder SRY was ineffective in this activation. Because of theability of both SOX9 and SF-1 to cooperate in AMH proximalpromoter activation, it was tempting to investigate if this co-operation involved a protein-protein interaction event. Fivedifferent and complementary approaches, namely, two-hybridanalysis, GST pulldown assays, EMSA, coimmunoprecipitationexperiments, and immunolocalization studies, all indicate thatSOX9 and SF-1 can interact with each other and may be partof a multimeric protein complex. We then performed a pre-liminary mapping of the amino acid sequence responsible forthis interaction. Dissection of the transcription factor SOX9and subsequent coprecipitation assays with GST-SF-1 showedthat the conserved N-terminal region of the SOX9 DNA bind-ing domain is required for its interaction with SF-1. This regionof SOX9 contains several amino acid sequence stretches thatare conserved between different members of the SOX familyand might provide the basis for interaction of SF-1 with othermembers of the family. Interestingly, another Sox gene prod-uct, Sox2, was recently shown to cooperate with the octamer-binding protein Oct-3 in order to synergistically activate thefibroblast growth factor 4 enhancer (1, 52). This activation wasdependent both on the protein-protein interaction involvingthe HMG domain of Sox2 and on the presence of DNA-binding sites for both Sox2 and Oct-3 transcription factors (1).A similar result was obtained with the SOX9/SF-1 couple. Wepropose that SOX9 could, by its DNA-bending activity (30),induce a local architectural modification of the DNA target,allowing the formation of a transcription complex includingSF-1. The SOX9–SF-1 interaction could then stabilize the re-sulting protein-DNA complex. This hypothesis is now underinvestigation. On the other hand, the region of SF-1 that wascharacterized in the two-hybrid experiment described herein asbeing required for the interaction starts after the proline-richregion of the receptor and extends into the ligand-binding anddimerization domain including both activation domains ofSF-1 (6). This region has been previously reported as support-ing the cell specificity of SF-1 activation of AMH reporterconstructs (43) as well as SF-1 activation by oxysterol (29); italso permits interaction with the nuclear receptor DAX-1 (5).However, despite the possible overlap between the two regionsof SF-1 involved in oxysterol activation and SOX9 binding,the SF-1–SOX9 interaction was not modified in the presenceof increasing 25-hydroxycholesterol concentrations (data notshown). The ability of this SF-1 sequence region to interactwith the coactivator SRC-1 was also recently shown (6). Thisinteraction would allow bridging of the transcription factorssuch as SOX9 and SF-1 that govern the tissue-specific expres-sion of AMH and the basal transcriptional machinery.

The absence of AMH expression before 12.5 days postco-itum in the mouse embryo despite coincident expression ofboth SF-1 and Sox9 could be questionable (34). This apparent

FIG. 7. Coimmunoprecipitation of endogenous SOX9 and SF-1 from meta-bolically 35S-labeled NT2/D1 cells. After labeling, equal amounts of NT2/D1 cellextracts (prepared as described in Materials and Methods) were immunopre-cipitated. (A) Immunoprecipitation with SOX9 antibodies (SOX9-Ab.) or pre-immune (pre-im.) antibodies. (B) Immunoprecipitation with SF-1 antibodies(SF-1-Ab.) or preimmune (pre-im.) antibodies. (C) Western blot analysis with aSOX9-specific rat antibody of an SF-1 immunoprecipitate (SF-1-Ab.). As acontrol, an unrelated antibody (unrel-Ab.) was introduced in the precipitationstep before revelation with the SOX9 antibody. First-step immunoprecipitationwith preimmune (pre-im.) antibodies is also shown. (D) Similar experimentscomparing two buffers containing two different detergents, TBST (lanes 1 to 3)or TLB (lanes 4 to 6) (see Materials and Methods), and including or notincluding ethidium bromide. Lanes 1 and 4 show immunoprecipitation withpreimmune (pre-im.) antibodies as controls.


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contradiction with our data could be the result either of theabsence of an elusive partner or of a missing positive regula-tion affecting one of the two transcription factors before thisdevelopmental stage. Another possibility is a regulation of thesubcellular localization of one or more transcription factorscontributing to the control of the onset of AMH expression. Inthis respect, mouse Sox9 expression studies have revealed apredominant cytoplasmic expression in cells of the genitalridge prior to 11.5 days postcoitum, i.e., prior to the sex deter-mination event. At later stages, Sox9 appeared fully nuclear inmale embryonic gonads (32). As recently suggested (45), thechange in Sox9 localization could be achieved via its interac-tion with putative protein partner(s) that would mask the Sox9nuclear localization signal(s). As an alternative, or in parallel,the mechanism used in this cytoplasm-nucleus trafficking couldresult from the existence of a leucine motif in the HMG do-main of Sox9 (10, 47), a motif conserved across SOX9 evolu-tion but also specific for this particular SOX protein (39). Thiskind of motif has been reported in many cases to act as anuclear export signal (49). This hypothesis will not requirefurther investigations.

Other gene products, namely, the Wilms’ tumor WT1 andthe nuclear receptor DAX-1, are also implicated in mamma-lian sexual development. Recently, WT1 in its 2KTS isoformhas been shown to interact and synergize with SF-1 (35). In thesame report, DAX-1 was also shown to antagonize this syn-ergy, even when present at low concentration. The inhibitoryaction of DAX-1 was accompanied by its ability to interactdirectly with the SF-1 ligand-binding domain. Interestingly,SOX9- and DAX-1-binding sites could overlap with respect totheir interaction with SF-1. Furthermore, in the same report,both WT1 and DAX-1 expression levels appear rather similar

between male and female mouse embryos 13.5 days postco-itum, i.e., at a time when AMH expression is on (35). There-fore, the only difference between the sexes remains in theexpression of the putative transcription factors SRY andSOX9. If, as mentioned above, SRY does not provide an at-tractive candidate, the high levels of SOX9 observed specifi-cally in male embryos (32) could compete with DAX-1 for itsbinding to SF-1 and thus could permit WT1 action. This hy-pothesis would justify the rather low level of AMH stimulationobserved in the present data when using SF-1 and SOX9 only.

It is well established that transcriptional regulation of agiven gene is the result of combinatorial interactions betweenmultiple proteins forming a higher-order complex based onprotein-protein and protein-DNA interactions. We now sug-gest that AMH gene regulation is no exception to this generalrule and that both SF-1 and SOX9 are members of the complexregulating the onset of AMH expression during embryogenesisbeyond WT1 and DAX-1. This statement will now requireintroduction of these four transcription factors in the same assayas well as in vivo characterization. The two proteins SOX9 andSF-1 appear to bind independently to separate DNA sites andcould, especially because of the DNA-bending capability ofSOX9 (30), facilitate the functional interaction of other regu-latory proteins, leading to the formation of the appropriatetranscription complex triggering Sertoli-specific AMH expres-sion. Interestingly, another well-conserved binding site in theAMH proximal promoter has homology to the in vitro canon-ical binding site for the GATA transcription factor family,WGATAR (Fig. 1A). Expression of GATA-1 or GATA-2 fac-tors in Sertoli cells reinforces this hypothesis and makesGATA factors attractive candidates to contribute to the regu-

FIG. 8. Immunolocalization of SOX9 and SF-1 protein in cultured human NT2/D1 cells. Immunostaining of SF-1 in green (A and D) and of SOX9 in red (B andE) or both (panel F) is shown. As a control, AMH expression was checked by using the corresponding rabbit antibody (in red) along with a counterstaining of cell nucleiwith Hoechst 33258 in blue (C). Scale bars, 10 mm.


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lation of AMH expression (38, 51), a hypothesis that we arenow attempting to test.

ACKNOWLEDGMENTS

We thank V. Laudet for the gift of the c-erbA construction plasmid,R. Rey for the rabbit anti-AMH antibody, J.-Y. Picard for pEMBL8-AMH plasmid, and A. Goldsborough and V. Laudet for comments onand corrections of the manuscript. We are grateful to Catherine Me-jean for protein production and purification assistance and to SandrineFaure for his constant support.

This investigation was supported by Biomed 2 grant BMH4-CT96-0790 from the European Economic Community.

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direct interaction of sry-related protein sox9 and steroidogenic

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