For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter 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 of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.

Haploinsufficiency for SOX9 has recently been identified as the cause for both campomelic dysplasia (CD), a human skeletal malformation syndrome, and the associated autosomal XY sex reversal. SOX9 contains a putative DNA-binding motif known as the high-mobility group (HMG) domain characterizing a whole class of transcription factors. We show in cell transfection experiments that SOX9 can transactivate transcription from a reporter plasmid through the motif AACAAAG, a sequence recognized by other HMG domain transcription factors. By fusing all or part of SOX9 to the DNA-binding domain of yeast GAL4, the transactivating function was mapped to a transcription activation (TA) domain at the C terminus of SOX9. This non-acidic TA domain is evolutionarily conserved and rich in proline, glutamine and serine. With one exception, all SOX9 nonsense and frame shift mutations described so far in CD/sex reversal patients lead to truncation of the TA domain, suggesting that impairment of gonadal and skeletal development in these cases results, at least in part, from loss of transactivation of genes downstream of SOX9.

Mammalian sex determination and early gonadal differentiation is a developmental process involving a cascade of regulatory gene interactions. Only a few of these genes, all encoding transcription factors, are known (reviewed in Ref. 1), among them the related genes SRY and SOX9. SRY encodes the Ychromosomal testis-determining factor as shown by XY sex reversal in human individuals mutant for SRY (2, 3) and by the demonstration of testis formation in chromosomally female mice transgenic for mouse Sry (4). SOX9 on chromosome 17 is an autosomal gene essential for testis development as mutations in and around this gene cause XY sex reversal in association with the skeletal malformation syndrome campomelic dysplasia (5, 6).Both SRY and SOX9 contain an 80-amino acid DNA-binding motif known as the high-mobility group (HMG) 1 domain that characterizes a whole class of transcription factors (reviewed in Ref. 7). SRY binds to the sequence AACAAT and variants thereof (8) and induces a sharp bend in the DNA (9). The three-dimensional solution structure of the SRY HMG domain complexed with its target sequence has been solved (10), as has a similar complex of the related factor LEF-1 (11). In cell transfection studies, some evidence for transcriptional activation of testis-specific genes by SRY has been presented (12). We have shown in similar transfection assays that SOX9 also functions as a transcription factor, contains a C-terminal transactivation domain (13) and can bind via its HMG domain to the motif AACAAT (14). Recently, mouse Sox9 was found to be expressed in the gonadal anlage of both sexes, with expression increasing in the developing testis and decreasing in the developing ovary, consistent with a role for SOX9/Sox9 in Sertoli cell differentiation (15, 16).As transcription factors, SRY and SOX9 must gain access to the nucleus. Studies on nuclear localization indicate that transport across the nuclear envelope is an active process mediated by one or more nuclear localization signal sequences (NLSs), usually present in the protein itself or in a cofactor (for review, see Refs. 17 and 18). With some exceptions, two main types of NLS motifs exist. One is a short cluster of mainly basic amino acids (arginine and/or lysine), its prototype found in the simian virus 40 large tumor antigen (19). The other is a bipartite NLS motif that comprises two basic amino acids, a spacer of about 10 -15 residues consisting of any amino acid, followed by generally three basic residues, as first described for nucleoplasmin (17). Specialized NLS-binding transporter proteins that carry NLS-containing proteins through the nuclear pore complex into the nucleus have been identified recently (20).Karyophilic NLS sequences are generally identified by their ability to direct an otherwise cytoplasmic protein to the nucleus when fused to it genetically or biochemically and by the effects of deletion or point mutations on nuclear entry (18). Using these approaches with ␤-galactosidase as a reporter protein, we have identified two independent NL...

The SOX genes form a gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. We have cloned and sequenced the SOX10 and Sox10 genes from human and mouse, respectively. Both genes encode proteins of 466 amino acids with 98% sequence identity. Significant expression of the 2.9-kb human SOX10 mRNA is observed in fetal brain and in adult brain, heart, small intestine and colon. Strong expression of Sox10 occurs throughout the peripheral nervous system during mouse embryonic development. SOX10 shows an overall amino acid sequence identity of 59% to SOX9. Like SOX9, SOX10 has a potent transcription activation domain at its C-terminus and is therefore likely to function as a transcription factor. Whereas SOX9 maps to 17q, a SOX10 cosmid has previously been mapped by us to the region 22q13.1. Mutations in SOX10 have recently been identified as one cause of Waardenburg-Hirschsprung disease in humans, while a Sox10 mutation underlies the mouse mutant Dom, a murine Hirschsprung model.

Sex determination in mammals is controlled by various transcription factors. Following the identification of SRY on the Y chromosome, several other factors have been identified. They can normally be identified as being involved in sex determination by the identification of sex reversal mutations or deletions, functional studies, and also by male-specific expression patterns in embryos. Here, it is shown that DMRT1, recently demonstrated to be deleted in 9p monosomies associated with sex reversal, is specifically expressed during sex determination in the genital ridge of human male, but not female, embryos, similar to SRY.