Despite 12 yr since the discovery of SRY, little is known at the molecular level about how SRY and the SRY-related protein, SOX9 [SRY-related high-mobility group (HMG) box 9], initiate the program of gene expression required to commit the bipotential embryonic gonad to develop into a testis rather than an ovary. Analysis of SRY and SOX9 clinical mutant proteins and XX mice transgenic for testis-determining genes have provided some insight into their normal functions. SRY and SOX9 contain an HMG domain, a DNA-binding motif. The HMG domain plays a central role, being highly conserved between species and the site of nearly all missense mutations causing XY gonadal dysgenesis. SRY and SOX9 are architectural transcription factors; their HMG domain is capable of directing nuclear import and DNA bending. Whether SRY and SOX9 activate testis-forming genes, repress ovary-forming genes, or both remains speculative until downstream DNA target genes are identified. However, factors that control SRY and SOX9 gene expression have been identified, as have a dozen sex-determining genes, allowing some of the pieces in this molecular genetic puzzle to be connected. Many genes, however, remain unidentified, because in the majority of cases of XY females and in all cases of XX males lacking SRY, the mutated gene is unknown.

The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with ␣-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal ␣-helix that pack together to form a single globular domain. Interestingly, the ␣-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.ATR-X syndrome ͉ NMR structure ͉ zinc finger A TRX was identified when the gene encoding this protein was shown to be mutated in a form of X-linked mental retardation (ATR-X syndrome) in young males (1, 2). Furthermore, null mutations in mice are lethal at the embryonic stage of development (3). Because ATRX mutations reduce ␣-globin synthesis, causing ␣-thalassemia, it seems likely that ATRX normally plays a role in the regulation of globin gene expression (2, 4). The complexity of the disease also suggests that ATRX could be involved in the regulation of other as yet unidentified genes. ATRX is a large (2,492 residue; Ϸ280 kDa) nuclear protein predominantly localized to heterochromatin and nuclear PML bodies (5, 6). It contains two highly conserved domains, and missense mutations that give rise to ATR-X syndrome fall within these. At the C terminus is a helicase/ATPase domain, which characterizes ATRX as a member of the SNF2 (SWI/SNF) family of chromatin-associated proteins. Experimental evidence shows that ATRX acts as a DNA-dependent ATPase and as a DNA translocase, and it confers modest chromatin-remodeling activity in vitro (6). Thus, it seems likely that ATRX exerts its function by targeting chromatin.Of the missense mutations identified in the ATRX gene, 50% are located in the N terminus of the ATRX protein, which represents just 4% of the coding sequence (Fig. 1a) (7). This region is highly cysteine-rich and contains two different types of zinc-finger motif. It was first noticed that a region of the sequence shares homology with the plant homeodomain (PHD)-type zinc fingers (8). Mutations in other PHD-containing proteins (WSTF and AIRE) are also associated with human disease (9, 10). PHD fingers are found in nuclear proteins, and a...

In humans, mutations in SOX9 result in a skeletal malformation syndrome, campomelic dysplasia (CD). The present study investigated two major classes of CD mutations: 1) point mutations in the high mobility group (HMG) domain and 2) truncations and frameshifts that alter the C terminus of the protein. We analyzed the effect of one novel mutation and three other point mutations in the HMG domain of SOX9 on the DNA binding and DNA bending properties of the protein. The F12L mutant HMG domain shows negligible DNA binding, the H65Y mutant shows minimal DNA binding, whereas the A19V mutant shows near wild type DNA binding and bends DNA normally. Interestingly, the P70R mutant has altered DNA binding specificity, but also bends DNA normally. The effects of the point mutations were interpreted using a molecular model of the SOX9 HMG domain. We analyzed the effects upon transcription of mutations resembling the truncation and frameshift mutations in CD patients, and found that progressive deletion of the C terminus causes progressive loss of transactivation. Maximal transactivation by SOX9 requires both the C-terminal domain rich in proline, glutamine, and serine and the adjacent domain composed entirely of proline, glutamine, and alanine. Thus, CD arises by mutations that interfere with DNA binding by SOX9 or truncate the C-terminal transactivation domain and thereby impede the ability of SOX9 to activate target genes during organ development.In humans, mutations in SOX9 cause campomelic dysplasia (CD), 1 a skeletal malformation syndrome that is often associated with XY sex reversal (1). Other tissues affected include kidney, heart, and brain, consistent with the expression pattern of Sox9 in developing mouse (2, 3). There are four major classes of mutations causing CD: 1) amino acid substitutions in the HMG domain (Fig. 1A), 2) truncations or frameshifts that alter the C terminus of SOX9 (Fig. 1B), 3) mutations at splice junctions, and 4) chromosomal translocations, of which classes 1 and 2 are investigated here. Most CD patients are heterozygous for wild type and mutant alleles of SOX9. CD appears to result from haploinsufficiency; presumably, a critical dose of SOX9 is required to switch on the appropriate genes during development. The present study reports the identification in a CD patient of a novel amino acid substitution mutation (H65Y) in the HMG domain of SOX9. We report the effects of this and three other point mutations (F12L, A19V, and P70R) on the DNA binding and DNA bending activities of the HMG domain.SOX proteins represent a large class of transcription factors related to SRY, the testis-determining factor, through their HMG domains that bind and bend DNA in a sequence-specific manner. Expression of these proteins in defined cell types at specific stages of development appears to govern cell fate decisions. SOX9 activates expression of type II and type XI collagen in vivo (4 -6), consistent with a role in bone development.SOX proteins fall within a larger group of HMG domain proteins comprising two clas...