Sex-determining region Y-related protein SOX13 is a diabetes autoantigen expressed in pancreatic islets.

The SOX (sex-determining region [SRY]-type high mobility group [HMG] box) family of transcription factors play key roles in determining cell fate during organ development. In this study, we have identified a new human SOX gene, SOX13, as encoding the type 1 diabetes autoantigen, islet cell antigen 12 (ICA12). Sequence analysis showed that SOX13 belongs to the class D subgroup of SOX transcription factors, which contain a leucine zipper motif and a region rich in glutamine. SOX13 autoantibodies occurred at a significantly higher frequency among 188 people with type 1 diabetes (18%) than among 88 with type 2 diabetes (6%) or 175 healthy control subjects (4%). Deletion mapping of the antibody epitopes showed that the autoantibodies were primarily directed against an epitope requiring the majority of the protein. SOX13 RNA was detected in most human tissues, with the highest levels in the pancreas, placenta, and kidney. Immunohistochemistry on sections of human pancreas identified SOX13 in the islets of Langerhans, where staining was mostly cytoplasmic. In mouse pancreas, Sox13 was present in the nucleus and cytoplasm of beta-cells as well as other islet cell types. Recombinant SOX13 protein bound to the SOX consensus DNA motif AACAAT, and binding was inhibited by homodimer formation. These observations-along with the known molecular interactions of the closely related protein, rainbow trout Sox23-suggest that SOX13 may be activated for nuclear import and DNA binding through heterodimer formation. In conclusion, we have identified ICA12 as the putative transcription factor SOX13 and demonstrated an increased frequency of autoantibody reactivity in sera from type 1 diabetic subjects compared with type 2 diabetic and healthy control subjects.

Understanding alpha-globin gene regulation: Aiming to improve the management of thalassemia.

Over the past 50 years, many advances in our understanding of the general principles controlling gene expression during hematopoiesis have come from studying the synthesis of hemoglobin. Discovering how the alpha- and beta-globin genes are normally regulated and documenting the effects of inherited mutations that cause thalassemia have played a major role in establishing our current understanding of how genes are switched on or off in hematopoietic cells. Previously, nearly all mutations causing thalassemia have been found in or around the globin loci, but rare inherited and acquired trans-acting mutations are being found more often. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression that, in turn, may open up new avenues for improving the management of patients with common types of thalassemia.

Genomic characterisation and fine mapping of the human SOX13 gene.

SOX13 is the member of the SOX (Sry related HMG BOX) family of transcription factors which encodes the type-1 diabetes autoantigen, ICA12, and is expressed in a number of tissues including pancreatic islets and arterial walls. By fluorescence in situ hybridisation, radiation hybrid mapping and YAC analysis we determined that the human SOX13 gene maps to Chromosome 1q31.3-32.1 near the marker D1S504, a region associated with type-1 diabetes susceptibility and familial dilated cardiomyopathy. Mouse Sox13 maps to the syntenic region near the marker D1Mit57. The human SOX13 gene spans >15.5kb of genomic DNA and is composed of 14 exons with introns interrupting regions encoding the HMG DNA binding domain and the leucine zipper/glutamine-rich dimerisation domain. Comparison with the mouse Sox13 gene suggests the existence of long and short forms of the SOX13 protein which may arise by differential splicing during different stages in embryogenesis. The high sequence conservation between human SOX13 and mouse, Xenopus and trout orthologues implies a conserved function in vertebrates. SOX13 belongs to SOX Group D members which contain a leucine zipper/glutamine-rich region. Phylogenetic analyses of SOX proteins suggest that such domains were acquired after the initial divergence of groups A to G.

Linkage studies of SOX13, the ICA12 autoantigen gene, in families with type 1 diabetes.

SOX13 is a member of the SOX family of transcription factors that encodes the type 1 diabetes autoantigen, ICA12. The SOX13 gene maps at chromosome 1q31.3-32.1 near a region containing a susceptibility locus for type 1 diabetes. SOX13 was assessed as a candidate susceptibility gene. Analysis of the SOX13 gene identified a number of single nucleotide polymorphisms and a polymorphic CA dinucleotide repeat. Linkage and association studies indicate that SOX13 is unlikely to make a substantial contribution to type 1 diabetes susceptibility.

Functional and structural studies of wild type SOX9 and mutations causing campomelic dysplasia.

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.

Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation.

Human mutations in the transcription factor SOX9 cause campomelic dysplasia/autosomal sex reversal. Here we identify and characterize two novel heterozygous mutations, F154L and A158T, that substitute conserved "hydrophobic core" amino acids of the high mobility group domain at positions thought to stabilize SOX9 conformation. Circular dichroism studies indicated that both mutations disrupt alpha-helicity within their high mobility group domain, whereas tertiary structure is essentially maintained as judged by fluorescence spectroscopy. In cultured cells, strictly nuclear localization was observed for wild type SOX9 and the F154L mutant; however, the A158T mutant showed a 2-fold reduction in nuclear import efficiency. Importin-beta was demonstrated to be the nuclear transport receptor recognized by SOX9, with both mutant proteins binding importin-beta with wild type affinity. Whereas DNA bending was unaffected, DNA binding was drastically reduced in both mutants (to 5% of wild type activity in F154L, 17% in A158T). Despite this large effect, transcriptional activation in cultured cells was only reduced to 26% in F154L and 62% in A158T of wild type activity, suggesting that a small loss of SOX9 transactivation activity could be sufficient to disrupt proper regulation of target genes during bone and testis formation. Thus, clinically relevant mutations of SOX9 affect protein structure leading to compound effects of reduced nuclear import and reduced DNA binding, the net effect being loss of transcriptional activation.