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V R Harley

Publications and source records attributed to V R Harley.

At least 19 recordsLinked to original sources
PubMed2003

Turning on the male--SRY, SOX9 and sex determination in mammals.

The decision of the bi-potential gonad to develop into either a testis or ovary is determined by the presence or absence of the Sex-determining Region gene on the Y chromosome (SRY). Since its discovery, almost 13 years ago, the molecular role that SRY plays in initiating the male sexual development cascade has proven difficult to ascertain. While biochemical studies of clinical mutants and mouse genetic models have helped in our understanding of SRY function, no direct downstream targets of SRY have yet been identified. There are, however, a number of other genes of equal importance in determining sexual phenotype, expressed before and after expression of SRY. Of these, one has proven of central importance to mammals and vertebrates, SOX9. This review describes our current knowledge of SRY and SOX9 structure and function in the light of recent key developments.

Animals↗
PubMed2003

Forward mandibular positioning up-regulates SOX9 and type II collagen expression in the glenoid fossa.

Regulatory factors governing the formation of bone in the glenoid fossa in response to functional appliance therapy have not been identified. Therefore, the purpose of this study was to investigate the temporal pattern of expression of two key chondrogenesis markers-SOX9 and its target gene, type II collagen-in the glenoid fossa by immunostaining in a 35-day-old Sprague Dawley rat model during both natural growth and forward mandibular positioning. The expression of both factors was up-regulated when the mandible was positioned forward, indicating an enhancement of chondrocyte differentiation and chondroid matrix formation. Our results indicate that chondroid bone formation in the glenoid fossa in response to forward mandibular positioning is regulated by molecular markers indicative of endochondral ossification.

Analysis of Variance↗
PubMed2001

Localisation of the SRY-related HMG box protein, SOX9, in rodent brain.

Human mutations in the transcription factor gene, SOX9, cause campomelic dysplasia (CD), a severe dwarfism associated with brain abnormalities including dilation of lateral ventricles, hypoplasia of the corpus callosum and cerebellum defects. To improve our understanding of how SOX9 contributes to the molecular genetic pathway of brain development we sought to investigate the distribution of SOX9 protein in rat and mouse brain. The regions of SOX9 expression identified in this study correlated with the sites of reported brain abnormalities in CD patients. SOX9 immunoreactivity was observed in nuclei of scattered cells throughout the brain, in the ependymal layer and cells of the choroid plexus. In the forebrain most SOX9-immunoreactive nuclei co-localised with the glial astrocyte marker S-100. In the cerebellum, SOX9 was observed mostly in cells surrounding Purkinje cells, which were identified, by electron microscopy, as Golgi epithelial cells, also known as Bergmann glia. Using SOX9 antibody as a marker for the precursors of Bergmann glia, we traced their origin during mouse development. At embryonic day (E)14.5 and E16.5, SOX9 immunoreactivity was present mainly in the primordial choroid plexus, and ventricular zone. By E18.5, SOX9 was observed in the granular cell and Purkinje cell layers but no labelling was detectable in the external granular layer. These results suggest that SOX9 immunoreactivity is a marker for Bergmann cells during development and favour the proposed origin of the secondary glial scaffold arising from Bergmann cells derived exclusively from the ventricular zone.

Animals↗
PubMed2001

Identification of an interaction between SOX9 and HSP70.

The campomelic dysplasia/autosomal sex reversal protein SOX9 is an important developmental transcription factor, required for correct bone and testis formation. Through in vitro and in vivo studies we have identified the heat shock protein HSP70 as an interacting partner for SOX9 in chondrocyte and testicular cell lines. HSP70 forms a ternary complex with DNA-bound SOX9. The interaction between HSP70 and SOX9 is ATP-independent and involves a highly conserved region of SOX9 hitherto of unknown function and the C-terminal region of HSP70. Our results implicate HSP70-SOX9 interactions in the assembly of multi-protein complexes during SOX9-mediated transcription.

Adenosine Triphosphate↗
PubMed2001

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.

Abnormalities, Multiple↗
PubMed2001

Temperature regulates SOX9 expression in cultured gonads of Lepidochelys olivacea, a species with temperature sex determination.

Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.

Amino Acid Sequence↗
PubMed2001

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.

Autoantigens↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Sox9 protein in rat sertoli cells is age and stage dependent.

We studied the location of Sox9 protein in the embryonic, juvenile, and adult rat testis by immunohistochemistry and immunoblotting. Sox9 belongs to a family of Sox proteins that are transcription factors and important in several developmental processes. In the incipient embryonic testis, Sox9 was prominently present in the gonadal blastema. With further embryonic differentiation, Sox9-positive cells arranged in the periphery of the testicular cords, showing the location of the Sertoli cells. Thereafter the immunoreaction for Sox9 gradually declined and was only weakly detectable in the 2-day-old postnatal rat testis. This situation remained for some period of time. In the 15-day-old rat testis, Sox9 protein strongly reappeared in the testicular cords. In the adult, the Sertoli cells of most regions of the seminiferous tubules were positive for Sox9. The strongest reaction for Sox9 was found in the dark zone. However, clearly negative or only weakly positive spermatogenic stages for the protein were also found, as seen for example in the pale zone. In fertile 1-year-old rats this basic situation was still detectable. Analyzed rat ovaries were all negative for Sox9, showing the sex-specific nature of Sox9. The results showed that Sox9 protein is distinctly present in the developing and mature Sertoli cells, but that its presence and amount is dependent on the age and the spermatogenetic stage within the seminiferous tubuli. The prominent presence of Sox9 in the incipient testis and at puberty suggests that this protein is needed at important phases of aggregation and reorganization of the Sertoli cells. The age and stage-specific presence of Sox9 in the testicular cords and in the seminiferous tubules of the adult suggests that Sox9 also may have a pivotal role in germ cell differentiation.

Aging↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

Differential expression of SOX9 in gonads of the sea turtle Lepidochelys olivacea at male- or female-promoting temperatures.

In mouse and chick embryos, the SOX9 gene is down-regulated in genetic females whereas in genetic males it remains in the Sertoli cells. We studied the distribution of SOX9 protein in developing genital ridges of embryos of the sea turtle Lepidochelys olivacea incubated at male- or female-promoting temperatures, using the antibody for detection. At stages 22-24, cells in medullary cords show SOX9 positive nuclei, while coelomic epithelial cells appear negative. At stage 25 however, most medullary cells are SOX9 negative and at the female-promoting temperature, and from stage 26 onwards, SOX9 protein is not detected. At the male-promoting temperature, medullary cords remain SOX9-positive at all stages. These results suggest that SOX9 is up-regulated in Sertoli cells irrespective of primary sex-determining switch. Sex is irreversibly determined at stage 24 or 26 at the male- or female-promoting temperature, respectively (Merchant-Larios et al.,'97). The present results suggest that there is a correlation between SOX9 expression and sex determination in the olive ridley. At the male-promoting temperature, Sertoli cells expressing SOX9 become committed at stage 24 and male sex is determined, whereas at the female-promoting temperature, SOX9 is down-regulated at stage 26 and female sex is determined. J. Exp. Zool. 284:705-710, 1999.

Amino Acid Sequence↗
PubMed1999

The DNA-binding specificity of SOX9 and other SOX proteins.

SOX (SRY-related HMG box) proteins are transcription factors that have critical roles in the regulation of numerous developmental processes. They share at least 50% homology in their HMG domains, which bind the DNA element AACAAT. How different SOX proteins achieve specific regulation of target genes is not known. We determined the DNA-binding specificity of SOX9 using a random oligonucleotide selection assay. The optimal SOX9 binding sequence, AGAACAATGG, contained a core DNA-binding element AACAAT, flanked by 5' AG and 3' GG nucleotides. The specific interaction between SOX9 and AGAACAATGG was confirmed by mobility shift assays, DNA competition and dissociation studies. The 5' AG and 3' GG flanking nucleotides enhance binding by SOX9 HMG domain, but not by the HMG domain of another SOX factor, SRY. For SRY, different 5' and 3' flanking nucleotides are preferred. Our studies support the notion that SOX proteins achieve DNA sequence specificity through subtle preferences for flanking nucleotides and that this is likely to be dictated by signature amino acids in their HMG domains. Furthermore, the related HMG domains of SOX9 and Sox17 have similar optimal binding sites that differ from those of SRY and Sox5, suggesting that SOX factors may co-evolve with their DNA targets to achieve specificity.

Base Sequence↗
PubMed1997

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.

3T3 Cells↗
PubMed1996

The HMG box of SRY is a calmodulin binding domain.

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. By affinity chromatography, native gel electrophoresis and fluorescence spectroscopy, we show the calcium-dependent binding of SRY to CaM. Binding occurs via the HMG box and an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and we propose that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors.

Amino Acid Sequence↗
PubMed1995

Interaction of normal and mutant SRY proteins with DNA.

In mammals, sex determination is caused by the Y-chromosome gene SRY. The DNA-binding domain of human SRY protein is similar to those of the chromatin protein HMG1. Like HMG1, SRY binds to kinked DNA structures, and bends linear DNA sharply upon binding. We analysed the biochemical properties of mutant SRY proteins from five patients with complete gonadal dysgenesis: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally, and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence, thus discriminating different target sites. We suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY in chromatin is essential for the expression of genes involved in testis differentiation.

Base Sequence↗
PubMed1994

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.

Base Sequence↗