SOX10 structure-function analysis in the chicken neural tube reveals important insights into its role in human neurocristopathies.

The HMG-domain containing transcription factor Sox10 is essential for neural crest (NC) development and for oligodendrocyte differentiation. Heterozygous SOX10 mutations in humans lead to corresponding defects in several NC-derived lineages and to leukodystrophies. Disease phenotypes range from Waardenburg syndrome and Waardenburg-Hirschsprung disease to Peripheral demyelinating neuropathy, Central dysmyelination, Waardenburg syndrome and Hirschsprung disease (PCWH). The phenotypic variability can partly be explained by the action of modifier genes, but is also influenced by the mutation that leads to haploinsufficiency in some and to mutant SOX10 proteins with altered properties in other cases. Here, we used in ovo electroporation in the developing neural tube of chicken to determine which regions and properties of SOX10 are required for early NC development. We found a strict reliance on the DNA-binding activity and the presence of the C-terminal transactivation domain and a lesser influence of the dimerization function and a conserved domain in the center of the protein. Intriguingly, dominant-negative effects on early NC development were mostly observed for truncated SOX10 proteins whose production in patients is probably prevented by nonsense-mediated decay. In contrast, mutant SOX10 proteins that occur in patients were usually inactive. Any dominant negative activity which some of these mutants undoubtedly possess must, therefore, be restricted to single NC-derived cell lineages or oligodendrocytes at later times. This contributes to the phenotypic variability of human SOX10 mutations.

Activation of Krox20 gene expression by Sox10 in myelinating Schwann cells.

The high-mobility group domain transcription factor Sox10 is believed to influence myelination in Schwann cells by directly activating myelin genes and by inducing Krox20 as a pivotal regulator of peripheral myelination. Krox20 induction at this stage is thought to be mediated by the myelinating Schwann cell element 35 kb downstream of the Krox20 transcriptional start site and requires cooperation with Oct6. Here, we prove for the first time in vivo that Schwann cell-specific Krox20 expression indeed depends on Sox10. We also provide evidence that Sox10 functions through multiple, mostly monomeric binding sites in the myelinating Schwann cell element in a manner that should render the enhancer exquisitely sensitive to Sox10 levels. Synergistic activation of the enhancer by Sox10 and Oct6 furthermore does not involve cooperative binding to closely spaced binding sites in defined composite elements. Nevertheless, the POU domain of Oct6 and the high-mobility group domain of Sox10 as the two DNA-binding domains were both essential indicating that each transcription factor has to bind independently to DNA. Whereas the POU domain was the only important region of Oct6, two further Sox10 domains were required for synergistic Krox20 activation. These were the carboxyterminal transactivation domain and the conserved K2 domain in the central portion of Sox10. All required regions are conserved in several closely related POU and Sox proteins thus explaining why Oct6 and Sox10 can be replaced by their relatives during Krox20 induction in myelinating Schwann cells.

Replacement of related POU transcription factors leads to severe defects in mouse forebrain development.

Related transcription factors of the POU protein family show extensive overlap of expression in vivo and exhibit very similar biochemical properties in vitro. To study functional equivalence of class III POU proteins in vivo, we exchanged the Oct-6 gene by Brn-1 in the mouse. Brn-1 can fully replace Oct-6 in Schwann cells and rescue peripheral nervous system development in these mice. The same mice, however, exhibit severe defects in forebrain development arguing that Oct-6 and Brn-1 are not functionally equivalent in the central nervous system. The cause of the observed forebrain phenotype is complex, but anteriorly expanded Wnt1 expression contributes. Oct-6 normally represses Wnt1 expression in the early diencephalon and replacement by Brn-1 as a weaker inhibitor is no longer sufficient to maintain the necessary level of repression in the mouse mutant. The extent of functional equivalence between related transcription factors is thus strongly dependent on the analyzed tissue.