Analysis of the human SOX10 mutation Q377X in mice and its implications for genotype-phenotype correlation in SOX10-related human disease.

Human SOX10 mutations lead to various diseases including Waardenburg syndrome, Hirschsprung disease, peripheral demyelinating neuropathy, central leukodystrophy, Kallmann syndrome and various combinations thereof. It has been postulated that PCWH as a combination of Waardenburg and Hirschsprung disease, peripheral neuropathy and central leukodystrophy is caused by heterozygous SOX10 mutations that result in the presence of a dominantly acting mutant SOX10 protein in the patient. One such protein with postulated dominant action is SOX10 Q377X. In this study, we generated a mouse model, in which the corresponding mutation was introduced into the Sox10 locus in such a way that Sox10 Q377X is constitutively expressed. Heterozygous mice carrying this mutation exhibited pigmentation and enteric nervous system defects similar to mice in which one Sox10 allele was deleted. However, despite presence of the mutant protein in Schwann cells and oligodendrocytes throughout development and in the adult, we found no phenotypic evidence for neurological defects in peripheral or central nervous systems. In the nervous system, the mutant Sox10 protein did not act in a dominant fashion but rather behaved like a hypomorph with very limited residual function. Our results question a strict genotype-phenotype correlation for SOX10 mutations and argue for the influence of additional factors including genetic background.

Sox8 and Sox10 jointly maintain myelin gene expression in oligodendrocytes.

In Schwann cells of the vertebrate peripheral nervous system, induction of myelination and myelin maintenance both depend on the HMG-domain-containing transcription factor Sox10. In oligodendrocytes of the central nervous system, Sox10 is also essential for the induction of myelination. Its role in late phases of myelination and myelin maintenance has not been studied so far. Here, we show that these processes are largely unaffected in mice that lack Sox10 in mature oligodendrocytes. As Sox10 is co-expressed with the related Sox8, we also analyzed oligodendrocytes and myelination in Sox8-deficient mice. Again, we could not detect any major abnormalities. Expression of many myelin genes was only modestly reduced in both mouse mutants. Dramatic reductions in expression levels and phenotypic disturbances became only apparent once Sox8 and Sox10 were both absent. This argues that Sox8 and Sox10 are jointly required for myelin maintenance and impact myelin gene expression. One direct target gene of both Sox proteins is the late myelin gene Mog. Our results point to at least partial functional redundancy between both related Sox proteins in mature oligodendrocytes and are the first report of a substantial function of Sox8 in the oligodendroglial lineage.

Transcription factors Sox10 and Sox2 functionally interact with positive transcription elongation factor b in Schwann cells.

Sox proteins are mechanistically versatile regulators with established relevance to different developmental processes and crucial impact on chromatin structure, DNA conformation, and transcriptional initiation. Here, we show that Sox2 and Sox10, two Sox proteins important for Schwann cell development, also have the capability to activate transcriptional elongation in a Schwann cell line by recruiting the positive transcription elongation factor b. Recruitment is mediated by physical interaction between the carboxyterminal transactivation domains of the two Sox proteins and the Cyclin T1 subunit of positive transcription elongation factor b, with interaction interfaces for the two Sox proteins being mapped to adjacent regions of the central part of Cyclin T1. Supporting the relevance of this interaction to Schwann cell development, transcription of myelin genes appears regulated at the level of elongation. Our results thus add a new facet to the activity of Sox proteins and expand the functional repertoire of this important group of developmental regulators. Sox transcription factors are important regulators of nervous system development. While they are known to regulate transcription by recruiting and stabilizing the RNA polymerase II preinitiation complex directly or with help of the Mediator complex, this study provides evidence that Sox10 and Sox2 additionally influence transcription in glial cells at the elongation stage by recruiting P-TEFb. Cdk9, cyclin-dependent kinase 9; P-TEFb, positive transcription elongation factor b; Pol II, RNA polymerase II; Sox, Sox2 or Sox10 protein.

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption.

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.