Einfluss von Sox-Proteinen auf transkriptionelle Elongation und Homöostase in Säuger-Gliazellen

Zusammenfassung Die Entwicklung der Myelin-bildenden Gliazellen des zentralen (ZNS) und peripheren Nervensystems (PNS) in Vertebraten wird durch ein Netzwerk an Transkriptionsfaktoren gesteuert, in dem verschiedene Sox-Proteine wie Sox10 und Sox2 essentielle Funktionen besitzen. Während bekannt ist, dass Sox-Proteine dabei die Initiationsphase der Transkription beeinflussen, wurde in dieser Arbeit erstmalig über Luziferase-Aktivitätstests gezeigt, dass Sox2 und Sox10 auch die Phase der transkriptionellen Elongation stimulieren. Sie rekrutieren den positiven Transkriptions-Elongationsfaktor b (P-TEFb) zur pausierenden RNA-Polymerase II und begünstigen eine Wiederaufnahme der Transkription. Eine solche Regulation der Sox-vermittelten Genexpression auf Ebene der Elongation wurde beispielhaft für die Myelingene Mpz, Connexin-32 und Mbp in Schwann-Zellen nachgewiesen. Interaktionsstudien ergaben, dass die physikalische Rekrutierung von P-TEFb über die Transaktivierungsdomäne der beiden Sox-Proteine erfolgt und auf Seiten von P-TEFb unterschiedliche, zentral gelegene Bereiche der Cyclin T1-Einheit erfordert. Ausführlich analysiert sind die Aufgaben von Sox10 während der Entwicklung, terminalen Differenzierung von oligodendroglialen Zellen und der damit einhergehenden Initiation der Myelinisierung. Dagegen ist der Einfluss von Sox10 auf den Erhalt des Myelins bisher ungeklärt. Aus diesem Grund wurden in dieser Arbeit die Auswirkungen einer postnatalen Sox10-Deletion in reifen, Myelin-bildenden Oligodendrozyten des ZNS der Maus untersucht. Die Analyse der Mausmutante ergab keine Veränderungen der Motorik und des Verhaltens. Auch Zahl und Verteilung von oligodendroglialen Zellen und Myelingen-Transkripten im Rückenmark waren unverändert. Sox8/Sox10-doppelt-defiziente Mutanten hingegen zeigten starke motorische Einschränkungen bis zu ihrem verfrühten Tod im Alter von zwei Monaten. Die Ursache lag in einem Myelinisierungsdefekt begründet, der mit einer starken Reduktion der Expression des oligodendroglialen Transkriptionsfaktors Myrf und der Myelingene Mbp und Plp einhergeht. Die Ergebnisse dieser Analyse lassen den Schluss zu, dass in ausgereiften Oligodendrozyten Sox8 und Sox10 redundant agieren und gemeinsam für den Erhalt des Myelins verantwortlich sind. Diese Hypothese könnte an einem in dieser Arbeit erzeugten Mausmodell weiter bestätigt werden, in dem das normale Sox10 durch eine möglicherweise dominant wirkende Sox10-Mutante ersetzt wurde, die im Menschen zentralnervöse Dysmyelinisierung auslöst.Summary Development of myelin forming glial cells of the central and peripheral nervous systems in vertebrates is controlled by a network of transcription factors. In this network several Sox proteins, for example Sox2 and Sox10, play essential roles and are known to influence initiation of transcription. In this thesis, it could be shown for the first time using luciferase assays that Sox2 and Sox10 also stimulate transcriptional elongation. Both Sox proteins recruit the positive transcription elongation factor b (P-TEFb) to paused RNA polymerase II to activate the transition into productive elongation. This regulation of Sox-mediated gene expression during elongation was established for the myelin genes Mpz, connexin-32 and Mbp in Schwann cells. Interaction studies indicate that the physical recruitment of P-TEFb is mediated by the C-terminal transactivation domain of the Sox proteins and by different central regions of the Cyclin T1 subunit of P-TEFb. The function of Sox10 during development and terminal differentiation as well as during initiation of myelination of oligodendroglial cells has been described in detail. However, the role of Sox10 in myelin maintenance has not been analysed before. Therefore, the effect of a postnatal Sox10 deletion in mature myelin producing oligodendrocytes of the CNS was studied in this thesis. The analysis of corresponding mutant mice showed no obvious impairement of motor function and behavior. In addition, number and distribution of oligodendroglial cells and myelin gene transcripts in the spinal cord was unaffected. In contrast, Sox8/Sox10 double deficient mutants exhibited strong motor disturbances which led to an early death at the age of two month. This phenotype is caused by a myelination defect concomitant with a strong reduction in the expression of the oligodendroglial transcription factor Myrf and the myelin genes Mbp and Plp. The results of this analysis allow the conclusion that in mature oligodendrocytes Sox8 and Sox10 act in a redundant manner and are jointly responsible for myelin maintenance. To confirm this hypothesis a new mouse model was generated during this thesis where normal Sox10 was replaced by a putative dominant Sox10 mutant. In human patients this mutation leads to central dysmyelination

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

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

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