Olig2 regulates Sox10 expression in oligodendrocyte precursors through an evolutionary conserved distal enhancer

The HMG-domain transcription factor Sox10 is expressed throughout oligodendrocyte development and is an important component of the transcriptional regulatory network in these myelin-forming CNS glia. Of the known Sox10 regulatory regions, only the evolutionary conserved U2 enhancer in the distal 5′-flank of the Sox10 gene exhibits oligodendroglial activity. We found that U2 was active in oligodendrocyte precursors, but not in mature oligodendrocytes. U2 activity also did not mediate the initial Sox10 induction after specification arguing that Sox10 expression during oligodendroglial development depends on the activity of multiple regulatory regions. The oligodendroglial bHLH transcription factor Olig2, but not the closely related Olig1 efficiently activated the U2 enhancer. Olig2 bound U2 directly at several sites including a highly conserved one in the U2 core. Inactivation of this site abolished the oligodendroglial activity of U2 in vivo. In contrast to Olig2, the homeodomain transcription factor Nkx6.2 repressed U2 activity. Repression may involve recruitment of Nkx6.2 to U2 and inactivation of Olig2 and other activators by protein–protein interactions. Considering the selective expression of Nkx6.2 at the time of specification and in differentiated oligodendrocytes, Nkx6.2 may be involved in limiting U2 activity to the precursor stage during oligodendrocyte development

Evolution of the Toarcian (Early Jurassic) carbon-cycle and global climatic controls on local sedimentary processes (Cardigan Bay Basin, UK)

The late Early Jurassic Toarcian Stage represents the warmest interval of the Jurassic Period, with an abrupt rise in global temperatures of up to ∼7 °C in mid-latitudes at the onset of the early Toarcian Oceanic Anoxic Event (T-OAE; ∼183 Ma). The T-OAE, which has been extensively studied in marine and continental successions from both hemispheres, was marked by the widespread expansion of anoxic and euxinic waters, geographically extensive deposition of organic-rich black shales, and climatic and environmental perturbations. Climatic and environmental processes following the T-OAE are, however, poorly known, largely due to a lack of study of stratigraphically well-constrained and complete sedimentary archives. Here, we present integrated geochemical and physical proxy data (high-resolution carbon-isotope data (δ13C), bulk and molecular organic geochemistry, inorganic petrology, mineral characterisation, and major- and trace-element concentrations) from the biostratigraphically complete and expanded entire Toarcian succession in the Llanbedr (Mochras Farm) Borehole, Cardigan Bay Basin, Wales, UK. With these data, we (1) construct the first high-resolution biostratigraphically calibrated chemostratigraphic reference record for nearly the complete Toarcian Stage, (2) establish palaeoceanographic and depositional conditions in the Cardigan Bay Basin, (3) show that the T-OAE in the hemipelagic Cardigan Bay Basin was marked by the occurrence of gravity-flow deposits that were likely linked to globally enhanced sediment fluxes to continental margins and deeper marine (shelf) basins, and (4) explore how early Toarcian (tenuicostatum and serpentinum zones) siderite formation in the Cardigan Bay Basin may have been linked to low global oceanic sulphate concentrations and elevated supply of iron (Fe) from the hinterland, in response to climatically induced changes in hydrological cycling, global weathering rates and large-scale sulphide and evaporite deposition

Analyses dealing with the control of expression and the molecular mode of action of the transcription factor Sox10 in murine glial cells

Der Transkriptionsfaktor Sox10 ist für die Entwicklung vieler glialer Zelltypen des zentralen (ZNS) und peripheren Nervensystems (PNS) in Wirbeltieren essentiell. Die Kontrolle seiner Genexpression sowie die Regulationsmechanismen seiner Zielgene sind bisher weitgehend ungeklärt. In dieser Arbeit wurde die Kontrolle der oligodendrozytären Sox10-Expression durch Analyse des Sox10-Enhancers U2 untersucht. Diese Untersuchungen ergaben, dass die Sox10-Expression in Oligodendrozyten-Vorläuferzellen durch den bHLH-Transkriptionsfaktor Olig2 aktiviert wird. Dabei bindet Olig2 in vitro und in vivo an den U2-Enhancer und kann diesen allein und synergistisch mit dem bHLH-Faktor E47 aktivieren. Eine von drei in vitro identifizierten Bindestellen erwies sich in Zellkultur und im transgenen Maus-Modell als essentiell für die U2-Aktivierung. Als negativer Regulator wirkte der Transkriptionsfaktor Nkx6.2, der mit Olig2 interagiert und dessen Wirkung reprimiert. Im zweiten Teil der Arbeit wurden die Mechanismen der Sox10-abhängigen Zielgen-Regulierung in Schwann-Zellen am Beispiel des Dhh-Gens untersucht. Mittels vergleichender Genom- und Reportergen-Analysen wurde im Intron I des murinen Dhh-Gens eine konservierte Region mit Schwann-Zell-spezifischer Enhancer-Funktion identifiziert. Innerhalb dieses Enhancers liegen drei monomere Sox-Bindestellen, deren Bindung durch Sox10 in Gelretardierungsexperimenten und Chromatin-Immunpräzipitationen (ChIP) aufgezeigt wurde. In Mutations-Analysen erwiesen sich diese Bindungen in vitro und in vivo als funktional und essentiell für die Sox10-Aktivierung des Dhh-Enhancers. Außerdem wurde in vivo die Abhängigkeit der Schwann-Zell-spezifischen Aktivität dieses Enhancers von Sox10 nachgewiesen. Da Dhh ein extrazelluläres, von Schwann-Zellen sezerniertes Signalmolekül darstellt, das für die Entwicklung der Nervenscheide essentiell ist, wurde in dieser Arbeit der Mechanismus aufgezeigt, wie Sox10 die Bildung der Nervenscheide auf nicht-zellautonomen Weg beeinflusst. An zwei weiteren Schwann-Zell-spezifischen Enhancern, die durch Sox10 reguliert werden, konnte im dritten Teil der Arbeit der molekulare Wirkmechanismus von Sox10 näher bestimmt werden. Es ließ sich nachweisen, dass Sox10 Brg1, die zentrale Komponente des BAF-Chromatin-Remodeling-Komplexes, an diese Enhancer rekrutiert. Der Nachweis wurde durch Kombination von ChIP mit shRNA-Behandlung beziehungsweise Cre-abhängiger Gendeletion in kultivierten Zellen und in peripheren Nerven der Maus zum Zeitpunkt der Schwann-Zell-Differenzierung erbracht. Sox10 scheint somit seine genregulatorische Funktion zumindest teilweise durch die Rekrutierung Chromatin-remodellierender Faktoren auszuüben und so die von ihm regulierten Gene für die Transkriptionsmaschinerie zugänglich zu machen.In vertebrates the transcription factor Sox10 plays essential roles during development of glial cell types of the central (CNS) and peripheral nervous systems (PNS). Until now the regulation of its own gene expression remains largely unknown. The same holds for the regulatory mechanisms it uses to control expression of its target genes. In this thesis the control of oligodendroglial Sox10 expression was studied by analysis of the Sox10 enhancer U2. Sox10 expression in oligodendrocyte precursor cells was shown to be activated by the bHLH transcription factor Olig2. Olig2 binds in vitro and in vivo to the U2 enhancer and is able to activate it on its own or synergistically with the bHLH factor E47. In cell culture and in transgenic mice, one out of three identified bHLH binding sites turned out to be essential for U2 activation. Additionally the transcription factor Nkx6.2 acted as a negative regulator of U2 activity and was shown to interact with Olig2 and repress its activating effect. The second part of this thesis addressed the mechanisms of Sox10-dependent target gene regulation in Schwann cells by analyzing the Dhh gene. By means of comparative genomic studies and reporter gene analyses a conserved region with Schwann-cell-specific enhancer activity was identified within intron I of the murine Dhh gene. Within this enhancer three monomeric Sox binding sites were mapped, which all showed binding by Sox10 in gel retardation and Chromatin immunoprecipitation (ChIP) assays. In vitro and in vivo analyses of enhancer variants that harbored binding site mutations clearly showed that these sites were functional and essential for activation of the Dhh enhancer by Sox10. Additionally the Schwann-cell-specific enhancer activity was shown to dependent on Sox10 in vivo. Dhh, an extracellular signaling molecule secreted by Schwann cells, exerts an essential role during nerve sheath formation. By illustrating the influence of Sox10 on Dhh expression this study provides a mechanistic explanation for the non-cell-autonomous function of Sox10 in nerve sheath formation. The molecular mechanism of Sox10 function was further elucidated in the third part of this thesis by investigating two additional Schwann-cell-specific, Sox10-dependent enhancers. Using a combination of ChIP and shRNA treatment in cultivated cells or a combination of ChIP and Cre-mediated gene deletion in murine peripheral nerve tissue, Sox10 was shown to recruit Brg1, the core subunit of the BAF chromatin-remodeling complex, to these enhancers at the time of Schwann cell differentiation. Therefore Sox10 appears to exert its gene regulatory function at least partially by recruitment of chromatin-remodeling factors thereby enhancing the accessibility of Sox10-regulated genes for transcription

Crazy Little Thing Called Sox—New Insights in Oligodendroglial Sox Protein Function

In the central nervous system, oligodendrocytes wrap axons with myelin sheaths, which is essential for rapid transfer of electric signals and their trophic support. In oligodendroglia, transcription factors of the Sox protein family are pivotal regulators of a variety of developmental processes. These include specification, proliferation, and migration of oligodendrocyte precursor cells as well as terminal differentiation to mature myelinating oligodendrocytes. Sox proteins are further affected in demyelinating diseases and are involved in remyelination following damage of the central nervous system. Here we summarize and discuss latest findings on transcriptional regulation of Sox proteins, their function, target genes, and interaction with other transcription factors and chromatin remodelers in oligodendroglia with physiological and pathophysiological relevance

Transcription factor Zfp276 drives oligodendroglial differentiation and myelination by switching off the progenitor cell program

In oligodendrocytes of the vertebrate central nervous system a complex network of transcriptional regulators is required to ensure correct and timely myelination of neuronal axons. Here we identify Zfp276, the only mammalian ZAD-domain containing zinc finger protein, as a transcriptional regulator of oligodendrocyte differentiation and central myelination downstream of Sox10. In the central nervous system, Zfp276 is exclusively expressed in mature oligodendrocytes. Oligodendroglial deletion of Zfp276 led to strongly reduced expression of myelin genes in the early postnatal mouse spinal cord. Retroviral overexpression of Zfp276 in cultured oligodendrocyte precursor cells induced precocious expression of maturation markers and myelin genes, further supporting its role in oligodendroglial differentiation. On the molecular level, Zfp276 directly binds to and represses Sox10-dependent gene regulatory regions of immaturity factors and functionally interacts with the transcriptional repressor Zeb2 to enable fast transition of oligodendrocytes to the myelinating stage

Chromatin remodeler Ep400 ensures oligodendrocyte survival and is required for myelination in the vertebrate central nervous system

Differentiating oligodendrocytes generate myelin to ensure rapid saltatory conduction in the vertebrate central nervous system. Although oligodendroglial differentiation and myelination are accompanied by dramatic chromatin reorganizations, previously studied chromatin remodelers had only limited direct effects on the process. To study the functional significance of chromatin changes for myelination and identify relevant remodelers, we deleted Ep400, the central ATP-hydrolyzing subunit of the TIP60/EP400 complex, at defined times of mouse oligodendrocyte development. Whereas Ep400-deficient oligodendrocyte precursors develop normally, terminal differentiation and myelination are dramatically impaired. Mechanistically, Ep400 interacts with transcription factor Sox10, binds to regulatory regions of the Myrf gene and is required to induce this central transcriptional regulator of the myelination program. In addition to reduced and aberrant myelin formation, oligodendrocytes exhibit increased DNA damage and apoptosis so that numbers never reach wildtype levels during the short lifespan of Ep400-deficient mice. Ep400 deletion in already mature oligodendrocytes remains phenotypically inapparent arguing that Ep400 is dispensable for myelin maintenance. Given its essential function in myelin formation, modulation of Ep400 activity may be beneficial in conditions such as multiple sclerosis where this process is compromised

Elevated In Vivo Levels of a Single Transcription Factor Directly Convert Satellite Glia into Oligodendrocyte-like Cells

International audienceOligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dor-sal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies

OLE is a Sox10-dependent oligodendroglial enhancer in vivo.

<p><b>(A-H)</b> The expression pattern of the lacZ reporter was followed during embryonic and early postnatal development in the spinal cord of <i>OLE-lacZ</i> (A-D) and <i>OLEa-lacZ</i> (E-H) transgenic animals by X-gal staining of transverse thoracic sections at E13.5, E15.5, E18.5 and P3. Size bar in A, valid for A-H: 200 μm. <b>(I-P)</b> Co-IHC was performed on spinal cord tissue of perinatal <i>OLE-lacZ</i> (I, J, M, N) and <i>OLEa-lacZ</i> (K, L, O, P) transgenic animals using antibodies directed against ß-galactosidase (in green) in combination with antibodies directed against Olig2 (I, K), Mbp (J, L), NeuN (M, O) and GFAP (N, P) (all in red). Pictures were taken from the ventral mantle zone for M, O and from the ventral marginal zone for all other panels. Size bar in I, valid for I-P: 20 μm. <b>(Q-T”)</b> Additionally, co-IHC was performed on DRG of mice that carried the <i>OLE-lacZ</i> (Q-R”) or the <i>OLEa-lacZ</i> (S-T”) transgene on a wildtype (Control) or 2TetSox10 background using antibodies directed against ß-galactosidase (green) in combination with antibodies directed against Olig2 (red). Nuclei were counterstained with Dapi (blue). Shown are single fluorescences for ß-galactosidase (Q-T) and Olig2 (Q’-T’) and the merge (Q”-T”) for each IHC. Size bar in Q, valid for Q-T”: 50 μm.</p