Galiñanes García, L.; (2010) Molecular mechanisms underlying Mash1 function in oligodendrogenesis. Doctoral thesis , UCL (University College London).

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Abstract

Members of the basic helix-loop-helix (bHLH) proneural family of proteins, including Mash1, are crucial transcription factors (TFs) in neurogenesis. More recently, a role for Mash1 in the specification of oligodendrocyte precursor cells (OPCs) has been demonstrated. Here we investigate the role of Mash1 in lineage commitment of neural progenitors and more specifically the mechanisms underlying Mash1 activity in oligodendroglial cell fate specification. We use an in vitro cell culture system to perform Mash1 locational analysis. Mouse OPCs were cultured as oligospheres that expressed Mash1, a proportion of which also coexpressed the early OPC marker platelet-derived growth factor receptor \alpha (PDGFR\alpha) and oligodendrocyte promoting TFs including the bHLH TF Olig2 and the high mobility group (HMG) TF Sox9. We use a chromatin immunoprecipitation (ChIP)-on-chip strategy and found that Mash1 protein binds to proximal genomic regions of early OPC genes such as Olig1 and Sox8, late oligodendrocyte genes including myelin oligodendrocyte glycoprotein (Mog) and oligodendrocyte myelin glycoprotein (Omg), and other genes of interest including Brevican (Bcan), Notch1 and Sulfatase1 (Sulf1). Mash1 also bound distal genomic regions of Olig2 and Sox9 in oligosphere cultures. To formulate a TF combinatorial code for the activation of these putative enhancers, TF synergy were analysed with luciferase reporter assays. Furthermore, to isolate genomic regions with activity in the oligodendroglial lineage in vivo we used mouse transient transgenics. We hypothesise that Mash1 interacts with either neuronal- or oligodendroglial-specific cofactors, and that these interactions modulate Mash1 activity. To address this question we performed Sox9 and Olig2 ChIP and found that some Mash1 bound elements were also occupied by these TFs in oligosphere cultures. In conclusion, using an in vitro cellular system and ChIP-on-chip technology to interrogate proximal promoter regions bound by Mash1, we can begin to elucidate the molecular mechanisms of Mash1 function in oligodendroglial cell fate specification.

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