Drechsel, D.; (2012) Genome-wide analysis of two transcriptional programmes of neurogenesis. Doctoral thesis , UCL (University College London).

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Abstract

Transcription factors (TFs) of the basic helix-loop-helix (bHLH) family, so called 'proneural proteins', are key regulators of neuron generation in mammals. During embryonic development, Ascl1/Mash1 and Neurogenin2, the two main proneural factors in the mammalian CNS, activate programmes of neuronal differentiation that control the generation of inhibitory and excitatory neuronal populations in the arising forebrain. The cellular functions of these TFs during neurogenesis are well understood. Yet, the identity and overlap of their targets, and how these are regulated, has not been addressed in depth. Here, we introduce an in vitro model that allows studying neurogenesis downstream of the proneural TF Mash1/Ascl1 and Neurogenin2 in a temporally specific manner. Ascl1/Mash1 and Neurogenin2, fused to the modified ligand-binding domain of the estrogen receptor (ERT2), were expressed in a neural stem cell line, NS5. In the presence of the ERT2-ligand, 4-hydroxytamoxifen (4-0HT), these inducible proneural-fusion constructs are able to bind their targets and induce gene expression. A retroviral transgene delivery system with a selection marker allowed us to generate large, homogenous cultures of NS cells that can be induced to undergo Ascl1/Mash1 and Ngn2- specific neuronal differentiation in a synchronous manner. A combination of time-course expression analysis after proneural Gain-of-Function (GoF) and localization analysis of genomic proneural binding sites using genome-wide approaches allowed, firstly, to describe and compare the gene expression programme induced by Ascl1/Mash1 and Neurogenin2 in NS5 cells in a time-specific manner, and secondly, to catalogue Ascl1/Mash1 and Ngn2 target genes along with their expression kinetics as well as their proneural-binding regulatory regions on the chromatin. An analysis of these data showed that both Ascl1/Mash1 and Neurogenin2 regulate targets with different expression kinetics i.e. early as well as late expressed genes, and up- as well as down- regulated genes. Further analyses regarding putative mechanisms of this different temporal expression patterns in Ascl1/Mash1 target genes specifically identified enriched DNA motifs and putative co-regulator binding sites around regulatory regions that relate to different expression kinetics of the corresponding genes. Additionally, binding of Mash1 close to the transcription start site, and clustering of Mash1-binding sites are related to an early onset of Mash1 target gene expression. NS5 is a neural stem cell line that expresses ventral markers. Within this biological context' this study found that Ascl1/Mash1 regulates more direct target genes than Ngn2. Nevertheless' this study identifies common molecular pathways of Ascl1/Mash1 and Neurogenin2, as well as their molecular mediators, and gives first insights into their mode of regulation. Thereby, it defines a 'core neurogenesis' programme' which includes pathways involved in neuronal migration (Lis1- and Reelin pathways), patterning (Wnt/TFG-beta pathways) or neural progenitor as well as astrogenic signaling pathways that are shut down or repressed during neurogenesis (EGF-pathway, SMAD-signalling). Further, this study identified specific programmes for Ascl1/Mash1 and Ngn2, such as a role of Ascl1/Mash1 in progenitor cell cycle regulation. Thus' this work provides a starting point to elucidate of the molecular underpinnings of the cellular common and distinct functions of Ascl1/Mash1 and Neurogenin2 during neurogenesis. It further represents a first step towards the identification of the molecular regulatory logic of the lineage determining factors proneural factors in the CNS.

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