Minieri, C; (2014) Regulation of neural enhancer activity by Ascl1 and Sox factors. Doctoral thesis , UCL (University College London).

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Abstract

Transcriptional enhancers and their associated transcription factors (TFs) have a profound impact in gene regulation during development. The TFs Ascl1 and the Sox factors play important roles in many aspects of neurogenesis in vertebrates. Ascl1 induces neuronal differentiation in neural progenitor cells. Sox2 is an essential TF in the maintenance of neural stem cell characteristics, but also functions in the correct specification of neuronal subtypes. SoxC factors have a fundamental role in the establishment of neuronal traits in differentiating neurons. Data previously generated in our lab have identified genomic regions bound by both Ascl1 and Sox2 in close proximity through a ChIP-seq approach, raising questions about possible interactions between these TFs as transcriptional regulators. Here, I have characterised these regions as neural enhancers regulated by Ascl1, Sox2, and SoxC factors using NS5 cells as an in vitro model of neural stem cells through luciferase assays. Enhancers activated by overexpression of Ascl1 were classified as neuronal enhancers, since overexpression of this TF induces neuronal differentiation in NS5 cells. Cotransfection experiments and luciferase assays demonstrated that Ascl1 and Sox2 counteract each other in the regulation of the enhancers, whereas Ascl1 and SoxC factors synergistically activate the neuronal enhancers. Mutations of the binding motifs of these TFs and comparisons between the transcriptional activity of the wt and mutant enhancers suggest direct binding of Ascl1 on all the enhancers activated by this TF, but different mechanisms of regulation exist for the Sox factors on the different enhancers analysed. I have shown direct binding of Ascl1 on one of these enhancers, MSB4, by EMSA. In conclusion, I have identified a novel transcriptional regulatory network in the regulation of neuronal differentiation where Ascl1 and SoxC factors synergistically activate neuronal enhancers, and Sox2 counteracts Ascl1 in this activation.

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