Exploring the role of Genome Architecture in Neuronal Development using an in vitro model system (360G-Wellcome-211596_Z_18_Z)

The research aim is to explore the role of genome organisation and three-dimensional configuration in regulating transcriptional responses during neuronal differentiation. To do so, expression of co-regulated candidate genes during differentiation from neuronal progenitor cells (NPCs) to post-mitotic neurons (PMNs) will be identified with qRT-PCR. NPCs will be dissected from E12.5 mouse cortices and cultured in basic Fibroblast Growth Factor (bGFG). Differentiation into PMNs will be induced by adding neurotrophin-3 (NT-3). The nuclear localisation of pairs of co-regulated genes will be detected using double fluorescence in situ hybridisation (D-FISH), assessing whether these genes relocate to transcriptionally active regions, like transcription factories, or transcriptionally repressive regions, like the nuclear periphery. Results will also compare transcriptional changes in neuronal differentiation with nuclear re-organisation patterns in the expression of activity-regulated genes (ARGs), like c-Fos and Gadd45b. This will allow the identification of genome architecture changes specific to cortical development. Possible gene co-localisation, genomic-wide contacts and loci interactions will be studied with 4C technology, which combines chromosome conformation capture (3C) methodology with high-throughput sequencing.