Progression of 3D genome folding in neurons during ageing

In vivo model: Sorted neurons from Castaneus/C57Bl6 hybrid female and male mice, at different stages (young, middle-aged and old), from hippocampus (brain region vulnerable to ageing and affected in Alzheimer’s disease) and cerebellum (resistant). Proposed experiments: HiC (genome folding), bisulfite sequencing (epigenetic chromatin state – methylation had been used as a biomarker for ageing-) and RNA-seq from each individual, to determine age-related alterations in 3D genome organisation, assess their relation to the epigenetic drift of the chromatin state and to gene expression. The HiC analysis and the 3D modelling of this data will be done with the help of the Cavalli (P8) and Giorgetti (P4) labs respectively. We will identify specific changes at loci of particular interest (notably at genes associated with cognitive performance or neurodegenerative disorders) that we will investigate further to test the causality between genome folding and gene expression changes. Prior to this molecular analysis, mice will undergo spatial navigation tests (Morris water maze – hippocampus-related task), to evaluate whether the extent of genome folding aberrations could correlate with cognitive performance. To assess the impact of genome folding alterations on expression of the genes of interest, we will derive neural stem cells from young and old animals, and use dCas9 approaches such as manipulation of looping and genetic screens with lentiviral libraries targeting architectural protein genes in particular. We will also carry out molecular analyses (HiC, RNA seq) and live cell imaging to evaluate chromosome and chromatin mobility using H3K27acetylation or H3K27me3 mintbodies, and dCas9-FP targeting to minisatellite regions in the neural stem cells.