To study the alterations of 3D genome folding in mouse neurons during ageing, in relation to aberrant gene expression and cognitive decline.
During ageing, chromatin undergoes a progressive epigenetic drift and a global loss of transcriptional silencing. Very little is known about the brain-specific changes, however the erosion of chromatin organisation might have major impact, it could lead to aberrant expression patterns during ageing and more generally to cognitive decline or to the onset of neurodegenerative disorders. Based on the long-standing expertise of the Heard lab in mammalian epigenetics and its complementarity with the expertise of the Cavalli lab in HiC analysis, using recent and innovative approaches, we will investigate the dynamics of the 3D genome folding during ageing in the mouse brain (in both males and females, and in an allele-specific manner), we will also evaluate whether it is related to molecular changes (chromatin, gene expression), and to cognitive decline.
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.
The combination of experiments described here should provide solid insights on the genome folding dysregulations occurring during brain ageing and better understand cognitive decline. The present study should also demonstrate how genome folding could affect the expression of genes associated with neurodegenerative disorders.
CNRS, France (3 months):
Modelling HiC data into comprehensive models of nuclear organisation.
BYFACILITY, Spain (2 weeks):
Creation of conversational interfaces about 3D genome folding of mouse neurons.
Enrolment in doctoral programs
University of Heidelberg.
Giorgetti, L. et al. Structural organization of the inactive X chromosome in the mouse. Nature 535, 575-579, doi:10.1038/nature18589 (2016).
Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381-385, doi:10.1038/nature11049 (2012).