Chromatin rearrangement during differentiation of mesenchymal stem cells in the context of nuclear envelope-linked diseases

The nuclear envelope contributes to regulating genome function by interacting with DNA and chromatin. Mutations in a subset of nuclear envelope proteins, the lamins, cause debilitating diseases including lipodystrophies, muscle dystrophies and premature a ging. The link between lamin mutations and disease is unknown, but may involve a disruption of the interaction of lamins with DNA. Experimental evidence suggests that lamin mutations affect chromatin organization and gene expression. The diseases predomin antly affect mesodermal tissues including fat, with an onset in young adults, suggesting a differentiation defect in mesenchymal stem cells (MSCs). Studies suggest that if lamin mutations occur in MSCs, they affect mesodermal differentiation pathways. We hypothesize that lamin-linked diseases affecting mesodermal tissues arise from epigenetic defects in MSCs. To test this hypothesis, the project combines our expertise in the nuclear envelope, MSC biology, imaging and epigenetics with that of our collabora tors in laminopathies, chromatin biology, gene expression arrays, and bioinformatics. AIM 1 of the project addresses chromatin reorganization during differentiation of ASCs using a tagged histone variant as an active chromatin marker; this will be done b y combining chromatin imaging and epigenomic approaches. AIM 2 will determine how a lipodystrophic lamin A mutation affects adipogenic differentiation and how differentiation deficiencies relate to global and promoter-specific chromatin rearrangements. AI M 3 examines chromatin organization in fibroblasts from patients, while AIM 4 addresses molecular mechanisms leading to lamin-dependent chromatin remodelling. The experimental strategy combines high-resolution quantitative chromatin imaging with single-l ocus and genome-wide epigenetic approaches, in the context of adipogenic differentiation of adipose-derived MSCs expressing wild-type and mutant, disease-causing, nuclear lamins.