Integrated structural analysis of eukaryotic chromatin in situ. Are nucleosome conformation and local order related to chromatin functional state? – CRYOCHROM

Eukaryotic chromosomes are complex polymorphic and dynamic objects. Centimeters to meters of DNA condense into a micron-scale nucleus though multiple levels of structural organisation, while interacting with a multitude of factors driving functional compartmentalization. Their basic beads-on-string nucleosome structure plays a key role in major cellular processes from regulation of gene expression and epigenetics to DNA replication and repair, cell differentiation and aging, but little is known about its ultrastructure in situ. Using cryo electron tomography of vitreous sections,we recently demonstrated the feasibility of imaging nucleosomes and linker DNA in situ at a level of details that now allows us to access their conformation, local order and fibre fold in the cellular context.
CRYOCHROM proposes an integrated structural approach to unveil nucleosome conformation and local organisation in relationship with chromatin functional compartmentalization. It relies on a multidisciplinary consortium, with specialists of chromatin biophysics and biology, experts in cryo-EM image analysis and of continuous conformational variability analysis, and a polymer physicist specialist of chromatin modelling. Heterochromatin versus euchromatin compartments in nuclei of Drosophila embryos and cell lines will be targeted by cryo correlative light and electron microscopy to benchmark chromatin in active and repressed states. We will i) characterize nucleosome structural variability landscape by sub-tomogram classification to determine different conformations in the cellular context and by normal mode analysis to delineate continuous conformational transitions, ii) determine the local chromatin fold by exploiting the DNA linker path visible after contrast enhancement by Volta phase plates, and iii) decipher the interplay between nucleosome conformation and chromatin fold. Linking chromatin ultrastructure and functional compartmentalization, we will uncover molecular bases of epigenetic silencing in the cellular context, with manifold impacts ranging from gene regulation during embryogenesis and aging, to stem cell research and genome reprogramming during cancer.