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Internship and PhD proposal

par Guillaume Tresset - publié le

NUCLEOSOME CONFORMATION AND LOCAL ORDER EXPLORED BY CRYO-ELECTRON TOMOGRAPHY IN RELATIONSHIP WITH CHROMATIN FUNCTIONAL COMPARTMENTATION


 

Eukaryotic chromosomes are complex polymorphic objects. Centimeters to meters of DNA condense into a micron-scale nucleus though multiple levels of organisation, while interacting with multiple factors driving functional compartmentalization. DNA is regularly associated with histones to form the so-called « bead-on-string » filament of nucleosomes. Chromatin is not uniform in space and time. It can be described as a hetero-polymer with structurally and functionally defined domains : the less compact, gene-rich and transcriptionally active euchromatin (EuC), and more compact and little transcribed heterochromatin domains, including constitutive heterochromatin (cHC), gene-poor and enriched in repetitive sequences. Despite spectacular advances in the knowledge of chromosome dynamics and large-scale organisation, at the nucleosome level, structural information in situ is dramatically lacking.

Using cryo electron microscopy (cryo-EM) and tomography (cryo-ET) of vitreous sections, we have recently succeeded, for the first time, in imaging nucleosomes in their native nuclear context at a level of detail sufficient to follow the DNA molecule wrapped around and analyse their conformation by comparison with available crystallographic structures (Eltsov et al, 2018). Our objective is now to determine the nucleosome conformation and local chromatin fold inside the interphase cell nucleus, in relationship with the functional state of chromatin.

For this purpose, we will use Drosophila embryonic brain as a model. This model presents many practical advantages, in particular a distinct segregation of large cHC domains from EuC. We will
implement a cryo-correlative light and electron microscopy (cryo-CLEM) workflow to target GFP-labelled heterochromatin 1 proteins (HP1) in cHC. We will thus explore, in relationship with chromatin functional compartmentalization : i) the nucleosome conformation and its structural variability landscape, ii), the local chromatin fold, and iii) the interplay between nucleosome conformation and chromatin fold.

This experimental M2 internship in structural biology will be devoted to specimen preparation (vitrification, cryo-sectionning), cryo-EM/ET data acquisition/reconstruction, and test of cryo-CLEM devices, including access to high end cryo-EM facility at IGBMC (Strasbourg). This project is part of an ANR-funded collaborative project including specialists in image analysis and polymer physics.

References
Leforestier A., Dubochet J. Livolant F. (2001) Biophys. J. 81, 2414-2421
Leforestier A., Lemercier N. Livolant F. (2012) Proc. Natl. Acad. Sci. USA, 109, 959-8964.
Eltsov, M., Grewe, D., Lemercier, N., Frangakis, A., Livolant, F., & Leforestier, A. (2018). Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron microscopy of vitreous sections. Nucleic acids research, 46(17), 9189-9200.

 

Announcement HERE