CHROMAG

The nucleus, far from solely being the repository of genomic information, is increasingly viewed as a complex physical entity, mechanically and chemically coupled to its cellular environment. It is now clear that the three-dimensional architecture and the physical properties of the genome play a central role in its genomic and non-genomic functions. In recent years, novel approaches based on chromatin conformation capture techniques, complementing genome-wide assays and fixed-cell imaging tools have revealed multiple layers of conformational organization closely linked to genome function, including gene regulation, DNA replication, chromosome segregation, differentiation and others. Yet, despite the rapid evolution in our understanding of genome architecture in the past years, several roadblocks remain, that critically hold up progress in genome biology. First, experiments are predominantly performed on population of cells or in fixed conditions and there is a clear need to carry out measurements at the level of single living cells in order to address the conformational dynamics of chromatin at all scales, from the formation of loops to that of domains, compartments and territories. Second, most experiments (notably imaging measurements) are based on the passive observation of chromatin, possibly combined with global alterations such as gene knock-down. As a result, it is often difficult to distinguish between correlation and causality. Third, the dynamics and conformation of chromatin is governed by many energy-driven processes, and understanding the properties of chromatin as active matter is essential. Finally, chromosomes are mechanical objects with a conformation that can be dynamically modified as a function of the mechanical stresses exerted on the nucleus. Overall there is a clear need for a comprehensive description of the genome that account for the physical properties of chromosomes, notably including the out-of-equilibrium and mechanical processes that contribute to their organisation and dynamics in the nucleus. To address this challenge, we propose a novel approach that consists in mechanically perturbing the genome architecture, at the single chromosome level and with genomic specificity, and directly assessing the associated effects on nuclear organization and genomic functions. To do so, we will leverage our consortium’s unique expertise in cell biophysics, genome editing and nanoprobe chemistry to design an innovative nanotoolbox for chromosome imaging and manipulation. Specifically, we will develop a highly versatile and scalable strategy to decorate genomic loci in live cells with fluorescent quantum dots or magnetic nanoparticles via specific targeting using recombinant catalytically inactive CRISPR/Cas9 proteins, complexed with synthetic gRNAs. These novel functionalized nanoprobes will allow us to image and physically manipulate individual chromosomes at specific genomic sites in mammalian cells and to record the effect of these mechanical perturbations (in the pN range) on chromosomal conformations and functions. By doing so, we will directly assess: (i) the rheological and elastic properties of individual chromosomes in interphase and the role of out-of-equilibrium processes in their multiscale conformations, (ii) how mechanical forces affect transcriptional activity. Our approach, which goes well beyond the state-of-the art in genome research, will contribute to bridge the gap between population biochemical assays and the biophysical models of chromatin. Thereby, we will achieve a more integrative understanding of nuclear organization and dynamics and we will open many prospects for the study of a variety of scientific questions on different genome functions (transcription, repair, replication) and on the alteration of genome organization during processes as diverse as development, differentiation, disease or ageing.