Cellular responses to environmental stimuli often require rapid transcriptional induction of specific gene subsets, which are somehow “targeted” within minutes despite representing a tiny fraction of the entire DNA sequence within the nucleus. The genome is highly spatially organized within eukaryotic cell nuclei, and a large body of work has correlated gene locations relative to nuclear landmarks, or certain genome configurations, with regulation of DNA metabolism. Active and inactive compartments form topologically separate domains exhibiting signatures of chromatin chemical modifications and factors recruited by them. Within the domains, enhancers can contact promoters specifically or selectively over short or long genomic distances, usually in cis. Enhancers, including super-enhancers, are known to interact stochastically, a feature particularly relevant during cellular differentiation. However it is still unclear whether transcription per se drives spatial reorganization and how we can use this information to gain a better understanding of cell responses in normal and pathological contexts. Moreover, the dynamics of such processes are poorly understood and mainly correlative. By live cell imaging we have recently demonstrated that transcription initiation rapidly confines the transcribed locus and that chromatin dynamics occur in large domains of coordinated movement with abrupt boundaries only in actively transcribing cells. To achieve this, we have developed two complementary new methods, one for particle tracking, the proprietary ANCHOR technology to fluorescently tag up to three specific DNA loci, and one for flow determination of fluorescent proteins and DNA in the entire nucleus. These approaches give us an unprecedented opportunity to assess the mechanism by which chromatin topology evolves and contributes to transcriptional control in living cells. We propose a multiscale approach probing chromatin domain conformation and dynamics of rapidly inducible estrogen target genes in mammary tumor cells. Live cell imaging will be combined with promoter capture Hi-C and polymer modeling to ask how structural reorganization regulates transcriptional output, and how this may be perturbed. Insights into chromosome biology and technological developments will foster new applications for diagnostics and drug discovery.

As the population in modern societies ages and the risks of bone diseases or bone accidents has increased, the need for a new generation of materials with superior biocompatibility and adequate mechanical properties is a challenge. CoCoA-bio combines two innovative metallurgical concepts to provide a material solution for the intended application, positioning itself on an essential public health issue. To this end, multicomponent and complex concentrated alloys (HEA/CCAs) based on TiNbZr-X (X = Mo, Ta) system will be fabricated via additive manufacturing (AM), namely by Selective Laser Melting (SLM). After each stage of development by SLM and microstructure optimization by Hot Isostatic Pressing (WP1), a full microstructure characterization (WP2) and mechanical behavior analyses of the resulting samples, under different loading conditions, including fatigue (WP3) will be carried out. A mechanical surface functionalization step carried by machining with metrological monitoring (WP4) will be conducted before a chemical functionalization for the suitability of materials developed for the intended application (WP5). Two additional WPs, WP0 (coordination) and WP6 (regulation and socio-economic aspect) further complete the structure of the proposal which will run for 48 months. In addition to the raw pre-alloyed powders, which will mainly be developed for us as part of an external service, the heart of the project is made up of WP1, WP4 and WP5 having as pillars WP2, WP3 and WP6, which allow screening of the envisaged solution. Indeed, the architecture of the project was designed to identify several alloys candidates for the desired application, then to converge on the most promising solution(s), using tools adapted to each task. At the end, dental implant prototypes, as a model implant, will be proposed to validate our experimental approach. The consortium is confident that the CoCoA-Bio project will participate in requalifying the HEAs from promising materials to truly candidates to replace the material solutions currently applied (for lack of anything better), in our case alloys for bio-implants. The cross expertise and resources of the consortium on additive manufacturing, the design and characterization of HEAs and their surface functionalization are important assets for achieving the objectives of the CoCoA-Bio proposal.

In eukaryotic cells, how chromatin and epigenetic marks are imposed, inherited and/or changed is a central issue for cellular functions and cell fate maintenance. Understanding the logic behind their dynamics is of critical importance to determine the molecular mechanisms behind the maintenance of cellular functions, changes during cell differentiation, and how this can go awry during disease onset and progression. In the CELLECTCHIP proposal, we aim at characterizing dynamics of histone variants and histone post-translational modification (PTMs) marks genome wide in three contexts: establishment, maintenance and erasure. We will exploit two biological situations during which chromatin organization and chromatin marks are remodeled by dynamic mechanisms, but with two distinct outcomes: (i) the dynamics of chromatin marks during S-phase of somatic cell lines for maintenance and (ii) paternal X-chromosome inactivation and reactivation during early mouse development for change. We will develop novel ChIP-seq technologies based on microfluidics and single-cell DNA barcoding that enables ChIP-Seq from very few cells (hundreds) as well as an ability to discriminate post-sequencing the sequence reads originating from individual cells. We will combine these technologies with engineered biological systems allowing the discrimination of (1) parental from newly assembled nucleosomes at defined times time during S-phase progression and (2) paternal X expressing and non-expressing cells from mouse pre-implantation embryos. This proposal should advance the scientific field of epigenetics in an original way (1) by bringing analysis to the level of individual cells and (2) by going beyond a static description of the chromatin/epigenetic landscape since it aims at deciphering the dynamics of histone variants and histone PTMs at particular times in the cell cycle (S-phase) and during development. This dynamic view should contribute to our understanding of epigenetic mechanisms at an unprecedented level and enable development of new technologies in epigenetics for drug discovery and personalized medicine.