Staphylococcus aureus is responsible for many human and animal diseases whose treatment is often complicated by the emergence of multidrug resistant strains. Our project aims at identifying and characterizing bacterial regulatory RNAs (sRNAs) that impact antibiotic resistance and virulence in S. aureus. Regulatory networks are subject to many input effectors, and sRNAs contribute to the "fine-tuning" of gene expression. Consequently, sRNA-dependent phenotypes are, in general, difficult to detect. For example, no phenotype has been associated with the absence of RsaE, an sRNA widely conserved in bacteria which down-regulates the Krebs cycle and folate metabolism. Minor sRNA-mediated phenotypes conferring modest advantages, however, may emerge as dominant traits after a few generations under selective pressure such as growth within the host or in presence of antibiotics. In our project, the first step will be to identify the phenotypes associated with sRNA gene disruptions. Thereby, the presence of a phenotype will be the main criterion for further study of the implicated sRNAs. Bacterial fitness is the ability to optimize survival and growth in a particular condition. sRNA mutations affecting fitness could be determined by measuring growth rates in batch cultures but tiny differences are difficult to determine with this method. However, sRNA-mediated phenotypes are expected to emerge during competitive fitness experiments by growing several strains together (each strain bears a single sRNA deletion) and evaluating their proportions in the population at different times. In addition, bacterial growth in the presence of a mixture of strains reflects a native context, as the fitness of a mutated strain may be affected by the genetically different surrounding strains. Therefore, these experiments could reveal patterns of gene interactions, or epistasis, among different mutants and possibly whether particular combinations of sRNA mutants interact synergistically or antagonistically to each other. Finding phenotypes associated with sRNA gene deletions usually require testing of many conditions for each mutant, with no assurance of success. We propose an innovative alternative to this problem. Introducing specific DNA sequences in each engineered sRNA mutant will allow monitoring of many mutants simultaneously. These DNA barcoded sequences will be quantitatively detected within the mixed cultures. This technique developed in yeast was also used in bacteria. In previously published fitness protocols, the genomic DNA from mixed populations was extracted, the tags were PCR-amplified and their ratios were determined by hybridizing the labeled PCR products on dedicated DNA arrays. These experiments are tedious and expensive, as each tested condition requires at least one array. The protocol was adapted to use deep sequencing technology and primers specific for each experiment were designed. Preliminary results show that the technology is time saving, increases response linearity, and has a drastically reduced cost by allowing multiple conditions to be assayed simultaneously, contrary to the initial protocol. We already tested 13 growth conditions in triplicate with 40 tagged S. aureus sRNA mutants. We identified 13 sRNA deletion mutants that resulted in the accumulation or disappearance of strains carrying them in at least one of the tested conditions. Competition fitness experiments will evaluate the effects of sRNA mutations by growing mutants in presence of different antibiotics and in two animal models of infections. These experiments will lead to the identification of sRNAs influencing/adapting to antibiotic resistance and S. aureus virulence. The targets of these sRNAs will be identified and the molecular mechanism of their action determined by innovative techniques that we recently developed. The sRNA-associated gene networks will be deciphered.

Cell fate choice and maintenance require a precise balance of gene activation and repression. An essential component in these processes is the selective formation and maintenance of repressive heterochromatin domains with long-term stability and minimal variation among individual cells. I and others have found that different types of mammalian facultative heterochromatin adopt specific 3D organizations within the cell nucleus. At the global scale, H3K9me2-marked heterochromatin is found at the repressive nuclear periphery, whereas the H3K27me3 mark clusters in distinct foci within the active nuclear interior. At the local scale, H3K9me2 and H3K27me3-marked domains form 3D compartments that are physically separated from their surroundings. A defining function of these types of facultative heterochromatin therefore is the formation of repressive microenvironments at different positions in the nucleus. Currently though, a precise understanding of how 3D organization is mechanistically linked to the repressive function of facultative heterochromatin, if there are functional differences between different types of facultative heterochromatin and if the 3D organization plays a role in minimizing repressive variation within the cell population remains poorly understood. My long-term objectives are to dissect the function of 3D genome organization in facultative heterochromatin function, stability and consistency and to determine how changes in 3D heterochromatin organization support correct cell fate choice and maintenance. To address these questions, I propose two research projects: [1] Mechanistic dissection of 3D facultative heterochromatin organization using a newly developed cellular platform that allows targeted modification of 3D genome organization at specific gene loci. [2] High-resolution studies of the link between 3D facultative heterochromatin organization and consistency of repression in individual cells, using a novel nanopore-based single-cell 4C-seq assay. Using the @RAction funding, a 48-months work-program will be established at the CNRS-CGM / future I2BC (Gif-sur-Yvette) in which a post-doc, a PhD student, a technician and I will use cutting edge techniques like 4C-seq, microfluidic transcript profiling and ChIP-seq to mechanistically link 3D genome organization and facultative heterochromatin function, both within the cell population and in individual cells. An important aspect of this program is the development of two novel technological platforms: a nanopore sequencing-based assay for high-resolution study of 3D genome organization in individual cells and a cell system that allows the systematic determination of regulatory protein function in 3D genome organization at defined places in the genome. The outcome of these studies will provide important insights into our understanding of epigenetics, nuclear organization and transcriptional regulation. Moreover, the development of two technological platforms will open up opportunities for collaboration, with early access for the French research community. Additionally, the single-cell 4C-seq assay may be exploited for commercial use in a clinical diagnostics setting. As a result, this project will importantly strengthen the position of the CGM / I2BC and the French research community in the field of 3D genome organization.

Phagocytosis is the mechanism of internalization of large particles of several microns in size and therefore, as other cellular functions dealing with large scales, it involves important mechanical constraints that have been poorly investigated. Phagocytosis is an ideal cellular function to understand how cells adapt to the mechanical properties of their environment because it is a local and inducible process that internalizes particles of variable mechanical properties. Many surface receptors capable to mediate phagocytosis belong to the integrins family of cell adhesion receptors that bind to extracellular matrix ligands, cell surface ligands and soluble ligands. Integrins signal through the plasma membrane in both directions. An “inside-out” signal induces a conformational change of the integrin that is necessary to activate its binding to extracellular ligands while the “outside-in” signal couples the ligand binding adhesion sites to the cytoplasmic tail, which links to the cytoskeleton and downstream signaling pathways. Our working hypothesis is that actin-binding proteins (ABPs), and their associated regulators, are mechanoensitive hubs at the center of feedback loops that control anchoring to the actin cytoskeleton and activation of the integrins during phagocytosis. Our proposal aims at understanding how these actin-associated mechanosensitive machineries, involved in particle adhesion to the CR3 receptor (CD11b/CD18 or integrin aMß2) and the subsequent anchoring of the actin cytoskeleton, sense and respond to mechanical parameters. The objectives of this project are the following: (i) to dissect the mechanisms by which mechanosensitive protein machineries sense force and modulate actin dynamics and anchoring during phagocytosis (ii) to determine the importance of the same protein machineries in the regulation of the adhesive properties of CR3. To this end, the complementary expertise of two groups, who have separately made important contributions in their fields and have already collaborated, will be brought together. They will combine investigation of actin remodeling downstream of integrins during phagocytosis using experimental models to follow phagosome completion with improved resolution and in vitro reconstitution with pure proteins of the dynamics of mechanosensitive protein machineries associated with integrin-mediated adhesion. The sequential recruitment of the proteins at phagocytic sites in living macrophages will be used to refine the in vitro system, while the interactions between molecular partners characterized in vitro will be used to generate mutants and analyze their functions in living cells. This work will bring new insight into the overlooked force-dependent CR3/integrin-mediated actin dynamics and adhesion associated with phagocytosis. Our project will contribute to expand scientific knowledge and could provide therapeutic strategies, because integrin-mediated phagocytosis is important in a variety of normal and pathological contexts such as the turnover and remodelling of tissues, disposal of dead cells and bacteria clearance. Integrin-mediated phagocytosis could also serve as a reference model to reveal new concepts associated with the regulation of integrins in other mechnanosensitive processes like cell adhesion or migration. Therefore this work will contribute to bring knowledge and approaches to the emerging field of mechanobiology.