Our objective is to decipher the structure and the function of the transcriptional coactivator NuA4 and its nucleosome remodeling partner SWR1 by combining molecular biology and biochemical approaches, structural biology and particularly electron cryo-microscopy (cryo-EM), and chemical biology. The regulation of gene expression at the transcriptional level is a vital mechanism that determines cell fate and influences all physiological and pathophysiological aspects of life. Transcription of protein coding genes is tightly controlled and requires the coordinated action of a large number of proteins that trigger the assembly of the Pre-Initiation Complex (PIC) at the promoter of the transcribed genes, leading to correct transcription initiation. Sequence-specific transcriptional activators and post-translational modifications of nucleosomal histones contribute to the recruitment of multi-subunit coactivator complexes acting as bridging factors between the transcriptional activators and the PIC. Co-activators modify the chromatin structure around the promoter region and coordinate PIC assembly, epigenetic chromatin modifications and activator-mediated cellular signaling events. Our goal is to understand the functional role, the structural organization and the mode of action of the yeast transcriptional co-activator NuA4, a multi protein complex containing 13 different subunits with for a total molecular weight of 1.0 MDa. NuA4 integrates epigenetic signaling by reading and writing post-translational histone modifications, interacts with transcriptional activators and incorporates specific histone variant. The Esa1 subunit of NuA4 is an acetyl transferase capable of modifying histone H4 and is essential for yeast growth. The large 430 kDa Tra1 subunit is involved in transcriptional activator binding thus making the link with the cellular signaling pathways. NuA4 also participates in the recruitment of the histone variant H2A.Z, a chromatin marker that bookmarks active gene promoters. To perform this function NuA4 interacts with the chromatin remodeler SWR1. In humans, the homologous NuA4 and SWR1 are physically associated to form the TIP60/p400 complex, which encompasses both histone acetyltransferase (TIP60) and histone exchange (p400) activities. Despite their key biological importance, the structural organization of the full NuA4 and SWR1 complexes is poorly understood and we aim at solving their high-resolution structure by cryo-EM. The mechanism by which these factors are recruited to gene promoters is currently unknown and we will form supramolecular complexes between NuA4 and, both transcriptional activators and nucleosomes to understand the network of interactions that target NuA4 to active genes. With the help of chemists we will measure binding constants of NuA4 with small molecule inhibitors, histone tails and activation domains in order to quantify the interaction network of the NuA4 complex. To stabilize the conformation of these molecular machineries, a prerequisite to achieve near atomic resolution, we will synthesize bi-functional chemical reagents in order to establish connections between flexible protein domains. Finally, even though the histone H2A.Z exchange activity is central for regulated gene expression and for DNA repair, the association of NuA4 with the chromatin remodeler SWR1 has not been studied so far at the structural level. We will analyze the structure of the NuA4/SWR1 complex by cryo-EM as well as structural intermediates of the exchange reaction. We will also analyze the structure and the function of the homologous human TIP60/p400 complexes in order to better understand the H2A.Z exchange mechanism.

Nucleic acids (NA) form an essential class of macromolecules for storage, transmission and expression of the genetic information of a cell. While deoxyribonucleic acid (DNA) encodes the information cells need to make proteins, ribonucleic acid (RNA), conformationally more variable, play multiple cellular roles, among which protein synthesis is the main one. Protein-nucleic acids (P-NA) interactions are thus involved in many biological processes. For instance, protein-RNA interactions form ribonucleoprotein complexes that mediate translation, intracellular transport, stress response, virulence of pathogens and are involved in different diseases (cancers, neurodegenerative or infectious diseases). Protein-DNA interactions are key for the functionality (replication, transcription, repair and recombination) and stability of the genome. The identification of P-NA interaction sites at the residue level (nucleotide and amino acid) is therefore of great biological and clinical significance. However, while high throughput methods for individual DNA/RNA/proteins identification are widespread (genomics/transcriptomics/proteomics), there are only few approaches that afford precise and systematic identification of RNA/DNA interaction sites at the single residue level. A variety of biophysical techniques has been developed for structural characterization of P-NA interactions: i) atomic-resolution techniques (crystallography, NMR or single-particle electron microscopy) that face typical challenges like sample requirements, size limitations and/or unfavourable crystallization/solubility ; ii) lower resolution techniques (e.g. small-angle X-ray scattering) that still fall short in elucidating P-NA conformational changes at the single residue level. Structural mass spectrometry (MS) approaches, including native MS, ion mobility and labelling (H/D exchange) or cross-linking MS (XL-MS) tools, have emerged as key techniques for the in vitro characterization of P-NA complexes, providing information on stoichiometries, dynamics and regions of close proximities. XL-MS appears as the most potent method to gain information on both protein and nucleotide sides. Photochemical XL using UV-light (UV-XL-MS) is the most popular method to study P-NA complexes in vitro, generating nearly ‘zero-length’ cross-linked peptide-oligonucleotide conjugates. However, while XL-MS has gained popularity in protein-protein interaction studies, it is still scarcely used for providing information on P-NA complexes, mostly because of the complexity of both analytical and data processing workflows. Indeed, all steps of the P-NA XL-MS workflow present limitations, explaining why XL-MS for P-NA stays the prerogative of a handful of experts worldwide. The aim of the NAProt-XLMS project is to provide new chemical cross-linking reagents and analytical MS-based proteomic workflows for the study of P-NA complexes through the : 1) synthesis of new chemical XL reagents based on the repurposing of drugs used in chemotherapy as P-NA cross-linking multifunctional probes; 2) development of sensitive, robust analytical LC-MS/MS proteomic workflows for in vitro and in vivo P-NA cross-linking; and 3) application of the new P-NA XL-MS methods on two well-characterized systems for proof-of-concept (nuclear receptor/DNA and ribonucleoproteins/RNA complexes). Altogether, the NAProt-XLMS project will provide new chemical tools along with improved proteomic analytics and bioinformatics for more accurate and straightforward characterization of P-NA machineries. This project will thus benefit to the whole community of biologists by providing tools for a more exhaustive and comprehensive characterization of the role of P-NA interaction in biological processes, opening new perspectives for large scale system wide P-NA interaction mapping.

Giant congenital diaphragmatic hernia is a rare congenital malformation with a high morbidity and mortality rate even today despite the fabulous progress of neonatal resuscitation and antenatal follow-up. These advances have made possible to bring increasingly severe forms of hernia to surgery, requiring the development of new research and progress in the field of surgical repair. Currently, these hernias are treated surgically during the first week of life in specialized hospital and university centers by interposition of a prosthesis in the vast majority of cases. Almost 30% of these children will present a release of this prosthesis during their childhood significantly increasing the morbidity and mortality of this congenital malformation throughout the development of the child; this makes it a priority axis of the national rare disease plan with the designation of reference center (CHU de Lille, collaborator of this project) and competence centers (CHU Strasbourg, project leader). In recent years, we have been able to prove the link between recurrence by releasing the prosthesis and certain non-optimal biomechanical characteristics of prostheses currently used in hospitals: insufficient colonization and lack of neoangiogenesis covering these prostheses, mechanical properties unsuitable for growing organisms. The DIAPID project aims to develop new prostheses responding to this double challenge: - better adapted mechanical properties: better stretchability thus adapting to the growth of the child while maintaining good mechanical resistance thanks to the electrospinning process of materials meeting the requirements of a medical device) - and a monolithic bifacial structure with a dual objective: to limit the colonization of the implant by the host on the abdominal side of the prosthesis and to optimize this colonization and rapid neoangiogenesis on the thoracic side for lasting tissue integration. The encouraging preliminary results allow us to propose a design of prostheses with a fibrous and functionalized face on the thoracic side and optimal biomechanical characteristics theoretically limiting the risks of release during childhood and therefore the morbidity and mortality of this pathology. The first results in mechanical and biological analysis must be deepened by additional experimental tests ex vivo and then in vivo on large animals. The strength of this project lies in having been able to combine the skills of laboratories and researchers in complementary fields of expertise with, on the one hand, pediatric surgeons recognized for their expertise in the field of this rare disease, researchers specializing in electrospinning, in biomechanics, bioengineering, biological functionalization and finally the reference center for diaphragmatic hernias having developed an animal model of diaphragmatic hernia.

Lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are major causes of morbidity and death worldwide for which therapies with efficient and prolonged local activity are much needed. Though it is considered as a promising approach to treat lung diseases, gene therapy still is reliant on the development of efficient and safe nucleic acid carriers. Poor gene transfer into the lungs has been attributed to numerous causes (limited cellular uptake, unproductive intracellular trafficking, carrier toxicity and immunogenicity…). A less elucidated bottleneck however is the adhesive and hyper viscoelastic airway mucus that prevents transfection particles from freely reaching the epithelium, thereby limiting gene transfer to the lungs. The LuTher research program aims at the development of nucleic acid carriers for administration to the airways. Sulfur-containing carriers will be designed so they display intrinsic mucolytic properties allowing transfection particles to overcome the mucus barrier in chronic inflammatory airway diseases associated with mucus hypersecretion such as asthma and COPD. They will be engineered to display biodegradability under various stimuli so as to liberate thiols in situ. These thiols are likely to disrupt disulfide bridges in the mucus biopolymer and reduce its viscoelasticity, allowing better penetration of the nucleic acid containing nanoparticles towards the targeted lung epithelium. The deeper and quicker the particles penetrate the mucus layer, the less they are subjected to elimination by mucociliary clearance and coughing. This in fine improves delivery of the therapeutic nucleic acid to the airway epithelial cells, which is mandatory for high transfection efficiency. Structure-activity relationship analysis is central to the project and will be conducted via biophysical and biological evaluations of the mucolytic carriers so as to determine the key parameters for efficiency and safety, and for improving the compounds through a feedback loop. Evaluations of the mucolytic carriers will include rating of transport and stability of transfection particles in mucus in vitro and ex vivo, and assessment of their efficiency in delivering pDNA and siRNA in both in vitro and in vivo models for asthma and COPD. Gene transfer efficiency will be determined using pDNA coding for the Luciferase protein whereas gene silencing experiments will be performed with p65 and MMP-12 targeting siRNAs, two potential targets for the treatment of asthma and COPD. The dual-function nucleic acid carriers proposed herein have no counterpart to date. Combining mucolytic activity and transfection properties in a single drug delivery system constitutes a strategy that has not yet been investigated for improving airway transfection. The intimate collocation of the transfection particles and in situ generated mucolytic agents is expected to boost the diffusion of transfection particles through the mucus towards the lung epithelial cells, provoking only local mucus alteration and thus keeping microviscosity of the latter to a minimum. A large number of nucleic acid delivery platforms, including viral and non-viral systems, have been developed to treat lung diseases. Poor gene transfer has been attributed to numerous biological barriers, including limited cellular uptake across the apical membrane, unproductive intracellular trafficking, carrier toxicity and immunogenicity. A less elucidated bottleneck however is the adhesive and hyperviscoelastic mucus that prevents gene carriers from freely reaching the epithelium, thereby limiting nucleic acid transfer. Consequently, overcoming the mucus barrier should be considered as an essential design criterion in developing a delivery platform capable of achieving clinical end points for lung gene therapy. This is the very purpose of the LuTher proposal.

The pharmacokinetics of many drugs, namely their resorption, distribution, metabolism and elimination depend on the hour of administration. As the consequence, these drugs are more effective and/or better tolerated if taken at appropriate time. Synchronization of drugs administration with the circadian cycle is not always possible or convenient. For instance, the anti-cancer drug 5-FU (5 Fluorouracile) should be taken at 4 a.m. to allow for a 50 percent improvement with respect to the non-chronomodulated treatment. Non-uniform distribution of medicaments in oral dosage forms (tablets or capsules) constitutes an advanced approach to programming of optimized diffusion-controlled drug release. However, creation of such systems with desired 3D-distribution of the drugs is a challenging issue. Within the framework of this research project, we propose a simple and cheap method to create biopolymer capsules with arbitrary complex spatial distributions of the drugs in them, and aim to explore the potential of the method for chronotherapies. A centimeter long, a few millimeter thick biopolymer capsules will be formed by spontaneous or assisted rolling up of thin strips of cellulose, chitosan, alginate, gelatin, or derivatives of these polymers. Prior to rolling, drug patterns will be formed on the strips (by inkjet printing or by stamping. Rolling up will transform the planar distribution of the drug along a strip in the 3D one inside the capsule according to a well defined relation, following from the archimedean spiral form of the rolls. Multidrug capsules can be also produced by this approach via printing a multidrug pattern on a strip and rolling up. In vitro release kinetics of drugs (carbamazepine, ranitidine, heparin, nonsteroidal anti-inflammatory drugs (NSAIDs), 5-fluorouracil, lorazepam, synthetic anti-inflammatory glucocorticoid medications, like methylprednisolone, triamcinolone, and prednisolone) from the capsules immersed in the classical physiological media defined in the 9th European Pharmacopeia (phosphate buffers, etc) will be monitored by fluorimetry, pH-metry, and high performance liquid chromatography. The experimental research will be accompanied by theoretical Fourier-Bessel analysis and random-walk simulation of the drug release processes from the capsules. Numerical and analytical solutions will be compared with the experimental data, and the models will be refined. The method’s potential for chronotherapy and chronopharmacology will be explored for drugs, known for their chronokinetics effects, on animal models. The in vitro and in vivo tests will be realized in accordance with the official EU and FDA guidelines. This project will have a significant impact on the materials engineering sector and on the health sector. When realized, the technology for time-programmed drug release can lead to considerable improvements in quality of life and the wellbeing of the patients (less of adverse and/or undesirable, toxic effects) who should take medicaments known for their chronopharmacokinetic properties. The new approach will also allow the patients with rare diseases to profit the advantages of chronomodulated therapies, and will bring new possibilities for personalized medicine.