Histone variants act through the replacement of conventional histones by dedicated chaperones. They confer novel structural properties to nucleosomes and change the chromatin landscape. The functional and physiological requirement of the replacement of conventional histones by histone variants during organ formation and post-natal life remains poorly described. The incorporation of the histone variant H3.3 into chromatin is DNA-synthesis independent and relies on two different chaperone complexes, HIRA and DAXX/ATRX, which have different genomic deposition domains. While most epigenetic studies are performed in vitro, we intend to study them in an in vivo context where cell behavior can be properly addressed and where consequences for tissue formation, growth, homeostasis and repair can be fully investigated. Skeletal muscle provides the possibility to address yet poorly explored biochemical, cell biology, and developmental aspects of chromatin biology during development and postnatal life. Based on published and preliminary data from the three partners involved in this project, we hypothesize that: (i) HIRA and DAXX play a key role in muscle stem cells identity and muscle fibers organization (ii) H3.3 contributes to genome stability and prevents premature aging in adult muscle fibers (iii) a third H3.3 chaperone exists, which allows H3.3 incorporation into chromatin in the absence of HIRA and DAXX. Therefore, the main objectives of this proposal are defined in three work packages as follows: WP1: Conserved and divergent functions of H3.3 and DAXX-ATRX/HIRA pathways in muscle progenitors: we have recently shown that in the absence of HIRA, the muscle stem cell pool is lost during muscle regeneration. In addition, conditional HIRA inactivation in muscle progenitors during development have reduced myoblast numbers and smaller muscle size. In this context, our investigations will be extended to DAXX and H3.3. Our preliminary results indicate that DAXX is regulates myogenic gene expression via its histone chaperone activity. WP2: Role of H3.3 and DAXX-ATRX/HIRA pathways in adult myofibers structure and function: H2A.Z inactivation in adult muscle causes accelerated aging due to accumulation of DNA damage consecutive defective DNA repair by non-homologous end joining (NHEJ). H3.3 is also required for NHEJ. We therefore predict that H3.3 inactivation in muscle fibers will cause DNA damage and premature aging. Many evidences indicate that H3.3 regulates gene expression. We will determine if similarly to H2A.Z, H3.3 function in muscle fibers is restricted to DNA repair or if it also regulates gene expression. Finally, the roles of H3.3 chaperones have not yet been investigated in post-mitotic muscle fibers. To address these points H3.3, HIRA and DAXX will be inactivated in muscle fibers. We have recently shown that muscle fibers contain several myonuclear domains with specific identity and function defined by nuclei-specific expression profiles. The epigenetic landscape and myonuclei identity will be evaluated by single nuclei RNA seq and ATAC seq in the KO muscles. WP3: characterization of a new H3.3 deposition pathway that can bypass DAXX-ATRX/HIRA: H3.3 Chip-seq in Hira KO and Daxx KO myoblasts show HIRA and DAXX independent H3.3 deposition at specific loci, suggesting the presence of a third chaperone. Like other chaperones, this new chaperone should be part of a large multiprotein complex. We will isolate this complex from myoblasts and identify its composition. The complex will then be reconstituted with recombinant proteins to analyze its deposition properties. We will also invalidate the expression of some of the important components of the new deposition complex in vivo and we will determine the presumably perturbed H3.3 distribution pattern and the resulting cell phenotype at molecular level. Taken collectively, the expected data should shed in depth light on the intimate mechanism of H3.3 deposition and H3.3 function.

Tight regulation of energy homeostasis at multiple levels is instrumental for organisms to cope with changes in food availability. The Central Nervous System (CNS) orchestrates a complex array of processes mediating energy intake and expenditure. Hormonal, neuronal and nutritional signals according to changes in food absorption, energy storage and energy consumption in different organs reach the CNS which in turn triggers corresponding changes in feeding behavior and peripheral cellular metabolism. Orexigenic neuropeptide Y (NPY) and agouti-related peptide (AgRP)-expressing AgRP/NPY neurons and anorexigenic proopiomelancortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus are primarily involved in the regulation of energy homeostasis. To control appetite and peripheral metabolism, these neurons are regulated by several hormones. Among others, leptin, ghrelin and insulin emerged as key players in this context. Both leptin and insulin receptors are expressed in these neurons and both insulin and leptin have been found to activate POMC and to inhibit AgRP/NPY neurons. Ghrelin enhances the activity of AgRP/NPY neurons via its receptor, while it decreases the action of POMC neurons through a ghrelin receptor independent mechanism. Dysfunction of these neuronal circuits is known to contribute to overnutrition and obesity that eventually culminates in life-threatening type 2 diabetes (T2D) and/or cardiovascular diseases. Recent genome-wide association studies (GWAS) and GWAS meta-analyses revealed that they represent complex polygenic diseases. In fact more than ~250 genetic loci have been identified for monogenic, syndromic, or common forms of T2D and/or obesity-related traits. Despite this remarkable success, the contribution of most obesity- and T2D-associated single nucleotide polymorphism (SNPs) to the pathogenesis of these diseases remains largely elusive. CDC123 (cell division cycle protein 123)/CaMK1D (calcium/calmodulin-dependent protein kinase ID) represents one such locus on chromosome 10 strongly associated with T2D. Fine mapping identified a predominant SNP within this locus enhancing CaMK1D gene transcription. Thus, CaMK1D expression might be enhanced in and contribute to T2D. Substantial work in the past including our own efforts established a concept in which canonical stress kinase signaling interferes with physiologic metabolic pathways contributing to obesity-related insulin resistance and beta cell dysfunction, two main hallmarks of T2D. Keeping the focus and expertise on kinase-mediated signaling, we recently started to center our efforts on CaMK1D and its role in this disease context. In the last years, we have generated results directing us to hypothesize that CaMK1D primarily acts in AgRP neurons in the hypothalamus to control appetite and energy expenditure in response to ghrelin. The objective will be to explore more fundamentally the role of CaMK1D in AgRP neurons, opening a new avenue going beyond our previous research activity and expertise. To this end, we initiated a national collaborative project with the laboratory of Dr. Serge Luquet at the Functional and Adaptive Biology Unit at University of Paris specialized in central control of feeding behaviour and energy expenditure. Our partner laboratory will provide state-of-the-art neurometabolic tools and the necessary complementary expertise to pinpoint a role of CaMK1D in AgRP neurons. Our comprehensive experimental approach covering basic aspects of cellular signaling, physiology and neuroscience will thus set the stage for a new concept explaining as to how CaMK1D controls body weight and how its deregulation may contribute to obesity and T2D.

Since 2018, the Institute of Genetics and Molecular and Cell Biology (IGBMC) has been committed to adopt a new approach to drive global changes in scientific data management and follow FAIR principles (Findable, Accessible, Interoperable, Reusable). To meet this challenge, the IGBMC relies on digital tools set up at the national level like OPIDoR data management plan dashboard or the LabGuru electronic laboratory notebook, but also local data management solution like OMERO storage, visualization and annotation tools and an institutional data warehouse that enable researchers to archive their work in a sustainable and secure manner. To date, we have identified two major parameters that limit the effectiveness of our organization. First, heterogeneous digital tools made available to research teams cause a scattering of data and loss of metadata. Second, beyond the support that can be offered to researchers to use these tools, their adoption or daily use remains complex because it requires a constant and diligent follow-up to keep in track with the FAIR principles. In this context, the IGBMC aims to associate its IT department, its imaging center and three research teams to carry out an experiment dedicated to the development of 1) a central and transversal gateway tool for the functional integration of the existing data management tools and 2) a methodology for monitoring projects and their data. The gateway tool, in the form of a web application, will facilitate the transversal identification of projects and their associated data, from the data management plan created on OPIdoR, to the publication, through the LabGuru electronic lab notebook and data processing tools such as OMERO. The aim is to streamline the transfer of data from production to archiving, while automatically enriching metadata. This tool will automate the publication of the archived data in official catalogs and reference them in indexes such as datacite (datacite.org) for sharing the research outputs. Keeping track of metadata for each dataset is an essential aspect to be compliant with FAIR principles. In parallel, the development of a methodology for monitoring projects will aim to support the research teams to show them the benefits of developing a data management plan from the beginning of a project, raise awareness of data storage management costs and train them in the FAIR principles. This experimentation will be carried out on light microscopy image data before extending it to all types of biology data (omics, phenotyping, etc.) for the next stage of the project. All connectors and gateways as well as dashboards will be designed to be usable in any research lab and released in standard package format under an OpenSource license.

The ribosomal DNA (rDNA) is a highly repetitive genomic region which is essential to initiate ribosome biogenesis. In budding yeast, frequent DNA damages within the rDNA (rDNA damages) triggers the formation of extra-chromosomal rDNA circles (ERCs), which are implicated in replicative aging through a mechanism that is still not clearly understood so far. The objective of this project is to establish which proteins interact specifically with the rDNA sequences during rDNA damages and ERC formation and accumulation. To achieve this goal, we will develop a unique methodology where proteins in close proximity to the rDNA are labelled by biotinylation. Then, we will analyze the biotinylated proteins by mass spectrometry. Hence, we will identify the molecular players involved in rDNA damage repair and in ERC excision and accumulation. This proteomic screen will be complemented by experiments combining fluorescence microscopy and microfluidics. This will allow us to validate the candidates identified by mass spectrometry. In addition, we will monitor, by time-lapse imaging, the dynamics of rDNA damage repair and the accumulation of ERCs in single cells. These results will help to understand: in which cases rDNA damages generate ERCs, why ERCs are toxic to the cell, and how they trigger a loss of nuclear homeostasis during replicative aging. Finally, this work focusing on ERCs in budding yeast will provide a general framework to study the extra-chromosomal DNA circles observed in human cells.

Increasing attention is being focused on the role of protein structural dynamics in crucial cellular signaling pathways and modulating structural dynamics is becoming an important avenue of exploitation for the discovery of new therapeutic compounds. However, there remains a serious paucity of experimental techniques that permit one to obtain relevant data related to structural dynamics on appropriate timescales. New approaches are needed to both elucidate and measure physical properties directly related to structural dynamics. Various physical chemical methods have been used to probe the structural dynamics, but each technique has it own limitations. So the search for new methods, complementary to existing ones, is still underway. In this proposal, our primary objective is to develop THz/far infrared absorption spectroscopy for the ligand binding events. Ligand binding will influence the underlying collective motions in this frequency range, which can be captured by far-IR experiments and interpreted through molecular simulations and structural analysis. Previously, we combined far-IR and molecular dynamics simulations to study the response of a PDZ domain to the binding of a small peptide ligand to elucidate the mechanism of allostery. We showed that exploitable information concerning changes in low frequency collective motions could be obtained even for proteins where there is no substantial conformational change upon ligand binding. This integrated approach allowed us to quantify a mechanism of allostery in a PDZ domain. In this project, we aim to enlarge the field of application and characterize the allosteric behavior in nuclear receptor (NR) proteins, which constitute a superfamily of proteins that function as DNA-binding, ligand dependent transcription factors. Being a larger, more complex protein than the one used in our preliminary study, they are implicated in the transcriptional cascade underlying many physiological phenomena making them one of the major signal transduction paradigms in metazoans. Indeed, evidence suggests that there exist multiple mechanisms exploiting structural dynamics and allostery that implicate ligand, DNA, co-activator and co-repressor binding, as well as post-translational modifications. Central to these mechanisms is the ligand binding domain (LBD), which acts as an allosteric hub, transmitting binding events to other protein interfaces and domains. We will focus on Peroxisome Proliferator-Activated Receptor gamma (PPAR-gamma) and its heterodimeric partner, RXRa, a nuclear receptor complex that is a particularly important target for development of therapeutic compounds for multiple diseases, including diabetes and cancer. Our project will begin with a study of the LDBs in both wild-type and mutant forms. We will characterize the effects of ligands, including agonists, antagonists and co-regulator peptides by far infrared absorption spectroscopy, molecular dynamics simulations and biophysical/structural characterization and interpret the results in the context of allosteric effects. Following the study of the LDB, we will expand our study towards heterodimer structures. A second objective is to gain molecular insight into the mechanisms of selective modulation of NRs activity by small-molecule ligands and ultimately make the link with their pharmacological profile. The consortium is comprised of members complementary in their expertise coming from fields of physical and computational chemistry, biophysics and structural biology. Through the fundamental research to be carried out by this high-level multi-disciplinary unit, a novel approach for the study of ligand binding in biomolecular systems will emerge. This, we believe, makes it a completely original methodological approach that we plan to further develop in the context of this call.