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.