DIvA

Maintaining genome integrity is of crucial importance for multicellular organisms, as illustrated by the variety of human diseases associated with DNA repair defects. Among the types of damage, DNA Double Strands Breaks (DSBs) are the most deleterious since they can lead to various mutations and chromosomes rearrangements. DSBs are repaired by two main pathways, namely Non Homologous End Joining (NHEJ) and Homologous Recombination (HR), both essential for survival. Over the past few years it has become evident that chromatin is the real substrate for all DNA related processes and plays a decisive role in DNA repair. Repair into this chromatin context raises several questions that we aim to address in this proposal: Firstly, we (Aymard et al, 2014) and others (reviewed in (Soria et al, 2012)) have accumulated evidences that the chromatin environment where a break occurs influences the choice of repair pathway. Since the different DSB repair mechanisms can lead to very different "scar" on the genome, further studies are clearly required to elucidate how chromatin structure regulates the targeting of DSB repair machineries. Secondly, while many DSB-induced chromatin modifications have been identified recently, a complete picture of the chromatin landscape set up at DSB is still awaited. More specifically the set of histone modifications associated with each repair pathway ("repair histone code") is still completely unknown. Finally, once repair has been completed, the initial chromatin landscape must be faithfully restored in order to maintain epigenome stability and cell fate. Until recently, the lack of systems that induce breaks at defined places on chromatin has completely precluded these studies, and this aspect of DSB repair is thus yet uncovered. We have recently developed a new experimental system, based on a restriction enzyme (AsiSI-ER), which generates multiple sequence-specific and unambiguously positioned DSBs widespread across the genome (Iacovoni et al, 2010; Massip et al, 2010). This system provides the unique opportunity to study simultaneously, with a high resolution and at a molecular level, the chromatin changes and repair events occurring at many DSBs located in various chromatin contexts. In addition, we have recently improved our DSB-inducible system to also be able to turn off the enzyme activity in a controlled manner (Aymard et al, 2014): one can now investigate the events that occur once DNA has been repaired. With such systems in hand, we propose to proceed in an investigation of the several uncovered aspects of the relationship between chromatin structure and DSB repair cited above. Using high-throughput genomic and proteomic technologies we will try (i) to understand the contribution of chromatin in the DSB repair pathway choice (PRIME), (ii) to describe more thoroughly the chromatin remodeling events that occur concomitantly to DSB to promote adequate repair (REPAIR) (iii) to elucidate the processes that are at work to restore epigenome integrity after DSB repair (RESTORE).