ESR2: “Epigenetic information recovery after DSB repair”

CNRS (G. Legube group): Epigenetic information recovery after DSB repair

i. Objective of research: To study how chromatin modifications established during DSB repair are erased following repair completion, in order to recover proper epigenetic information.

ii. Current state of the art: Amongst the types of damage that can challenge the DNA molecule, 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 groups of pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) that are both essential for survival. Over the past few years it has become evident that chromatin, that packages DNA in eukaryotes nuclei and that is encoding epigenetic information, is the real substrate for all DNA related processes and plays a decisive role in DNA repair. Following break induction chromatin is locally modified and recently, using ChIP-seq, we have been able to draw a more comprehensive picture of the chromatin landscape induced around DSBs, and more specifically to decipher the “histone code” associated with each repair pathway (HR or NHEJ). However, in order to maintain cell fate, this specific chromatin landscape established during DSB repair must be removed following repair, in order to restore the proper epigenetic information.

iii. Research methodology and approach: A prerequisite to investigate how chromatin is restored following DSB repair, is to induce DSBs at well identified loci where chromatin can be analyzed. Hence, we will use the experimental system that we developed a few years ago, where multiples sequence-specific DSBs, widespread across the genome can be rapidly induced using a restriction enzyme whose activity can be turned ON and OFF by adding respectively hydroxytamoxifen (4OHT) and auxin (IAA) to the culture medium. ChIP-seq will be performed against histone modifications and histone variant identified as either induced/deposited (H4K20 trimethylation, macroH2A, γH2AX) or removed (H3K79me2, H2AZ, H1), before DSB induction, after DSB induction (with 4OHT) and at different time points after auxin addition. These experiments will also be performed in synchronized cells in order to address the cell cycle stage specificity for chromatin recovery, and in various siRNA backgrounds (against chromatin modifiers). Functional consequences on cell survival will be further investigated.

iv. Originality and innovative aspects of the ESR project: While chromatin modifications establishment and function have received considerable attention over the past few years, the information on kinetics and mechanisms for chromatin recovery following repair are still very sparse, although these events are necessary to maintain cell fate. The project will give insights into this essential step of DSB repair.

v. Integration of the ESR project to the overall research programme: Our ESR will collaborate with the Polo group on microscopy after UV damage, with the Crosetto group on the genome-wide detection of DSBs, with Genevia and Norgenotech on the analysis of ChIP-seq data and the development of novel assays for DSB detection.