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DIvA Report Summary

Project ID: 647344
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - DIvA (Chromatin function in DNA Double Strand breaks repair: Prime, repair and restore DSB Inducible via AsiSI)

Reporting period: 2015-07-01 to 2016-12-31

Summary of the context and overall objectives of the project

Maintaining genome integrity is of crucial importance for multicellular organisms. This is illustrated by the variety of human diseases, associated with DNA repair defects. Among the lesions that can occur on the genome, DNA Double Strand break (DSB) are the most deleterious since they can trigger genome rearrangements such as translocations. Over the past few years it has become evident that chromatin, being the real substrate for DNA related processes, plays a decisive role in DSB repair. Therefore, understanding how DSB repair is affected by chromatin structure is an outstanding challenge nowadays.

While ChIP followed by high throughput sequencing (ChIP-seq) is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DSB repair has been hindered by the limitations of the available damage induction methods. Indeed, genotoxic drugs or radiation, usually used to generate DSBs, induce breaks at random positions throughout the genome, which are not suitable for subsequent ChIP analyses. We previously developed a new experimental system, based on the use of a restriction enzyme fused to the ligand binding domain of the oestrogen receptor that generate multiples sequence-specific and unambiguously positioned DSBs across the genome, therefore compatible with ChIP-seq.

In this project we aim at deciphering the relationship that exists between chromatin and DSB repair, by using this novel cell line combined with various technologies based on high throughput sequencing. More specifically we want to investigate whether and how chromatin dictates the choice of repair pathways, how the chromatin is modified following break detection and how it is faithfully restored following repair completion to maintain cell fate.

The knowledge acquired with the completion of this project should help in a better understanding of the processes that lead to genome instability and rearrangement, which lie at the heart of cancer onset and progression.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first period of the project, we focused on the spatial reorganization of chromosomes following DSB generation. More specifically we addressed whether multiples DSBs can coalesce (or cluster) together within the nucleus, which may therefore promote translocation (occurring when two DSB are juxtaposed). We discovered that DSB can indeed cluster within foci but only when they are induced in the transcribing fraction of the genome (active genes). Importantly we also found that clustering coincides with delayed DSB repair and that it is enhanced during G1. Finally we started to decipher the mechanisms that promote clustering and found that ATM (one of the main kinase activated following DSB), the MRN complex (a well-known player involved in repair), but also some component of the cyto/nucleoskeleton (namely Formin 2 and the LINC complex) all contribute to DSB clustering.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Recent data indicate that active genes likely represent a fragile fraction of our genome, and experience high breakage frequency. In addition most of the cells in multicellular organisms are non-proliferative and arrested in G1.
The discovery that damaged active genes are prone to cluster, especially in G1 cells, may reveal of particular importance to understand the genomic instability associated with cancer apparition. In addition, the possibility that active mechanisms could be involved in translocations will deserve close attention as the cytoskeleton is a well-known and robust target for cancer therapy
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