Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - DIvA (Chromatin function in DNA Double Strand breaks repair: Prime, repair and restore DSB Inducible via AsiSI)

Teaser

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...

Summary

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

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 (Caron et al, 2015) but only when they are induced in the transcribing fraction of the genome (active genes) (Aymard et al, 2017). 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 (Caron et al, 2015; Aymard et al, 2017).
Hence, during these studies we found that active genes, when broken, exhibit a very specific behaviour, being clustered and unrepaired in G1 and repaired by homologous recombination in G2

In an attempt to further determine the other players that could contribute to active gene repair, we next identified senataxin, a protein mutated in a rare genetic disease, as critical for repair of DSB induced in active genes. We found that senataxin removes RNA:DNA hybrids that form at DSB, promotes the use of homologous recombination and couteracts illegitimate rejoining of distant DNA ends, a process involved in the generation of translocations (Cohen et al, 2018)

Final results

\"Recent data indicate that active genes likely represent a fragile fraction of our genome, and experience high breakage frequency. Altogether our data tends to demonstrate that a specific repair pathway is at work to repair these broken active genes, a pathway that is coordinated with transcription extinction of the damaged gene.
Hence we now intend to caracterize this new \"\"Transcription coupled DSB repair pathway\"\" both in term of repair mechanisms and chromatin changes.
We are currently caracterizing in a comprehensive manner the chromatin landscape that is specifically set up at DSBs, and we will further try to decipher how these newly deposited marks are removed following repair,in order to properly reestablish the epigenetic informtaion and resume transcription.


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