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Chromatin function in DNA Double Strand breaks repair: Prime, repair and restore DSB Inducible via AsiSI

Periodic Reporting for period 4 - DIvA (Chromatin function in DNA Double Strand breaks repair: Prime, repair and restore DSB Inducible via AsiSI)

Reporting period: 2020-01-01 to 2020-12-31

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 aimed 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 wanted 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.
Using genomic technologies, we reported the most comprehensive landscape available to date of histone modifications induced around DSBs, as well as the 3D genome folding and changes in chromosome organization following DSB induction on the human genome. Our work also allowed to identify a novel repair pathway that handles DSB induced in transcriptionally active loci.
The knowledge acquired with the completion of this project helps to better understand the process that lead to genome instability and rearrangement, which lie at the heart of cancer onset and progression.
During this project, we investigated the contribution of chromatin during the response to DNA Double strand break.
First we found that pre-establish chromatin is a key determinant for the repair process. Indeed, altogether our work revealed that when damaged, active genes exhibit a very peculiar behavior compared to the rest of the genome and undergo a specific repair pathway that we named TC-DSBR (Transcription Coupled DSB Repair). We wrote a number of reviews to discuss this novel pathway (Clouaire et al., 2017; Marnef and Legube, 2017; Marnef et al., 2017; Puget et al., 2019). In brief, we found that these DSB induced in active genes, are clustered and mostly left unrepaired during the G1 phase of the cell cycle (Caron et al, 2015, Aymard et al, 2017) and are repaired by homologous recombination in G2, in a manner that involves the Senataxin RNA:DNA helicase (Cohen et al, 2018). Deficiency of this pathway enhances translocations frequency (Cohen et al, 2018). Moreover, we also reported new insights on how DSB occurring on the most transcribed part of our genome, i.e. the ribosomal DNA, are repaired. Unexpectedly we discovered that the nuclear envelope is somehow involved in this repair process, and identified a chromatin modifying complex (the HUSH complex) as involved in ribosomal break repair (Marnef et al, 2019).

Second, during this project we provided the most comprehensive view of the chromatin structure that is induced around DSBs: We described the chromatin landscape induced at DSBs, and identified a “repair histone code” (Clouaire et al, 2018). We also reported how chromosome folding is modified post break induction using chromosome conformation capture, followed by high throughput sequencing. This allowed us to discover that chromosome folding, and more specifically the process of “loop extrusion” is instrumental for the formation of the DNA Damage response foci (Arnould et al, 2021).
These findings that DSBs occurring in active genes display a particular fate represent a key advance in our understanding of the mechanisms that ensure genome stability. Indeed, recent high-throughput genomic approaches revealed that endogenous DSBs occur far more frequently than previously thought, mainly within the transcribed regions of the genome. Such DSBs were shown to occur under physiological conditions and also in pathological contexts such as upon oncogenic stress. Our plan is now to build on our past work, and to characterize in-depth this novel pathway, its function in maintaining genome integrity and its potential as a therapeutic target for cancer therapy.
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