Our Research interests

 

The aims of our research are to understand how DNA double-strand breaks (DSBs) are repaired in the context of chromatin and what dictates the repair pathway choice. DSBs are mainly repaired by either Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). When inaccurately used, these repair pathways have dramatically different consequences on the genome; with for examples translocations mainly caused by Alt-NHEJ, or repeat amplifications provoked by the use of unequal HR. The choice between all the available pathways is thus a critical aspect of DSB repair. However, how this choice is executed is far from being understood.

 

​To address the molecular mechanisms at play, we developed the DIvA cell line which is a powerful experimental cell system that enables the creation of DSBs at well known positions across the genome in various chromatin contexts and which has the advantage of detaining statistical power over a large range of DSBs.

Questions adressed

Tools to induce DSBs

 

As their name suggests, DIvA cells, for DSB Inducible via AsiSI, rely on the expression of the AsiSI restriction enzyme, which relocates from the cytoplasm into the nucleus thanks to an oestrogen receptor ligand-binding domain activated by OHT addition. This relocalization allows the cutting of DNA at ~100 annotated positions across the human genome (Iacovoni et al, 2010; Massip et al, 2010, Aymard et al, 2017, Clouaire et al, 2018).

 

In order to study the DSB repair kinetics, we additionally developped the AID-DIvA cell line where AsiSI is fused to an auxin-inducible degron enabling its degradation.

Questions in details

 

How does the chromatin state influence the recognition of DSB and the repair pathway choice?

In eukaryotes, DSB repair occurs within chromatin which is the structure that tightly packages and regulates DNA metabolism. A fair amount of studies, including ours, have suggested a role of chromatin in addressing the repair pathway. Our hypothesis is that a "repair histone code" exists, where specific histone marks present before the appearance of a DSB, could specifically help to recruit and/or stabilize one or the other repair pathway (Clouaire, et al, 2015). And indeed, chromatin influences DSB repair at various steps, from the break detection to sequence recovery, with for example the histone mark H3K36me3 being important for channeling DSB induced in active genes towards HR (Aymard et al, 2014). Importantly, chromatin can also be altered during the repair process, and this at various scales, in a manner that relates to the repair pathway used (Clouaire et al. 2018).


In order to decipher the chromatin landscape induced at DSBs, we mainly used BLESS, ChIP-chip/seq and RNA seq to draw high resolution profiles of DSB-induced chromatin modifications and DNA repair complexes around breaks (Iacovoni et al, 2010; Massip et al, 2010; Caron et al, 2012; Clouaire et al, 2018).

 

How are active genes repaired?

Recent work revealed that transcriptionally active loci are prone to breakage and our work suggested that when damaged, active genes display a very peculiar behavior compared to the rest of the genome: they experience a strong repair delay and undergo clustering in an actin/LINC and MRN-dependent manner in G1 (Aymard et al, 2017) while they are mostly repaired by homologous recombination in a Senataxin-dependent manner in post-replicative cells (Cohen et al, 2018). We are trying to characterize in more details this novel transcription-coupled DSB Repair (TC-DSBR) pathway (reviewed in Marnef et al, 2018).

 

How does chromosome conformation changes following DSB?

Our previous work deciphered the histone modifications landscape induced at DSBs. Yet, the structure of chromatin (nucleosome density, folding) in cis around DSB remains to be determined. Using chromosome conformation capture, we try to understand the structure in 3D of the chromosomes following breakage (such as (Aymard et al. 2017) where genome-wide mapping of long-range contacts using Capture-HiC enable us to unveil the clustering of DNA double-strand breaks at damaged active genes.

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