Team
Team manager: Legube Gaelle
Presentation
Since its creation in 2011, the lab is dedicated to better understand chromatin biology during DNA Double Strand break repair (legubelab). To tackle this question, we developed the DIvA system, a human cell line that allows to induce approximately one hundred DSBs at annotated positions. Combined to a large range of genome wide technologies (ChIP-seq, DRIP-seq, RNA-seq, Hi-C …), this cell line allowed us to uncover unexpected features of DSB repair processes, such as (i) unequal DSB repair depending on the genomic /epigenomic context, (ii) a repair pathway histone code, (iii) the existence of a DSB repair pathway specific to the transcribed genome (TC-DSBR) and (iv) and the role of chromosome architecture in these processes.
Project 1
We try to understand how the chromatin structure that pre-exists where a break occurs can control the repair pathway used.
Our work revealed that transcriptionally active genes tend to be repaired by Homologous Recombination in G2 (Aymard et al., 2014) in contrast to untranscribed loci that rather undergo NHEJ even during G2. As a follow up, we investigated the status of R-Loops, three stranded structures, that can stably form and decorate transcribed loci. Using ChIP-seq and DRIP-seq, we found that Senataxin (an RNA:DNA hybrid helicase) and RNA:DNA hybrids removal are critical factors in the repair of DSBs produced in transcriptionally active genes (Cohen et al., 2018).
We also investigated repair of DSB induced in ribosomal DNA which represents the most transcribed part of the genomes. We found that, DSBs induced on rDNA trigger transcriptional repression in a cohesin, CTCF and HUSH (a H3K9 methylation complex) dependent manner, at all phases of the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway (Marnef et al., 2019).
We are following up on these projects to fully delineate the repair mechanisms taking place at active loci.
Project 2
We also aim at drawing a more comprehensive picture of the chromatin landscape induced around DSBs. We described the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq and identified a “repair histone code”, i.e. the NHEJ and HR specific chromatin events (Clouaire et al., 2018).
Moreover, we are also trying to identify how chromosome architecture is modified following DSB and how this contributes to repair processes.
Using HiC, we found that chromosome folding, and more specifically the process of “loop extrusion” mediated by the cohesin complex, is instrumental for the assembly of DNA damage foci and proposed a model whereby H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin (Arnould et al, 2021).
Using super-resolution, time-lapse microscopy, Capture Hi-C and Hi-C, we also reported that DSBs cluster in mammalian nuclei. We discovered that upon DSB induction, a novel chromatin compartment forms, that we called the D-compartment (for DSB-induced compartment), which comprises not only in clustered DSBs but also on a subclass of DNA Damage responsive genes. We further found that the physical localization of these DDR genes in the D-compartment contributes to activate the DNA Damage response (Arnould, et al, 2023).
We are following up on these critical aspects of DSB repair by combining live imaging, high-throughput microscopy and 3C based techniques.
Team members
– Arnould C#, Rocher V#, Saur F, Bader AS, Muzzopappa F, Collins S, Lesage E, Le Bozec B, Puget N, Clouaire T, Mangeat T, Mourad R, Ahituv N, Noordermeer D, Erdel F, Bushell M, Marnef A and Legube G. Chromatin compartmentalization regulates the response to DNA damage. Nature. 2023 Oct 18. doi: 10.1038/s41586-023-06635-y.
– Cohen S#, Guenolé A#, Lazar I#, Marnef A, Clouaire T, Vernekar DV, Puget N, Rocher V, Arnould C, Aguirrebengoa M, Genais M, Firmin N, Shamanna RA, Mourad R, Bohr VA, Borde V and Legube G. A POLD3/BLM dependent pathway handles DSBs in transcribed chromatin upon excessive RNA:DNA hybrid accumulation. Nat Commun. 2022 Apr 19;13(1):2012. doi: 10.1038/s41467-022-29629-2.
– Marnef A, Legube G. R-loops as Janus-faced modulators of DNA repair. Nat Cell Biol. 2021 Apr;23(4):305-313. doi: 10.1038/s41556-021-00663-4.
– Arnould C, Rocher V, Finoux AL, Clouaire T, Li K, Zhou F, Caron P, Mangeot PE, Ricci EP, Mourad R, Haber JE, Noordermeer D and Legube G. Loop extrusion as a mechanism for formation of DNA damage repair foci. Nature 2021 Feb;590(7847):660-665. doi: 10.1038/s41586-021-03193-z.
– Marnef A, and Legube G.m6A RNA modification as a new player in R-loop regulation. Nat Genet. 2019 Dec 23. doi: 10.1038/s41588-019-0563-z.
– Marnef A, Finoux AL§, Arnould C§, Guillou E, Daburon V, Rocher V, Mangeat T, Mangeot PE, Ricci EP and Legube G. A Cohesin/HUSH and LINC-dependent pathway controls ribosomal DNA Double Strand Break repair. Genes Dev. 2019 Sep 1;33(17-18):1175-1190.
– Clouaire T, Legube G.A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks. Trends Genet. 2019 May;35(5):330-345. Review.
– Clouaire T, Rocher V, Lashgari A, Arnould C, Aguirrebengoa M, Biernacka A, Skrzypczak M, Aymard F, Fongang B, Dojer N, Iacovoni JS, Rowicka M, Ginalski K, Côté J, Legube G Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures. Mol Cell. 2018 Oct 18;72(2):250-262.e6.
– Cohen S, Puget N, Lin Y-L, Clouaire T, Aguirrebengoa M, Rocher V, Pasero P, Canitrot Y and Legube G. Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations. Nature Communications 2018 Feb 7;9(1):533.
– Aymard F, Aguirrebengoa M, Guillou E, Javierre BM, Bugler B, Arnould C, Rocher V, Iacovoni JS, Biernacka A, Skrzypczak M, Ginalski K, Rowicka M, Fraser P, and Legube G. Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes. Nature structural & molecular biology 2017 Apr;24(4):353-361.
– Aymard F, Bugler B, Schmidt C. K, Guillou E, Caron P, Briois S, Iacovoni J.S, Daburon V, Miller K. M, Jackson S. P, and Legube G. Transcriptionally active chromatin channels DNA double strand breaks to homologous recombination. Nature structural & molecular biology. 2014 21(4):366-74 PMC4300393
– Lacovoni JS, Caron P, Lassadi I, Nicolas E, Massip L, Trouche D,Legube G. High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 2010 Apr 21;29(8):1446-57.
All publications list here
