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Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks


Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation–sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation–associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

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Figure 1: A subset of AsiSI-induced DSBs recruits RAD51.
Figure 2: RAD51 recruitment at RAD51-bound DSBs occurs mainly in G2.
Figure 3: RAD51-bound AsiSI-DSBs are resected and repaired by a RAD51-dependent pathway.
Figure 4: RAD51-bound DSBs lie within transcribed units.
Figure 5: Transcriptional activation of an unexpressed gene leads to repair by HR.
Figure 6: H3K36me3 enrichment on chromatin correlates with the use of HR.
Figure 7: The H3K36me3-LEDGF axis targets RAD51 at DSBs induced in active genes.
Figure 8: Targeting of HR at transcriptionally active loci.

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We thank New England Biolabs for providing AsiSI genomic DNA. We thank B. Lopez (Institut de Cancérologie Gustave-Roussy) for RG37-HR I-SceI GFP cells. We thank V. Benes and the Solexa team at the European Molecular Biology Laboratory Genomic Core Facility, the Beijing Genomic Institute (BGI) and the Genomics core facility at the Cancer Research Institute (CRI) in Cambridge for high-throughput sequencing. We thank the Flow Cytometry platform from the Fédération de Biologie de Toulouse at the Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP-FRBT). F.A. is supported by a grant from the Ligue Nationale Contre le Cancer (LNCC); P.C. is supported by a grant from the Association Contre le Cancer (ARC); and S.B. and E.G. are supported by grants from the Fondation pour la Recherche Médicale (FRM). Research in S.P.J.'s laboratory is supported by grants from Cancer Research UK (C6/A11226), the European Research Council and the European Community's Seventh Framework Program (DDResponse) and by core infrastructure funding from Cancer Research UK and the Wellcome Trust. K.M.M. was funded by a Wellcome Trust Project grant and C.K.S. by a Return-to-Europe Federation of European Biochemical Societies fellowship. S.P.J.'s salary is provided by the University of Cambridge and supplemented by Cancer Research UK. Funding to G.L. was provided by grants from the ARC, Agence Nationale pour la Recherche (ANR-09-JCJC-0138), Canceropole Grand Sud Ouest (GSO) and Research Innovation Therapeutic Cancerologie (RITC).

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F.A., P.C., E.G. and V.D. performed experiments. B.B. developed the AID-DIvA cell line. C.K.S. suggested and performed immunofluorescence studies on laser-induced damage. J.S.I. and S.B. performed bioinformatic analyses of the ChIP-seq data. K.M.M. performed XRCC4 ChIP library preparation and sequencing. G.L. conceived and analyzed experiments. F.A., J.S.I., K.M.M., S.P.J. and G.L. wrote the manuscript.

Corresponding author

Correspondence to Gaëlle Legube.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 γH2AX profile around AsiSI-induced DSBs.

ChIP-seq against γH2AX (Epitomics) performed in 4OHT treated DIvA cells. a. A representative γH2AX domain is presented (top panel, chromosome position are indicated in Mbp). A magnification of the γH2AX enrichment at the proximity of the DSB is shown (bottom panel, chromosomal positions are in bp). The DSB is indicated by an arrow. b. Average γH2AX signal on a 20kb window around each annotated AsiSI site on the human genome. c. The γH2AX signal obtained by ChIP-seq, around the DSBs presented Fig. 1b is shown. DSBs are indicated by arrows. RAD51-bound DSBs are indicated in red, and RAD51-unbound DSBs in blue.

Supplementary Figure 2 Binding of RAD51 and XRCC4 at AsiSI-induced DSBs.

a. Duplicate ChIP-seq experiment in 4OHT treated DIvA cells using an anti XRCC4 antibody (Abcam) or an anti-RAD51 antibody (Santa Cruz). The averaged XRCC4 (blue) and Rad51 (red) signal on 10kb around annotated AsiSI sites on the human genome are shown. b. Signals obtained around the four DSBs presented Fig. 1b are shown. Positions are indicated in bp. c. ChIP experiments performed in DIvA cells, treated (filled circles) or not (empty circles) with 4OHT, using an anti-XRCC4 antibody (blue) or an anti-RAD51 antibody (red). ChIP efficiencies (as percent of input immunoprecipitated) were measured by RT-qPCR at 80, 200 and 800bp from an AsiSI induced DSB (DSB-III). Mean and S.e.m (technical replicates n=4) of representative experiment is shown.

Supplementary Figure 3 Measure of cleavage efficiency at selected AsiSI sites, throughout the cell cycle.

Cleavage efficiency (using the protocol described in the online methods) was measured from untreated or 4OHT-treated cells by RT-qPCR for five AsiSI sites either identified as RAD51-bound or RAD51-unbound. The same representative experiment is presented in a, b and c. a. Raw qPCR data, as percent of input pulled down by the streptavidin purification are shown. b. The data presented in (a) were further normalized using an internal control constituted by in vitro AsiSI-digested plasmid added in the ligation reaction. This allows calculating the cutting efficiency at each specific sites as percent of cleavage (the in vitro digested plasmid representing the 100% cleavage). c. Ratios between treated and untreated cells are presented (using the normalized data presented in b). This last representation will be used throughout the manuscript. d. Pull own efficiency was measured by RT-qPCR for the eight AsiSI sites analyzed for the presence of ssDNA in Fig. 3a. Ratios between treated and untreated cells are presented. Mean and s.e.m (technical replicates, n=4) of a representative experiment is shown.

Supplementary Figure 4 Kinetics of DSB induction and disappearance in the AID-DIvA cell line.

a. HA and γH2AX staining in AID-DIvA treated as indicated. b. Measure of cleavage efficiency, at two AsiSI-induced DSBs, in AID-DIvA cells treated as indicated. c. γH2AX staining in AID-DIvA cells treated (or not) with 4OHT for 4h and further incubated with auxin (or not) as indicated. d. γH2AX foci observed in AID-DIvA cells treated as in c, were counted using images aquired with an Array-Scan device. The mean and s.e.m (n=5, biological replicates, about 200 acquired nuclei per experiment) are presented. e. Repair analysis of AsiSI-induced RAD51-unbound (in blue, right panel) or RAD51-bound (in red, left panel) DSBs in AID-DIvA cells transfected with control, RAD51, or XRCC4. A biological replicate from Fig. 3f is shown.

Supplementary Figure 5 HR-prone DSBs are enriched in active chromatin marks and in PolII-S2P.

a. ChIP-seq data of various histones marks, associated with active (top and middle panels) or inactive (bottom panels) chromatin in U20S and K562 cells were retrieved from the ENCODE project, over 4kb surrounding each DSB from the RAD51-bound (red) and RAD51-unbound subsets (blue). The name of the Institute that generated the data are indicated in parentheses. The average and s.e.m are shown. p values (Mann-Whitney) are shown above each graphs. b. Average PolII–S2P signal obtained by ChIP-seq in DIvA cells, around the transcription start sites (TSS) of all genes from the human genome (hg18). c. Similar analysis than for Fig.4b except that all cleaved DSBs (100 best) were divided in two categories of 50 DSBs each based on their ratio RAD51/XRCC4. d. The Pol II–S2P signal obtained around two RAD51-bound DSBs, but far from any annotated gene (RefSeq, indicated in brown) are shown. DSBs are indicated by arrows, and positions are in bp. Feature from in lincRNA (large intergenic non coding RNAs) and TUCP (transcripts of uncertain coding potential) database (UCSC)(left panel), or from annotated pseudogene and non coding RNA (UCSC)(right panel) are indicated in purple.

Supplementary Figure 6 Effect of DRB and actinomycin D on transcription, RAD51 binding and AsiSI-mediated break induction.

ChIP using a PolII-S2P antibody in 4OHT-treated DIvA cells incubated with DRB (a) or actinomycin D (b) PolII-S2P levels were analyzed within a 1kb region around DSBs shown Fig. 4c-d, i.e. three RAD51-bound DSBs (DSB-I; -II; -III), one RAD51-unbound DSB (DSB-1), and two DSBs located far from any gene (DSB-5, -6). c. XRCC4 (blue) and RAD51 (red) ChIP in 4OHT-treated DIvA incubated with Actinomycin D or DRB as indicated. XRCC4 and RAD51 enrichments were respectively analyzed close to (<100bp) and at 800bp from selected DSBs, by RT-qPCR. The mean and s.e.m (n=4 technical replicates) of % of input immunoprecipitated for three DSBs (one RAD51-bound: DSB-III, one RAD51-unbound: DSB-1, and one intergenic: DSB-5) are shown. d. Analysis of cleavage efficiency in DIvA cells incubated with Actinomycin D (or not) (see online Methods). e. Analysis of cleavage efficiency in DIvA cells in G1 or G2 synchronized cells, and in presence or absence of DRB treatment as indicated, at an AsiSI site either identified as RAD51-bound (in red), or RAD51-unbound (in blue). Mean and s.e.m (n=4, technical replicates) of the raw qPCR data of a representative experiment, as percent of input pulled down by the streptavidin purification are shown.

Supplementary Figure 7 Effect of LEDGF and SETD2 siRNAs on H3K36me3, RAD51, XRCC4 and PolII-S2P levels.

DIvA cells were transfected using a control siRNA, a siRNA directed against LEDGF (a-d) or a siRNA directed against SETD2 (e-h). a,e. Relative mRNA level assessed by reverse transcription followed by qPCR. b,f. H3K36me3 ChIP analysed by qPCR at selected sites. c,g XRCC4 (blue) and RAD51 (red) ChIP after 4OHT-treatment respectively analyzed close to (<100bp) and at 800bp from selected DSBs. ChIP efficiencies (as % of input) are shown for 3 DSBs (RAD51 bound (DSB-III), RAD51 unbound (DSB-1), and intergenic (DSB-5)). The mean and s.e.m (n=4, technical replicate) of a representative experiment are presented. d, h PolII-S2P ChIP analyzed within a 1kb window around DSBs. The mean and s.e.m (n=4, technical replicates) of a representative experiment are presented. p values are indicated, ns non-significant (unpaired student test, two sided).

Supplementary Figure 8 Uncropped blots presented in Figure 3.

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Supplementary Figures 1–8 and Supplementary Tables 1–4 (PDF 943 kb)

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Aymard, F., Bugler, B., Schmidt, C. et al. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nat Struct Mol Biol 21, 366–374 (2014).

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