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Activation of homologous recombination in G1 preserves centromeric integrity

Abstract

Centromeric integrity is key for proper chromosome segregation during cell division1. Centromeres have unique chromatin features that are essential for centromere maintenance2. Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements3, little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle4. Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA–RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of the RAD51–BRCA1–BRCA2 complex5 and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity.

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Fig. 1: Centromeric DSBs recruit RAD51 throughout the cell cycle.
Fig. 2: H3K4me2 supports DNA-end resection by promoting centromeric transcription and increased R-loop formation in response to DSBs.
Fig. 3: The USP11–HJURP–CENP-A axis licenses HR at centromeric DSBs in G1.
Fig. 4: Licensing of HR at centromeric breaks in G1 promotes centromeric integrity.

Data availability

Source data are provided with this paper.

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Acknowledgements

We thank K. Caldecott, D. Durocher, Y. Dalal for critical reading of the manuscript and members of the Soutoglou laboratory for comments; A. Nussenzweig for the BRCA1 antibody; A. Sfeir for the polθ−/− MEFs; A. Aguilera and K. Mekhail for RNAseH plasmids; D. Skalnik and G. Stewart for the SETD1A and ΔSET plasmids; Y. Dalal for the GFP–CENPA and GFP–HJURP plasmids; Daniel Foltz for the mCherry–LacI–HJURP plasmid; and the IGBMC Imaging Center for support. D.Y. was supported by the Ministerial fellowship from the UdS doctoral school and the Fondation pour la Recherche Medicale (FRM). The E.S. laboratory was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation program (ERC-2015-COG-682939) and by ANR-10-LABX-0030-INRT, managed by the Agence Nationale de la Recherche under the program Investissements d’Avenir labelled ANR-10-IDEX-0002-02 and the Academy of Medical Sciences (AMS). M.A. acknowledges research funding from the Swiss National Science Foundation (PP00P3_179057).

Author information

Authors and Affiliations

Authors

Contributions

E.S. and D.Y. conceived the study. Most of the experiments and data analysis were performed by D.Y. Genomic instability in WT and Polθ−/− MEF and related experiments were performed by K.M. QIBC experiments and quantifications were performed by A.L. and Y.W. under the supervision of M.A. A.F. provided technical support. B.R.-S.-M provided Cas9 vectors and designed guide RNAs. E.S., D.Y., M.A. and B.R.-S.-M. wrote the manuscript.

Corresponding author

Correspondence to Evi Soutoglou.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Nausica Arnoult and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of HR factors recruitment at centromeric and pericentromeric DSBs in G1 and G2.

a, Immunofluorescence confocal analysis of (upper part) NIH3T3 (EdU- for G1 and RO-3306 arrested for G2) cells expressing GFP-CENP-A and (lower part) U2OS cells (thymidine arrested for G1 and RO-3306-arrested for G2) stained with DAPI and antibodies specific for the DNA damage marker γ-H2AX and the anti-centromere CREST in G1 and G2 phases of the cell cycle. On the right, percentage of cells with γ-H2AX colocalizing with GFP-CENP-A or CREST, corresponding to cells with at least one centromere colocalizing with γ-H2AX. b, Western blot analysis of GFP, γ-H2AX, phospho-ATM (pATMS1981) and tubulin in cells expressing dCas9-GFP, Cas9-GFP with a gRNA targeting minor satellite repeats (mi gRNA) or treated with the indicated concentrations of Neocarzinostatin (NCS). c, Cell cycle analysis by flow cytometry using propidium iodide and EdU in NIH3T3 (left panel) and U2OS cells (right panel), either untreated or treated with double thymidine or RO-3306. d, e, Immunofluorescence confocal analysis of (d) NIH3T3 cells expressing Cas9 + mi gRNA and GFP-CENP-A and (e) U2OS cells expressing Cas9 + gRNA targeting alpha satellite repeats stained with DAPI and antibodies specific for 53BP1 (used as a damage marker), CREST and RPA in G1 and G2. On the right, percentage of cells with RPA colocalizing with GFP-CENP-A or CREST. f, g, Immunofluorescence confocal analysis of (f) NIH3T3 cells expressing Cas9 + mi gRNA and GFP-CENP-A and (g) U2OS cells expressing Cas9 + gRNA targeting alpha satellite repeats stained with DAPI and antibodies specific for 53BP1, CREST and BRCA1 in G1 and G2. On the right, percentage of cells with BRCA1 colocalizing with GFP-CENP-A or CREST. h, Percentage of cells with RAD51 colocalizing with γ-H2AX and CREST in HeLa and RPE1 cells expressing Cas9 + gRNA targeting alpha satellite repeats in G1. i, Percentage of cells with RAD51, RPA and BRCA1 recruitment at pericentromeric DSBs in NIH3T3 cells expressing Cas9 + gRNA targeting the major satellite repeats in G1 and G2. For confocal images, scale bars represent 5 µm for G1 cells and 10 µm for G2 cells. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

Source data

Extended Data Fig. 2 Recruitment of HR factors at centromeres is specific to DSB induction.

a, b, Immunofluorescence confocal analysis of NIH3T3 cells expressing Cas9 + mi gRNA stained with DAPI and (a) RAD51 antibody in 568nm or (b) γ-H2AX antibody in 568nm. Images in the other two channels (488nm and 647nm) demonstrate that the RAD51 signal is very specific, and it is not a bleed through signal from different antibody staining. c, Immunofluorescence of super resolution analysis of NIH3T3 cells expressing Cas9 + mi gRNA stained with DAPI, CREST, RAD51 and BRCA1 antibodies demonstrate that RAD51 and BRCA1 were detected simultaneously on the same centromere. d, Immunofluorescence confocal analysis of NIH3T3 cells expressing dCas9 + mi gRNA stained with DAPI, 53BP1, RPA or BRCA1 antibodies. e, Immunofluorescence confocal analysis of U2OS cells expressing dCas9 + gRNA targeting the alpha satellite repeats stained with DAPI, 53BP1, RPA or BRCA1 antibodies. For confocal images, scale bars represent 5 µm. f, g, Immunofluorescence confocal analysis of (f) EdU- NIH3T3 cells expressing GFP-CENP-A and (g) thymidine treated U2OS cells for G1 and RO-3306-arrested cells for G2 treated with Neocarzinostatin (NCS, 200 ng/ml), stained with DAPI and antibodies specific for γ-H2AX, CREST and RAD51. Percentage of cells with DSBs (γ-H2AX) and RAD51 colocalizing with GFP-CENP-A or CREST, corresponding to cells with at least one centromere colocalizing with γ-H2AX and RAD51, is shown on the right as mean ± SD. For confocal images, scale bars represent 5 µm for G1 cells, 10 µm for G2 cells. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

Source data

Extended Data Fig. 3 The role of H3K4me2 in HR factors recruitment at centromeric DSBs in G1 and G2.

a, Quantification of SETD1A mRNA by RT-qPCR in NIH3T3 cells depleted of SETD1A (siSETD1A), normalized to GAPDH and expressed as relative to control (siSCR). b, Western blot analysis of LSD1 and tubulin in NIH3T3 cells expressing GFP-dCas9-LSD1 and control cells. c, Chromatin immunoprecipitation analysis of H3K4me2 enrichment at centromeres over the Input and relative to the no-antibody control in siSETD1A vs siSCR NIH3T3 cells. d, Chromatin immunoprecipitation analysis of H3K4me2 enrichment at centromeres over the Input and relative to the no-antibody control in cells expressing dCas9 or dCas9-LSD1 and a gRNA targeting the minor satellite repeats (mi gRNA). e, Immunofluorescence confocal analysis of NIH3T3 cells expressing Cas9 + mi gRNA and dCas9 or dCas9-LSD1 and stained with DAPI and antibodies specific for γ-H2AX, 53BP1, RPA, BRCA1 and RAD51 in G1. f, Quantification of fold change of RPA, BRCA1 and RAD51 recruitment at centromeric DSBs in siSETD1A vs siSCR NIH3T3 cells in G2 and expressing Cas9 + mi gRNA. g, Quantification of fold change of RPA, BRCA1 and RAD51 recruitment at centromeric DSBs in NIH3T3 cells in G2, expressing Cas9 + mi gRNA and co-expressing dCas9-LSD1 or dCas9 alone. h, Western blot analysis of flag-SETD1A and flag-SETD1A-ΔSET in NIH3T3 cells using flag antibody. i, Chromatin immunoprecipitation analysis of H3K4me2 enrichment at centromeres over the Input and relative to the no-antibody control in cells depleted for SETD1A (siSETD1A) and reconstituted with WT SETD1A or with a truncated catalytically inactive mutant (SETD1A-ΔSET) or in control cells (siSCR). j, Quantification of fold change of RAD51 recruitment in G2 at pericentromeric DSBs in NIH3T3 cells expressing Cas9 + mi gRNA and siSETD1A relative to siSCR. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

Source data

Extended Data Fig. 4 The role of centromeric RNA in HR factors recruitment at centromeric DSBs in G1.

a, b, Quantification by RT-qPCR of fold change of centromeric RNA in NIH3T3 cells expressing dCas9 (left panels) or Cas9 (right panels) + gRNA targeting the minor satellite repeats (mi gRNA) (a) depleted of SETD1A (siSETD1A) relative to control (siSCR) and (b) expressing dCas9 or dCas9-LSD1 and mi gRNA. c, Western blot analysis of H3K4me2, Cas9 and Lamin A in NIH3T3 cells expressing either dCas9 + mi gRNA or Cas9 + mi gRNA. d, Chromatin immunoprecipitation analysis of H3K4me2 enrichment at pericentromeric heterochromatin over the input and relative to the no-antibody control in cells expressing Cas9 + mi gRNA. e, Immunofluorescence confocal analysis of G1 NIH3T3 cells expressing dRNAseH-GFP and co-expressing dCas9 + mi gRNA or Cas9 + mi gRNA and stained with DAPI and antibodies specific for γ-H2AX and CENP-A. f, Quantification of fold change of dRNAseH recruitment in G1 at centromeric DSBs in cells expressing Cas9 + mi gRNA and expressing siSETD1A vs siSCR or expressing dCas9 vs dCas9-LSD1. g, Quantification of fold change of RAD51 recruitment at centromeric DSBs in cells expressing Ca9 + mi gRNA and RNAseH vs dRNAseH. h, Quantification of fold change of 53BP1 and γ-H2AX intensity at centromeric DSBs co-stained with RPA, BRCA1 and RAD51 presented in Fig. 2h and Extended Data Fig. 4g, in cells expressing RNAseH vs dRNAseH. i, Chromatin immunoprecipitation analysis of H3K4me2 enrichment at centromeres over the input and relative to the no-antibody control in cells expressing GFP alone vs GFP-RNAseH. For confocal images, scale bars represent 5 µm. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

Source data

Extended Data Fig. 5 The role of HJURP and CENPA in eliciting RAD51 recruitment at centromeric DSBs in G1.

a, Quantification of CENP-A, HJURP and MIS18 mRNA levels by RT-qPCR in NIH3T3 cells expressing siCENP-A, siHJURP or siMIS18, normalized to GAPDH and expressed as relative to siSCR control. b, Quantification of fold change of RPA recruitment in G1 at centromeric DSBs in NIH3T3 cells expressing siHJURP, siCENP-A, siHJURP+siCENP-A or siMIS18 relative to siSCR and expressing Cas9 + mi gRNA. c, Quantification of fold change of RAD51 recruitment at centromeric DSBs in NIH3T3 cells depleted of CENP-C and CENP-N, components of the CCAN, relative to siSCR and expressing Cas9 + mi gRNA. d, Quantification of CENP-C and CENP-N mRNA by RT-qPCR in cells expressing siCENP-C or siCENP-N, normalized to GAPDH and relative to siSCR control. e, Quantification of fold change of RAD51 recruitment at lacO locus in NIH3T3 lacO-Isce-I-tet cells expressing mCherry-LacI or mCherry-lacI-HJURP and co-expressing I-SceI. f, Immunofluorescence confocal analysis of NIH3T3 lacO-Isce-I-tet cells expressing mCherry-LacI or mCherry-lacI-HJURP and co-expressing I-SceI and stained with DAPI, and antibodies against γ-H2AX and RAD51. g, Quantification of fold change of RAD51 recruitment at centromeric DSBs in NIH3T3 cells in G2, expressing Cas9 + mi gRNA and co-expressing siHJURP or siCENP-A vs siSCR. h, Western blot analysis of USP11, GFP and GFP-HJURP after GFP-IP in siUSP11 vs siSCR NIH3T3 cells in G1 or G2. The Input was 1 % of the extract used for the IP. i, Immunofluorescence confocal analysis of U2OS lacO-Isce-I-tet cells expressing mCherry-LacI or mCherry-lacI-HJURP and co-expressing GFP-USP11 and stained with DAPI. On the right, quantification of percentage of cells with colocalization of GFP-USP11 with LacO locus in G1. j, Quantification of percentage of cells with colocalization of GFP-USP11 with LacO locus in G2 arrested NIH3T3 lacO-Isce-I-tet cells expressing mCherry-LacI or mCherry-lacI-HJURP and co-expressing GFP-USP11. k, Quantification of USP11 mRNA by RT-qPCR in cells expressing siUSP11, normalized to GAPDH and relative to siSCR control. l, Immunofluorescence confocal analysis of NIH3T3 cells expressing Cas9 + mi gRNA and co-expressing dCas9 or dCas9-USP11 and stained with DAPI and antibodies specific for γ-H2AX and RAD51 in G1. On the right, fold change of percentage of cells with colocalization of RAD51 with γ-H2AX positive pericentromeric repeats. m, Western blot analysis of BRCA1, RAD51, RPA and tubulin in cells expressing siSETD1A, siUSP11, siCENPA, or siHJURP vs siSCR. n, Quantification of γ-H2AX intensity at centromeres in NIH3T3 cells expressing Cas9 + mi gRNA and siSETD1A, siUSP11, siCENPA, or siHJURP vs siSCR. For confocal images, scale bars represent 5 µm. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

Source data

Extended Data Fig. 6 Characterization of USP11 role in licensing of HR at centromeric DSBs in G1.

a, Western blot analysis of Ubiquitin (FK2 antibody) after GFP-IP under denaturing conditions in NIH3T3 cells expressing GFP or GFP-CENPA and co-expressing USP11-Flag vs Flag. The input was 1% of the extract used for the IP. b, Western blot analysis of ubiquitin (FK2 antibody) after GFP-IP under denaturing conditions in NIH3T3 cells expressing GFP or GFP-HJURP and siUSP11 vs siSCR. The input was 1% of the extract used for the IP. c, 2 biological replicates of western blot analysis showing the interaction of CENPA with HJURP in siUSP11 vs siSCR NIH3T3 cells (left panel). Quantification from western blot of CENPA interaction with HJURP in siUSP11 vs siSCR NIH3T3 cells (right panel). d, Quantification of fold change of CENP-A recruitment at LacO in cells expressing LacI-HJURP and co-expressing siUSP11 vs siSCR. e, Schematic representation of the SNAP technology. f, Western blot analysis of USP11, GFP and tubulin in HeLa cells expressing GFP-HJURP or not and co-expressing siUSP11 or siHJURP vs siSCR. g, Immunofluorescence confocal analysis of de novo deposited CENP-A-SNAP (TMR Star in Red) and total CENPA (Green) in Hela expressing CENP-A-SNAP and co-expressing siHJURP or siUSP11 vs siSCR.  h, Cell cycle analysis by flow cytometry using propidium iodide and EdU in HeLa CENP-A-SNAP cells depleted of USP11 relative to siSCR control. i, Chromatin immunoprecipitation analysis of USP11 enrichment at pericentromeric heterochromatin over the Input and relative to the no-antibody control, in NIH3T3 cells expressing Cas9 + mi gRNA vs dCas9 + gRNA (left panel). Enrichment of USP11 at GAPDH promoter over the Input and relative to the no-antibody control, in NIH3T3 cells expressing Cas9 + gRNA targeting GAPDH promoter vs dCas9 + gRNA (right panel). j, Chromatin immunoprecipitation analysis of CENPA (left) and Histone H3 (right) enrichment at centromeres over the Input and relative to the no-antibody control, in NIH3T3 cells expressing Cas9 + mi gRNA vs dCas9 + mi gRNA. k, IF confocal analysis of de novo deposited CENP-A-SNAP in Hela expressing CENP-A-SNAP and GFP-dCas9 (left) or Cas9 and stained with γ-H2AX antibody (right). l, Quantification of fold change of RAD51 recruitment in G1 at pericentromeric DSBs in NIH3T3 cells expressing Cas9 + gRNA targeting major satellite repeats, co-expressing dCas9-CENPA (upper panel) or dCas9-HJURP (lower panel) and depleted of USP11 (siUSP11) vs siSCR. For confocal images, scale bars represent 5 µm. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

Source data

Extended Data Fig. 7 Assessment of genomic instability associated with lack of HR at centromeric DSBs in G1.

a, Immunofluorescence confocal analysis of NIH3T3 cells treated with RAD51 inhibitor (RAD51i) and irradiation (IR) and stained with DAPI and antibodies specific for γ-H2AX and RAD51; RAD51i was washed out or not after IR. b, Quantification of the number of translocations per 50 metaphase spreads in cells expressing Cas9 + mi gRNA and depleted of RAD51 (siRAD51), or of RAD51 and RAD52 (siRAD51+siRAD52) and compared to siSCR control.  c, Quantification of RAD51 and RAD52 mRNA by RT-qPCR in NIH3T3 cells expressing siRAD51 or siRAD52, normalized to GAPDH and relative to siSCR. d, Quantification of the number of chromosomes with broken centromeres (lacking or with free centromeres) for 50 metaphase spreads in cells expressing Cas9 + mi gRNA and co-expressing siRAD51, or siRAD51+siRAD52 compared to siSCR. e, Quantification of fold enrichment of centromeric DNA after qPCR analysis in U2OS cells expressing Cas9 + gRNA targeting the alpha satellite repeats and siRAD51 alone or together with siRAD52 relative to siSCR control. f, Quantification of RAD52 mRNA by RT-qPCR in siRAD52 U2OS cells, normalized to GAPDH and relative to siSCR (left panel). Western blot analysis of RAD51 in siRAD51 U2OS cells compared to siSCR (right panel). g, Immunofluorescence confocal analysis of NIH3T3 cells expressing Cas9 + mi gRNA and RAD52-GFP, depleted of RAD51 (siRAD51) compared to siSCR and stained with DAPI and antibodies specific for γ-H2AX and RAD51. h, Quantification of the number of translocations in WT and PolQ−/− MEFs expressing Cas9 + mi gRNA and treated with RAD51i or DMSO as a control.  For confocal images, scale bars represent 5 µm. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.

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Extended Data Fig. 8 Schematic representation of the step-wise model for the licensing of HR in G1 at centromeres.

For details see text.

Supplementary information

Supplementary Information

This file contains Supplementary Fig. 1 (the uncropped western blots), Supplementary Fig. 2 (gating strategy for flow cytometry and cell cycle analysis) and Supplementary Tables 1–6 (siRNA references, guide RNA sequences and lists of plasmids, primers, antibodies and FISH probes used).

Reporting Summary

Video 1

Z-stack from immunofluorescence confocal analysis of a NIH 3T3 cell expressing GFP-CENP-A and stained with DAPI and antibodies specific for γ-H2AX (Alexa 568) and RAD51 (Alexa 647) in G1 phase of the cell cycle.

Video 2

Z-stack from immunofluorescence confocal analysis of a NIH 3T3 cell expressing GFP-CENP-A and stained with an antibody specific for RAD51 (Alexa 647) in G1 phase of the cell cycle.

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Yilmaz, D., Furst, A., Meaburn, K. et al. Activation of homologous recombination in G1 preserves centromeric integrity. Nature 600, 748–753 (2021). https://doi.org/10.1038/s41586-021-04200-z

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