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MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end resection

Nature volume 521, pages 537–540 (2015)Cite this article

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Abstract

Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and ageing1,2. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3′ telomeric overhangs, indicating that MAD2L2 inhibits 5′ end resection. End resection blocks NHEJ while committing to homology-directed repair, and is under the control of 53BP1, RIF1 and PTIP3. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5′ end resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end resection at irradiation-induced DNA double-strand breaks and promotes end-joining of DNA double-strand breaks in several settings, including during immunoglobulin class switch recombination. These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis4. Together, our data establish MAD2L2 as a crucial contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5′ end resection downstream of RIF1.

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Figure 1: A functional genetic screen identifies MAD2L2 as a critical factor in telomere-driven genomic instability.
Figure 2: MAD2L2 facilitates telomeric G-overhang degradation, NHEJ-mediated telomere fusion, repair and CSR.
Figure 3: MAD2L2 inhibits end resection and promotes telomere-induced genomic instability in a CTIP- and EXO1-dependent manner.
Figure 4: MAD2L2 localizes to DSBs, inhibits end resection and promotes telomere-NHEJ in a 53BP1- and RIF1-dependent manner.

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Acknowledgements

We thank A. Konishi, G. Celli and T. de Lange for TRF2(Ile468Ala), Trf2flox/− p53−/− MEFs and Trf2flox/− p53−/− Lig4−/− MEFs, P. Bouwman, M. Pieterse, T. Halazonetis, O. Kallioniemi, B. Gerritsen, B. Morris and P. Halonen for composing and providing the DDR TRC shRNA sub-library, members of the NKI Genomics Core Facility for technical support, A. van Kessel for MAD2L2 cDNA, E. Hendrickson for repair plasmids, R. Chapman and S. Boulton for mRIF1 antibody, 53bp1−/− and Rif1−/− MEFs, E. Callen and A. Nussenzweig for 53BP1 and PTIP expression vectors and for Ptip−/− MEFs, N. de Wind for Rev1−/− and Rev3−/− MEFs, B. van den Broek for help with telomere mobility analysis, I. de Krijger, Z. Yalcin and M. Simonetta from the Jacobs group for contributing to end resection and interaction analysis and members of the R. Medema laboratory for discussion. This work was supported by grants from the European Research Council (ERCStG 311565) and Dutch Cancer Society (KWF-NKI2007-3907) to J.J.L.J., a grant from the Canadian Institutes for Health Research (CIHR MOP89754) to D.D., a La Caixa fellowship to S.S.B. and a CIHR post-doctoral fellowship to A.O. D.D. is a Canada Research Chair (tier 1) in the Molecular Mechanisms of Genome Integrity and J.J.L.J. is an EMBO Young Investigator.

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Author notes

  1. Vera Boersma and Nathalie Moatti: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands,

    Vera Boersma, Nathalie Moatti, Sandra Segura-Bayona, Marieke H. Peuscher, Jaco van der Torre, Brigitte A. Wevers & Jacqueline J. L. Jacobs

  2. The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada,

    Alexandre Orthwein & Daniel Durocher

  3. Department of Molecular Genetics, University of Toronto, Ontario M5S 3E1, Canada,

    Daniel Durocher

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Contributions

J.J.L.J., V.B., N.M. and S.S.-B. designed the experiments and analysed the data. M.H.P. and J.v.d.T. performed the TIGIR-screen, analysed the screen results and performed initial validation. V.B., N.M., S.S.-B. and B.A.W. performed all experiments, with the exception of assessment of CSR and sister-telomere fusion upon repair activation in mitosis, which were performed and analysed by A.O. and D.D. S.S.-B., M.H.P. and J.v.d.T. contributed equally. J.J.L.J. wrote the manuscript.

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Correspondence to Jacqueline J. L. Jacobs.

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Extended data figures and tables

Extended Data Figure 1 Related to Fig. 1.

a, A functional genetic screen for TIGIRs identifies independent shRNAs against Mad2l2 and the previously identified regulators of NHEJ-mediated telomere fusion Atm, Nbs1 (also known as Nbn), Rad50, 53bp1 and Rnf8. Listed are the independent shRNAs enriched >1.5-fold in at least two out of three TIGIR screens, with their average ratio of enrichment over all three screens. Ratios reflect shRNA abundance after 12 days of telomere uncapping at 39 °C followed by 4 days recovery at 32 °C versus shRNA abundance after growth for 4 days at 32 °C. b, Additional shRNAs targeting Mad2l2 or known TIGIRs that were enriched >1.5-fold in one out of three screens, with their ratio. Not shown are additional shRNAs against factors not previously implicated in control of telomere fusion, that were considerably enriched in these TIGIR screens but await validation. c, qRT–PCR analysis of Mad2l2 expression levels in TRF2ts MEFs transduced with four independent shRNAs targeting Mad2l2 and used in Fig. 1b (error bars denote s.d.). d, Survival assay of TRF2ts cells infected with control or Mad2l2 sh4 shRNAs, complemented with empty control or RNAi-resistant Flag-MAD2L2RR and grown as indicated. e, Western blot showing expression of endogenous MAD2L2 and exogenous Flag–MAD2L2 in TRF2ts cells used in d and in Fig. 1c. f, Photograph of shRNA-transduced TRF2ts cells grown for 12 days at 39 °C. Scale bar, 100 μm. g, None out of ten shRNAs targeting Rev1 or Rev3 were substantially enriched in any of three independent DDR TIGIR screens. h, shRNA-mediated Rev1 knockdown does not increase survival after prolonged telomere uncapping in survival assays of TRF2ts MEFs. Of note, Rev3 knockdown compromised viability and was therefore not informative in these assays. i, qRT–PCR analysis of mouse Rev1 expression levels of cells shown in h (error bars denote s.d.).

Extended Data Figure 2 Related to Fig. 2.

a, Representative metaphase spreads of TRF2ts MEFs transduced as indicated, collected after 24 h at 39 °C for telomere FISH. Original magnifications, ×63. b, Representative telomeric single-stranded G-overhang (ss TTAGGG) and total telomere (total TTAGGG) analysis in TRF2ts MEFs at 32 °C and after 48 h at 37 °C or 39 °C. c, d, Mad2l2 knockdown does not affect cell cycle parameters in TRF2ts MEFs. Cell cycle phase analysis was based on propidium iodide staining of asynchronously growing cells (c), as well as on 1 h incubation with BrdU, followed by detection of BrdU incorporation and propidium iodide staining for DNA content (d) (n = 3, mean ± s.d.). e, MAD2L2 is required for sister-telomere fusion upon activation of DNA repair in mitosis. Sister-telomere fusions were quantified in IMR90 cells expressing exogenous wild-type 53BP1 and RNF8, or 53BP1 (Thr1608Ala/Ser1618Ala) (TASA) and RNF8(Thr198Ala) (TA) mutant alleles, and depleted for endogenous RNF8 and 53BP1, as well as depleted for MAD2L2, RIF1 or PTIP (n = 4). f, Examples of DNA content profiles of TRF2ts cells transduced with the indicated shRNAs, grown at 32 °C, or for 48 h at 39 °C and stained with propidium iodide. Analysis of the fraction of cells with 8N (tetraploid) or >4N (aneuploid) DNA content was done on corresponding dotplots of which the results are shown in Fig. 2c.

Extended Data Figure 3 Related to Fig. 2.

a, qRT–PCR analysis of MAD2L2 expression levels in U2OS cells infected with control or MAD2L2 shRNAs, and used in the repair assays shown in Fig. 2d (error bars denote s.d.). b, c, qRT–PCR analysis of RAD51 (b) and MAD2L2 (c) expression levels in RAD51-depleted, E6E7-expressing U2OS cells used in the assays shown in Fig. 2e (error bars denote s.d.). d, Clonogenic survival assays of U2OS cells transduced with non-targeting control or 53BP1, RIF1 or MAD2L2 shRNAs and treated with the indicated doses of ionizing radiation (n = 3–4, mean ± s.e.m.). e, Western blot analysis of 53BP1, MAD2L2 and RIF1 in U2OS cells transduced with the indicated shRNAs. f, CSR in shRNA-transduced primary B cells (n = 2, mean ± s.d.). g, Western blot analysis of MAD2L2 and 53BP1 in CH12F3-2 B cells and mouse primary B cells transduced with the indicated shRNAs. h, MAD2L2 depletion does not affect cellular proliferation in murine B cells. CH12F3-2 cells transduced with control, 53bp1 or Mad2l2 shRNAs were loaded with CFSE and analysed by flow cytometry at 0, 24 and 48 h after stimulation. Profiles from all time points are plotted in the same histogram. i, MAD2L2 depletion does not affect the transcription of critical genes implicated in CSR. RT–PCR analysis of Aid (Aicda) mRNA, IgM and IgA germline transcript (GLT) levels using twofold serial dilutions of cDNA made from activated CH12F3-2 B cells transduced with the indicated shRNAs. Gapdh was used as a control for transcript expression.

Extended Data Figure 4 Related to Fig. 3.

a, Representative images of immunofluorescence detection of p-ATM(S1981), γH2AX, 53BP1 and RIF1 in TRF2ts MEFs transduced with control or Mad2l2 shRNAs and grown at 32 °C or for 12 h at 39 °C to induce telomere uncapping. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Original magnifications, ×40. b, Quantification of the number of p-ATM, γH2AX, 53BP1 and RIF1 foci per cell in TRF2ts MEFs transduced with control or Mad2l2 shRNAs and grown at 32 °C or for 3 and 12 h at 39 °C (n = 2, mean ± s.d.). c, Quantifications of 53BP1, RIF1 and PTIP foci in U2OS cells transduced with control or MAD2L2 shRNAs, 3 h after 5 Gy (n = 2, mean ± s.d.).

Extended Data Figure 5 Related to Fig. 3.

a, Telomeric single-stranded G-overhang assay of TRF2ts MEFs transduced with control or Mad2l2 sh4 shRNAs, showing that the increase in overhang signal upon Mad2l2 knockdown is due to 3′ terminal sequences because the signal is removed by treatment with Escherichia coli 3′ exonuclease Exo1. b, Mad2l2 knockdown causes increased single-stranded telomeric G-overhang signals in TRF2ts Lig4−/− MEFs. c, Quantification of relative telomeric G-overhang signals in TRF2ts Lig4−/− MEFs transduced with control or Mad2l2 shRNAs and grown at 32 °C or for 12 or 24 h at 39 °C (n = 2, mean ± s.e.m.). d, qRT–PCR analysis of Mad2l2 expression levels in TRF2ts Lig4−/− MEFs infected with control or Mad2l2 shRNA lentivirus (error bars denote s.d.). e, Survival assays of TRF2ts MEFs transduced with control or Mad2l2 shRNAs and subsequently with control, Ctip or Exo1 shRNAs. f, Quantification of the survival assays shown in e. g, qRT–PCR analysis of Ctip and Exo1 expression levels of cells shown in e, f and h and in Fig. 3f (error bars denote s.d.). h, Growth curves at 39 °C of TRF2ts MEFs transduced with non-targeting control or Mad2l2 shRNAs and subsequently with control, Ctip or Exo1 shRNAs (error bars denote s.e.m.).

Extended Data Figure 6 Related to Figs 3 and 4.

a, TSCE analysis in shRNA-transduced TRF2ts MEFs grown at 32 °C or for 12 h at 39 °C to uncap telomeres (n = 2, mean ± s.d., counting >1,000 chromosomes per condition, per experiment). TSCE frequency in control cells is set at 1 (corresponding to an average of 6.9% of chromosomes with a TSCE event). Shown on the right are examples of chromosomes without and with TSCE in cells quantified on the left. Original magnifications, ×63. b, qRT–PCR analysis of Mad2l2 expression levels in 53bp1−/−, Rif1−/−, Ptip−/− and Ptip+/+ MEFs used in the chromosome fusion analysis shown in Fig. 4a and in c (error bars denote s.d.). c, Percentage of chromosomes fused upon TRF2 inhibition in the Ptip+/+ MEFs matching with the Ptip−/− MEFs shown in Fig. 4a (n = 2, mean ± s.e.m.). d, Analysis of different types of telomere fusions in Rif1−/− MEFs. Depletion of MAD2L2 in Rif1−/− MEFs does not reduce inter-chromosomal telomere fusions induced by TRF2 inhibition, indicating epistasis. However, irrespective of TRF2 inhibition, MAD2L2 depletion in Rif1−/− MEFs induces association between sister telomeres, causing an increase in total fusions scored for MAD2L2-depleted Rif1−/− MEFs, as also visible in Fig. 4a (n = 2, mean ± s.e.m., >1,300–2,000 chromosomes were analysed per condition, per independent experiment). e, Explanation of scoring different types of telomere fusions shown in d.

Extended Data Figure 7 Related to Fig. 4.

a, Schematic overview of C-terminal and N-terminal deletion mutants of MAD2L2. b, c, Expression analysis in U2OS cells of GFP-tagged wild-type MAD2L2 and C- and N-terminal MAD2L2 deletion mutants (b) and of Flag-tagged wild-type MAD2L2 and MAD2L2(Cys70Arg) and MAD2L2(Leu186Ala) (c). d, Analysis of DDR foci formation of GFP-tagged wild-type MAD2L2 and C- and N-terminal MAD2L2 deletion mutants by immunofluorescence detection of GFP and γH2AX in U2OS at 3 h after ionizing radiation (n = 2 for N-terminals, n = 3 for C-terminals, mean ± s.e.m.). e, Analysis of wild-type, Cys70Arg and Leu186Ala MAD2L2 accumulation into DDR foci by immunofluorescence detection of MAD2L2 and 53BP1 in U2OS at 3 h after ionizing radiation (n = 2, ± s.e.m.). f, g, Expression analysis in TRF2ts MEFs of GFP-tagged MAD2L2 and C- and N-terminal MAD2L2 deletion mutants (f) and of Flag-tagged wild-type MAD2L2 and MAD2L2(Cys70Arg), MAD2L2(Leu186Ala), MAD2L2(Thr103Ala) and MAD2L2(Thr103Asp) (g). h, Quantification of chromosome fusions after 24 h of telomere deprotection at 39 °C in TRF2ts MEFs transduced with control or Mad2l2 shRNAs and complemented with empty vector control or RNAi-resistant GFP-tagged wild-type MAD2L2 and C- or N-terminal MAD2L2 deletion mutants (error bars denote s.e.m.). i, Quantification of survival assays of TRF2ts MEFs transduced with control or Mad2l2 shRNAs and subsequently with empty vector control, wild-type MAD2L2, MAD2L2(Cys70Arg), MAD2L2(Leu186Ala), MAD2L2(Thr103Ala) or MAD2L2(Thr103Asp) retroviruses (n = 2, mean ± s.e.m.).

Extended Data Figure 8 Related to Fig. 4.

a, Representative images of immunofluorescence detection of endogenous MAD2L2 and γH2AX or 53BP1 in U2OS cells transduced with control, 53BP1, RNF8, RNF168, RIF1, PTIP or REV3 shRNA lentiviruses, irradiated with 5 Gy and processed for immunofluorescence after 3 h (quantifications are shown in Fig. 4e). b, Western blot or qRT–PCR analysis of PTIP, RNF8, RNF168 and REV3 levels in shRNA-transduced U2OS cells (error bars denote s.d.). c, Quantification and representative images of immunofluorescence detection of γH2AX and GFP–MAD2L2 in Ptip+/+ or Ptip−/− MEFs, 3 h after 5 Gy (n = 2, mean ± s.d.). d, Representative images of immunofluorescence for 53BP1 and exogenous Flag–MAD2L2 wild-type, Cys70Arg or Leu186Ala mutants in U2OS cells depleted for endogenous MAD2L2 with lentiviral shRNA, processed for immunofluorescence 3 h after 5 Gy (quantifications are shown in Extended Data Fig. 7e). e, Quantification and representative immunofluorescence images of GFP–MAD2L2 localization to uncapped telomeres in TRF2ts MEFs transduced with control or 53bp1 shRNAs (n = 2, mean ± s.d.). Original magnifications, ×63 with zoom factor 5.

Extended Data Figure 9 Related to Fig. 4.

a, Schematic representation of the 53BP1 alleles with wild-type and substituted S/TQ sites used to address MAD2L2 IRIF localization dependence on 53BP1. Representative immunofluorescence images are displayed that show colocalization of endogenous MAD2L2 with 53BP1-DB-WT and -8A, but not with 53BP1-DB-7A or -28A. Quantifications are provided in Fig. 4f. b, Top, representative immunofluorescence images showing colocalization of endogenous RIF1 with 53BP1-DB-WT and -8A, but not 53BP1-DB-7A or -28A. Quantifications are presented in Fig. 4f. Bottom, representative immunofluorescence images showing colocalization of PTIP–GFP with 53BP1-DB-WT and -7A, but impaired colocalization of PTIP–GFP with 53BP1-DB-8A or -28A. c, Schematic representation of GFP-tagged wild-type RIF1 and GFP-tagged RIF1 lacking the N-terminal HEAT repeats (ΔHEAT), used to address MAD2L2 IRIF localization dependence on the HEAT repeats of RIF1. Representative immunofluorescence images are shown. Quantifications are presented in Fig. 4g. d, Cell cycle phase distributions of the RPE cells used in Fig. 4h to address cell cycle dependence of endogenous RIF1 and MAD2L2 localization to IRIFs (n = 3, mean ± s.d.). Original magnifications, ×63 with zoom factor 5.

Extended Data Figure 10 Related to Fig. 4.

a, Frequency distribution (left) and scatter plot (right) of total distances travelled by uncapped telomeres in control or Mad2l2 knockdown cells. b, qRT–PCR analysis of Mad2l2 expression levels and western blot analysis of 53BP1 proteins levels in TRF2ts cells used in the experiments shown in a and c (error bars denote ± s.d.). c, Distance travelled by 10 representative uncapped telomeres for each condition. While multiple uncapped telomeres in 53BP1-depleted cells have reduced mobility, this is not seen for uncapped telomeres in MAD2L2-depleted cells. d, Model of the role of MAD2L2 in promoting NHEJ.

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Boersma, V., Moatti, N., Segura-Bayona, S. et al. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end resection. Nature 521, 537–540 (2015). https://doi.org/10.1038/nature14216

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