Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN

Abstract

Classical non-homologous end joining1 (cNHEJ) and homologous recombination2 compete for the repair of double-stranded DNA breaks during the cell cycle. Homologous recombination is inhibited during the G1 phase of the cell cycle, but both pathways are active in the S and G2 phases. However, it is unclear why cNHEJ does not always outcompete homologous recombination during the S and G2 phases. Here we show that CYREN (cell cycle regulator of NHEJ) is a cell-cycle-specific inhibitor of cNHEJ. Suppression of CYREN allows cNHEJ to occur at telomeres and intrachromosomal breaks during the S and G2 phases, and cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside the G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell-cycle-dependent inhibitor of cNHEJ that promotes error-free repair by homologous recombination during cell cycle phases when sister chromatids are present.

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Figure 1: CYREN depletion reactivates cNHEJ in phases S and G2 at deprotected telomeres.
Figure 2: CYREN inhibits NHEJ at intrachromosomal breaks.
Figure 3: CYREN interaction with Ku in S and G2 inhibits cNHEJ.
Figure 4: CYREN prevents overhang processing.

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Acknowledgements

All data are archived at the Salk Institute. We thank E. Hendrickson, J. Stark and D. A. Ramsden for support and N. O’Reilly for peptide arrays. N.A. was supported by the Human Frontiers Science Program (LT000284/2013) and N.A. and A.M. by the Paul F. Glenn Center for Biology of Aging Research. J.M. is supported by a Larry Hillblom Foundation Fellowship Grant. S.J.B. is supported by a Wellcome Trust Senior Investigator Award and the Francis Crick Institute (Cancer Research UK), the UK Medical Research Council (FC0010048), and the Wellcome Trust (FC0010048). A.S. is supported by NIH (R01 GM102491), the NCI Cancer Center Support Grant P30 (CA014195), The Leona M. and Harry B. Helmsley Charitable Trust (#2012-PG-MED002), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals. The Salk Institute Cancer Center Core Grant (P30CA014195), the NIH (R01GM087476, R01CA174942), the Donald and Darlene Shiley Chair, the Highland Street Foundation, the Fritz B. Burns Foundation and the Emerald Foundation support J.K.

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Experiments were designed and performed by N.A. (all except Fig. 3c, Extended Data Figs 7b–d, 9), J.M. (Fig. 3c, Extended Data Figs 7b–d, 9), S.J.B. (Extended Data Figs 7a, 9), A.S. (Fig. 3c, Extended Data Figs 7b–d, 9), A.M. (Fig. 2a, b) and J.K. Experiments were performed by A.C. (Figs 2c, d, 3d, f–h, 4, Extended Data Figs 3c, 4, 6, 7f, 8), M.T. (Fig. 1b, c, f, Fig. 3b, Extended Data Figs 1, 3a, b, f, 5), S.G.G. (Extended Data Fig. 9) and M.M. (Extended Data Fig. 9), C.W.B. analysed data (Fig. 4b, c, Extended Data Fig. 8) and N.A. and J.K. wrote the manuscript.

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Correspondence to Jan Karlseder.

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Extended data figures and tables

Extended Data Figure 1 CYREN depletion leads to chromatid-type fusions at deprotected telomeres.

a, qRT–PCR measurement of CYREN isoforms expression for Fig. 1b–d. Normalized to ACTB qRT–PCR. CYREN-1, PCR primers target mRNA transcript variant 1. CYREN-2, PCR primers target mRNA transcript variants 2, 3, 4 and 5. CYREN-3, PCR primers target mRNA transcript variant 7. b, Western blot showing TRF2 depletion. For gel source data, see Supplementary Fig. 1. c, Experimental outline of Fig. 1c. HT1080 6TG cells stably transduced with an inducible control shLuci or one of three shCYREN RNAs were infected with shControl or shTRF2 on day 0. shTRF2-transduced cells were selected with puromycin and shCYREN expression was induced with doxycycline on day 2. Cells were collected for fusion analysis on day 5. d, Partial metaphase spreads of functional (shControl) and deprotected (shTRF2) telomeres after CYREN depletion. Green arrows, chromosome-type fusions. Blue arrows, chromatid-type fusions. e, Percentage of cells with fusions ± upper and lower value of 95% confidence intervals, Wilson–Brown test. ****P < 0.0001, ***P < 0.001. Fisher’s exact test, two-sided. n, number of metaphases analysed. f, Mean percentage of chromosome ends fused by sister telomere associations. Error bars, s.e.m. One-way ANOVA, Sidak’s multiple comparison test. n, number of metaphases analysed. g, Number of metaphases analysed, total telomere and fusions counted.

Extended Data Figure 2 Chromatid-type fusions induced by CYREN depletion in IMR90 fibroblasts.

a, Western blot showing TRF2 depletion. For gel source data, see Supplementary Fig. 1. b, qRT–PCR measurement of CYREN isoforms expression after siRNA knockdown. Normalized to ACTB qRT–PCR. CYREN-1, PCR primers target mRNA transcript variant 1. CYREN-2, PCR primers target mRNA transcript variants 2, 3, 4 and 5. CYREN-3, PCR primers target mRNA transcript variant 7. c, Representative images of partial metaphase spreads of functional (shControl) and deprotected (shTRF2) telomeres after CYREN depletion. d, Mean percentage of fused chromosome ends per metaphase. Error bars, s.e.m. **P < 0.01. One-way ANOVA, Sidak’s multiple comparison test. n, number of metaphases analysed.

Extended Data Figure 3 CYREN prevents cNHEJ in S and G2 at deprotected telomeres.

a, Schematic of CO-FISH. Chromatid-type fusions involving leading and lagging strands. b, Percentage of fusions ± upper and lower value of 95% confidence intervals, Wilson–Brown test. 126 fusions counted. c, Western blot showing knockdown of ATM, ligase 4, DNA-PKcs and ligase 3 in Fig. 2c. For gel source data, see Supplementary Fig. 1. d, Experimental timeline for Fig. 2c. CYRENWT and CYRENKO HT1080 cells were infected with shTRF2 on day 0, followed by transfection with siRNAs on day 2. On day 3, shTRF2-infected cells were selected with puromycin and cells were collected for fusion analysis on day 5. e, Experimental timeline for panel f. HT1080 6TG cells were stably transduced with shTRF2 on day 0, followed by transfection with non-targeting (NT) or CYREN siRNAs on day 2. On day 3, shTRF2-infected cells were selected with puromycin and inhibitors were added. Cells were collected for fusion analysis on day 5. f, Percentage of cells with fusions ± upper and lower value of 95% confidence intervals, Wilson–Brown test. Cells were treated for 48 h with DMSO or the following inhibitors: ATMi (KU-55933) 10 μM, DNA-PKcsi (NU-7441) 1 μM, PARPi (olaparib) 10 μM, RAD51i (RI-1) 20 μM. ****P < 0.0001, NS, not significant. Fisher’s exact test, two-sided. Experiment shown is representative of two biological replicates.

Extended Data Figure 4 CYREN regulates the DSB pathway choice at intrachromosomal breaks.

a, Experimental timeline for Fig. 2a, b. CYRENWT and CYRENKO clonal HT1080 were synchronized by double thymdine block, and irradiated at 2 Gy at 2 h, 6 h or 10 h after thymidine release, corresponding to the S, G2 or G1 phases of the cell cycle, respectively. Cells were arrested for immunofluorescence or chromosome spreads 26 h after thymidine release. b, Cell cycle profiles of cells used in Fig. 2a, b, 2 h, 6 h and 10 h after thymidine release. 20,000 cells were analysed. c, Representative flow cytometry controls for the DSB repair reporter. One million cells per sample were analysed. d, Experimental outline of Fig. 2d. A single clone of HT1080 cells transduced with the DSB repair reporter was isolated and transfected with Cas9 and sgCYREN. Single clones were isolated and genotyped. Selected CYRENWT and CYRENKO clones were then transfected with ISce1 and the HR donor, followed by flow cytometry analysis 48 h later. e, Cell cycle distribution of the CYRENWT and CYRENKO clones obtained by flow cytometry of propidium iodide and BrdU-labelled cells. 80,000 cells were analysed.

Extended Data Figure 5 CYREN does not regulate repair of replication-induced DSBs.

a, Representative images of chromosomes from b. b, Percentage of metaphases with radial chromosomes ± upper and lower value of 95% confidence intervals, Wilson–Brown test. n, number of metaphases analysed. Experiment shown is representative of two biological replicates. c, Per cent survival with increasing concentrations of PARP inhibitor olaparib.

Extended Data Figure 6 CYREN isoforms 1 and 2 inhibit cNHEJ.

a, Anti-Flag western blot on whole-cell extracts of HT1080 6TG cells expressing Flag–CYREN isoforms used in Fig. 4b. Asterisk indicates two nonspecific bands. For gel source data, see Supplementary Fig. 1. b, Experimental outline of Fig. 4b. HT1080 6TG cells were stably transfected with pcDNA3 empty Flag vector or pcDNA3 expressing CYREN-1–Flag, CYREN-2–Flag or CYREN-3–Flag. Cells were selected and infected with shTRF2 on day 0, followed by transfection with a control pool of non-targeting (NT) siRNAs or a pool of siRNAs targeting the 3′ UTR of CYREN. shTRF2-infected cells were selected on day 3 and cells were collected for fusion analysis on day 5. c, Schematic representation of N-terminal 3×Flag endogenous tagging of CYREN-1 and CYREN-2. d, Sequencing of the N-terminal 3×Flag–CYREN tagged allele. e, Anti-Flag western blot of whole-cell extracts from HT1080 cells and HT1080 cells with endogenous N-terminally 3×Flag-tagged exon 1 of C7orf49. Upper band, CYREN-1. Lower band, CYREN-2. Increasing amounts of protein extract were loaded (5, 10, 15, 20 μl). For gel source data, see Supplementary Fig. 1. f, Schematic representation of C-terminal 3×Flag endogenous tagging of CYREN-1 and CYREN-3. g, Sequencing of the C-terminal CYREN–3×Flag tagged allele. h, Flag western blot of HT1080 6TG cells without and with endogenous tagged C-terminal CYREN–3×Flag. Increasing amounts of protein extracts were loaded (5, 10, 15, 20 μl). For gel source data, see Supplementary Fig. 1. i, Flag western blot of 3×Flag–CYREN tagged HT1080 cells following CYREN knockdown by siRNA. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 CYREN interaction with Ku in S and G2 inhibits cNHEJ.

a, Immunoblotting of a peptide binding array of full-length CYREN-1. Each dot represents 20 amino acids of CYREN-1 with a 19-amino acid overlap with the previous and following peptides. Upper panel, Ponceau stain. Middle panels, duplicate incubation with Ku70/80 recombinant proteins and immunoblotting with anti-Ku70 antibody. Lower panel, control immunoblotting with anti-Ku70 antibody without incubation with recombinant Ku70/80. For gel source data, see Supplementary Fig. 1. b, Alanine scan of CYREN-1 on residues 9–46. Flag immunoprecipitation of protein extracts from HEK293T cells transfected with pCDNA3.1 plasmids expressing wild-type CYREN-1–Flag or each of the single residues mutated to alanine. Total lysate and Flag-immunoprecipitate were then immunoblotted with anti-Flag or Ku70 antibodies. For gel source data, see Supplementary Fig. 1. c, Protein alignment of CYREN-1 KBM among vertebrates. d, Photo-crosslinked pulldown of a BPA–BIO–CYREN(2–24) peptide in HEK293T cell protein extract, followed by immunoblotting with anti-Ku70 and Ku80 antibodies. Single and double plus symbols, 25 μM and 50 μM of BPA–BIO–CYREN, respectively. 100 μM of CYREN(2–24) free peptide was used as a competitor. For gel source data, see Supplementary Fig. 1. e, Experimental outline of Fig. 3d. HT1080 6TG cells stably expressing an inducible control GFP or wild-type or mutant CYREN-1–3×Flag were transduced with shTRF2 on day 0 and transfected with a control (NT) pool of siRNAs or a pool of siRNAs targeting the 3′ UTR of CYREN on day 2. Expression of wild-type or mutant CYREN-1 was induced on day 3 and cells were collected on day 5. f, Flag western blot of endogenous N-terminal 3×Flag-tagged CYREN-1 and CYREN-2 cells following double thymidine synchronization. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 8 CYREN inhibits cNHEJ preferentially at breaks with overhangs by preventing processing.

a, Percentage of cells using HR to repair Cas9-induced breaks. Details of four CYRENWT and four CYRENKO clones used in Fig. 4b. b, Deletion profiles of repair of Cas9-induced breaks. Detail profiles of four CYRENWT and four CYRENKO clones used in Fig. 4c. Blue line in blunt ends: break site. Blue area: overhang region created by the pair of sgRNAs. c, Average percentage of mClover+ cells in four CYRENWT clones, four CYRENKO clones, four CYRENKO clones complemented with wild-type CYREN and four CYRENKO clones complemented with mutant CYREN (RPW-AAA), in three independent experiments, normalized to wild-type. Tukey box and whiskers; box represents 25th to 75th percentiles, upper whiskers represent 75th percentile plus 1.5 interquartile distance, lower whiskers represent 25th percentile minus 1.5 interquartile distance. *P < 0.05. Unpaired t-test. In each experiment, 100,000 cells per sample were analysed. d, Deletion profiles of repair of Cas9 breaks. Average percentage of deletion in CYRENWT clones, CYRENKO clones, CYRENKO clones complemented with wild-type CYREN and CYRENKO clones complemented with mutant CYREN (RPW-AAA). Error bars, s.e.m.

Extended Data Figure 9 CYREN does not promote cNHEJ in vitro.

a, In vitro cNHEJ assay using CYRENWT and CYRENKO cells. Left, immunoblots of extracts used in the assay. Middle, in vitro ligation assay. Right, quantification. Error bars, s.d., three independent experiments. For gel source data, see Supplementary Fig. 1. b, In vitro cNHEJ assay using CYRENWT cells and increasing amounts of recombinant wild-type CYREN and CYRENΔKu mutant. Right, quantification. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 10 Maps of plasmids used in Figs 1, 2, 3, 4.

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Arnoult, N., Correia, A., Ma, J. et al. Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN. Nature 549, 548–552 (2017). https://doi.org/10.1038/nature24023

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