The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres

Abstract

Repair of DNA double-stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non-homologous end-joining (NHEJ) or error-free homologous recombination. NHEJ precedes either by a classic, Lig4-dependent process (C-NHEJ) or an alternative, Lig4-independent one (A-NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere-binding proteins are recognized as DSBs. In this report, we studied which end-joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end-to-end chromosome fusions mediated by the C-NHEJ pathway. In contrast, removal of Tpp1-Pot1a/b initiated robust chromosome fusions that are mediated by A-NHEJ. C-NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A-NHEJ is a major pathway to process dysfunctional telomeres.

Figure 1

53BP1 domains required for telomere localization and repair. (A) γ-H2AX-positive TIFs in 53BP1^+/+ and 53BP1^−/− MEFs after Trf2 depletion. (B) SV40LT immortalized 53BP1^+/− and 53BP1^−/− MEFs were treated with control vector or Trf2 shRNA for 120 h, metaphases were prepared and telomere fusions were visualized by Tam-OO-(CCCTAA)₄ telomere peptide nucleic acid (PNA; red) and 4,6-diamidino-2-phenylindole (DAPI; blue). (C) Schematic of 53BP1 domains and its deletion and point mutants used in this study. (D) 53BP1 TIFs and chromosome fusions in 53BP1^−/− MEFs reconstituted with full-length 53BP1 and the indicated 53BP1 deletion or point mutants. For TIFs, cells stably infected with Trf2 shRNA were fixed after 72 h and stained with anti-53BP1 antibody (green), telomere-PNA FISH (red) and DAPI (blue). For telomere fusions, metaphases prepared from reconstituted cell lines infected with Trf2 shRNA after 120 h were visualized by telomere PNA-FISH (red) and DAPI (blue). Arrows point to representative fused chromosomes. (E) Quantification of 53BP1 TIFs. A minimum of 100 cells were examined and cells with >4 TIFs were scored as TIF positive. Mean values were derived from at least three experiments. Error bars: s.d. (F) Quantification of telomere fusion frequencies. A minimum of 1600 chromosomes were analysed and mean values derived from at least three experiments presented. Error bars: s.d.

Figure 2

Chromosome fusions in the absence of Tpp1–Pot1a/b are mediated by alternative NHEJ. (A) SV40LT immortalized 53BP1^+/+ or 53BP1^−/− MEFs were treated with the indicated DNA constructs for 120 h, metaphases prepared and telomeres visualized by telomere FISH (FITC-OO-(TTAGGG)₄ (green, leading strand) and Tam-OO-(CCCTAA)₄ (red, lagging strand)). Arrows point to fused chromosomes. (B) Quantification of the chromosome fusion frequencies in 53BP1^+/+ MEFs observed in (A). A minimum of 2000 chromosome ends were analysed per cell line. Error bars: s.d. ^**P<0.001 calculated using a two-tailed Student's t-test. (C) Quantification of the chromosome fusion frequencies in 53BP1^−/− MEFs observed in (A). A minimum of 2500 chromosome ends were analysed per cell line. Error bars: s.d. ^*P<0.65, ^**P<0.001 calculated using a two-tailed Student's t-test. (D) Quantification of T-SCEs in 53BP1^+/+ MEFs. A minimum of 1500 chromosome ends were scored per cell line. Error bars: s.d. (E) Quantification of T-SCEs in 53BP1^−/− MEFs. A minimum of 2100 chromosome ends were scored per cell line. Error bars: s.d. (F) SV40LT immortalized Lig4^−/− MEFs were treated with the indicated DNA constructs for 120 h, metaphases prepared and telomeres visualized by CO-FISH (FITC-OO-(TTAGGG)₄ (green) and Tam-OO-(CCCTAA)₄ (red)). Arrows point to fused chromosomes. (G) Quantification of the chromosome fusion frequencies observed (F). A minimum of 1500 chromosome ends were analysed per cell line and the mean value derived from three independent experiments are given. Error bars: s.d.

Figure 3

Ku70 represses A-NHEJ of telomeres devoid of Tpp1–Pot1a/b. (A) Ku70^−/− cells were treated with DNA constructs as indicated, metaphases prepared and telomeres visualized by telomere FISH (FITC-OO-(TTAGGG)₄ (green) and Tam-OO-(CCCTAA)₄ (red)). Arrows point to representative fused chromosomes. (B) Ku70^−/− cells stably expressing shLig4 were treated with DNA constructs as indicated, and metaphases prepared as in (A). Arrows point to representative fused chromosomes. (C) Quantification of the chromosome fusion frequency observed in (A, B). A minimum of 2000 chromosome ends were analysed per cell line and the mean value derived from two independent experiments are given. Error bars: s.d. ^*P<0.4, ^**P<0.001 calculated using a two-tailed Student's t-test. (D) 53BP1^−/− or Lig4^−/− cells stably expressing shRad51 were treated with shTrf2 and Tpp1^ΔRD, and metaphases prepared as in (A). Almost all the chromosomes are fused. (E) Quantification of the chromosome fusion frequency observed in (D). A minimum of 2000 chromosome ends were analysed and the mean value derived from three independent experiments are given. Error bars: s.d.

Figure 4

ATR and CtIP are required for A-NHEJ of telomeres lacking Tpp1–Pot1a/b. (A) Metaphase spreads prepared from ATM^−/− MEFs or 53BP1^−/− MEFs stably expressing shATR were treated with the indicated DNA constructs, metaphases prepared and analysed by telomere FISH (FITC-OO-(TTAGGG)₄ (green) and Tam-OO-(CCCTAA)₄ (red)). Arrows point to representative fused chromosomes. (B) Quantification of the chromosome fusion frequencies observed in ATM^−/− MEFs. A minimum of 2100 chromosomes were analysed and the mean value derived from two independent experiments are given. Error bars: s.d. (C) Quantification of the chromosome fusion frequencies observed in shATR treated 53BP1^−/− and ATR^Δ/− 53BP1^+/+ MEFs (Guo et al, 2007). A minimum of 1200 chromosomes were analysed per cell line and the mean value derived from two independent experiments are given. Error bars: s.d. (D) Metaphase spreads prepared from 53BP1^−/− MEFs expressing the indicated DNA constructs were analysed by CO-FISH [FITC-OO-(TTAGGG)₄ (green) and Tam-OO-(CCCTAA)₄ (red)]. Arrows point to representative fused chromosomes. (E) Quantification of the chromosome fusion frequencies observed in (D). A minimum of 2500 chromosomes were analysed per cell line and the mean value derived from two independent experiments are given. Error bars: s.d. (F) Quantification of T-SCE frequencies observed in shATR treated 53BP1^−/− MEFs. A minimum of 1500 chromosome ends were scored per cell line. Error bars: s.d.

Figure 5

C-NHEJ is dispensable for chromosome fusions of naturally shortened telomeres. (A) Kaplan–Meier (KM) tumor-free survival curve of 53BP1^+/+ (wt), 53BP1^−/−+5 Gy IR, G1–2 mTerc^−/− 53BP1^+/+ and G1–2 mTerc^−/−53BP1^−/− mice. (B) KM tumor-free survival curve of G4 mTerc^−/− 53BP1^+/+ and G4 mTerc^−/−53BP1^−/− mice. (C) Quantification of anaphase bridges and (D) apoptotic bodies observed in the intestinal crypts of mice of the indicated genotypes. Error bars: s.d. (E, F) Representative images of metaphase spreads derived from bone marrow (BM) of G4 mTerc^−/−53BP1^+/− and G4 mTerc^−/−53BP1^−/− mice. Arrows point to fused chromosomes. A minimum of 30 metaphases were analysed per genotype. (G) Quantification of number of chromosome fusions per metaphase in bone marrow and (H) thymic lymphomas of mice of the indicated genotypes. A minimum of 30 metaphases were analysed per genotype. Error bars: s.d.

Figure 6

Telomere-FISH analysis of chromosome fusion sites. (A) Metaphases prepared from 53BP1^+/+ MEFs depleted of Trf2, Lig4^−/− MEFs depleted of both Tpp1 and Trf2, and 53BP1^−/− MEFs depleted of Tpp1 were analysed by CO-FISH (FITC-OO-(TTAGGG)₄ (green, to detect the leading strand) and Tam-OO-(CCCTAA)₄ (red, to detect the lagging strand)). Metaphases from G4mTerc^−/−53BP1^−/− and G4mTerc^−/−53BP1^+/− MEFs were analysed by Tam-OO-(CCCTAA)₄ telomere FISH (red). Telomere intensities at chromosome fusion sites were scored. White arrows: robust telomeric signals at fusion sites, with approximately equal intensity lagging- and leading-strand telomeres; yellow arrow: attenuated telomere signals (either leading- or lagging-strand telomere) at fusion sites; red arrows: fusion sites devoid of telomeric signals. (B) Quantification of telomere signals at chromosome fusion sites. A minimum of 18 metaphases were analysed per cell type. Error bars: s.d.

Figure 7

Model of how telomeres are protected from engaging in inappropriate end-joining reactions. Telomeres are normally protected by the TRF2-RAP1 and TPP1–POT1 from engaging in inappropriate fusion reactions. Removal of TRF2-RAP1 initiates downstream DDR events to activate ATM/53BP1 for Lig4-mediated C-NHEJ. Removal of TPP1–POT1 stimulates ATR and CtIP to activate A-NHEJ. Natural telomere attrition results in chromosome fusions that do not require factors involved in C-NHEJ. We speculate that A-NHEJ is used to fuse naturally shortened telomeres.