The Mre11 Complex and the Response to Dysfunctional Telomeres

MEF derivation, transformation and culture.Primary MEFs were generated from appropriate crosses as previously described (43). For simian virus 40 (SV40) transformation, early-passage MEFs were transfected with the p129-Mertz SV40 plasmid by using Lipofectamine 2000 (Invitrogen) in accordance with manufacturer's instructions. For 3T3 transformation and subsequent culture of both SV40- and 3T3-transformed cells, cells were counted and replated every 3 days in accordance with the 3T3 protocol (44). Cumulative population doubling levels (cPDLs) were calculated using the formula ΔPDL = log(n_f/n_o)/log(2), where n_o is the initial number of cells and n_f is the final number of cells (16).

Telomere fluorescence in situ hybridization (FISH).Metaphases were prepared from cultures treated with colcemid (2 × 10⁻⁷ M) for 1 h or less for rapidly proliferating cultures. Cells were then trypsinized and hypotonically swelled (0.075 M KCl) for 7 min at 37°C, fixed, washed in ice-cold methanol-acetic acid (3:1), dropped onto slides, and air dried overnight. Slides were rehydrated in phosphate-buffered saline (PBS), dehydrated through a series of ethanol washes (70%, 90%, and 100%), and air dried. Hybridization mixture (10 mM Tris-Cl, pH 7.2, 70% [vol/vol] formamide, 0.5% [wt/vol] block reagent [Dupont NEN], 1:1,000 TelC-fluorescein isothiocyanate [FITC] peptide nucleic acid probe [Applied Biosystems]) was added to slides and denatured at 80°C for 3 min and then hybridized at room temperature for 2 h in a dark, humid chamber. Slides were washed two times for 15 min (70% formamide, 10 mM Tris-Cl, pH 7.2, 0.1% bovine serum albumin) and then three times for 5 min (100 mM Tris-Cl, pH 7.2, 150 mM NaCl, 0.08% Tween, with DAPI [4′,6-diamidino-2-phenylindole] added to the second wash), dehydrated in ethanol as described above, dried, and mounted. A total of >1,000 metaphase chromosomes from a minimum of 25 spreads were analyzed.

qFISH.Quantitative FISH (qFISH) was carried out as described in reference 35. Briefly, staining was carried out as for telomere FISH, with the following modifications. After air drying overnight, slides were rehydrated in PBS for 15 min, then fixed in 4% formaldehyde in PBS for 2 min, and washed three times for 5 min in PBS. Slides were treated with 1 mg/ml pepsin in 0.01 N HCl, pH 2.0, at 37°C for 10 min, washed two times for 2 min in PBS, then refixed in 4% formaldehyde in PBS, washed three times for 5 min in PBS, and dehydrated in an ethanol series (70%, 90%, and 100%) for 5 min each. Hybridization, washing, and mounting were done as described for telomere FISH.

For image capture and analysis, a reference slide of 0.2-μm FITC fluorescent beads (Molecular Probes) was used to generate control images for calibration of the microscope (see reference 35 for details). Image capture and analysis were carried out using an Axiovert 300 M (Zeiss) microscope and Volocity software (Improvision). Captured images did not contain saturated pixels. Telomere fluorescence units (TFU) were measured using Volocity software. Telomere regions to be measured were manually assigned and intensity was measured by Volocity to obtain the average number of TFU for each telomeric region. Data from all slides were pooled to give telomere length profiles for the cell population.

CO-FISH.Chromosome orientation-FISH (CO-FISH) was done as described for telomere FISH above, with the following modifications. Infected cells were grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% cosmic calf serum (CCS) containing a 3:1 ratio of 5′-bromo-2′-deoxyuridine (BrdU)-5′-bromo-2′-deoxycytidine (BrdC) (Sigma) at a final concentration of 1 × 10⁻⁵ M for 17 h, with colcemid (2 × 10⁻⁷ M) added for the last hour. Metaphase spreads were prepared as described above, except that care was taken not to denature the DNA after the chromosomes were dropped. Slides were rehydrated in PBS, then treated with 0.5 mg/ml RNase A in PBS at 37°C for 10 min, stained with 0.5 μg/ml Hoechst 33258 (Sigma) in 2× standard saline citrate (SSC) (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 15 min at room temperature, and exposed to 365 nm UV light (Stratalinker 1800 UV irradiator) in 2× SSC for 30 min. The BrdU/BrdC-substituted DNA was digested with exonuclease III (10 U/μl; Promega) in buffer supplied by manufacturer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl₂, and 5 mM dithiothreitol) for 10 min at room temperature. Slides were then rinsed in PBS and dehydrated through an ethanol series as before. Hybridization mixture (10 mM Tris-Cl, pH 7.2, 70% [vol/vol] formamide 0.5% [wt/vol] block reagent [Dupont NEN], 1:10,000 TelG-Cy3 peptide nucleic acid probe [Applied Biosystems]) was added, and TelG probe was hybridized at room temperature for 2 h in a dark, humid chamber. Slides were quickly rinsed in wash I (70% [vol/vol] formamide, 10 mM Tris-Cl, pH 7.2, 0.1% [wt/vol] bovine serum albumin), and then hybridization mixture with 1:1,000 TelC-FITC probe was applied for 2 h in a dark, humid chamber. Slides were then washed twice with wash I for 30 min each and three times with wash II for 5 min each at room temperature, with DAPI added to the second wash. Slides were dehydrated and mounted as described above.

Telomere restriction fragment (TRF) Southern blotting (telomere blots).Clamped homogenous electric field gel electrophoresis of mouse DNA was performed essentially as described previously (48). Cells were resuspended in PBS and mixed 1:1 (vol/vol) with 2% (wt/vol) pulsed-field gel grade agarose (Bio-Rad) to obtain 1 × 10⁶ cells per agarose plug. Plugs were digested with 1 mg/ml proteinase K in TENS buffer (100 mM EDTA, 0.2% [wt/vol] sodium deoxycholate, 1% [vol/vol] sodium lauryl sarcosine) and washed extensively in TE buffer (10 mM Tris, pH 8, 1 mM EDTA). Plugs were incubated overnight at 37°C with 60 U MboI in 0.5 ml buffer. Following digestion, plugs were washed in TE buffer and equilibrated in 0.5× Tris-borate-EDTA (TBE) before being loaded into a 1% [wt/vol] agarose-0.5× TBE gel. The gel was run using a CHEF-DRII pulsed-field gel electrophoresis apparatus (Bio-Rad) in 0.5× TBE for 18 h with the following settings: initial pulse, 5 s; final pulse, 5 s; 5.4 V/cm²; and 14°C. In-gel hybridization with a [γ-³²P]ATP end-labeled (CCCTAA)⁴ oligonucleotide and subsequent denaturation and hybridization steps were performed as described previously (18). Gels were exposed onto a PhosphorImager screen, and lanes were quantified with ImageGauge software.

Conditional deletion of TRF2 from MEFs.Lentiviral production, concentration, and determination of titers were carried out using established methods (10, 25). SV40-transformed MEFs were infected with either a cre-green fluorescent protein vector-based lentivirus or the same virus with a deleted cre locus as a negative control. Cells were infected in suspension at 1 × 10⁶ cells/ml at a multiplicity of infection of 10 in DMEM plus 10% CCS with 5 μg/ml polybrene. Tubes were spun at 1,900 rpm (∼600 × g) for 90 min, with occasional stops to manually resuspend cells. After viral infection, cells were plated and grown in DMEM containing 10% CCS.

Western blotting.Protein lysates for Rap1 were prepared by subjecting cells to three freeze-thaw cycles in high-salt-concentration TNG buffer (50 mM Tris, pH 7.5, 400 mM NaCl, 0.1% Tween, and 0.5% NP-40 plus protease inhibitors) and transferred onto a nitrocellulose membrane. To prepare extracts for γ-H2AX detection, the chromatin pellet from regular lysates was further incubated for 30 min in 0.1 M HCl, and the resulting supernatant was used for immunoblotting. Binding and washing steps were done with 5% milk and PBS-0.05% Tween 20 buffer. Rabbit anti-Rap1 polyclonal (1:10,000; obtained from T. de Lange), antiactin mouse monoclonal (1:1,000; AC-40; Sigma), anti-γ-H2AX mouse monoclonal (1:500; Ser139; Upstate), and anti-total histone H2A rabbit (1:500; Upstate) primary antibodies and horseradish peroxidase-conjugated species-specific secondary antibodies (Pierce) were used. Horseradish peroxidase signal was detected with ECL Plus reagent (Amersham).

Immunofluorescence-FISH.Cells were grown on coverslips, fixed for 15 min in 4% paraformaldehyde, washed in PBS, and blocked for 30 min in blocking solution (1 mg/ml bovine serum albumin, 3% goat serum, 0.1% Triton X-100, 1 mM EDTA). Coverslips were incubated at room temperature for 1 h with polyclonal anti-53BP1 rabbit antibody (1:1,000 diluted in blocking solution; Novus), washed, incubated with anti-rabbit secondary antibody (Rhodamine, diluted in blocking solution 1:200) for 30 min, fixed for 5 min in 4% paraformaldehyde, and washed in PBS. Coverslips were dehydrated through a series of ethanol washes (70%, 90%, and 100%), hybridized and denatured for 5 min at 80°C, and then hybridized for 2 h at room temperature by using a FITC-TelC probe (1:1,000; Applied Biosystems). Coverslips were washed twice each for 15 min in FISH wash solution (70% formamide, 10 mM Tris-Cl, pH 7.2) and then three times in PBS, with DAPI added to the second wash, and were dried and mounted.

SKY.Spectral karyotyping (SKY) of metaphase chromosomes was carried out as described previously (43).

Statistical analyses.Statistical significance was determined by Fisher's exact test (for 3T3 fusions, cre-induced fusions, and telomere dysfunction-induced focus [TIF] formation), the Wilcoxon rank sum test (for qFISH), and the chi-square goodness-of-fit test (for orientation of fusions), using Excel or Mstat software (Norman Drinkwater, McArdle Laboratory for Cancer Research).

The Mre11 complex in mammalian telomere length regulation.Cells from the hypomorphic mouse mutant Mre11^ATLD1/ATLD1 exhibit defects in the ATM-dependent DNA damage response (29, 39, 42). Cells from this mutant, as well as an additional Mre11 complex hypomorph, the Nbs1^ΔB/ΔB mouse, which is also defective in ATM activity (49), were used to examine the telomeric functions of the Mre11 complex in mammals. Mre11^+/ATLD1 mice were crossed with telomerase heterozygous (Tert^+/Δ) mice to create double heterozygous mutant mice, and these animals were then crossed together to generate Mre11^ATLD1/ATLD1Tert^+/+, Mre11^+/+Tert^Δ/Δ, and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs.

MEFs were isolated and passaged by following the 3T3 protocol (44), and cPDLs were calculated for each genotype throughout the course of the experiment. Individual embryo cultures were transformed both by SV40 infection and by classic 3T3 transformation. Although 3T3 transformation was slower than SV40 transformation (approximately passage 20 for 3T3 cells versus approximately passage 7 for SV40 cells) (Fig. 1A), no overt differences in the efficiency of transformation of any genotype or in the growth rate of the cells in culture were observed (Fig. 1A). To ensure that meaningful cPDL comparisons could be made, the rates of BrdU incorporation and cell death were determined throughout the course of the experiment by flow cytometry. For 3T3-transformed cultures, 49% of cells were BrdU positive on average throughout the experiment. For SV40-transformed cultures, an average of 56% of cells were BrdU positive (Table 1). For both transformation methods, cells with sub-G₁ DNA content (correlated with cell death) were generally less than 2% of the culture (Table 1). These parameters confirm that the potentially confounding effects of differing cell death rates among the various genotypes did not skew the calculated cPDLs and that the calculated values reflected comparable numbers of cell divisions among the genotypes examined.

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FIG. 1.

Telomere shortening and fusions in Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs. (A) Cell proliferation in 3T3-cultured Mre11^ATLD1/ATLD1, Mre11^+/+Tert^Δ/Δ, and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs are presented, showing passage number versus cPDL. SV40-transformed MEFs undergo transformation faster than 3T3-transformed MEFs, p7 versus p20. No differences are observed in the growth rates of the various genotypes. (B) Telomere FISH on 3T3-cultured, SV40-transformed cPDL 300 MEFs. Metaphase spreads (cPDL 300) were hybridized with an FITC-labeled telomeric probe (TelC; green) and stained with DAPI (pseudocolored red). Lower panels show enlarged images of the indicated genotypes. Note the strong telomere signal in Mre11^ATLD1/ATLD1 cells, compared to the increase in signal-free ends and telomere fusions in both Mre11^+/+Tert^Δ/Δ and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs. (C) qFISH measurement of telomere length. Telomere length distribution in cPDL 70 (left) and cPDL 300 (right) MEFs of the indicated genotypes is shown. Telomeres were hybridized with a fluorescent TelC probe (as shown in panel B), and fluorescence was measured as a readout of telomere length. No telomere shortening was observed in Mre11^ATLD1/ATLD1 cells (top row, compare left and right profiles). Both Mre11^+/+Tert^Δ/Δ and Mre11^ATLD1/ATLD1Tert^Δ/Δ cells show significant telomere shortening (P < 10⁻⁶) (compare the left and right profiles of the middle and bottom panels; the profiles shift to the left at cPDL 300, indicating telomere shortening). (D) Quantitation of fusion formation. No telomere fusions in cPDL 70 MEFs of any genotype were observed. Reduced chromosome fusions were observed in cPDL 300 Mre11^ATLD1/ATLD1Tert^Δ/Δ cells (gray bar, middle) (5%), compared to the level for cPDL 300 Mre11^+/+Tert^Δ/Δ cells (white bar, middle) (8%), despite equivalent telomere shortening (P < 0.001). Percents telomere fusion per chromosome are calculated per total number of chromosomes, and >1,000 chromosomes were scored for each set. (E) Distribution of telomere fusions in cPDL 300 MEFs. Predominance of p-p fusions in Mre11^+/+Tert^Δ/Δ and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs. Few p-q fusions, q-q fusions, unknown fusion types, and multiple fusions were observed.

It has previously been reported that wild-type (WT) (telomerase-proficient) MEFs display minimal telomere shortening upon continual passage in tissue culture (4). To determine the effect of Mre11 complex hypomorphism, metaphase spreads were analyzed following hybridization of a FITC-labeled telomeric probe (TelC) at cPDLs 70, 300, and 400 and were assessed for the appearance of signal-free ends and telomere fusions. At cPDL 70, none of the genotypes displayed marked loss of telomere signal or telomere fusion formation. At late passage (cPDL 300), Mre11^ATLD1/ATLD1 cells did not show loss of telomere signal, nor did we observe the appearance of chromosome fusions (Fig. 1B and D).

As expected, the telomeres of Tert^Δ/Δ and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs underwent progressive shortening upon passage through culture (4, 23), resulting in the appearance of signal-free ends and chromosomal fusions at high cPDLs (Fig. 1B and D). Telomere shortening in cPDL 70 and 300 MEFs was quantified by qFISH (19). We did not observe telomere shortening in Mre11^ATLD1/ATLD1 MEFs at cPDL 300 (Fig. 1C, top row), indicating that Mre11 complex hypomorphism does not appear to impair the recruitment of telomerase. In Tert^Δ/Δ MEFs, marked telomere shortening was observed, from an average TFU count of 766 at cPDL 70 to an average TFU count of 520 TFU at cPDL 300. Mre11^ATLD1/ATLD1Tert^Δ/Δ cultures also showed significant telomere shortening at cPDL 300, and there was no difference in telomere length at this cPDL between Mre11^+/+Tert^Δ/Δ (520 TFU) and Mre11^ATLD1/ATLD1Tert^Δ/Δ (517 TFU) MEFs (Fig. 1C, bottom two rows).

These results were confirmed using TRF Southern blotting, which showed that equivalent levels of shortening were observed between Mre11^+/+Tert^Δ/Δ and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs (data not shown). TRF analysis also confirmed that maintenance of the G-strand overhang was unaffected (data not shown). These data support the interpretation that the murine Mre11 complex does not influence telomere length maintenance in either telomerase-proficient or telomerase-deficient mouse cells.

Mre11 complex and telomere fusions.As described above, Mre11^+/+Tert^Δ/Δ and Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs showed diminished telomere signals due to telomere shortening and the appearance of chromosomal fusions (Fig. 1B and D). The observed fusions did not contain cytologically detectable telomeric sequences, suggesting that they result from telomere attrition-dependent uncapping. Fusions were predominantly p-p fusions (short arm) (Fig. 1E), which may be a reflection of the increased stability of p-p fusions in mouse cells in culture. Short arm fusions are likely to be more stable because of the acrocentric nature of mouse telomeres; thus, p-q and q-q fusions create dicentric telomeres that are more likely to undergo breakage upon cell division (27). Analysis by SKY of cPDL 300 spreads demonstrated that the observed fusions were not clonal, as we did not detect rearrangements common to multiple cells (Table 2). This suggests that fusion of critically short telomeres is ongoing in late passage cells.

The rate of telomere fusion was reduced in Mre11^ATLD1/ATLD1Tert^Δ/Δ cells. At cPDL 300, 5% of all chromosomes were fused in Mre11^ATLD1/ATLD1Tert^Δ/Δ, in comparison to 8% of all chromosomes in Tert^Δ/Δ (P < 0.001). Fewer fusions were also observed in cPDL 400 Mre11^ATLD1/ATLD1Tert^Δ/Δ than in cPDL 400 Mre11^+/+Tert^Δ/Δ (P < 0.0003) (Fig. 1D). These data suggest a role for the Mre11 complex in NHEJ of dysfunctional telomeres.

Mre11 complex and repair of dysfunctional telomeres.The Mre11 complex associates with mammalian telomeres and has been suggested to recognize uncapped telomeres as DNA damage following replication (47, 54), resonant with its role in the recognition of interstitial DSBs and the subsequent activation of ATM (21, 39). It has recently been shown that loss of the shelterin component Trf2 in the absence of ATM results in a significant decrease in telomere fusion formation (8). On these bases, we hypothesized that the Mre11 complex is required for recognition of dysfunctional telomeres and activation of ATM; hence, the observed decrease in telomere fusions in late-passage Mre11^ATLD1/ATLD1Tert^Δ/Δ MEFs would reflect the decrement in ATM activity attendant to the Mre11^ATLD1 allele.

To test this hypothesis, we utilized the Trf2 conditional allele, Trf2^Flox, which provides an alternative means of generating telomere dysfunction, and which may elicit a response distinct from that seen at eroded telomeres. Introduction of lentiviral cre recombinase into Trf2^F/Δ or Trf2^F/F cells (which were used interchangeably here) generates Trf2-null cells (Trf2^Δ/Δ) and results in acute telomere dysfunction due to loss of Trf2 and subsequent telomere uncapping (5). The uncapped telomere activates the DNA damage response and leads to chromosome fusions and the formation of TIF (41). We generated Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F MEFs and assessed their responses to acute telomere dysfunction.

Trf2^F/F cells were infected with lentivirus expressing the cre recombinase, and depletion of Trf2 protein was monitored by assessing the level of Rap1. Mouse Trf2 antisera recognize a nonspecific band that obscures Trf2 on Western blots. As Rap1 is destabilized upon loss of Trf2, Rap1 is used as a surrogate marker for Trf2 depletion (5) (Fig. 2A). As previously shown, depletion of Trf2 in WT cells results in the formation of multiple chromosome fusions with retention of telomere signal at the fusion junction (Fig. 2B) (20). In this study, 43.3% of the total chromosomes were fused in Trf2^Δ/Δ cells (chromosome plus chromatid type fusions) (Fig. 2D). Consistent with the reduction in fusions seen in cells with critically short telomeres, both Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ MEFs exhibited significant reductions in total fusions, to 18.3% (P < 0.0003 for comparison with Trf2^Δ/Δ) and 7.0% (P < 0.0001 for comparison with Trf2^Δ/Δ), respectively (Fig. 2D).

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FIG. 2.

Reduced chromosome fusions in cre-infected Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F MEFs upon acute telomere dysfunction. (A) Depletion of TRF2 induces acute telomere dysfunction. Cell extracts were prepared from each genotype following infection with empty lentiviral vector (−) and lentiviral cre-recombinase (+). Trf2 deletion was measured by degradation of RAP1 protein, which is destabilized in the absence of TRF2. Actin was used as a loading control. (B) Chromosome fusions upon acute telomere dysfunction. Representative telomere FISH images from metaphase spreads following Trf2 deletion are shown. Chromosome fusions were observed in the majority of Trf2^F/F chromosomes upon cre recombination (bottom left panel). Chromosome fusions were significantly reduced in Mre11^ATLD1/ATLD1Trf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) and Nbs1^ΔB/ΔBTrf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) MEFs (bottom panels, middle and right). Insets in the bottom panels show chromatid fusions in Trf2-deficient Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB. (C) TRF Southern blotting following acute telomere dysfunction induced by loss of TRF2. The left panel shows a nondenatured blot displaying the 3′ G overhang, which is lost in Trf2^F/F cells following deletion of TRF2 (lane 2). In contrast, the G overhang remains intact in Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F MEFs following Trf2 deletion (left panel, lanes 4 and 6). Following denaturation (right panel), Trf2^Δ/Δ cells show a high degree of fusion formation, as evidenced by the high-molecular-weight smear. Although fusion formation is decreased, Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ MEFs also show the appearance of some fusions. (D) Quantification of chromosome and chromatid fusions observed by telomere FISH following Trf2 deletion. Black bars represent the fraction of chromosome fusions (expressed as a percentage), dark gray bars represent the fraction of chromatid fusions per chromosome, and light gray bars represent the sum of both (total fusions scored). Mre11^ATLD1/ATLD1Trf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) and Nbs1^ΔB/ΔBTrf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) MEFs show drastically reduced chromosome fusions but increased proportions of chromatid fusions (P < 0.0001 and P < 0.0001, respectively, for comparison with Trf2^Δ/Δ).

Assessment of telomere fusions was also carried out using TRF Southern blots. TRF length analysis showed increased fusion formation in Trf2^Δ/Δ cells, as evidenced by the higher-molecular-weight smear indicated on the blot and the loss of the WT telomere bulk. In contrast, Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ MEFs showed reduced fusion formation (Fig. 2C, right panel, lanes 10 and 12), consistent with the data obtained from metaphase cells by using telomere FISH. The single-stranded G overhang (Fig. 2C, left panel, lanes 4 and 6) remained intact in both cre-infected Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F cells, showing that 3′ overhang loss does not occur upon acute telomere dysfunction in either Mre11^ATLD1/ATLD1Trf2^Δ/Δ or Nbs1^ΔB/ΔBTrf2^Δ/Δ cells.

Chromatid fusions predominate in Mre11 complex mutant cells.Although overall fusions were markedly reduced in cre-infected Mre11^ATLD1/ATLD1Trf2^F/Δ or Nbs1^ΔB/ΔBTrf2^F/F, a substantial fraction of the residual fusions in those mutants were events in which only one chromatid is fused. Less than 2.0% of the total fusions in Trf2^Δ/Δ were chromatid fusions, whereas 48.5% of Mre11^ATLD1/ATLD1Trf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) and 29.3% of Nbs1^ΔB/ΔBTrf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) fusions were of this class (Fig. 2B).

To examine the basis of the observed bias toward chromatid type fusions, these events were characterized using CO-FISH, which allows for identification of the leading (5′) and lagging (3′) strands (2). If fusion formation were a random event, a 1:2:1 ratio of leading-leading, leading-lagging, and lagging-lagging fusions would be observed (Fig. 3A).

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FIG. 3.

CO-FISH analysis of metaphase chromosomes following acute telomere dysfunction reveals a predominance of leading-leading-strand chromatid fusions in cre-infected Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F MEFs. (A) Leading-leading, leading-lagging, and lagging-lagging chromatid fusions as identified by CO-FISH. (B) CO-FISH metaphases showing a predominance of leading-leading chromatid fusions (open yellow arrowheads) in Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F MEFs following Trf2 deletion (middle and right panels). cre-infected Trf2^F/F MEFs (left panel) show predominantly chromosome fusions. (C) Quantification of chromatid fusion type as a percentage of total chromatid fusions in each genotype following Trf2 deletion. Results are shown for leading-leading, leading-lagging, and lagging-lagging chromatid fusions. Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ MEFs show significant increases in leading-leading-strand fusions (P < 0.0001).

The rare chromatid type fusions seen in Trf2^Δ/Δ cells (fewer than one per spread) were equally distributed among leading-leading, leading-lagging, and lagging-lagging chromatid fusions (Fig. 3A and B). It is likely that this departure from expectation is an artifact of the small sample size available. In contrast, Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ MEFs exhibited a striking tendency toward leading-leading-strand fusions (Fig. 3B and C and Table 3). In Mre11^ATLD1/ATLD1Trf2^Δ/Δ MEFs, 96.1% of chromatid fusions and 87.3% of Nbs1^ΔB/ΔBTrf2^Δ/Δ MEFs were leading-leading-strand fusions (P < 0.0001 for comparison with Trf2^Δ/Δ). These data indicate that fusions involving the lagging strand are strongly inhibited in Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB cells.

Signaling damage at dysfunctional telomeres.Telomeres protect chromosome ends from being recognized as DNA DSBs. Accordingly, telomere dysfunction in mammalian cells induces a DNA damage response that can be measured through the formation of TIF, consisting of telomeric 53BP1 or γ-H2AX foci (5). Given the role of the Mre11 complex in promoting both the activation and the activity of ATM, we asked whether the Mre11 complex is required for the response to dysfunctional telomeres. TIF formation in cre-infected Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F cells was examined, and as previously seen in Atm^−/− cells, both showed drastically reduced TIF formation (Fig. 4A). As expected, 60.0% of Trf2^Δ/Δ MEFs were TIF positive (defined as cells having at least five telomeric 53BP1 foci) following Trf2 deletion. In contrast, only 16.8% and 24.0% of cells were TIF positive in Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ, respectively (P < 0.0001 for comparison with Trf2^Δ/Δ) (Fig. 4B). The formation of γ-H2AX induced by Trf2 depletion was similarly impaired in Mre11 complex mutants (Fig. 4C). These observations suggest that the Mre11 complex's DNA damage recognition and signaling functions, required for the response to interstitial DNA damage, are also required for the detection or signaling of acute telomere dysfunction. Moreover, the data underscore the intimate relationship of the DNA damage response pathway and telomere function.

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FIG. 4.

Reduced TIF formation in cre-infected Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/F MEFs despite acute telomere dysfunction. (A) TIF formation in MEFs following Trf2 deletion by cre recombinase. TIF formation is scored by immunofluorescence-FISH measuring colocalization of 53BP1 foci and a TelC-FITC telomeric probe. We define TIF-positive cells as those showing colocalization of at least five 53BP1 and TelC foci per cell. Deletion of Trf2 induces TIF formation (TIF-positive cells) in Trf2^F/F MEFs (column 2). Mre11^ATLD1/ATLD1Trf2^F/Δ and Nbs1^ΔB/ΔBTrf2^F/Δ cells show reduced 53BP1 foci following Trf2 deletion (columns 4 and 6, middle row), resulting in reduced numbers of TIF-positive cells (P < 0.0001). (B) Quantification of TIF formation for each genotype. Each symbol (diamond, Trf2^F/F; square, Mre11^ATLD1/ATLD1Trf2^F/Δ; and triangle, Nbs1^ΔB/ΔBTrf2^F/F) represents the average percentage of TIF-positive cells following Trf2 deletion in one experiment. Black bars represent the average of results from all experiments for each genotype. Both Mre11^ATLD1/ATLD1Trf2^Δ/Δ and Nbs1^ΔB/ΔBTrf2^Δ/Δ cells show reduced numbers of TIF-positive cells (P < 0.0001 for comparison with Trf2^Δ/Δ). (C) Western blot analysis of γ-H2AX abundance following infection of cells with the indicated genotypes with cre-expressing lentivirus (+) or with control virus lacking cre (−).