The mre11 complex and the response to dysfunctional telomeres

Abstract

In this study, we examine the telomeric functions of the mammalian Mre11 complex by using hypomorphic Mre11 and Nbs1 mutants (Mre11(ATLD1/ATLD1) and Nbs1(Delta)(B/)(DeltaB), respectively). No telomere shortening was observed in Mre11(ATLD1/ATLD1) cells after extensive passage through culture, and the rate of telomere shortening in telomerase-deficient (Tert(Delta)(/)(Delta)) Mre11(ATLD1/ATLD1) cells was the same as that in Tert(Delta)(/)(Delta) alone. Although telomeres from late-passage Mre11(ATLD1/ATLD1) Tert(Delta)(/)(Delta) cells were as short as those from Tert(Delta)(/)(Delta), the incidence of telomere fusions was reduced. This effect on fusions was also evident upon acute telomere dysfunction in Mre11(ATLD1/ATLD1) and Nbs1(Delta)(B/)(DeltaB) cells rendered Trf2 deficient by cre-mediated TRF2 inactivation than in wild-type cells. The residual fusions formed in Mre11 complex mutant cells exhibited a strong tendency toward chromatid fusions, with an almost complete bias for fusion of telomeres replicated by the leading strand. Finally, the response to acute telomere dysfunction was strongly impaired by Mre11 complex hypomorphism, as the formation of telomere dysfunction-induced DNA damage foci was reduced in both cre-infected Mre11(ATLD1/ATLD1) Trf2(F/)(Delta) and Nbs1(Delta)(B/)(DeltaB) Trf2(F/F) cells. These data indicate that the Mre11 complex influences the cellular response to telomere dysfunction, reminiscent of its influence on the response to interstitial DNA breaks, and suggest that it may promote telomeric DNA end processing during DNA replication.

FIG. 1.

Telomere shortening and fusions in Mre11^ATLD1/ATLD1 Tert^Δ/Δ MEFs. (A) Cell proliferation in 3T3-cultured Mre11^ATLD1/ATLD1, Mre11^+/+ Tert^Δ/Δ, and Mre11^ATLD1/ATLD1 Tert^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/ATLD1 Tert^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/ATLD1 Tert^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/ATLD1 Tert^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/ATLD1 Tert^Δ/Δ MEFs. Few p-q fusions, q-q fusions, unknown fusion types, and multiple fusions were observed.

FIG. 1.

Telomere shortening and fusions in Mre11^ATLD1/ATLD1 Tert^Δ/Δ MEFs. (A) Cell proliferation in 3T3-cultured Mre11^ATLD1/ATLD1, Mre11^+/+ Tert^Δ/Δ, and Mre11^ATLD1/ATLD1 Tert^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/ATLD1 Tert^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/ATLD1 Tert^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/ATLD1 Tert^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/ATLD1 Tert^Δ/Δ MEFs. Few p-q fusions, q-q fusions, unknown fusion types, and multiple fusions were observed.

FIG. 2.

Reduced chromosome fusions in cre-infected Mre11^ATLD1/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^Δ/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^Δ/Δ (P < 0.0001 for comparison with Trf2^Δ/Δ) and Nbs1^ΔB/ΔB Trf2^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).

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/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^Δ/Δ and Nbs1^ΔB/ΔB Trf2^Δ/Δ MEFs show significant increases in leading-leading-strand fusions (P < 0.0001).

FIG. 4.

Reduced TIF formation in cre-infected Mre11^ATLD1/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^F/Δ and Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^F/Δ; and triangle, Nbs1^ΔB/ΔB Trf2^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/ATLD1 Trf2^Δ/Δ and Nbs1^ΔB/ΔB Trf2^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 (−).

FIG. 5.

Summary figure. (A) DNA damage-dependent checkpoints and telomeric end resection. Uncapped telomeres (asterisks; red represents the leading strand, and green represents the lagging strand) elicit checkpoint responses in WT cells. The smaller asterisk in Mre11 complex mutants reflects impaired signaling, which leads to defective checkpoint activation and the accumulation of unfused uncapped chromatids in metaphase cells. Red in the WT cell cycle phase diagram represents potential sites of accumulation of cells with uncapped telomeres, and green in the analogous positions reflects the failure to arrest in Mre11 complex mutants. Defective checkpoints are denoted by an unfilled font. (B) Mre11 complex at functional versus nonfunctional telomeres: a role in 3′ overhang formation. On the left side, the Mre11 complex recognizes normal (i.e., functional) telomeres and promotes the resection of the telomeric ends to create the 3′ overhang en route to t-loop formation. On the right side, the Mre11 complex's role at interstitial DNA breaks is recapitulated at the telomere; the complex recognizes and signals the presence of dysfunctional telomeres, leading to activation of ATM as well as promoting their “repair” (i.e., fusion). The complex may also influence the degradation of the 3′ overhang prior to or during the fusion process. This is not required for ATM activation.