Fig 1.
Altered repair of telomeric DSBs after ATRX deletion.
(A) Immunoblots for FokI-TRF1 (6 days) in ATRXF/F MEFs treated with Cre (4 days). Actin serves as a loading control. (B) PFGE of telomeric DNA. Left, ethidium bromide–stained gel and right, detection of telomeric restriction fragments by native in-gel hybridization with a [CCCTAA]4 probe for the telomeric overhang. The ratio of short versus bulk telomere signal (mean from 3 experiments) is given below the blot, with values reported relative to FokIDA-TRF1 (set at 100). (C) TIFs detected by IF-FISH for 53BP1 (IF, green) and telomeres (FISH, red). DNA was stained with DAPI. (D) Quantification of the TIF response as in (C). Bars: means and SDs from at least 4 experiments of >100 cells each. (E) APBs (arrows) detected by IF-FISH for PML (IF, green) and telomeres (FISH, red). DNA was stained with DAPI. (F) Quantification of the percentage of cells with ≥10 APBs, as assayed in (E). Bars: means and SDs of at least 3 experiments of ≥100 cells each. (G) CO-FISH on metaphase spreads. Telomeres replicated by leading strand DNA synthesis were labeled by PNA-FISH with an Alexa-647-[TTAGGG]3 probe (red) and lagging strand telomeres with a Cy3-[CCCTAA]3 probe (green). Chromosome ends with telomere exchanges are indicated by an “x” and ECTSs are marked by arrows. (H) Quantification of telomere exchanges detected by CO-FISH. Each data point represents the percentage of chromosome ends with telomere exchanges in one metaphase spread. Bars: means and SDs of >60 metaphases from 8 experiments (23 metaphases from 3 experiments for FokIDA-TRF1+Cre). (I) Quantification of ECTSs from metaphase spreads as in (G, H). Each data point represents the number of ECTSs per chromosome in one metaphase spread. (J) Representative G-circle assay using 30 ng gDNA from ALT+ U2OS and ATRXF/F MEFs expressing FokI-TRF1. The amplified products were detected by in-gel hybridization with an end-labeled 32P-[TTAGGG]4 probe. (K) Quantification of G-circle amplification signals from the indicated MEFs, reported relative to ALT+ U2OS (set at 100). Bars: means and SDs from 4 experiments. All p-values were derived from a one-way ANOVA with Tukey correction. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Data. ALT+, alternative lengthening of telomeres-positive; APB, ALT-associated PML body; ATRX, alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler; ATRXF/F, female embryo with two floxed ATRX alleles; CO-FISH, chromosome orientation fluorescence in situ hybridization; Cre, recombinase acting on Lox sites; DA, nuclease dead FokI-TRF1; DSB, double-strand break; ECTS, extrachromosomal telomeric signal; FISH, fluorescence in situ hybridization; FokI-TRF1, FokI nuclease domain and telomeric repeat binding factor 1 fusion protein; gDNA, genomic DNA; IF, immunofluorescence; MEF, mouse embryonic fibroblast; PFGE, pulsed-field gel electrophoresis; PML, promyelocytic leukemia; PNA-FISH, peptide nucleic acid fluorescence in situ hybridization; SD, standard deviation; TIF, telomere dysfunction–induced foci; U2OS, human osteosarcoma cell line; WT, wild-type FokI-TRF1; 53BP1, tumor protein p53-binding protein 1.
Fig 2.
Loss of ATRX causes a telomere-specific cohesion defect.
(A) Immunoblot showing loss of ATRX in conditional KO MEFs treated with Cre (4 days). γtubulin serves as a loading control. Fragments of the same original image were spliced together to remove unnecessary lanes. (B) FISH of the arm (RP23-453P21, red) and subtelomeric (RP23-71E10, green) probes on Chromosome 10 in interphase cells. (C) Quantification of the Chromosome 10 FISH signals observed as doublets. Bars from ATRXF/F MEFs: means and SEMs of 2 experiments in which ATRX deletion was mediated by Hit&Run Cre or pWZL-Cre. Bars from ATRXF/y MEFs: means and SDs of at least 4 experiments. (D) Immunoblots showing shRNA knockdown of SA1 in ATRXF/y MEFs treated with Cre (4 days). Fragments of the same original image were spliced together to remove unnecessary lanes. (E) FISH staining of interphase cells from MEFs described in (D) with probes targeting the arm (red) and subtelomeric (green) regions of Chromosome 10. (F) Quantification of the FISH signals observed as doublets. Bars: means and SEMs from 2 experiments utilizing two distinct shRNAs. (G) FISH of the arm (RP11-442P12, red) and subtelomeric (RP11-326O23, green) probes on human Chromosome 4 in telomerase-positive and ALT cell lines. (H) Quantification of the Chromosome 4 FISH signals observed as doublets. Bars: means and SDs from at least 3 experiments. The p-values were derived by comparing each ALT cell line to the collective results of all telomerase-positive cells (dotted line). Bold text denotes ALT cell lines with ATRX mutations, and gray indicates undetectable ATRX protein by immunoblot. (I) Immunoblot for ectopic expression of ATRX in 2 U2OS clones. Actin serves as a loading control. Fragments of the same original image were spliced together to remove unnecessary lanes. (J) Quantification of the Chromosome 4 FISH signals observed as doublets. Bars: means and SDs of at least 3 experiments collected over approximately 1 month. Pairwise comparisons in panel C were derived from a two-tailed, unpaired t test. All other p-values were derived from a one-way ANOVA with Tukey or Dunnett’s correction. Symbols as in Fig 1. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Data. ALT, alternative lengthening of telomeres; ATRX, alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler; ATRXF/F, female embryo with two floxed ATRX alleles; ATRXF/y, male embryo with a single floxed ATRX allele; Cre, recombinase acting on Lox sites; FISH, fluorescence in situ hybridization; KO, knockout; MEF, mouse embryonic fibroblast; pWZL, retroviral vector; SA1, stromal antigen 1; SD, standard deviation; SEM, standard error of the mean; shRNA, short hairpin RNA; U2OS, human osteosarcoma cell line.
Fig 3.
Telomere cohesion defects promote interactions between nonallelic telomeres.
(A) Representative images of CO-FISH staining on a normal metaphase chromosome, the sister telomere associations frequently observed after deletion of TPP1, and the occasional nonallelic telomere associations observed after TPP1 loss that are suppressed by ATRX- and SA1-mediated telomere cohesion. (B) Immunoblots showing Cre-mediated deletion of ATRX (4 days) and SA1 targeting by CRISPR/Cas9 in the indicated MEFs. pop, an SA1-targeted population of cells; c#, SA1 KO clone. Phosphorylation of MCM2 S108 in TPP1-deficient cells is representative of DNA damage signaling and efficient deletion of TPP1. γtubulin serves as a loading control. (C) Representative images of CO-FISH staining on metaphase spreads (as in Fig 1G) from the indicated cell lines. Telomere associations are highlighted by asterisks (*, sister association; **, nonallelic association). (D, E) Quantification of sister (D) and nonallelic (E) telomere associations in Cre-treated cells detected by CO-FISH. Data points represent the percentage of long arm chromosome ends displaying sister associations and the percentage of all chromatids associated with nonallelic telomeres in one metaphase spread. Bars: means and SDs of >25 metaphases from 3 experiments. All p-values were derived from a one-way ANOVA with Tukey correction. Symbols as in Fig 1. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Data. ATRX, alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler; c#, SA1 KO clone number; Cas9, CRISPR associated protein 9; CO-FISH, chromosome orientation fluorescence in situ hybridization; Cre, recombinase acting on Lox sites; CRISPR, clustered regularly interspaced short palindromic repeats; KO, knockout; MCM2, minichromosome maintenance complex component 2; MEF, mouse embryonic fibroblast; ns, not significant; P-S108, phospho-serine 108; pop, SA1-targeted population; SA1, stromal antigen 1; SD, standard deviation; sgSA1, single guide RNA for SA1; TPP1, ACD shelterin complex subunit and telomerase recruitment factor.
Fig 4.
SA1 deletion exacerbates the TIF response to telomeric DSBs but does not induce ALT phenotypes.
(A) Immunoblots showing expression of FokI-TRF1 (6 days) in control and CRISPR/Cas9-targeted SA1 KO ATRXF/F MEFs infected with Cre (4 days). p1/p2, two separate SA1-targeted populations of cells; c4, SA1 KO clone; Ctrl, nonspecific band used as a loading control. (B) TIFs in SA1 KO ATRXF/F MEFs expressing FokIWT-TRF1 detected by IF-FISH as in Fig 1C. (C) Quantification of the TIF response in FokIWT-TRF1–expressing cells, as assayed in (B). Bars: means and SDs from at least 3 experiments of >100 cells each. (D) APBs in SA1 KO ATRXF/F MEFs expressing FokIWT-TRF1 detected by IF-FISH as in Fig 1E. (E) Quantification of the percentage of cells with ≥10 APBs in MEFs expressing FokIWT-TRF1, as assayed in (D). Bars: means and SDs from at least 3 experiments of >100 cells each. (F) CO-FISH staining of metaphase spreads from SA1 KO c4 ATRXF/F MEFs expressing FokIWT-TRF1 as in Fig 1G. (G) Quantification of telomere exchanges detected by CO-FISH in control and SA1 KO MEFs expressing FokI-TRF1. Each data point represents the percentage of chromosome ends with telomere exchanges in one metaphase spread. Bars: means and SDs of >25 metaphases from at least 3 experiments. All p-values were derived from a one-way ANOVA with Tukey correction. Symbols as in Fig 1. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Data. ALT, alternative lengthening of telomeres; APB, ALT-associated PML body; ATRXF/F, female embryo with two floxed ATRX alleles; Cas9, CRISPR associated protein 9; CO-FISH, chromosome orientation fluorescence in situ hybridization; Cre, recombinase acting on Lox sites; CRISPR, clustered regularly interspaced short palindromic repeats; DA, nuclease dead FokI-TRF1; DSB, double-strand break; FISH, fluorescence in situ hybridization; FokI-TRF1, FokI nuclease domain and telomeric repeat binding factor 1 fusion protein; IF, immunofluorescence; KO, knockout; MEF, mouse embryonic fibroblast; ns, not significant; PML, promyelocytic leukemia; SA1, stromal antigen 1; SD, standard deviation; sgSA1, single guide RNA for SA1; TIF, telomere dysfunction–induced foci; WT, wild-type FokI-TRF1; 53BP1, tumor protein p53-binding protein 1.
Fig 5.
Deletion of DAXX does not alter telomeric DSB repair.
(A) Immunoblots showing FokI-TRF1 expression (6 days) in control and CRISPR/Cas9-targeted DAXX KO ATRXF/y MEFs treated with Cre (4 days). pop, a DAXX-targeted population of cells; c1/c5, DAXX KO clones; Ctrl, nonspecific band used as a loading control. (B) Detection of TIFs in 2 DAXX KO ATRXF/y clones expressing FokIWT-TRF1 by IF-FISH as in Fig 1C. (C) Quantification of the TIF response in FokIWT-TRF1–expressing cells, as assayed in (B). Bars: means and SDs from 3 experiments for DAXX KO MEFs (means and SEMs from 2 experiments for control MEFs) of >100 cells each. (D) APBs in 2 DAXX KO ATRXF/y clones expressing FokIWT-TRF1 detected by IF-FISH as in Fig 1E. (E) Quantification of the percentage of cells with ≥10 APBs in control and DAXX KO MEFs expressing FokIWT-TRF1, as assayed in (D). Bars: means and SDs from 3 experiments of >100 cells each for DAXX KO MEFs (means and SEMs from 2 experiments for control MEFs). (F) CO-FISH staining of metaphase spreads from DAXX KO c5 ATRXF/y MEFs expressing FokI-TRF1, as in Fig 1G. (G) Quantification of telomere exchanges detected by CO-FISH in DAXX KO ATRXF/y MEFs expressing FokI-TRF1. Each data point represents the percentage of chromosome ends with telomere exchanges in one metaphase spread. Bars: means and SDs of >20 metaphases from 2–3 experiments. Pairwise comparisons in panels C and E were derived from a two-tailed, unpaired t test. All other p-values were derived from a one-way ANOVA with Tukey correction. Symbols as in Fig 1. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Data. APB, ALT-associated PML body; ATRXF/y, male embryo with a single floxed ATRX allele; Cas9, CRISPR associated protein 9; CO-FISH, chromosome orientation fluorescence in situ hybridization; Cre, recombinase acting on Lox sites; CRISPR, clustered regularly interspaced short palindromic repeats; DA, nuclease dead FokI-TRF1; DAXX, death domain-associated protein; DSB, double-strand break; FISH, fluorescence in situ hybridization; FokI-TRF1, FokI nuclease domain and telomeric repeat binding factor 1 fusion protein; IF, immunofluorescence; KO, knockout; MEF, mouse embryonic fibroblast; ns, not significant; PML, promyelocytic leukemia; SD, standard deviation; SEM, standard error of the mean; sgDAXX, single guide RNA for DAXX; TIF, telomere dysfunction–induced foci; WT, wild-type FokI-TRF1; 53BP1, tumor protein p53-binding protein 1.
Fig 6.
The combined loss of DAXX and telomere cohesion phenocopies ATRX loss.
(A) Immunoblots showing FokI-ERT2-TRF1 expression (6 days) in control and a CRISPR/Cas9-targeted DAXX-deficient population (pop) of ATRXF/F MEFs. Cre-mediated deletion of ATRX and shRNA knockdown of SA1 and SA2 are also shown (4 days). γtubulin serves as a loading control. (B) Detection of TIFs in DAXX-targeted cells expressing FokIWT-ERT2-TRF1 by IF-FISH for 53BP1 and telomeres as in Fig 1C. (C) Quantification of the TIF response in FokI-ERT2-TRF1–expressing cells, as assayed in (B). Bars: means and SDs from at least 3 experiments of >100 cells each. (D) APBs in DAXX-targeted MEFs expressing FokIWT-ERT2-TRF1 detected by IF-FISH as in Fig 1E. (E) Quantification of the percentage of cells with ≥10 APBs in cells expressing FokI-ERT2-TRF1, as assayed in (D). Bars: means and SDs from at least 3 experiments of >100 cells each. (F) CO-FISH staining of metaphase spreads from DAXX-targeted ATRXF/F MEFs expressing FokIWT-ERT2-TRF1 as in Fig 1G. (G) Quantification of telomere exchanges detected by CO-FISH in control and DAXX-deficient ATRXF/F MEFs expressing FokI-ERT2-TRF1. Each data point represents the percentage of chromosome ends with telomere exchanges in one metaphase spread. Bars: means and SDs of >40 metaphases from 5 experiments (29 metaphases from 3 experiments for sgDAXX pop+Cre). (H) Quantification of the ECTSs from metaphase spreads described in (F, G). Each data point represents the number of ECTSs per chromosome in one metaphase spread. All p-values were derived from a one-way ANOVA with Tukey correction. Symbols as in Fig 1. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Data. APB, ALT-associated PML body; ATRX, alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler; ATRXF/F, female embryo with two floxed ATRX alleles; Cas9, CRISPR associated protein 9; CO-FISH, chromosome orientation fluorescence in situ hybridization; Cre, recombinase acting on Lox sites; CRISPR, clustered regularly interspaced short palindromic repeats; DA, nuclease dead FokI-ERT2-TRF1; DAXX, death domain-associated protein; ECTS, extrachromosomal telomeric signal; FISH, fluorescence in situ hybridization; FokI-ERT2-TRF1, tamoxifen-inducible nuclear localized FokI-TRF1 fusion protein; IF, immunofluorescence; MEF, mouse embryonic fibroblast; ns, not significant; PML, promyelocytic leukemia; SA1, stromal antigen 1; SA2, stromal antigen 2; SD, standard deviation; sgDAXX, single guide RNA for DAXX; shRNA, short hairpin RNA; shSA1, short hairpin RNA for SA1; shSA2, short hairpin RNA for SA2; TIF, telomere dysfunction–induced foci; WT, wild-type FokI-ERT2-TRF1; 53BP1, tumor protein p53-binding protein 1.
Fig 7.
Model for how loss of ATRX promotes ALT.
(A) Proposed role for ATRX in repressing ALT through telomere cohesion and a DAXX-mediated activity. (B) Repair of telomeric DSBs in cells with intact ATRX and DAXX. Telomere cohesion is established during replication in an SA1-dependent manner and at a DSB by SA2-dependent cohesion. The close proximity and proper alignment of the chromatids by cohesin favors use of the sister telomere for DSB repair and ensures that repair results in equal sister telomere exchange or BIR using the sister telomere in-register, thus providing no mechanism for telomere extension. (C) ATRX loss changes telomeric DSB repair and promotes ALT through two separate pathways. First, the disruption of ATRX-mediated telomere cohesion allows unequal sister telomere recombination and the use of nonallelic telomeres for DSB repair. These types of interactions are necessary for telomere elongation and are consistent with the recombination events observed in ALT cells. Second, disruption of a DAXX-dependent function of ATRX is proposed to change the repair outcomes of telomeric DSBs, promoting BIR over other DSB repair pathways. ALT, alternative lengthening of telomeres; ATRX, alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler; BIR, break-induced replication; DAXX, death domain-associated protein; DSB, double-strand break; HDR, homology-directed repair; SA1, stromal antigen 1; SA2, stromal antigen 2.