ATRX affects the repair of telomeric DSBs by promoting cohesion and a DAXX-dependent activity
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.