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. 2020 Jan 2;18(1):e3000594. doi: 10.1371/journal.pbio.3000594

Fig 3. Telomere cohesion defects promote interactions between nonallelic telomeres.

Fig 3

(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.