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. 2020 Dec 1;9:e61235. doi: 10.7554/eLife.61235

Figure 5. Heterozygous TINF2 mutations induce telomere lengthening.

(A) Telomeric Southern blot of MboI/AluI-digested genomic DNA from immortalized and primary patient cells (lymphocytes), normal lung fibroblasts (MRC5), and HeLa1.3 cells. Median telomere length (MTL) is indicated. (B) Telomeric Southern blot of MboI/AluI-digested genomic DNA from control clones and clones with heterozygous c.604G > C mutations at the indicated PDs. Telomere length changes are indicated and were calculated over 48 PDs. (C) Quantification of median telomere length changes for control cells and cells with heterozygous c.604G > C mutations. Three cell lines per genotype were analyzed in two independent experiments (symbols denote the individual cell lines). (D) Telomeric Southern blot as in (B) for control clones and clones with heterozygous c.557del mutations. (E) Quantification of median telomere length changes for control cells and cells with heterozygous c.557del mutations as in (C). (F) Detection of telomeres in MboI/AluI-digested genomic DNA from control clones and clones with heterozygous c.557del mutation probed under native conditions with a telomeric probe for the 3’ overhang. The change in MTL over 28 PDs is indicated. (G) Telomeric southern blot as in (B) for control cells and TIN2+/- cells. The indicated telomere length changes were calculated over 42 PDs. (H) Quantification of median telomere length changes for control cells and TIN2+/- cells as in C. (I) Telomeric southern blot of MboI/AluI-digested genomic DNA from control and TIN2+/- hESCs. All clones were generated and propagated in parallel and telomere length was determined at 28 days after the CRISPR/Cas9 targeting. (J) Quantification of the median telomere length (as determined by blotting as in (I)) for control and heterozygous hESCs clones (control, n = 5; TIN2+/KO, n = 4). Bar graphs in (C), (E), (H), and (J) show means ± SDs. P-values are based on unpaired t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. ns, not significant. See also Figure 5—figure supplements 1–3.

Figure 5.

Figure 5—figure supplement 1. Long telomeres in TINF2 c.604G > C and c.557del patients and hESC CRISPR/Cas9 editing.

Figure 5—figure supplement 1.

(A) Median telomere length (MTL) in individuals with TINF2 truncations as measured by Flow FISH. (B) Schematic of the TINF2 locus showing landmarks relevant to CRISPR/Cas9 mediated targeting of the allele in hESCs. The single guide RNA (sgRNA) target regions and the primers used for genotyping (blue arrows) are indicated. (C) PCR genotyping of hESC clones showing the longer wild-type and shorter edited allele.
Figure 5—figure supplement 2. No evidence for increased telomere recombination in c.604G > C mutant and Tin2+/- cells.

Figure 5—figure supplement 2.

Quantification of telomeric sister chromatid exchanges (T-SCE) in RPE1 control cells, cells with heterozygous c.604G > C, or TIN2+/- cells (≥15 spreads per cell line, and two cell lines per condition, means ± SD and individual data points are shown). Unpaired t-test was was used to determine significance, ns, not significant.
Figure 5—figure supplement 3. Mutant and control RPE1 clones show similar telomerase activity.

Figure 5—figure supplement 3.

(A–C) TRAP assay using extracts from the indicated c.604G > C (A), control and c.557del (B), or TIN2+/- (C) cells together with the respective control cell lines. Serially diluted extracts (500, 100, 25 cells) were subjected to the TRAP assay. H, heat-treated extract from 500 cells; TSR8, positive control. Mutant and wild-type controls were harvested and analyzed in parallel and the TRAP products were analyzed on two gels (left and right half of each panel).