Fig. 2: MMSET induces NHEJ-mediated telomere fusion and G-overhang degradation.
From: H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres
a Chromosome fusions in TRF2ts MEFs and LigIV−/− TRF2ts MEFs transduced with control or Mmset-targeting shRNA (n = 2–3 independent experiments, mean ± s.d., unpaired t test: ns, not significant; *p ≤ 0.05). b Representative metaphase spreads of TRF2ts MEFs transduced as indicated, collected after 24 h at 39 °C for telomere-FISH. Scale bar, 10 μm. c Immunoblot of TRF2ts MEFs or LigIV−/− TRF2ts MEFs used in (a), γtubulin serves as loading control. d Immunoblot of MMSET and H3K36me2 levels in MEFs used in (e). e Chromosomal fusions in wild-type (WT) MEFs transduced with control or Mmset-targeting shRNA prior to treatment with control or Trf2-targeting shRNA for 5 days to induce telomere uncapping. Control transduced 53bp1−/− and Rif1−/− MEFs were included for comparison. Values were normalized for the control condition (n = 2 independent experiments, mean ± s.d., unpaired t test: **p ≤ 0.01). f Telomeric G-overhang quantification (n = 2 independent experiments, mean ± s.d., unpaired t test: *p ≤ 0.05). g Representative telomeric G-overhang assay in control and Mmset knockdown TRF2ts MEFs after 48 h at the nonpermissive temperature (37 °C). h Chromosome fusions in TRF1F/FTRF2F/FKu70−/−p53−/− MEFs treated with DMSO or PARPi (Olaparib, 0.5 µM), or transduced with control virus or shRNA targeting Mmset. Cells were treated with tamoxifen (4-OHT) for 24 h to induce deletion of floxed shelterin alleles and harvested after a total of 4 days. A minimum of 1500 chromosomes (−4OHT) or 2000 chromosomes (+4OHT) was counted per condition per individual experiment. Genotypes were blinded before manual scoring (n = 2 independent experiments, mean ± s.d., unpaired t test: ns, not significant; **p ≤ 0.01).
