Fig. 3: MMSET promotes NHEJ of uncapped telomeres through H3K36 dimethylation.
From: H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres
a Immunoblot of histone marks in TRF2ts MEFs transduced as indicated and kept at 39 °C for indicated times to induce telomere uncapping. γtubulin serves as loading control. b Immunoblot for H3K36me2 in TRF2ts MEFs transduced as indicated and used in Fig. 1e and Supplementary Fig. 1c, in the absence of telomere damage (32 °C). c Immunoblot of p53−/− MEFs transduced as indicated, untreated or 20 min post IR (10 Gy). γtubulin serves as loading control. d Relative enrichment of H3K36me2/H3 for the indicated target regions in control or shMmset transduced TRF2ts MEFs cultured for 3 h at 39 °C, determined by ChIP for H3 and H3K36me2 and qRT-PCR. QRT-PCRs of individual experiments were performed in technical triplicates and normalized to input DNA. H3K36me2 ChIP data was then normalized for total H3 (n = 3 independent ChIP experiments, mean ± SEM, unpaired t test: ns, not significant; *p ≤ 0.05; **p ≤ 0.01). e Schematic representation of different MMSET cDNA constructs used. H1143G, SET-domain mutant; PHD, plant homeodomain; PWWP, Pro-Trp-Trp-Pro motif; HMG, high mobility-group motif; MMSETRR, RNAi-resistant (RR) MMSET. f Survival assay of TRF2ts MEFs transduced with indicated shRNA and cDNA constructs and cultured as specified (representative of n = 2 independent experiments). g Immunoblot showing expression of the different MMSET cDNA constructs. The upper blot, probed with MMSET antibody, shows both full-length MMSET isoform II containing the catalytic domain and the smaller MMSET isoform I that lacks catalytic activity. Both isoforms are targeted by the Mmset shRNA used, while complementation with RNAi-resistant full-length MMSET cDNA (wild-type or H1143G mutant) only restores full-length MMSET isoform II expression. The PHD, PWWP and HMG truncated MMSET variants lose the MMSET antibody epitope and are detected by a Flag-antibody in de lower blot. h Immunoblot for H3K36me2 in histone extracts of TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 24 h at 39 °C. H3 serves as loading control (representative of 2 independent experiments).
