Fig 2: SMYD3 regulates the expression of MDC1 through methylating histone H3K4. (a) ChIP assay was performed in MCF7 cells using specific antibodies. The examined positions at the MDC1 locus were indicated, in which region S3 and region TA for the predicted SMYD3 and TATA box binding sites, respectively. (b–e) ChIP assays were performed with SMYD3-repressed MCF7 cells using specific antibodies shown at the top of the histogram. Fold enrichment of each antibody compared with IgG was shown. (f) Ratios of H3K4me3/H3 ChIP signals were displayed. In (b–f), immunoprecipitated chromatin was quantified by qRT-PCR. *P < 0.05. **P < 0.01. All values in the histograms were means ± SD of triplicates and data were representative of n ≥ 3 for each experiment.

Fig 3: dsRNA-dependent recruitment of 53BP1 and MDC1 to DSBs. Imaging of CTD Y1P, 53BP1 and MDC1 without permeabilization, upon permeabilization and incubation with bovine serum albumin (BSA, control) or structure-specific RNases prior to fixation, or upon add-back with total RNA purified from AsiSI-ER U2OS cells in presence (+) or absence (–) of 4OHT. Representative images are shown (top). Quantification of cells with foci staining double-positive for CTD Y1P and 53BP1 (bottom). The experiment was done in triplicates. 60 cells were counted per replicate. Cells were scored based on average number n < 10 of CTD Y1P/53BP1 double-positive foci (yellow) per cell. Asterisk, P-value < 0.05, one-tailed t-test. Error bar: mean ± SEM, n = 3.

Fig 4: DNA–RNA hybrid-dependent DSB foci formation and onset of DSB signalling. (A) Confocal imaging of MDC1 and GFP-RNaseH1 following transient transfection of pEGFP-M27 or non-transfected (mock) cells. n, number of cells with shown phenotype in %. (B) ChIP analysis of HA-53BP1 occupancy at DS1 using HA antibody (3F10) and site-specific primers. Asterisk, P-value < 0.05, two-tailed t-test. Error bar: mean ± SEM, n = 3. (C) Immunoblots detecting phospho-ATM, total ATM, ?H2A.X, phospho-Chk1 and p53 levels following fluorescence-activated cell sorting (FACS) in absence of 4OHT (left) and after pulse-chase 4OHT incubation kinetics (right). Ponceau S, control.