Fig. 6
JMJD6 modulates the H4K16ac level around DSBs. a Depletion of JMJD6 increases H4K16ac in U2OS cells after ionizing radiation. U2OS cells transfected with control or JMJD6 siRNAs were untreated or treated with 10 Gy of IR, and 1 h later, the cell lysates were extracted and subjected to immunoblot analysis using indicated antibodies. b JMJD6 is required for the recruitment of SIRT1 to chromatin after IR treatment. U2OS cells transfected with control or JMJD6 specific siRNAs were treated with 10 Gy of IR, and 1 h later, the nuclear-soluble fraction (N) and chromatin-bound proteins (P) of U2OS cells were extracted and subjected to western blot analysis using antibodies against the indicated proteins. c Knockdown of JMJD6 increases digestion by micrococcal nuclease in response to IR treatment. Control or JMJD6-depleted U2OS cells were treated with IR or not. The nucleosomes were digested by micrococcal nuclease and subjected to DNA gel electrophoresis. d JMJD6 overexpression leads to increased recruitment of SIRT1 and decreased level of H4K16ac around the DSB. U2OS-DR-GFP cells were transfected with indicated expression constructs. ChIP assays were performed using IgG, anti-SIRT1 or anti-H4K16ac, and the final DNA exactions were amplified by quantitative real-time PCR using primers that cover the DNA sequences around the I-SceI site. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t-test. **p < 0.01, *p < 0.05. e JMJD6 knockdown results in decreased recruitment of SIRT1 and increased level of H4K16ac around the DSB. U2OS-DR-GFP cells stably expressing control or JMJD6 shRNAs were transfected with empty vector or HA-I-SceI expression constructs. ChIP assays were performed using indicated antibodies. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t-test. *p < 0.05
