Figure 4. H3K36me2 recruits HP1α and promotes deposition of H3K9me3 at DSB locus in SIRT6-dependent manner. (A) SIRT6, but not KDM2A, is required for deposition of H3K9me3 at DSB locus. ChIP assay was performed 12 h post transfection with I-SceI vector using antibody against H3K9me3. (B) SIRT6, but not SUV39h1, is required for accumulation of H3K36me2 at DSB locus. Western blot shows depletion of SUV39h1. (C) HP1α binds H3K36me2 in vivo. Human skin fibroblast cells were irradiated with 3 Gy of γ-irradiation. Cell lysate was immunoprecipitated within 10 minutes after irradiation with antibody against HP1α and blotted against H3K36me2 (Top panel), HP1α (middle) and β-tubulin (lower panel). (D) HP1α binds H3K36me2 in vitro. Mono nucleosomes carrying either H3K36me2, H3K27me3, H3K9me3 or H3K9Ac groups were incubated with HP1α and immunoprecipitated with antibody against HP1α and blotted with H3 antibodies. (E) SIRT6, but not SUV39h1, is required for HP1α recruitment to DSB locus. (F) HP1α interacts with 53BP1 after γ-irradiation. Proximity ligation assay (PLA) was performed using antibodies against HP1α and 53BP1 and the PLA signal was detected within 10 minutes after irradiation. NS; not significant. All experiments were repeated three times. *p < 0.05; **p <0.01.