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Sirt1 deacetylates auto-acetylated Tip60
To investigate the mechanism of how Sirt1 represses acetylation of H2AX, we examined the effects of γIR on the physical interaction between Tip60 and Sirt1 (Fig. S5A). Unexpectedly, the amount of Sirt1-myc co-immunoprecipitated with Flag-Tip60 was unaffected by γIR. However, γIR strongly affected the level of acetylated Tip60, which is auto-acetylated or is acetylated by p300/CBP [20] and [21]; that BIBF 1120 is, the level of acetylated Flag-Tip60 was decreased gradually after approximately 2 h post-γIR and returned to the basal level within 4 h (Fig. S5A).
Additionally, we co-transfected Flag-Tip60 with either Sirt1-myc or H363Y Sirt1-myc and treated each cell culture with 5 Gy γ-ray (Fig. 3A). In the control experiment, acetylated Flag-Tip60 decreased within 2 h after γIR. Co-expression of Sirt1-myc accelerated the decrease of acetylated Flag-Tip60, whereas co-expression of H363Y Sirt1-myc delayed it. Furthermore, depletion of Sirt1 by shSirt1 increased acetylated Flag-Tip60 and recombinant GST-Sirt1-myc effectively deacetylated acetylated Flag-Tip60, confirming secondary compounds Sirt1 is required for the deacetylation of Tip60 (Fig. S5B and C).