Figure 1. Tip60’s chromodomain is required for acetyltransferase activity.

(a) HeLaS3 cells expressing HA-Tip60 or HA-Tip60 with the indicated mutations were exposed to bleomycin (5μM for 40 minutes) as indicated. Tip60 was immunopurified with HA antibody or IgG, and the associated acetyltransferase activity measured as described in methods. Each data point represents the average of 3 independent immunoprecipitation assays, results ± SD. (b) HeLaS3 cells expressing vector, Tip60 or the indicated Tip60 chromodomain mutants were untreated (−) or irradiated (5Gy: +), and ATM immunoprecipitated. Western blot analysis to detect ATM, phospho-ATM (pS1981) or Tip60-dependent acetylation of ATM (AcLys) was then carried out. (c) HeLaS3 cells expressing either HA-Tip60 or HA-Tip60^CD (Tip60^Y47A) were untreated (0Gy) or irradiated (5Gy). Cells were then fixed and immunofluorescent (IF) staining with antibodies to detect γH2AX or HA-Tip60 carried out. Scale bar equals 10μM. (d) HCT116 cells expressing either a non-targeting shRNA (shCon) or shRNA targeting Rad50 (shRad50) were transfected with HA-Tip60. Cells were either unirradiated (No IR) or exposed to 0.5Gy. Cells were fixed 15 or 40 minutes post-irradiation and immunofluorescent staining with antibodies to detect HA-Tip60 carried out. Scale bar equals 10μM. (e) Quantification of the images in (e). The number of cells with Tip60 foci (defined as cells with > 5 foci) was measured visually. Results ± SD, based on analyzing at least 80 cells per slide.