Abstract
TIP60 (HTATIP) is a histone acetyltransferase (HAT) whose function is critical in regulating ataxia-telangiectasia mutated (ATM) activation, gene expression, and chromatin acetylation in DNA repair. Here we show that under non-stressed conditions, activating transcription factor-2 (ATF2) in cooperation with Cul3 ubiquitin ligase promotes degradation of TIP60, thereby attenuating its HAT activity. Inhibiting either ATF2 or Cul3 expression by small interfering RNA stabilizes the TIP60 protein. ATF2 association with TIP60 on chromatin is decreased following exposure to ionizing radiation (IR), resulting in enhanced TIP60 stability and activity. We also identified a panel of melanoma and prostate cancer cell lines whose ATF2 expression is inversely correlated with TIP60 levels and ATM activation after IR. Inhibition of ATF2 expression in these lines restored TIP60 protein levels and both basal and IR-induced levels of ATM activity. Our study provides novel insight into regulation of ATM activation by ATF2-dependent control of TIP60 stability and activity.
Highlights
The nuclear protein kinase ataxia-telangiectasia mutated (ATM)2 belongs to the phosphatidylinositol 3-kinase-like kinase family [1], which is activated in response to genotoxic insults that elicit double-stranded DNA breaks (DSBs) [2]
The amount of activating transcription factor-2 (ATF2) bound to TIP60 significantly decreased following ionizing radiation (IR) (Fig. 1c), TIP60 levels remained unchanged (Fig. 1d). These findings suggest that ATF2 is part of the TIP60 complex and that association of the two proteins is reduced following IR-induced DNA damage
To map TIP60 domains required for association with ATF2, we generated three TIP60 fragments (Fig. 2a) predicted to retain their original conformation based on the crystal structure of the yeast TIP60 homologue ESA1 [30]
Summary
Cells—HeLa and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with calf serum (10%) and antibiotics. WM115, WM793, FEMX, HHMSX, WIDR, A549, PC3, LNcaP, ALav, and M12 cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and antibiotics. Plasmids and Transfection—Plasmids expressing both wild type and mutant forms of HA-ATF2 were described previously [8]. PCR-amplified TIP60 cDNA [17] was subcloned into the BamHI and NotI sites of the PEF-FLAG vector. TIP60 deletion mutants were generated by PCR and subcloned into PEF-FLAG vector. PcDNA3 Myc-tagged Cullin and pcDNA3 HA-ROC1 were. ATF2 Regulates TIP60 and ATM Activity a
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