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Overexpression of Sirt1 results in loss of cellular viability following γIR, which can be rescued by Tip60
Finally, to analyze the association of Sirt1 and Tip60 on cellular survival after γIR, HeLa SB207499 were stably transfected with HA-Tip60 or HA-Tip60-ad and either Sirt1-myc or H363Y Sirt1-myc as shown in Fig. 4B. Expression of HA-Tip60-ad sensitized cells to γIR, indicating that Tip60 activity was required for cellular survival after γIR. Moreover, overexpression of Sirt1-myc reduced the resistance of cells to γIR, indicating that an excess of Sirt1 had deleterious effects on DNA-damaged cells. The effect could be partially rescued by overexpression of HA-Tip60. These results are consistent with the hypothesis that Sitr1 negatively regulates DDR by repressing the activity of Tip60.
Discussion
We showed that Sirt1 physically interacts with Tip60 and monomer Sirt1 deacetylates auto-acetylated Tip60 and stimulates the proteasome-dependent degradation of Tip60. Although Tip60 plays an important role in DDR through acetylating H2AX on Lys5, Sirt1 represses acetylation of H2AX on Lys 5, which is acetylated by Tip60 in response to DNA damage. Furthermore, we showed that Tip60 and Tip60-mediated acetylation of H2AX are required for effective MDC1, BRCA1 and Rad51 foci formation. Overexpression of Sirt1 resulted in repressed H2AX acetylation. In addition, depletion of Sirt1 resulted in accelerated H2AX acetylation, leading to increased foci formation of MDC1, BRCA1 and Rad51. These findings strongly suggested that Sirt1 negatively regulates the formation of these foci after γIR, probably via negatively regulating Tip60-mediated H2AX acetylation on Lys 5.