The Regulation And Mechanism Of Tip60 On Cellular Response To DNA Damage

Tip60 was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat. It is one of the members of MYST family. The MYST family, which has the activity of acetyltransferase, can transfer acetyl group to the Lys residue of histones, through which it changes the conformation of chromatin. Tip60 can also create a mode of multiple-site modifications by combining with other forms of post-transcriptional modifications, which makes a lot of proteins, including transcription factors, much easier recognized and accessible.Tip60 has been broadly investigated in many facets such as signal transduction, DNA damage repair, cell cycle checkpoint, apoptosis, etc. Tip60 has dual roles on transcription regulation: transcription activation through its activity of acetyltranferase, and transcription repressing by recruiting histone deaceytyltransferase to the promoter of target genes. The data reveals that Tip60 interacts with TEL through its MYST domain, and displays as a co-repressor of TEL. Tip60 catalyzes the acetylation of androgen receptor(AR) and enhances transactivation of AR.Similarly Tip60 is critical for DNA damage repair. The evidence shows that the ability of DNA damage repair is abolished when Q377 and G380 sites, which locates in the domain of Tip60 interacting with the acetyl coenzyme A, are mutated. Tip60 causes acetylation ofγH2AX which is necessary for the ubiquitination ofγH2AX by UBC13 as well as in charge ofγH2AX exchanging with H2AX. The HAT activity of Tip60 is activated by interacting with H3K9me3. Tip60 is essential for the function of p53, and the proliferation inhibition mediated by p53 can be attenuated by down-regulation of Tip60. Tip60 acetylates p53 and regulates its transactivation activity on downstream target genes. There are two manners of Tip60 involving in DNA damage repair. On one hand, Tip60 acetylates K3016 of ATM, which in turn activates the phosphorylation of S1981 on ATM. ATM is then activated and phosphorylates its downstream factors related to DNA damage responses, such as p53, BRCA1 andγH2AX. On the other hand, NuA4-Tip60 complex mediates the chromatin remodeling in responding to DNA damage. The complex acetylates H4 and H2AX surrounding DNA double-strand breaks (DSB) sites, which attenuates the interaction between histones and relaxes and opens the condensed chromatin.More evidence shows that Tip60 is indispensable for DNA damage repair. We employed the cellular model of expressing exogenous Tip60 gene or its specific shRNA in a human osteogenic sarcoma cell line U2OS, to investigate the effect of Tip60 protein on cellular radiosensitivity and its related mechanisms, the main results were summarized as below:1. Overexpressing Tip60 in U2OS cells by transfecting vectors expressing exogenous Tip60 decreased the proliferation activity as compared with control cells;2. siRNA-mediated knock-down of Tip60 significantly increased the sensitivity of U2OS-Tip60si cells toγirradiation as compared with the control U2OS-Laczi cells. The comet assay, pulsed-field gel electrophoresis (PFGE) andγ-H2AX foci detection revealed a decreased capacity of repairing radiation-induced DNA DSB in Tip60-knockdown cells;3. In contrast, overexpressing Tip60 in U2OS cells by transfecting vectors expressing exogenous Tip60 increased the radio-resistance and repair capability of DNA DSBs;4. Tip60 can interact with DNA-PKcs but not another component Ku70 of DNA-PK complex in response to radiation-induced DNA damage;5. Anacardic acid, an inhibitor of Tip60 acetyltransferase activity, inhibits the phosphorylation of DNA-PKcs on T2609 site;6. Tip60-overexpressing cells exhibited a noticeable delay on the elimination of radiation-induced G2/M arrest as compared with control cells. The alteration of cyclin B1 expression was detected in Tip60-overexpressing cells and control cells. A decreased constitutive expression level of cyclinB1 was observed in Tip60-overexpressing cells. Moreover, the level of Cdk1 was also found to be downregulated in Tip60 overespressing cells.7. The involvement of Tip60 in a large-scale chromatin remodeling has been demonstrated under the situation with or withoutγ-ray irradiation. Chromatin relaxation was shown afterγirradiation. We also find that the point-mutated separately on S86A, S90A or Q377E/G380E sites does not significantly attenuate the remodeling activity of Tip60, but on S90D and S86D/S90D sites could inhibit its function on the chromatin remodeling.