Functional Analysis of the Tip60 Complex

描述(申请人提供)：癌症发生在致癌过程中，细胞暴露在遗传毒性的侮辱下会导致永久性的基因变化。细胞检测这种DNA损伤并激活适当的修复机制的能力对于细胞抑制致癌事件至关重要。Tip60组蛋白乙酰转移酶是细胞检测和修复这些DNA损伤能力的关键介质。Tip60是NuA4-Tip60染色质重塑复合体的关键成分。染色质重塑被用来解开更高有序的染色质结构，改变组蛋白-DNA相互作用，以促进DNA修复机制对DNA损伤的访问。我们的结果表明，NuA4-Tip60复合体对染色质的重塑需要Tip60‘S HAT活性。此外，Tip60含有一个染色域，这是一个可能与组蛋白上的甲基化赖氨酸残基相互作用的保守结构域，该结构域是激活Tip60‘S HAT活性和NuA4依赖的染色质重塑所必需的。长期目标是验证Tip60是一个新的信号转导途径的关键组成部分的假设，该信号转导途径将DNA损伤的检测与染色质结构的变化联系起来。我们将检验这一假设，即NuA4的催化成分，包括P400 ATPase活性和Tip60‘S HAT活性，是DNA损伤后染色质重塑所必需的。此外，我们认为Tip60的染色域与DNA损伤部位的组蛋白上暴露的甲基赖氨酸残基之间的相互作用介导了Tip60‘S HAT活性的激活。Tip60以三聚体形式存在，包含Tip60、epc1和ING3蛋白。在具体目标1中，将确定ING3调节蛋白在控制Tip60‘S HAT活性中的作用，并将确定NuA4被招募到DNA断裂的机制。在特定的目标2中，将确定NuA4的P400亚单位的ATPase活性在染色质解离中的作用，并确定Tip60在DNA损伤部位对组蛋白乙酰化的贡献。在具体目标3中，将确定Tip60的色域与组蛋白上的甲基化赖氨酸残基相互作用的特异性，并研究组蛋白甲基化在Tip60 DNA损伤依赖的激活中的作用。了解Tip60检测博莱霉素和相关药物引起的DNA链断裂的信号转导途径将为了解这些药物的细胞毒性和诱变特性提供重要的见解。对Tip60如何调节DNA损伤反应的更全面的了解可能会使新的治疗方法，包括合理设计新的治疗化合物，应用于癌症治疗和临床有益的干预措施，以降低遗传毒性应激的风险。最后，Tip60及其相关蛋白是开发可改变肿瘤细胞DNA损伤反应的治疗剂的潜在靶点。与公共健康相关：接触致癌物质，包括紫外线、烟草烟雾、重金属和其他遗传毒性物质等环境物质，是许多癌症的根本原因。因此，了解Tip60如何调节这种DNA损伤的修复对于改善公众健康是相关的，并将允许合理设计可应用于癌症治疗的新的治疗化合物，并对与接触致癌物相关的癌症风险进行临床有益的干预。此外，Tip60是开发治疗药物的潜在靶点，可以改变肿瘤细胞的DNA损伤反应，并使其对抗癌治疗敏感。

DESCRIPTION (provided by applicant): Cancer arises during the process of carcinogenesis, in which the exposure of cells to genotoxic insults leads to the introduction of permanent genetic changes. The ability of cells to detect this DNA damage and to activate appropriate repair mechanisms is crucial for the cell to suppress carcinogenic events. The Tip60 histone acetyltransferase is a key mediator of the cells ability to detect and repair these DNA lesions. Tip60 is a key component of the NuA4-Tip60 chromatin remodeling complex. Chromatin remodeling is utilized to unpack higher ordered chromatin structures and alter histone-DNA interactions to facilitate access of the DNA repair machinery to DNA lesions. Our results demonstrate that chromatin remodeling by the NuA4-Tip60 complex requires Tip60's HAT activity. Further, Tip60 contains a chromodomain, a conserved domain with the potential to interact with methylated lysine residues on histones, and this domain is required for the activation of Tip60's HAT activity and NuA4-dependent chromatin remodeling. The long term aims are to test the hypothesis that Tip60 is a key component of a novel signal transduction pathway which links the detection of DNA damage to alterations in chromatin structure. We will test the hypothesis that the catalytic components of NuA4, including the p400 ATPase activity and Tip60's HAT activity, are required for chromatin remodeling following DNA damage. Further, we propose that interactions between the chromodomain of Tip60 and exposed methyl-lysine residues on histones at sites of DNA damage mediate this activation of Tip60's HAT activity. Tip60 exists as a trimeric complex containing the Tip60, epc1 and ING3 proteins. In specific aim 1, the role of the ING3 regulatory protein in controlling Tip60's HAT activity will be defined, and the mechanism by which NuA4 is recruited to DNA breaks will be determined. In specific aim 2, the function of the ATPase activity of the p400 sub- unit of NuA4 in chromatin unwinding will be determined, and the contribution of histone acetylation by Tip60 at sites of DNA damage identified. In specific aim 3, the specificity of interaction between the chromodomain of Tip60 and methylated lysine residues on histones will be determined, and the role of histone methylation in the DNA-damage dependent activation of Tip60 examined. An understanding of the signal transduction pathway by which Tip60 detects DNA strand breaks caused by bleomycin and related agents will provide crucial insights into the cytotoxicity and mutagenic properties of these agents. A more complete understanding of how Tip60 regulates the DNA damage response may allow new therapies, including the rational design of novel therapeutic compounds, to be applied to cancer therapy and to clinically beneficial interventions for reducing the risk from genotoxic stress. Finally, Tip60 and its associated proteins are potential targets for developing therapeutic agents which can modify the DNA damage response of tumor cells. PUBLIC HEALTH RELEVANCE: Exposure to carcinogens, including environmental agents such as UV light, tobacco smoke, heavy metals and other genotoxic agents, are the underlying cause of many cancers. An understanding of how Tip60 regulates the repair of this DNA damage is therefore relevant to improving public health, and will allow for the rational design of novel therapeutic compounds which could be applied to cancer therapy, and for clinically beneficial interventions to the cancer risk associated with exposure to carcinogens. In addition, Tip60 is a potential target for developing therapeutic agents which can modify the DNA damage response of tumor cells and sensitize them to anti-cancer therapies.