Function of RVB1-Tip60 in the DNA damage response

描述(申请人提供)：人类细胞对DNA损伤反应的两个重要分子特征是组蛋白变异体H2 AX的磷酸化和转录程序的变化。我们实验室最近的结果表明，RVB1蛋白参与了细胞对DNA损伤的反应。人RVB是染色质重塑复合体的组成部分，该复合体含有组蛋白乙酰转移酶、Tip60、ATPase P400和其他几种细胞蛋白。RVB1或Tip60的缺失会导致DNA损伤后磷酸化H2AX的积累增加，并使癌细胞对DNA损伤剂敏感，这表明RVB ATPase抑制剂在化疗或放射治疗中可能是有用的。本项目将检验两个假设：1)RVB1需要中和P400对Tip60复合体的乙酰转移酶活性的抑制作用；2)Tip60的乙酰转移酶活性需要在DNA被细胞磷酸酶去磷酸化之前将含有核小体的磷酸H_2AX从DNA上动员出来。目的1探讨Tip60是否是下调磷酸H_2AX的主要染色质重构体，以及RVB2是否与RVB1在这一功能上合作。目的2验证RVB1和P400调控Tip60的假说是否适用于Tip60控制的启动子染色质重塑。目标3将测试与自身或与RVB2的ATP结合/水解和寡聚的作用。体外试验将被开发来探索RVB1如何激活Tip60乙酰转移酶复合体以及Tip60复合体如何在磷H_2AX去磷酸化之前动员含磷的核小体的分子基础。公共卫生相关性细胞对DNA损伤的反应既决定了环境DNA损伤剂如何导致突变和导致癌症，也决定了治疗性DNA损伤剂如何治疗癌症。RVB调控的染色质重构体既作用于DNA损伤部位，又作用于启动子，控制细胞的反应。因此，对RVB如何调节染色质重塑因子的分子理解对DNA损伤的最佳反应至关重要，这将有助于设计干扰RVB功能的化学物质，从而使细胞对化疗或放射治疗敏感。

DESCRIPTION (provided by applicant): Two important molecular signatures of a human cell's response to DNA damage are the phosphorylation of a histone variant H2AX and a change in the transcriptional program. Recent results from our laboratory have implicated the RVB1 protein in the cellular response to DNA damage. Human RVB is an integral part of a chromatin remodeling complex containing the histone acetyltransferase, TIP60, the ATPase p400 and several other cellular proteins. RVB1 or TIP60 depletion leads to an increase in phosphoH2AX accumulation after DNA damage and sensitizes cancer cells to DNA damaging agents, suggesting that inhibitors of RVB ATPase may be useful in chemo- or radiotherapy. This project will test two hypotheses: 1) RVB1 is required to neutralize the inhibitory action of p400 on the acetyltransferase activity of the TIP60 complex and 2) the acetyltransferase activity of TIP60 is required to mobilize phosphoH2AX containing nucleosomes off the DNA prior to its dephosphorylation by cellular phosphatases. Aim 1 will explore whether TIP60 is the primary chromatin remodeler involved in down modulating phosphoH2AX and whether RVB2 co-operates with RVB1 in this function. Aim 2 will test whether the hypothesis of TIP60 regulation by RVB1 and p400 is applicable to chromatin remodeling at promoters controlled by TIP60. Aim 3 will test the role of ATP binding/hydrolysis and oligomerization with self or with RVB2. In vitro assays will be developed to explore the molecular basis of how RVB1 activates the TIP60 acetyltransferase complex and how the TIP60 complex mobilizes phosphoH2AX-containing nucleosomes prior to dephosphorylation of phosphoH2AX. PUBLIC HEALTH RELEVANCE The cellular response to DNA damage determines both how environmental DNA damaging agents lead to mutations and cause cancers and how therapeutic DNA damaging agents treat cancers. Chromatin remodelers regulated by RVB act both at sites of DNA damage and at promoters to control the cell's response. Therefore a molecular understanding of how RVB regulates chromatin remodeling factors important for the optimal response to DNA damage will allow the design of chemicals that interfere with RVB function and thus sensitize cells to chemo- or radio-therapy.