Function of RVB1-Tip60 in the DNA damage response

描述（由申请人提供）：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，RVB 1蛋白参与了细胞对DNA损伤的反应。人RVB是含有组蛋白乙酰转移酶、TIP 60、ATP酶p400和几种其他细胞蛋白的染色质重塑复合物的组成部分。RVB 1或TIP 60耗竭导致DNA损伤后磷酸H2 AX积累增加，并使癌细胞对DNA损伤剂敏感，表明RVB ATP酶抑制剂可用于化疗或放疗。该项目将测试两个假设：1）需要RVB 1来中和p400对TIP 60复合物的乙酰转移酶活性的抑制作用，以及2）需要TIP 60的乙酰转移酶活性来在DNA被细胞磷酸酶去磷酸化之前将含有磷酸H2 AX的核小体从DNA上动员下来。目的1将探讨TIP 60是否是参与下调磷酸化H2 AX的主要染色质重塑因子，以及RVB 2是否与RVB 1在此功能中合作。目的2将检验RVB 1和p400调节TIP 60的假设是否适用于TIP 60控制的启动子处的染色质重塑。目的3将测试ATP结合/水解和与自身或与RVB 2的寡聚化的作用。将开发体外试验以探索RVB 1如何激活TIP 60乙酰转移酶复合物以及TIP 60复合物如何在磷酸化H2 AX去磷酸化之前动员含磷酸化H2 AX的核小体的分子基础。细胞对DNA损伤的反应决定了环境DNA损伤剂如何导致突变和导致癌症，以及治疗性DNA损伤剂如何治疗癌症。由RVB调节的染色质重塑在DNA损伤位点和启动子处起作用以控制细胞的反应。因此，对RVB如何调节染色质重塑因子的分子理解对于对DNA损伤的最佳反应很重要，这将允许设计干扰RVB功能的化学物质，从而使细胞对化疗或放疗敏感。

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.