Functional analysis of the Tip60 complex

描述（由申请人提供）： 细胞检测和修复电离辐射造成的DNA双链断裂（DSB）的能力可能受到病变周围染色质结构的限制。因此，细胞需要重塑DSB处的局部染色质结构，以进入该位点并加工和修复DSB。然而，染色质重塑复合物，驱动染色质组织在DSB的变化是不明确的。NuA 4-Tip 60重塑复合物被迅速募集到DSB，在那里它的功能是重组局部染色质结构， 开放的、灵活的结构域，对于DSB的检测和修复是必不可少的。NuA 4-Tip 60的功能是在DSB处将新的组蛋白变体交换到核小体上，并促进组蛋白的超乙酰化，这两者都有利于开放染色质的形成。重要的是，在DSB处的染色质的NuA 4定向重塑是控制组蛋白修饰、加载DSB修复蛋白、控制断裂的末端切除和修复途径的选择所必需的。中心假设是NuA 4-Tip 60在DSB处产生开放的、柔性的染色质，并调节受损DNA的末端加工。我们的方法是利用锌指核酸酶在紧凑的基因间区域和转录的基因中产生靶向DSB，以探索这些不同结构域之间DSB修复的差异。我们将确定NuA 4如何促进组蛋白乙酰化的扩散和组蛋白H2A.Z的交换，并确定这两个过程如何削弱相邻核小体之间的相互作用并促进开放的柔性染色质的形成。我们将确定NuA 4-Tip 60如何将核小体定位在DSB的两侧，以及这种定位如何调节末端切除、DSB的加工和DSB修复途径的选择。此外，我们将确定如何招聘的染色质结构蛋白Brd 2 DSB施加特定的功能顺序的染色质和调节细胞的放射敏感性。此外，我们将确定如何JQ 1，一种新的布罗莫结构域抑制剂，阻止Brd 2招募DSB，抑制染色质重组DSB和细胞电离辐射的敏感性。该项目将深入研究电离辐射产生的DSB如何通过NuA 4-Tip 60导致染色质的特定结构重组。此外，它将确定核小体重组如何影响基因和沉默的基因间区域中DSB修复的机制和保真度。此外，我们将确定JQ 1是否为一类新的抗癌药物提供了基础，这些药物的功能是阻断DSB处的染色质重组并使肿瘤对放射治疗敏感。最后，肿瘤细胞中的染色质景观经常改变，包括组蛋白修饰的独特模式、改变的染色质包装和改变的染色质调节蛋白（包括NuA 4-Tip 60复合物的组分）的表达/突变。这项工作将为NuA 4指导的染色质重组如何影响肿瘤细胞中的DSB修复提供新的见解，并定义肿瘤中染色质结构的改变如何影响肿瘤进展以及肿瘤对放疗和化疗的敏感性。

DESCRIPTION (provided by applicant): The ability of cells to detect and repair DNA double-strand breaks (DSBs) created by ionizing radiation can be limited by the chromatin structure surrounding the lesion. Cells therefore need to remodel the local chromatin structure at the DSB to gain access to the site and to process and repair the DSB. However, the chromatin remodeling complexes which drive changes in chromatin organization at DSBs are poorly defined. The NuA4-Tip60 remodeling complex is rapidly recruited to DSBs, where it functions to reorganize the local chromatin structure, creating open, flexible domains which are essential for the detection and repair of DSBs. NuA4-Tip60 functions to both exchange novel histone variants onto nucleosomes at the DSB and to promote hyperacetylation of histones, both of which favor the formation of open chromatin. Importantly, NuA4-directed remodeling of the chromatin at DSBs is required for controlling histone modification, loading of DSB repair proteins, controlling end resection of the break and choice of repair pathway. The central hypothesis is that NuA4-Tip60 creates open, flexible chromatin at DSBs and regulates end processing of the damaged DNA. Our approach is to utilize Zinc Finger Nucleases to create targeted DSBs in both compact intergenic regions and in transcribed genes to explore the differences in DSB repair between these distinct domains. We will determine how NuA4 promotes the spreading of histone acetylation and exchange of histone H2A.Z, and determine how these 2 processes weaken interaction between adjacent nucleosomes and promote the formation of open, flexible chromatin. We will determine how NuA4-Tip60 positions nucleosomes either side of the DSB and how this positioning regulates end resection, processing of the DSB and choice of DSB repair pathway. Further, we will determine how recruitment of the chromatin structural protein Brd2 to DSBs imposes specific functional order on the chromatin and regulates cellular radiosensitivity. In addition, we will determine how JQ1, a novel bromodomain inhibitor which blocks Brd2 recruitment to DSBs, inhibits chromatin reorganization at DSBs and sensitizes cells to ionizing radiation. This project will provide insigh into how DSBs created by ionizing radiation leads to specific structural re-organization of the chromatin by NuA4-Tip60. Further, it will determine how nucleosome reorganization impacts the mechanism and fidelity of DSB repair in both genes and silent, intergenic regions. In addition, we will determine if JQ1 provides the basis for a new class of anti-cancer agents which function to block chromatin reorganization at DSBs and sensitize tumors to radiation therapy. Finally, the chromatin landscape in tumor cells is frequently altered, including unique patterns of histone modifications, altered chromatin packing and altered expression/mutation of chromatin regulatory proteins, including components of the NuA4-Tip60 complex. This work will provide new insight into how NuA4-directed chromatin reorganization impacts DSB repair in tumor cells, and define how altered chromatin structure in tumors impacts tumor progression and the sensitivity of tumors to both radiation therapy and chemotherapy.