HYPERTHERMIA AND THE FUNCTIONS OF HSP70

Ku自身抗原是一种DNA结合蛋白，由70 kDa(Ku70)组成 和86 kDa(Ku80)亚基。最近的数据表明Ku80与 DNA双链断裂的修复和Ku70在抑制肿瘤生长中的作用 热诱导HSP70表达。这个项目的长期目标是 以了解细胞对应激反应的分子基础。 下一个批准期的工作将集中在机械和 Ku蛋白在调节细胞反应中的功能 热休克和碘化辐射。具体目标有三个： 具体目的一重点研究苦参素的分子机制(S) 抑制HSP70的热诱导。我们将检验一个假说 从我们的初步研究推断，Ku调节热度 通过调节转录因子与转录因子的结合来进行休克反应 它们各自在启动子区域的调控元件 HSP70基因。 通过体内基因组足迹法，研究Ku对血吸虫病模型的影响。 与HSP70启动子结合的蛋白质，特别是其对HSP70启动子的影响 热休克转录激活子HSF1与其他分子的结合 转录因子与其各自的结合部位将 下定决心。Ku与中特定元素结合的可能性 将对HSP70基因的调节区进行测试。此外 为了体内基因组足迹，突变将被引入到 连接到报告基因构建的HSP70的启动子，以检测 对于顺式元件(S)的存在，这种顺式元件一旦发生突变就会废除 Ku介导的报告基因热诱导抑制 表情。 在特定的目标二中，我们将研究结构和功能 参与HSP70基因调控的Ku结构域 表情。啮齿动物细胞系的稳定和组成性表达 各种突变的Ku基因将被建立。热休克反应 野生型和突变型细胞的生化特性 将对Ku亚基进行检查。 在特定目的III中，我们建议建立纯合子啮齿动物细胞 Ku70或Ku80的两个等位基因通过以下途径失活的品系 基因打靶诱变。这些“双淘汰赛”Ku70-/-和Ku8- /-细胞系将使我们能够研究不同的生理作用 Ku的各个亚基及其功能域的解剖 参与细胞对热休克和电离的反应 辐射。因为生物学上的重要性和多重 Ku的细胞作用、构建Ku70-/-的意义和 Ku80-/-LINES超越了澄清 Ku在HSP70基因调控和细胞敏感性中的作用 做X光检查。这些细胞系及其衍生细胞表达不同的 突变的Ku亚基可以直接用于分析任何 Ku的功能方面。为此选择胚胎干细胞 进一步的工作确保了这种研究可以很容易地扩展到 动物模型，通过构建基因敲除小鼠品系。

Ku autoantigen is a DNA binding protein consisting of 7O-kDa (Ku70) and 86-kDa (Ku80) subunits. Recent data have implicated Ku80 in the repair of DNA double strand breaks, and Ku70 in the suppression of heat-induced hsp70 expression. The long term goal of this project is to understand the molecular bases of the cellular response to stress. Work in the next granting period will focus on the mechanistic and functional aspects of Ku protein, in modulating cellular response to heat shock and to ioizing radiation. There are three specific aims: Specific Aim I focuses on the molecular mechanism(s) by which Ku suppresses heat-induction of hsp70. We will test a hypothesis inferred from our preliminary studies that Ku regulates the heat shock response by modulating the binding of transcription factors to their respective regulatory elements in the promoter region of the hsp70 gene. By in vivo genomic footprinting, the effects of Ku on the pattern of protein binding to the hsp70 promoter, especially its effects on the binding of the heat shock transcription activator HSF1 and other transcription factors to their respective binding sites will be determined. The possibility that Ku binds to a specific element in the regulatory region of the hsp70 gene will be tested. In addition to in vivo genomic footprinting, mutations will be introduced into the promoter of hsp70, linked to a reporter gene construct, to test for the presence of a cis-element(s), which when mutated abolishes the Ku-mediated suppression of heat-induction of reporter gene expression. In Specific Aim II we will study the structural and functional domains of Ku that are involved in the modulation of hsp70 gene expression. Rodent cell lines stably and constitutively expressing various mutant Ku genes will be established. The heat shock response of these cells and the biochemical properties of wild type and mutant Ku subunits will be examined. In Specific Aim III we propose to establish homozygous rodent cell lines in which both alleles of Ku70 or Ku80 are inactivated through gene-targeting mutagenesis. These "double knockout" Ku7O-/- and Ku8- /- cell lines will enable us to study various physiological roles of the individual subunits of Ku and to dissect the functional domains involved in the cellular response to heat shock and ionizing radiation. Because of the biological importance and the multiple cellular roles of Ku, the significance of constructing Ku70-/- and Ku80-/- lines extends beyond the immediate goal of elucidating the role of Ku in hsp70 gene regulation, and in the sensitivity of cells to x-rays. These cell lines and their derivatives expressing various mutant Ku subunits can be used directly in the analysis of any functional aspect of Ku. The choice of embryonic stem cells for this work further assures that such studies can be readily extended to animal models, through the construction of knockout mice strains.