Gene regulation by LEDGF

描述(申请人提供)：虽然，LEDGF存在于细胞核中 在大多数未受压力的细胞中，氧化和热应激会提高LEDGF的水平。 高表达LEDGF的非应激细胞也上调Hsp27的表达， α-晶体蛋白和AOP2，这些细胞获得了很强的抵抗力 抵抗环境压力。应激诱导的LEDGF升高可能是一种 细胞提高对环境耐受性的基本机制 压力。在晶状体上皮细胞(LECs)和COS7细胞中，应激不会升高 不仅有LEDGF，还有伴侣蛋白和其他应激相关蛋白(Hsp27， α-晶体蛋白和AOP2)。我们假设LEDGF与 上调压力相关基因。我们发现了相当多的证据 LEDGF与应激反应元件结合这一假设的支持 (STRE；共识序列A/TGGGGA/T)和热休克元件(HSE； 共识序列ngaan)，以上调压力相关基因。(见附录 8、9)。在上调的应激相关基因中，Hsp27、α-晶体蛋白、 AOP2与白内障的发生密切相关。Hsp27和α-晶状体蛋白， 这两种伴侣蛋白对复性变性蛋白都是必不可少的。AOP2， 一种相对较新的抗氧化蛋白质，保护细胞免受氧化应激的伤害。至 增加我们对LECs如何抵抗压力的理解，我们的研究重点是 LEDGF上调应激相关基因表达的研究我已经选择了 Hsp27、α-晶体蛋白和AOP2基因，并将研究 其启动子元件与LEDGF的相互作用与热休克 转录因子1(HSF1)。此外，我还将研究其功能角色 在年龄相关性白内障中，AOP2及其与LEDGF的关系。 本申请包含三个具体目标：1)描述和定义 LEDGF的DNA结合域、结合亲和力、功能 相互作用(S)和反式激活潜能对两种截然不同的调控 Hsp27和AOP2基因启动子的hse和stre元件。2)了解 HSE-HSF1和HSE-LEDGF相互作用的功能意义 在正常(非应激)细胞和Hsp27的应激激活中， α-晶体蛋白和AOP2基因。3)确定转录调控 LEDGF检测AOP2及其抗氧化活性的分子基础 氧化应激。这个应用程序将提供对 LEDGF调控应激反应基因的机制。它还将 扩大我们对这些基因和LEDGF在细胞生存中作用的理解 在压力下。特别是，我们对LEC的研究可能会增加我们的理解 年龄相关性白内障的发生和减缓这种致盲退变的方法 进程。

DESCRIPTION (provided by applicant): Although, LEDGF is present in the nucleus of most unstressed cells, oxidative- and heat-stress elevate LEDGF levels. Unstressed cells transfected to over-expressing LEDGF also up-regulated Hsp27, alphaB-crystallin, and AOP2, and these cells acquired a strong resistance against environmental stresses. Stress-induced elevation of LEDGF may be a basic mechanism by which cells increase their tolerance to environmental stress. In lens epithelial cells (LECs) and cos7 cells, stress elevates not only LEDGF but also chaperones and other stress-related proteins (Hsp27, alphaB-crystallin and AOP2). We hypothesized that LEDGF bound to and up-regulated stress-related genes. We have found considerable evidence in support of this hypothesis that LEDGF bound to the stress response elements (STRE; consensus sequence A/TGGGGA/T) and the heat shock element (HSE; consensus sequence nGAAn) to up-regulate stress-related genes. (See Appendices 8, 9). Among the up-regulated stress-related genes, Hsp27, alphaB-crystallin, and AOP2 are most relevant to cataractogenesis. Hsp27 and alphaB-crystallin, both chaperone proteins, are essential for refolding denatured proteins. AOP2, a relatively new antioxidant protein, protects cells from oxidative stress. To increase our understanding of how LECs resist stress, we have focused our study on the up-regulation of stress-related genes by LEDGF. I have selected the genes for Hsp27, alphaB-crystallin and AOP2 and will study the interaction between their promoter elements and LEDGF and heat shock transcriptional factor1 (HSF1). In addition, I will study the functional role of AOP2 and its relationship to LEDGF in the context of age related cataract. This application contains three Specific Aims; 1) To characterize and define the DNA-binding domains of LEDGF, their binding affinity, functional interaction(s) and transactivation potential to two distinct regulatory elements (HSE and STRE) of the Hsp27 and AOP2 gene promoter. 2) To understand the functional significance of the interactions between HSE-HSF1 and HSE-LEDGF in the normal (unstressed) cells and in the stress-activation of the Hsp27, alphaB-crystallin and AOP2 genes. 3) To determine transcriptional regulation of AOP2 by LEDGF and the molecular basis of antioxidant potency of AOP2 under oxidative stress. This application will provide novel insights into the mechanisms by which stress-response genes are regulated by LEDGF. It will also expand our understanding of roles of these genes and LEDGF in survival of cells under stress. In particular, our studies in LECs may increase our understanding of age-related cataractogenesis and means of slowing this blinding degenerative process.