Gene regulation by LEDGF

LEDGF 的基因调控

• 批准号: 6871191
• 负责人: DHIRENDRA P SINGH
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6871191
• 关键词: DNA binding protein; DNA footprinting; affinity chromatography; aging; binding sites; cataract; crystallins; enzyme linked immunosorbent assay; epithelium; fluorescence microscopy; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; growth factor receptors; heat shock proteins; immunocytochemistry; laboratory mouse; lens; northern blottings; organ culture; oxidative stress; polymerase chain reaction; protein protein interaction; protein structure function; site directed mutagenesis

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.

描述（由申请人提供）：虽然，LEDGF 存在于细胞核中 在大多数未受应激的细胞中，氧化应激和热应激会升高 LEDGF 水平。 转染过度表达 LEDGF 的无应激细胞也会上调 Hsp27， αB-晶状体蛋白和AOP2，这些细胞获得了很强的抵抗力 对抗环境压力。压力引起的 LEDGF 升高可能是 细胞增强环境耐受性的基本机制 压力。在晶状体上皮细胞 (LEC) 和 cos7 细胞中，压力不会升高 不仅包括 LEDGF，还包括分子伴侣和其他应激相关蛋白（Hsp27、 αB-晶状体蛋白和 AOP2)。我们假设 LEDGF 结合并且 上调应激相关基因。我们已经找到了相当多的证据 支持 LEDGF 与应激反应元件结合这一假设 （STRE；共有序列 A/TGGGGA/T）和热休克元件（HSE； 共有序列 nGAAn) 上调应激相关基因。 （见附录 8、9)。在上调的应激相关基因中，Hsp27、αB-晶状体蛋白、 和AOP2与白内障发生最相关。 Hsp27 和 αB-晶状体蛋白， 这两种伴侣蛋白对于变性蛋白的重折叠至关重要。 AOP2， 一种相对较新的抗氧化蛋白，可保护细胞免受氧化应激。到 为了增加我们对 LEC 如何抵抗压力的理解，我们重点研究了 LEDGF 对应激相关基因的上调。我已经选择了 Hsp27、αB-晶状体蛋白和 AOP2 基因，并将研究 它们的启动子元件与LEDGF之间的相互作用和热激 转录因子1 (HSF1)。另外我会研究一下功能角色 在年龄相关性白内障的背景下，AOP2 及其与 LEDGF 的关系。 该应用程序包含三个具体目标； 1) 表征和定义 LEDGF 的 DNA 结合域、它们的结合亲和力、功能 两种不同监管的相互作用和反式激活潜力 Hsp27 和 AOP2 基因启动子的元件（HSE 和 STRE）。 2) 理解 HSE-HSF1 和 HSE-LEDGF 之间相互作用的功能意义 在正常（无应激）细胞和 Hsp27 的应激激活中， αB-晶状体蛋白和 AOP2 基因。 3) 确定转录调控 LEDGF 的 AOP2 以及 AOP2 抗氧化效力的分子基础 氧化应激。该应用程序将为您提供新颖的见解 LEDGF 调节应激反应基因的机制。它还将 扩大我们对这些基因和 LEDGF 在细胞存活中的作用的理解 在压力下。特别是，我们对 LEC 的研究可能会增加我们的理解 与年龄相关的白内障发生以及减缓这种致盲退化的方法 过程。