Gene regulation by LEDGF

LEDGF 的基因调控

• 批准号: 6431185
• 负责人: DHIRENDRA P SINGH
• 金额: $ 34.48万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 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 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6431185
• 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）：尽管LEDGF存在于细胞核中， 对于大多数未受应激的细胞，氧化应激和热应激升高LEDGF水平。 转染过表达LEDGF的未应激细胞也上调Hsp 27， α B-晶状体蛋白和AOP 2，这些细胞获得了强的抗性 对抗环境压力。应激诱导的LEDGF升高可能是一种 细胞增加对环境的耐受性的基本机制 应力在透镜上皮细胞（LEC）和cos 7细胞中，应激升高，而不是 不仅是LEDGF而且还有分子伴侣和其它应激相关蛋白（Hsp 27， α B-晶状体蛋白和AOP 2）。我们假设LEDGF结合于 上调压力相关基因。我们发现了大量证据， 支持LEDGF与应激反应元件结合的假设 (STRE共有序列A/TGGGGA/T）和热休克元件（HSE; 共有序列nGAAn）来上调应激相关基因。(See附录 第8、9段）。在上调的应激相关基因中，热休克蛋白27，α B-晶状体蛋白， 和AOP 2与白内障发生最相关。Hsp 27和α B-晶状体蛋白， 这两种分子伴侣蛋白对于变性蛋白质的重折叠是必需的。AOP2， 一种相对较新的抗氧化蛋白，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，我们将研究重点放在了 LEDGF对应激相关基因的上调作用。我选择了 Hsp 27、alphaB-晶状体蛋白和AOP 2的基因，并将研究 它们的启动子元件与LEDGF的相互作用和热休克 转录因子1（HSF 1）。此外，我还将研究 在年龄相关性白内障的背景下，AOP 2及其与LEDGF的关系。 本申请包含三个具体目标：1）表征和定义 LEDGF的DNA结合结构域、它们的结合亲和力、功能性 相互作用和反式激活潜力，以两种不同的调节 Hsp 27和AOP 2基因启动子的HSE和STRE元件。2)了解 HSE-HSF 1和HSE-LEDGF相互作用的功能意义 在正常（非应激）细胞和应激激活的Hsp 27中， α B-晶状体蛋白和AOP 2基因。3)为了确定转录调控， LEDGF对AOP 2的抑制作用以及LEDGF作用下AOP 2抗氧化能力的分子基础 氧化应激该应用程序将提供新的见解， 应激反应基因受LEDGF调控的机制。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和减缓这种致盲性退行性疾病的方法 过程