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）：尽管核中存在LEDGF 在大多数无重的细胞中，氧化和热应力升高LEDGF水平。 未转染至过表达的LEDGF的无应力细胞也上调了HSP27， Alphab-Crystallin和AOP2，这些细胞获得了强烈的抗性 反对环境压力。压力引起的LEDGF升高可能是 细胞增加对环境的容忍度的基本机制 压力。在晶状体上皮细胞（LEC）和COS7细胞中，应力不提高 仅LEDGF，但伴侣和其他与应力相关的蛋白质（HSP27， alphab-crystallin和aop2）。我们假设LEDGF绑定到和 上调与应力相关的基因。我们在 支持与压力响应元素结合的这一假设 （stre;共有序列A/TGGGGA/T）和热激元（HSE; 共识序列ngaan）以上调与应力相关的基因。 （请参阅附录 8、9）。在上调与应力相关的基因中，hsp27，α-晶状体蛋白， AOP2与白内垂直生成最相关。 HSP27和Alphab-Crystallin， 两种伴侣蛋白，对于重折叠变性蛋白都是必不可少的。 AOP2， 一种相对较新的抗氧化剂蛋白，可保护细胞免受氧化应激。到 提高我们对LEC如何抵抗压力的理解，我们集中了研究 关于LEDGF对应激相关基因的上调。我选择了 HSP27，Alphab-Crystallin和AOP2的基因，将研究 启动子元素与LEDGF和热冲击之间的相互作用 转录因子1（HSF1）。此外，我将研究功能作用 AOP2及其与年龄相关白内障的关系的关系。 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，其结合亲和力，功能 相互作用和对两个不同调节的反式激活潜力 HSP27和AOP2基因启动子的元素（HSE和Stre）。 2）理解 HSE-HSF1与HSE-LEDGF之间相互作用的功能意义 在正常（无数强度）细胞中，在HSP27的应力激活中， α-晶状体和AOP2基因。 3）确定的转录调节 ledgf的AOP2和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 由LEDGF调节应力反应基因的机制。它也会 扩展我们对这些基因和LEDGF在细胞存活中的作用的理解 在压力下。特别是，我们在LEC中的研究可能会增加我们的理解 与年龄相关的白内垂质发生和减慢这种盲人退化的手段 过程。