ANALYSIS OF HUMAN VMD2: A MODEL FOR RPE GENE REGULATION

描述(摘自申请者摘要)：长期目标 这个项目是为了更好地了解其背后的分子机制。 视网膜色素上皮(RPE)特异性基因表达。RPE很重要 视网膜光感受器的功能和存活。心脏的异常情况 RPE与光感受器退行性变的发病机制有关 在RCS大鼠和人类老年性黄斑变性(AMD)中，领先的 导致美国老年人不可逆转的失明。此外，基因中的突变 RPD65、RLBP1、TIMP3和VMD2，四个特异或 在RPE中优先表达，已被证明引起Leber‘s 先天性黑便(LCA)、视网膜色素变性(RP)、Sorsby眼底 营养不良(SFD)和卵黄样黄斑营养不良(Best‘s病)， 分别进行了分析。然而，尽管RPD很重要，也很重要 在RPE中特异或优先表达的基因很少 已知调控RPE特异性基因的分子机制 表情。因此，这项提议的目标是开始澄清这种 以人类VMD2(Best‘s病基因)为模型系统的机制。 更具体地说，将解决以下问题：什么是 调控VMD2表达的顺式作用DNA元件？做不同的事情 RPE特异性基因有共同的调控元件吗？什么是变装？ 与DNA调控元件相结合的因素？RPE中是否存在突变 启动子和/或RPE转录因子与人类疾病有关？为 回答这些问题，VMD2启动子区域的DNA元件将是 通过与其他RPE特异性启动子的序列比较和通过 利用DNase I足迹和凝胶迁移率变化进行生化分析 (EMSA)检测。VMD2启动子区域的功能分析将使用 瞬时转基因和转基因小鼠相结合的方法。 最后，克隆和鉴定与 DNA调控元件中的VMD2启动子，我们将使用酵母单杂交 用人RPE基因文库进行克隆。 增加对RPE基因调控的理解应该有助于提供新的见解 涉及RPE基因表达异常的疾病。此外， 这些知识将有助于RPE靶向基因疗法的发展和 外源基因靶向表达转基因小鼠的建立 蛋白质到RPD。此外，由于贝斯特病具有一些临床和病理特征 AMD的组织学特征，对人类VMD2表达的研究可能有 对理解黄斑变性有重要意义。

DESCRIPTION (Adapted from Applicant's abstract): The long-term objective of this project is to better understand the molecular mechanisms that underlie retinal pigment epithelium (RPE)-specific gene expression. The RPE is important for the function and survival of retinal photoreceptors. Abnormalities in the RPE have been implicated in the pathogenesis of photoreceptor degeneration both in the RCS rat and in human age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Americans. In addition, mutations in RPD65, RLBP1, TIMP3, and VMD2, four genes that are specifically or preferentially expressed in the RPE, have been shown to cause Leber's Congenital Amaurosis (LCA), retinitis pigmentosa (RP), Sorsby's fundus dystrophy (SFD), and vitelliform macular dystrophy (Best's disease), respectively. However, despite the importance of the RPD, and the importance of genes that are specifically or preferentially expressed in the RPE, little is known about the molecular mechanisms that regulate RPE-specific gene expression. Therefore, the goal of this proposal is to begin elucidating such mechanisms using human VMD2 (Best's disease gene) as a model system. More specifically, the following questions will be addressed: What are the cis-acting DNA elements that regulate VMD2 expression? Do different RPE-specific genes share common regulatory elements? What are the trans-acting factors that bind to the DNA regulatory elements? Are mutations in RPE promoters and/or RPE transcription factors involved in human disease? For answering these questions, DNA elements in the promoter region of VMD2 will be identified by sequence comparison with the other RPE-specific promoters and by biochemical analysis using DNase I footprint and electrophoretic mobility shift (EMSA) assays. The promoter region of VMD2 will be functionally analyzed using a combination of transient transfection and transgenic mouse approaches. Finally, to clone and characterize cDNAs for the trans-acting that bind to the DNA regulatory elements in the VMD2 promoter, we will use the yeast one-hybrid approach with a human RPE cDNA library. Increased understanding of RPE gene regulation should help provide new insights into diseases involving abnormalities of RPE gene expression. In addition, the knowledge will be useful in the development of RPE-targeted gene therapy and the generation of transgenic mice with targeted expression of exogenous proteins to the RPD. Furthermore, since Best's disease shares some clinical and histological features with AMD, studies of human VMD2 expression may have important implications for understanding macular degeneration.