ANALYSIS OF HUMAN VMD2: A MODEL FOR RPE GENE REGULATION

描述（改编自申请人摘要）： 这个项目是为了更好地理解 视网膜色素上皮细胞（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。异常 RPE与光感受器变性的发病机制有关， 在RCS大鼠和人类年龄相关性黄斑变性（AMD）中， 导致美国老年人不可逆转的失明。此外， RPD 65、RLBP 1、TIMP 3和VMD 2，这四个基因特异性或 优先在RPE中表达，已被证明会导致Leber's 先天性黑蒙（LCA）、视网膜色素变性（RP）、Sorsby眼底 营养不良（SFD）和卵黄状黄斑营养不良（Best病）， 分别然而，尽管RPD的重要性，以及 在RPE中特异性或优先表达的基因中， 已知调节RPE特异性基因的分子机制 表情因此，本提案的目标是开始阐明 使用人类VMD 2（Best’s disease gene）作为模型系统， 更具体地说，将讨论以下问题： 调节VMD 2表达的顺式作用DNA元件？做不同 RPE特异性基因共享共同的调控元件？什么是反作用 与DNA调控元件结合的因子是视网膜色素上皮的突变 启动子和/或RPE转录因子参与人类疾病？为 为了回答这些问题，VMD 2启动子区的DNA元件将被 通过与其他RPE特异性启动子的序列比较以及通过 使用DNA酶I足迹和电泳迁移率变动的生化分析 （EMSA）测定。VMD 2的启动子区将使用 瞬时转染和转基因小鼠方法的组合。 最后，为了克隆和表征结合到蛋白质的反式作用的cDNA， 在VMD 2启动子中的DNA调控元件，我们将使用酵母单杂交 方法与人RPE cDNA文库。 增加对RPE基因调控的理解将有助于提供新的见解 涉及RPE基因表达异常的疾病。此外该 这些知识将有助于RPE靶向基因治疗的发展， 外源基因靶向表达转基因小鼠的建立 蛋白质到RPD。此外，由于贝斯特病与其他疾病有一些临床和 AMD的组织学特征，人VMD 2表达的研究可能具有 对了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.