Molecular Mechanisms Regulating VMD2 Expression

描述(申请人提供)：该项目的长期目标是更好地了解调节视网膜色素上皮(RPE)特异性基因表达的分子机制。RPE对于光感受器的功能和存活至关重要。几个RPE特异或优先基因的突变会导致遗传性人类视网膜营养不良。RPE异常也与老年性黄斑变性(AMD)的发病机制有关，AMD是美国老年人不可逆性失明的主要原因。然而，尽管RPE很重要，但相对较少的研究集中在RPE基因表达的调控上。这导致我们使用人类VMD2作为模型系统来研究这种机制，VMD2是导致卵黄样黄斑营养不良(最佳疾病)的基因。我们的研究确定了足以在转基因小鼠中引导RPE特异性表达的启动子区域，并确定小眼球相关转录因子(MITF)是一个潜在的正调控因子。这项提案的目标是扩大这些发现。 首先，为了确定MITF在VMD2调控中的作用，我们将通过定量聚合酶链式反应和细胞转染实验来检测不同MITF亚型的表达和功能活性。用染色质免疫沉淀法分析MITF与牛Vmd2启动子的体内结合，用定量聚合酶链式反应检测MITF突变小鼠Vmd2的表达，用体内电穿孔技术检测MITF突变RPE中Vmd2启动子的活性。然后，两个MITF家族成员TFE3和TFEB的作用将通过细胞转染和凝胶移位分析进行类似的研究。最后，使用VMD2启动子-LacZ构建的转基因小鼠研究将缩小RPE特异性表达所需的调控区域，并将使用酵母单杂交系统克隆与它们结合的因子。了解VMD2表达的详细机制将为RPE中基因表达的调控网络以及涉及RPE基因表达异常的疾病提供新的见解。

DESCRIPTION (provided by applicant): The long-term objective of this project is to better understand the molecular mechanisms that regulate retinal pigment epithelium (RPE)-specific gene expression. The RPE is critical for the function and survival of photoreceptors. Mutations in several RPE-specific or preferential genes cause inherited human retinal dystrophies. RPE abnormalities have also been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Americans. However, despite the importance of the RPE, relatively few studies have focused on the regulation of RPE gene expression. That led us to study such mechanisms using human VMD2, the gene responsible for vitelliform macular dystrophy (Best disease), as a model system. Our studies defined the promoter region sufficient to direct RPE-specific expression in transgenic mice and identified microphthalmia-associated transcription factor (MITF) as a potential positive regulator. The goal of this proposal is to extend these findings. First, to define the role of MITF in VMD2 regulation, expression and functional activities of different MITF isoforms will be examined by quantitative PCR and cell transfection assays. Binding of MITF to the bovine Vmd2 promoter in vivo will be analyzed by chromatin immunoprecipitation, Vmd2 expression in Mitf mutant mice will be examined by quantitative PCR, and the activity of the VMD2 promoter in Mitf mutant RPE will be measured using in vivo electroporation. Then, the role of TFE3 and TFEB, two MITF family members, will be similarly examined using cell transfection and gel shift assays. Finally, transgenic mouse studies with VMD2 promoter-lacZ constructs will narrow down the regulatory regions that are necessary for RPE-specific expression, and factors that bind to them will be cloned using the yeast one-hybrid system. Understanding of the detailed mechanisms of VMD2 expression should provide new insights into the regulatory networks for gene expression in the RPE as well as diseases involving abnormalities of RPE gene expression.