Molecular Mechanisms Regulating VMD2 Expression

描述（由申请人提供）：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。一些 RPE 特异性或优先基因的突变会导致遗传性人类视网膜营养不良。 RPE 异常也与年龄相关性黄斑变性 (AMD) 的发病机制有关，AMD 是美国老年人不可逆失明的主要原因。然而，尽管 RPE 很重要，但关注 RPE 基因表达调控的研究相对较少。这促使我们使用人类 VMD2（导致卵黄状黄斑营养不良（最佳疾病）的基因）作为模型系统来研究此类机制。我们的研究确定了足以指导转基因小鼠中 RPE 特异性表达的启动子区域，并确定了小眼相关转录因子 (MITF) 作为潜在的正调节因子。该提案的目标是扩展这些发现。 首先，为了明确 MITF 在 VMD2 调节中的作用，将通过定量 PCR 和细胞转染测定来检查不同 MITF 亚型的表达和功能活性。通过染色质免疫沉淀分析 MITF 与牛 Vmd2 启动子的体内结合，通过定量 PCR 检查 Mitf 突变小鼠中的 Vmd2 表达，并使用体内电穿孔测量 Mitf 突变 RPE 中 VMD2 启动子的活性。然后，将使用细胞转染和凝胶转移测定来类似地检查 TFE3 和 TFEB（MITF 家族的两个成员）的作用。最后，使用 VMD2 启动子-lacZ 构建体的转基因小鼠研究将缩小 RPE 特异性表达所需的调控区域范围，并且将使用酵母单杂交系统克隆与其结合的因子。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.