Molecular Mechanisms Regulating VMD2 Expression

描述（由申请人提供）：本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对光感受器的功能和存活至关重要。几个RPE特异性或优先基因的突变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的重要性，相对较少的研究集中在RPE基因表达的调控。这使得我们使用人类VMD2（负责卵黄状黄斑营养不良（Best疾病）的基因）作为模型系统来研究这种机制。我们的研究确定了足以指导转基因小鼠RPE特异性表达的启动子区域，并确定了小眼症相关转录因子（MITF）作为潜在的正调节因子。本提案的目标是扩展这些发现。 首先，为了确定MITF在VMD 2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同种型的表达和功能活性。通过染色质免疫沉淀分析体内MITF与牛Vmd2启动子的结合，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，TFE3和TFEB，两个MITF家族成员的作用，将类似地使用细胞转染和凝胶迁移试验进行检查。最后，转基因小鼠研究VMD2启动子lacZ结构将缩小RPE特异性表达所必需的调控区，并将使用酵母单杂交系统克隆与它们结合的因子。VMD2表达的详细机制的理解应该提供新的见解的基因表达的RPE以及涉及异常的RPE基因表达的疾病的调控网络。

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.