Molecular Mechanisms Regulating VMD2 Expression

调节 VMD2 表达的分子机制

• 批准号: 7909545
• 负责人: NORIKO ESUMI
• 金额: $ 5.85万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7909545
• 关键词: Age related macular degeneration; American; Binding; Biological; Biological Assay; Biological Models; Blindness; Cattle; Cells; Complementary DNA; Complex; Development; Disease; E-Box Elements; Elderly; Electrophoretic Mobility Shift Assay; Electroporation; Elements; Family; Family member; Gelshift Analysis; Gene Expression; Gene Transfer; Genes; Goals; Grant; Human; In Vitro; Inherited; LacZ Genes; Libraries; Location; Measures; Methods; Molecular; Mus; Mutant Strains Mice; Mutation; Nucleic Acid Regulatory Sequences; Pathogenesis; Pattern; Photoreceptors; Pigments; Play; Promoter Regions; Protein Isoforms; Rattus; Regulation; Research Personnel; Retinal Dystrophy; Retinal Photoreceptors; Reverse Transcription; Role; Screening procedure; Sequence Homology; Structure of retinal pigment epithelium; System; TFE3 gene; Testing; Trans-Activators; Transfection; Transgenic Mice; Vision; Vitelliform macular dystrophy; Yeasts; base; chromatin immunoprecipitation; combinatorial; in vivo; insight; melanocyte; member; microphthalmia-associated transcription factor; mutant; photoreceptor degeneration; programs; promoter; research study; transcription factor; yeast two hybrid system

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.

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对光感受器的功能和存活至关重要。几个rpe特异性或偏好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很重要，但关注RPE基因表达调控的研究相对较少。这导致我们使用人类VMD2（负责卵黄状黄斑营养不良（Best disease）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在转基因小鼠中指导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。本提案的目标是扩展这些发现。