CRX & ITS REGULATORY NETWORK IN RETINAL DEGENERATIONS

克雷克斯

• 批准号: 6363168
• 负责人: SHIMING CHEN
• 金额: $ 26.24万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363168
• 关键词: DNA binding protein; cone cell; cytogenetics; gel mobility shift assay; gene expression; gene mutation; genetic mapping; homeobox genes; nucleic acid sequence; protein binding; protein protein interaction; retina degeneration; rod cell; transcription factor; transfection; visual photoreceptor; yeast two hybrid system

Experiments proposed in this application will determine the importance of the Cone-Rod Homeobox (CRX) and its associated regulatory network in photoreceptor gene expression and photoreceptor degenerative diseases. CRX is a novel member of otd/Otx homeodomain transcription factor family that is specifically expressed in photoreceptors. CRX binds to and trans-activates the promoters of many photoreceptor-specific genes. Mutations in CRX are associated with photoreceptor degenerative diseases having a wide age of onset, including cone-rod dystrophy, retinitis pigmentosa and Leber's congenital amaurosis. Our hypothesis is that CRX serves as one-of the key molecules that control the expression of photoreceptor-specific genes and is essential for the normal development and maintenance of the photoreceptors. Two specific aims are proposed to test this hypothesis: Aim I will test how disease-related CRX mutations alter the way that CRX regulates photoreceptor gene expression. Five known CRX homeodomain mutations will be analyzed for their effects on the binding of CRX to DNA and trans-activation of the rhodopsin promoter. Aim 2 will identify and characterize other components of the CRX regulatory pathway, such as proteins that regulate the activity of CRX or co-factors required for CRX function. These proteins are hypothesized to physically interact with CRX and, therefore, will be isolated using protein-protein interaction screens in yeast cells. The resulting positive clones will be characterized by sequence homology to known proteins, expression patterns, and physical and functional interactions with CRX. We will also determine the chromosomal localization of the corresponding human and murine genes. These studies will provide information about the program controlling photoreceptor-specific gene expression and the development and maintenance of photoreceptor function. They are expected to provide information about the molecular basis of photoreceptor degenerative diseases and aid in the selection of targets for therapeutic approaches to these diseases.

本申请中提出的实验将确定视锥-视杆同源异型盒（CRX）及其相关调控网络在光感受器基因表达和光感受器变性疾病中的重要性。CRX是otd/Otx同源结构域转录因子家族的新成员，在光感受器中特异表达。CRX结合并反式激活许多光感受器特异性基因的启动子。CRX中的突变与具有广泛发病年龄的光感受器退行性疾病相关，包括视锥-视杆营养不良、色素性视网膜炎和Leber先天性黑蒙。我们的假设是CRX是控制光感受器特异性基因表达的关键分子之一，对光感受器的正常发育和维持至关重要。提出了两个具体的目标来验证这一假设：目的我将测试如何疾病相关的CRX突变改变的方式，CRX调节感光基因的表达。五个已知的CRX同源结构域突变将分析其对CRX与DNA的结合和视紫红质启动子的反式激活的影响。目标2将鉴定和表征CRX调节途径的其他组分，例如调节CRX活性的蛋白质或CRX功能所需的辅因子。假设这些蛋白质与CRX物理相互作用，因此将使用酵母细胞中的蛋白质-蛋白质相互作用筛选来分离。所得阳性克隆将通过与已知蛋白质的序列同源性、表达模式以及与CRX的物理和功能相互作用来表征。我们还将确定相应的人类和小鼠基因的染色体定位。这些研究将提供有关控制感光细胞特异性基因表达的程序以及感光细胞功能的发育和维持的信息。它们有望提供有关光感受器退行性疾病的分子基础的信息，并有助于选择这些疾病的治疗方法的靶点。