MOLECULAR MECHANISMS OF RETINA SPECIFIC GENE EXPRESSION

视网膜特异性基因表达的分子机制

• 批准号: 2608669
• 负责人: ANAND SWAROOP
• 金额: $ 21.52万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608669
• 关键词: DNA binding protein; RNase protection assay; SDS polyacrylamide gel electrophoresis; affinity chromatography; cell type; congenital eye disorder; gel mobility shift assay; gene expression; immunoprecipitation; in situ hybridization; molecular cloning; molecular genetics; molecular pathology; northern blottings; polymerase chain reaction; protein purification; protein structure function; retina; retina disorder; rhodopsin; tissue /cell culture; transcription factor; western blottings; yeast two hybrid system

Differential expression of genes in a particular tissue, such as retina, is achieved by combinatoriaI action of transcription factors. Nrl is an evolutionarily-conserved bZIP transcription factor, identified in our laboratory by subtraction cloning. In the adult, high levels of Nrl transcripts are detected only in the retina. The Nrl protein shows strong homology to the product of a transforming oncogene, v-maf. The proteins of the Maf-Nrl subfamily recognize a long AP-1 like DNA sequence element, are shown to heterodimerize with selected bZIP proteins in vitro, and implicated in tissue-specific gene regulation. Several lines of evidence suggest that Nrl is involved in rhodopsin gene regulation. The underlying hypothesis is that in the adult retina Nrl plays a major role in regulating the expression of gene products that are needed for the appropriate fiinctioning of photoreceptors and other neurons. Since protein-protein interactions are a major determinant of transcriptional activity and can generate tremendous flexibility in target site selection, the objective of this proposal is to identify the proteins that specifically interact with Nrl and to elucidate the biological relevance of these interactions. To accomplish this, Specific Aim 1 proposes to evaluate whether any of the known bZIP proteins of the Fos, Jun, Maf-Nrl and NF-E2 p45 subfamilies is expressed in the adult retina and interacts with Nrl to modulate gene expression. Specific Aim 2 focuses on the purification and characterization of retinal proteins that bind to the Nrl-response element in the rhodopsin promoter. Specific Aim 3 employs a yeast two- hybrid approach with the "Nrl-bZIP bait" to identify the proteins that productively heterodimerize with Nrl and may be involved in regulating different sets of genes in the retina. The long-term objective of P.I.'s research is to understand the molecular events involved in the pathogenesis of eye diseases and eventually assist in the design of gene-based therapeutic strategies. Transcription factors have become attractive targets for mechanism- based design of drugs, which can be used to modulate specific biochemical pathways. In that direction, the proposed studies are designed to identify transcriptional regulatory proteins that together with Nrl mediate tissue- or cell type- specific gene expression in the retina, and should provide mechanistic insights into the molecular basis of congenital and inherited retinal disorders.

基因在特定组织中的差异表达，例如 视网膜是通过转录因子的组合作用来实现的。 NRL是一种进化保存的BZIP转录因子， 通过减法克隆在我们的实验室中确定。在成年人中 仅在视网膜中检测到NRL转录本水平。 NRL 蛋白质与转化的产物表现出很强的同源性 癌基因，V-MAF。 MAF-NRL亚家族的蛋白质识别很长 AP-1之类的DNA序列元件被证明与 体外选定的BZIP蛋白，并与组织特异性基因有关 规定。几条证据表明NRL参与 视紫红质基因调节。 基本的假设是 成人视网膜NRL在调节表达中起重要作用 适当的基因产品 感受器和其他神经元。 由于蛋白质 - 蛋白质相互作用 是转录活动的主要决定因素，可以产生 目标站点选择的巨大灵活性，目的 建议是确定与特殊相互作用的蛋白质 NRL并阐明这些相互作用的生物学相关性。 为了实现这一目标，具体目标1提议评估是否有任何 FOS，JUN，MAF-NRL和NF-E2 P45的已知BZIP蛋白 亚家族在成人视网膜中表达，并与NRL相互作用 调节基因表达。特定目标2专注于净化 和与NRL反应结合的视网膜蛋白的表征 视紫红质启动子中的元素。特定目标3采用酵母两种 与“ NRL-BZIP诱饵”的混合方法，以识别蛋白质 有效地与NRL异二聚二聚体，可能参与调节 视网膜中的不同基因集。 P.I.研究的长期目标是了解 参与眼病的发病机理的分子事件和 最终协助设计基于基因的治疗策略。 转录因子已成为机制的有吸引力的目标 - 基于药物的设计，可用于调节特定 生化途径。在这个方向上，拟议的研究是 旨在确定共同的转录调节蛋白 NRL介导组织或细胞类型特异性基因表达 视网膜，应向分子提供机械洞察力 先天性和遗传性视网膜疾病的基础。