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: 6693055
• 负责人: ANAND SWAROOP
• 金额: $ 31.27万
• 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 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6693055
• 关键词: DNA binding protein; RNase protection assay; SDS polyacrylamide gel electrophoresis; affinity chromatography; electroretinography; gel mobility shift assay; gene expression; gene induction /repression; immunoaffinity chromatography; immunoprecipitation; laboratory mouse; molecular cloning; molecular genetics; molecular pathology; northern blottings; polymerase chain reaction; protein protein interaction; protein structure function; retina; rhodopsin; site directed mutagenesis; tissue /cell culture; transcription factor; western blottings; yeast two hybrid system

Differential expression of genes is fundamental to biological processes. It is accomplished by the combinatorial and synergistic (or antagonistic) action of a relatively small number of transcription factors. To elucidate transcriptional regulatory mechanisms in the retina, it is essential to identify specific activators (and repressors) and delineate how these regulators integrate the signaling pathways with basal transcription machinery to generate a transcriptional response. We have previously identified Nrl, a transcription factor of the basic motif-leucine zipper (bZIP) family, by subtraction cloning. Nrl regulates rhodopsin promoter activity synergistically with Crx, a photoreceptor-specific transcription factor. Mutations in the human CRX or NRL gene result in photoreceptor degeneration, suggesting a major role for these two transcription factors in modulating photoreceptor gene expression in vivo. Our studies also reveal that Nrl interacts with Crx, TATA-binding protein, and other as yet uncharacterized proteins in the retina. Because of its unique expression pattern, possible regulation by FGF-2, and involvement in rhodopsin regulation, Nrl appears to be a key mediator of transcriptional response in the developing and mature retina. We hypothesize that interaction of Nrl with other regulatory proteins in the context of DNA-binding sequences is responsible for spatial and temporal control of gene expression in the retina. The goals of this proposal are to identify Nrl- interacting proteins (NIPs) in the retina using genetic (yeast two-hybrid) and biochemical (immuno- and DNA-affinity chromatography) methods, with a focus on delineating rhodopsin regulation (Aims 1-3). To assess the role of Nrl in developing retina, we propose to isolate NIPs from fetal retinal libraries by a yeast two-hybrid approach (Aim 2) and directly examine Nrl function in mice using a gene-knockout strategy (Aim 4). Elucidation of transcriptional regulatory pathways should reveal significant new insights into retinal development and disease. Since mutations in retinal transcription factors and their target genes result in retinopathies, it might be possible to experimentally manipulate the function of specific transcription factor(s) to up- or down-regulate a particular target gene and correct a disease phenotype.

基因的差异表达是生物过程的基础。 它是通过相对较少的转录因子的组合和协同（或拮抗）作用来完成的。 为了阐明视网膜中的转录调节机制，必须识别特定的激活剂（和阻遏物），并描述这些调节器如何将信号通路与基础转录机械整合在一起以产生转录响应。我们先前通过减法克隆确定了基本基序拉链（BZIP）家族的转录因子（BZIP）家族的转录因子。 NRL与光感受器特异性转录因子CRX协同调节视紫红质启动子活性。 人CRX或NRL基因中的突变导致感光性变性，这表明这两个转录因子在调节体内的光感受器基因表达中起了主要作用。 我们的研究还表明，NRL与视网膜中的CRX，TATA结合蛋白和其他尚未表征的蛋白质相互作用。 由于其独特的表达模式，FGF-2的可能调节以及参与视紫红质调节，NRL似乎是发育中和成熟视网膜中转录反应的关键介体。 我们假设在DNA结合序列的背景下，NRL与其他调节蛋白的相互作用是导致视网膜中基因表达的空间和时间控制。 该提案的目标是使用遗传（酵母两杂种）和生化（免疫和DNA - 亲和力色谱）方法鉴定视网膜中NRL相互作用的蛋白质（NIP），重点介绍了可划定的若dopsin调节（目标1-3）。 为了评估NRL在开发视网膜中的作用，我们建议通过酵母双杂交方法将NIP从胎儿视网膜文库中分离出来（AIM 2），并使用基因敲除策略直接检查小鼠中NRL功能（AIM 4）。阐明转录调节途径应揭示对视网膜发育和疾病的重大新见解。由于视网膜转录因子及其靶基因的突变会导致视网膜病变，因此有可能通过实验操纵特定转录因子的功能，以上或下调特定的靶基因并纠正疾病表型。