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: 7171799
• 负责人: ANAND SWAROOP
• 金额: $ 48.02万
• 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 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171799
• 关键词: Affect; Animals; Biological Assay; Cells; Co-Immunoprecipitations; Code; Complex; Degenerative Disorder; Development; Electroretinography; Evolution; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Goals; Histology; Human; Human Genome; Leucine Zippers; Mammals; Meat; Mediating; Mediator of activation protein; Microarray Analysis; Molecular; Mus; Mutation; Nature; Neural Retina; Nuclear Receptors; Numbers; Opsin; Pathogenesis; Personal Communication; Phenotype; Photoreceptors; Phototransduction; Physiological; Play; Progress Reports; Proteins; Regulation; Regulator Genes; Regulatory Pathway; Retina; Retinal Cone; Retinal Degeneration; Retinal Diseases; Rhodopsin; Role; Signaling Molecule; Signaling Protein; Specific qualifier value; Staging; Time; Tissue-Specific Gene Expression; Transcription Regulatory Protein; Transgenic Mice; Transplantation; Validation; Yeasts; bZIP Domain; base; cell type; chromatin immunoprecipitation; combinatorial; comparative; day; disease phenotype; enhanced green fluorescent protein; fighting; fluorescence activated cell sorter device; immunocytochemistry; insight; mutant; novel; postnatal; promoter; retinal rods; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): Diverse cellular phenotypes and functions in metazoans are specified by differential expression of genes. Regulation of quantitatively precise expression of genes in the right cell type and at the fight time is mediated by the combinatorial and synergistic (or antagonistic) action of a limited number of transcription factors. Nrl, first identified by the PI, is a key basic motif-leucine zipper (bZIP) transcription factor, which is now established as a prime mediator of gene regulation in both developing and mature rod photoreceptors. Nrl interacts with Crx and other transcription regulatory proteins to synergistically (or antagonistically) modulate the expression of rhodopsin and many rod-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. We have shown that the deletion of Nrl by gene targeting in mice (Nrl -/-) results in complete lack of rod function and rod-specific gene expression; instead, there is enhanced S-cone function indicating a phenotypic transformation. Using the Nrl-promoter to drive enhanced green fluorescent protein (EGFP) in transgenic mice, we have demonstrated that Nrl expression during development corresponds to the genesis of rods. Continued expression of Nrl in mature rods suggests that it also plays a major role in maintaining appropriate expression of genes required for rod function. Our studies have identified Nr2e3 (photoreceptor specific nuclear receptor, PNR) as a direct transcriptional target of Nrl and revealed that Nr2e3 acts synergistically with Nrl and Crx in regulating rod phototransduction genes. In this renewal application, we propose to decipher the Nrl-mediated transcriptional regulatory network(s) in developing and mature rod photoreceptors. The specific aims of the project are as follows: Specific Aim 1: We will identify the genes that are directly regulated by Nrl ("direct targets") using a comprehensive strategy, involving microarray analysis, chromatin immunoprecipitation (CHIP) and promoter activity assays. Specific Aim 2: We will identify transcriptional regulatory proteins that interact with Nrl during early and late stages of rod development and validate the physiological relevance of selected interactions. Specific Aim 3: We will determine whether Nrl is sufficient to induce rod-specific gene expression and generate functional rods, by expressing Nrl in the Nrl -/- retina at temporally distinct stages. Specific Aim 4: We will delineate the function of Nr2e3, a direct target of Nrl, in developing and mature rod photoreceptors. Specific Aim 5: We will define the molecular mechanism(s) that underlie the pathogenesis of retinopathies, caused by mutations in the human NRL and NR2E3 genes. Since mutations in Nrl, its interacting proteins, and their target genes result in retinopathies, it is expected that our studies will reveal significant new insights into retinal diseases. A better understanding of transcriptional regulatory pathways may allow us to experimentally manipulate the expression of specific target gene(s) to correct a disease phenotype.

描述（由申请人提供）：后生动物中的不同细胞表型和功能由基因的差异表达指定。在正确的细胞类型中和在战斗时间对基因的定量精确表达的调节是由有限数量的转录因子的组合和协同（或拮抗）作用介导的。Nrl，首先由PI鉴定，是一个关键的碱性基序-亮氨酸拉链（bZIP）转录因子，现在被确定为发育和成熟的视杆细胞光感受器中基因调控的主要介质。Nrl与Crx和其他转录调节蛋白相互作用以协同地（或拮抗地）调节视紫红质和许多视杆细胞特异性基因的表达。人类NRL基因的突变与视网膜变性疾病有关。我们已经表明，在小鼠中通过基因靶向缺失Nrl（Nrl -/-）导致完全缺乏视杆功能和视杆特异性基因表达;相反，存在增强的S-视锥功能，表明表型转化。在转基因小鼠中使用Nrl启动子驱动增强型绿色荧光蛋白（EGFP），我们已经证明发育期间Nrl表达对应于视杆细胞的发生。Nrl在成熟视杆中的持续表达表明其在维持视杆功能所需的基因的适当表达中也起主要作用。我们的研究已经确定Nr 2 e3（photoreceptor specific nuclear receptor，PNR）为Nrl的直接转录靶点，并揭示了Nr 2 e3与Nrl和Crx在调节视杆细胞光转导基因中的协同作用。在该更新申请中，我们提出破译发育和成熟视杆光感受器中的Nrl介导的转录调控网络。该项目的具体目标如下：具体目标1：我们将使用一种综合策略，包括微阵列分析、染色质免疫沉淀（CHIP）和启动子活性测定，来鉴定由Nrl直接调控的基因（“直接靶点”）。具体目标二：我们将确定与Nrl在杆发育的早期和晚期阶段相互作用的转录调控蛋白，并验证所选相互作用的生理相关性。具体目标3：我们将通过在时间上不同的阶段在Nrl -/-视网膜中表达Nrl来确定Nrl是否足以诱导视杆特异性基因表达并产生功能性视杆。具体目标4：我们将描述Nr 2 e3（Nrl的直接靶标）在视杆细胞光感受器发育和成熟中的功能。具体目标五：我们将定义视网膜病变发病机制的分子机制，由人类NRL和NR 2 E3基因突变引起。由于Nrl、其相互作用蛋白及其靶基因的突变导致视网膜病变，因此预计我们的研究将揭示对视网膜疾病的重要新见解。更好地理解转录调控途径可能使我们能够通过实验操纵特定靶基因的表达来纠正疾病表型。