Molecular Mechanisms of Retina-specific Gene Expression

描述(由申请人提供)：后生动物中不同的细胞表型和功能是由基因的不同表达所指定的。在正确的细胞类型和战斗时间，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)作用所介导的。NRL最先由PI鉴定，是一个关键的基本基序-亮氨酸拉链(BZIP)转录因子，目前被认为是发育中和成熟的杆状感光细胞中基因调控的主要中介因子。NRL与CRX和其他转录调节蛋白相互作用，协同(或拮抗)调节视紫红质和许多视杆特异基因的表达。人类NRL基因突变与视网膜退行性疾病有关。我们已经证明，通过基因打靶在小鼠中缺失NRL(NRL-/-)导致完全缺乏杆功能和杆特异性基因表达；相反，存在增强的S锥体功能，表明表型转化。利用NRL启动子在转基因小鼠中驱动增强型绿色荧光蛋白(EGFP)，我们已经证明了NRL在发育过程中的表达与杆状突起的发生相对应。NRL在成熟杆中的持续表达表明，它在维持杆功能所需基因的适当表达方面也发挥了重要作用。我们的研究已经确定Nr2e3(光受体特异性核受体，PNR)是NRL的直接转录靶点，并揭示了Nr2e3与NrL和CRX在调控杆状光导基因方面的协同作用。在这一新的应用中，我们建议破译发育和成熟的杆状感光细胞中的NRL介导的转录调控网络(S)。该项目的具体目标如下：具体目标1：我们将采用综合策略，包括微阵列分析、染色质免疫沉淀(ChIP)和启动子活性分析，确定受NRL(直接靶标)直接调控的基因。具体目标2：我们将鉴定在杆状发育的早期和后期与NRL相互作用的转录调节蛋白，并验证所选相互作用的生理学相关性。具体目标3：我们将通过在时间上不同的阶段在NRL-/-视网膜表达NRL，来确定NRL是否足以诱导杆状特异性基因表达并产生功能棒。具体目标4：我们将描述NRL的直接靶标Nr2e3在发育和成熟的杆状感光细胞中的功能。具体目标5：我们将确定视网膜病变的分子机制(S)，它是由人类NRL和NR2E3基因突变引起的。由于NRL及其相互作用蛋白及其靶基因的突变会导致视网膜病变，预计我们的研究将揭示对视网膜疾病的重大新见解。对转录调控途径的更好理解可能使我们能够通过实验操纵特定靶基因(S)的表达来纠正疾病表型。

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.