Molecular Mechanisms of Retina-specific Gene Expression

描述（由申请人提供）：在后生动物中，不同的细胞表型和功能是由基因的差异表达指定的。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是一个关键的基本基序-亮氨酸拉链（bZIP）转录因子，现已被确定为发育和成熟杆光感受器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，协同（或拮抗）调节视紫红质和许多杆状特异基因的表达。人类NRL基因突变与视网膜退行性疾病有关。我们已经证明，在小鼠中通过基因靶向缺失Nrl （Nrl -/-）导致棒状细胞功能和棒状细胞特异性基因表达完全缺失；相反，s锥功能增强表明表型转化。利用Nrl启动子在转基因小鼠中驱动增强的绿色荧光蛋白（EGFP），我们已经证明Nrl在发育过程中的表达与杆状细胞的发生相对应。Nrl在成熟杆细胞中的持续表达表明，它在维持杆细胞功能所需基因的适当表达方面也起着重要作用。我们的研究发现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协同调节棒光转导基因。在这个更新的应用中，我们建议破译nrl介导的转录调控网络(s)在发育和成熟的视杆光感受器。该项目的具体目标如下：具体目标1：我们将使用包括微阵列分析、染色质免疫沉淀（CHIP）和启动子活性测定在内的综合策略，确定由Nrl直接调控的基因（“直接靶标”）。具体目标2：我们将确定在杆发育的早期和晚期与Nrl相互作用的转录调节蛋白，并验证所选相互作用的生理相关性。具体目标3：我们将确定Nrl是否足以诱导杆状细胞特异性基因表达并产生功能杆状细胞，通过在时间上不同的阶段在Nrl -/-视网膜中表达Nrl。具体目标4：我们将描述Nrl的直接靶点Nr2e3在杆状光感受器发育和成熟中的作用。具体目标5：我们将定义视网膜病变发病机制的分子机制，由人类NRL和NR2E3基因突变引起。由于Nrl及其相互作用蛋白及其靶基因的突变导致视网膜病变，我们的研究有望为视网膜疾病提供重要的新见解。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。

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.