MOLECULAR MECHANISMS OF RETINA-SPECIFIC GENE EXPRESSION

基因的差异表达是生物过程的基础。它是由相对较少的转录因子的组合和协同（或拮抗）作用完成的。为了阐明视网膜中的转录调控机制，必须确定特定的激活因子（和抑制因子），并描述这些调节因子如何将信号通路与基础转录机制整合以产生转录反应。我们之前已经通过减法克隆确定了基本基序-亮氨酸拉链（bZIP）家族的转录因子Nrl。Nrl与光受体特异性转录因子Crx协同调节视紫红质启动子活性。人类CRX或NRL基因的突变导致光感受器变性，表明这两种转录因子在体内调节光感受器基因表达中起主要作用。我们的研究还揭示了Nrl与视网膜中Crx、tata结合蛋白和其他尚未表征的蛋白相互作用。由于其独特的表达模式，可能受到FGF-2的调节，并参与视紫红质调节，Nrl似乎是发育和成熟视网膜转录反应的关键介质。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视网膜中基因表达的空间和时间控制的原因。本提案的目标是使用遗传（酵母双杂交）和生化（免疫和dna亲和层析）方法鉴定视网膜中的Nrl相互作用蛋白（NIPs），重点是描述视紫红质调控（Aims 1-3）。为了评估Nrl在视网膜发育中的作用，我们建议通过酵母双杂交方法（Aim 2）从胎儿视网膜文库中分离NIPs，并使用基因敲除策略直接检测Nrl在小鼠中的功能（Aim 4）。转录调控途径的阐明将揭示视网膜发育和疾病的重要新见解。由于视网膜转录因子及其靶基因的突变导致视网膜病变，因此有可能通过实验操纵特定转录因子的功能来上调或下调特定靶基因并纠正疾病表型。

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.