MOLECULAR MECHANISMS OF RETINA-SPECIFIC GENE EXPRESSION

基因的差异表达是生物过程的基础。它是通过相对少量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控机制，识别特定的激活物(和抑制物)并描述这些调控因子如何将信号通路与基础转录机制结合以产生转录反应是至关重要的。我们已经通过消减克隆的方法鉴定了NRL，它是一个碱性基序亮氨酸拉链(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基因突变导致光感受器变性，提示这两种转录因子在体内调节光感受器基因表达方面发挥重要作用。我们的研究还发现，NRL与CRX、TATA结合蛋白以及视网膜中其他尚未鉴定的蛋白相互作用。由于其独特的表达模式，可能受成纤维细胞生长因子-2的调节，以及参与视紫红质的调节，NRL似乎是发育和成熟的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与其他调控蛋白的相互作用负责视网膜基因表达的空间和时间控制。这项建议的目标是使用遗传(酵母双杂交)和生化(免疫和DNA亲和层析)方法识别视网膜中的NRL相互作用蛋白(NIP)，重点描述视紫红质调节(目标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从胚胎视网膜文库中分离NIP(目标2)，并使用基因敲除策略直接检测小鼠的NRL功能(目标4)。转录调控途径的阐明将揭示对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(S)的功能来上调或下调特定的靶基因，并纠正疾病的表型。

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.