MOLECULAR MECHANISMS OF RETINA SPECIFIC GENE EXPRESSION

基因在特定组织中的差异表达，例如 视网膜，是通过转录因子的联合作用而实现的。 NRL是一个进化保守的bZIP转录因子， 在我们实验室通过减法克隆鉴定。在成人中，高 只在视网膜中检测到NRL转录本的水平。NRL 蛋白质与转化产物有很强的同源性 癌基因，v-maf。Maf-NRL亚家族的蛋白质识别一个长的 AP-1类似的DNA序列元件被显示为与 体外筛选bZIP蛋白及其与组织特异性基因的关系 监管。有几条证据表明NRL参与了 视紫红质基因调控。潜在的假设是，在 成人视网膜NRL在调节血管紧张素转换酶的表达中起主要作用 基因产物是进行适当筛选所需的 光感受器和其他神经元。由于蛋白质-蛋白质相互作用 是转录活性的主要决定因素，并能产生 在目标地点选择方面具有极大的灵活性，其目标是 一项建议是确定特定与之相互作用的蛋白质 NRL并阐明这些相互作用的生物学相关性。 为了实现这一点，具体目标1建议评估是否有任何 Fos、Jun、Maf-NRL和NF-E2P45的已知bZIP蛋白 亚家族在成人视网膜中表达，并与NRL相互作用 调节基因表达。具体目标2侧重于提纯 和与NRL反应结合的视网膜蛋白的特征 视紫红质启动子中的元件。SPIRATIC AIM 3使用一种酵母菌- 用“NRL-bZIP诱饵”的混合方法来鉴定 与NRL高效异源二聚，可能参与调节 视网膜中的不同组基因。 P.I.S研究的长期目标是了解 参与眼科疾病发病机制的分子事件和 最终协助设计基于基因的治疗策略。 转录因子已成为有吸引力的机制靶点- 基于药物的设计，可用于调节特定的 生化途径。在这方面，建议进行的研究包括 旨在识别转录调节蛋白，这些蛋白一起 NRL介导组织或细胞类型特异性基因表达 视网膜，并应提供对分子的机械性见解 先天性和遗传性视网膜疾病的基础。

Differential expression of genes in a particular tissue, such as retina, is achieved by combinatoriaI action of transcription factors. Nrl is an evolutionarily-conserved bZIP transcription factor, identified in our laboratory by subtraction cloning. In the adult, high levels of Nrl transcripts are detected only in the retina. The Nrl protein shows strong homology to the product of a transforming oncogene, v-maf. The proteins of the Maf-Nrl subfamily recognize a long AP-1 like DNA sequence element, are shown to heterodimerize with selected bZIP proteins in vitro, and implicated in tissue-specific gene regulation. Several lines of evidence suggest that Nrl is involved in rhodopsin gene regulation. The underlying hypothesis is that in the adult retina Nrl plays a major role in regulating the expression of gene products that are needed for the appropriate fiinctioning of photoreceptors and other neurons. Since protein-protein interactions are a major determinant of transcriptional activity and can generate tremendous flexibility in target site selection, the objective of this proposal is to identify the proteins that specifically interact with Nrl and to elucidate the biological relevance of these interactions. To accomplish this, Specific Aim 1 proposes to evaluate whether any of the known bZIP proteins of the Fos, Jun, Maf-Nrl and NF-E2 p45 subfamilies is expressed in the adult retina and interacts with Nrl to modulate gene expression. Specific Aim 2 focuses on the purification and characterization of retinal proteins that bind to the Nrl-response element in the rhodopsin promoter. Specific Aim 3 employs a yeast two- hybrid approach with the "Nrl-bZIP bait" to identify the proteins that productively heterodimerize with Nrl and may be involved in regulating different sets of genes in the retina. The long-term objective of P.I.'s research is to understand the molecular events involved in the pathogenesis of eye diseases and eventually assist in the design of gene-based therapeutic strategies. Transcription factors have become attractive targets for mechanism- based design of drugs, which can be used to modulate specific biochemical pathways. In that direction, the proposed studies are designed to identify transcriptional regulatory proteins that together with Nrl mediate tissue- or cell type- specific gene expression in the retina, and should provide mechanistic insights into the molecular basis of congenital and inherited retinal disorders.