Kumar R, Chen S, Scheurer D, Wang Q L, Duh E, Sung C H, Rehemtulla A, Swaroop A, Adler R, Zack D J
Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9277, USA.
J Biol Chem. 1996 Nov 22;271(47):29612-8. doi: 10.1074/jbc.271.47.29612.
In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp. Activity of the -40 to -84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.
体外DNA结合试验以及用猴肾细胞进行的瞬时转染分析表明，bZIP转录因子的Maf-Nrl亚家族成员Nrl和Nrl反应元件（NRE）参与视紫红质表达的调控。我们现在进一步探究了NRE和周围启动子元件的作用。以整合的NRE和侧翼DNA为诱饵进行酵母单杂交筛选，获得的主要克隆是牛Nrl。筛选中截短克隆的回收表明，Nrl的羧基末端一半包含碱性和亮氨酸拉链结构域，足以进行DNA结合。为了从功能上剖析视紫红质启动子，我们用原代鸡视网膜细胞培养物进行了瞬时表达研究。缺失和突变分析确定了两个正调控序列：一个在-40至-84碱基对（bp）之间，另一个在-84至-130 bp之间。-40至-8区域的活性很大程度上归因于NRE。与NRL表达载体共转染时，含有完整NRE的视紫红质启动子构建体的活性增加了3至5倍，但对含有突变或缺失NRE的视紫红质启动子几乎没有影响。在刺激视紫红质启动子活性方面，Nrl比相关的bZIP蛋白c-Fos和c-Jun更有效。-84至-130 bp区域与NRE协同作用，增强基础表达水平和Nrl介导的反式激活程度。这些研究支持Nrl作为体内视紫红质表达的调节因子，确定了NRE上游的另一个调控区域，并证明了原代视网膜细胞培养物在表征调节光感受器基因表达的顺式作用反应元件和反式作用因子方面的实用性。
