Ret 4, a positive acting rhodopsin regulatory element identified using a bovine retina in vitro transcription system
Ret 4, a positive acting rhodopsin regulatory element identified using a bovine retina in vitro transcription system
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DOI：
10.1074/jbc.271.45.28549
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发表时间：
1996-11-08
影响因子：
4.8
通讯作者：
Zack, DJ
中科院分区：
文献类型：
--
作者：
Chen, SM;Zack, DJ
先前的转基因小鼠研究表明，牛视紫红质序列之间的-222和+70碱基对（bp）包含一个最小的启动子，这是足以指导感光细胞特异性表达的lacZ报告基因。为了更全面地确定参与调节视紫红质转录的DNA调控元件和蛋白质因子，我们已经开发了一种来自牛视网膜核提取物的体外转录系统。视网膜提取物相比，肝脏，HeLa，和果蝇胚胎细胞提取物，表现出优先活性的视紫红质启动子。跨越牛视紫红质上游区-590至+15 bp的模板显示相对于用含有-38至+15 bp模板的TATA盒观察到的基础活性的显著活化。缺失分析表明，-85和-38 bp之间的区域具有显著的正调控活性。用HeLa提取物未观察到这种活性，表明其可能是视网膜特异性的。对-64 ~-38 bp的亚区进行系统性定点突变，结果表明-60 ~-58 bp和-48 ~-40 bp的序列含有关键元件。前一个序列是视网膜特异性转录因子Nrl的结合位点的一部分，该转录因子与视紫红质调节有关。电泳迁移率变动分析表明，后者的序列（-48至-40 bp），和侧翼DNA，指定为Ret 4，结合视网膜特异性和普遍表达的蛋白质因子。突变体寡聚体的转移测定进一步确定了这些蛋白质因子的推定识别序列。总之，我们的研究结果表明，多个启动子元件和转录因子参与调节光受体特异性视紫红质转录。
Previous transgenic mouse studies demonstrated that the bovine rhodopsin sequence between -222 and +70 base pairs (bp) contains a minimal promoter, which is sufficient to direct photoreceptor cell-specific expression of a lacZ reporter gene. To more fully define the DNA regulatory elements and protein factors involved in regulating rhodopsin transcription, we have developed an in vitro transcription system derived from bovine retinal nuclear extracts. Retinal extracts, as compared to liver, HeLa, and Drosophila embryonic cell extracts, demonstrated preferential activity for the rhodopsin promoter. A template spanning the bovine rhodopsin upstream region from -590 to +15 bp showed significant activation relative to the basal activity seen with a TATA box containing -38 to +15 bp template. Deletion analysis indicated that the region between -85 and -38 bp contained significant positive regulatory activity. This activity was not observed with HeLa extracts, suggesting that it might be retina-specific. Systematic site-directed mutagenesis of the subregion from -64 to -38 bp indicated that sequences between -60 and -58 bp and between -48 and -40 bp harbor critical elements. The former sequence is part of the binding site for the retina-specific transcription factor Nrl, which has been implicated in rhodopsin regulation. Electrophoretic mobility shift assays showed that the latter sequence (-48 to -40 bp), and flanking DNA, designated Ret 4, is bound by both retina-specific and ubiquitously expressed protein factors. Shift assays with mutant oligomers further defined the putative recognition sequences for these protein factors. Together, our results suggest that multiple promoter elements and transcriptional factors are involved in regulating photoreceptor-specific rhodopsin transcription.