SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity.
SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity.
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DOI：
10.1016/s0021-9258(19)36544-5
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发表时间：
1993-09
期刊：
影响因子：
--
通讯作者：
M. Cornwell;D. Smith
中科院分区：
文献类型：
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作者：
M. Cornwell;D. Smith
为了确定影响转录的人多药耐药（MDR 1）启动子序列，我们使用荧光素酶作为报告基因测量了MDR 1启动子构建体的活性。缺失启动子序列至-121（相对于转录起始位点）对瞬时转染细胞中的启动子活性几乎没有影响。进一步缺失至-88使活性增加约4倍，而缺失至-51使活性降低约10倍。数据表明，在-121和-88之间以及在-73和-51之间是调节启动子活性的序列。由于这两个区域含有对其他启动子的功能很重要的富G序列，因此测量了富G区域（-110至-103和-61至-43）突变对MDR 1启动子活性的影响。-110 G-box的突变（-110到-103）导致启动子活性增加6倍，并抑制特异性核蛋白复合物的形成，表明该区域在循环细胞中作为转录“阻遏物”结合位点发挥作用。相比之下，-50 G盒的突变（-61至-43）使启动子活性降低5倍。DNA酶-I足迹和电泳迁移率变动分析表明，具体的，但不同的蛋白质-DNA复合物形成在这两个富含G的区域。-50 G盒含有SP1和EGR-1特异性结合的重叠位点。将MDR 1启动子构建体与SP1共转染到缺乏SP1的细胞（果蝇Schneider 2细胞）中，等同地激活了野生型MDR 1启动子和含有MDR 1 - 50 GC盒和MDR 1起始元件的合成启动子。
To define sequences in the human multidrug resistance (MDR1) promoter that influence transcription, we measured the activity of MDR1 promoter constructs using luciferase as a reporter gene. Deletion of promoter sequences to -121 (relative to the transcription initiation site) had very little effect on promoter activity in transiently transfected cells. Further deletion to -88 increased activity about 4-fold, while deletion to -51 decreased activity about 10-fold. The data indicated that between -121 and -88 and between -73 and -51 were sequences that modulated promoter activity. Because these two regions contain G-rich sequences, which are important for function in other promoters, the effect of mutation of the G-rich regions (-110 to -103 and -61 to -43) on MDR1 promoter activity was measured. Mutation of the -110 G-box (-110 to -103) resulted in a 6-fold increase in promoter activity and inhibited the formation of a specific nuclear protein complex, suggesting that this region functions as a transcriptional “repressor” binding site in cycling cells. In contrast, mutation of the -50 G-box (-61 to -43) reduced promoter activity 5-fold. DNase-I footprinting and electrophoretic mobility shift analysis indicated that specific but distinct protein-DNA complexes formed at each of these two G-rich regions. The -50 G-box contains overlapping sites to which both SP1 and EGR-1 bind specifically. Co-transfection of MDR1 promoter constructs with SP1 into cells that lack SP1 (Drosophila Schneider 2 cells) activated equivalently both the wild type MDR1 promoter and a synthetic promoter containing the MDR1 -50 GC box and MDR1 initiator element.