STRUCTURAL AND FUNCTIONAL ANALYSES OF THE PROMOTER OF THE MURINE MULTIDRUG RESISTANCE GENE MDR3/MDRLA REVEAL A NEGATIVE ELEMENT CONTAINING THE AP-1 BINDING-SITE
STRUCTURAL AND FUNCTIONAL ANALYSES OF THE PROMOTER OF THE MURINE MULTIDRUG RESISTANCE GENE MDR3/MDRLA REVEAL A NEGATIVE ELEMENT CONTAINING THE AP-1 BINDING-SITE
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DOI：
10.1089/dna.1991.10.639
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发表时间：
1991-11-01
影响因子：
3.1
通讯作者：
KUO, MT
中科院分区：
文献类型：
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作者：
IKEGUCHI, M;TEETER, LD;KUO, MT
我们以前已经表明，多药耐药/P-糖蛋白基因，mdr 3/mdr 1a，在小鼠肝细胞癌（HCC）中被激活。在这项研究中，我们表明在许多肝癌衍生细胞系（Hepa 1c 1 c、Hepa 1c 1 c-BprC 1和Hepa 1 -6）中，mdr 3以高水平表达。为了研究mdr 3在这些细胞中的转录调控，我们分离了含有小鼠mdr 3基因5'端部分的DNA片段，并对其启动子进行了功能分析。使用不同长度的启动子序列指导氯霉素乙酰转移酶（CAT）报告基因的表达的瞬时转染试验表明，位于小鼠mdr 3转录起始位点上游约94个核苷酸的序列在小鼠肝癌细胞中起负元件的作用。位于-117处的典型AP-1结合序列TGA-GTCA至少部分地负责来自以下观察结果的负面效应：（i）通过定点诱变改变该AP-1序列增强CAT表达。(ii)当含有AP-1序列而非突变序列的双链DNA用作共转染实验中的竞争者时，CAT报告基因的表达升高。(iii)CAT表达的增强也见于使用表达c-jun/c-fos蛋白的重组质粒DNA的共转染实验中，所述c-jun/c-fos蛋白与AP-1序列相互作用。有趣的是，仓鼠pgp 1启动子的近端区域与小鼠mdr 3基因的序列具有惊人的相似性，包括AP-1位点，但仓鼠启动子中的AP-1位点作为正调控因子。虽然以前的研究已经证明，积极和消极的转录因子可以通过与c-jun/c-fos的相互作用来调节基因表达，这是第一个研究表明，AP-1位点作为一个负顺式元件在基因表达的调控。
We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.