Novel Transcriptional Regulation of the Human CYP3A7Gene by Sp1 and Sp3 through Nuclear Factor κB-like Element
Novel Transcriptional Regulation of the Human CYP3A7Gene by Sp1 and Sp3 through Nuclear Factor κB-like Element
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Sp1 和 Sp3 通过核因子 κB 样元件对人类 CYP3A7 基因的新转录调控
DOI：
10.1074/jbc.m106130200
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发表时间：
2001
影响因子：
4.8
通讯作者：
T. Kamataki
中科院分区：
文献类型：
--
作者：
Tetsuya Saito;Yoshiki Takahashi;H. Hashimoto;T. Kamataki
人CYP 3A 7和CYP 3A 4基因5′端同源性达90%，但在胎儿和成人肝脏中均有表达。本研究的目的是阐明CYP 3A 7基因在具有胎儿表型的人肝癌HepG 2细胞中的转录调控机制。使用一系列CYP 3A 7或CYP 3A 4启动子-荧光素酶嵌合基因的转染研究发现，在核苷酸−2326和−2297之间存在一个核因子κB（NF-κB）样元件，该元件赋予CYP 3A 7基因的转录激活。CYP 3A 4基因相应区域内的1个碱基对错配足以产生差异增强子活性。使用HepG 2细胞核提取物的凝胶迁移试验表明，Sp1和Sp3与CYP 3A 7基因的NF-κ B样元件结合，但不与CYP 3A 4基因结合。通过将p3 A7 NF-κB或p3 A4 NF-κB报告基因与Sp1或Sp3表达质粒共转染缺乏内源性Sp家族的果蝇细胞，证实了Sp1和Sp3对CYP 3A 7启动子的特异性激活作用。此外，在肝细胞核因子3β、上游刺激因子1和近端启动子中的基本转录元件的结合位点引入突变，使荧光素酶活性降低至完整CYP 3A 7启动子水平的20%。因此，我们得出结论，CYP 3A 7基因在HepG 2细胞中的表达是由Sp1，Sp3，肝细胞核因子3β和上游刺激因子1协同调节的。
Abstract Human CYP3A7 and CYP3A4 are expressed in fetal and adult livers, respectively, although the 5′-flanking regions of the two genes show 90% homology. The purpose of this study was to clarify the mechanism(s) responsible for the transcriptional regulation of theCYP3A7 gene in human hepatoma HepG2 cells that showed fetal phenotypes. Transfection studies using a series of theCYP3A7 or CYP3A4 promoter-luciferase chimeric genes identified a nuclear factor κB (NF-κB)-like element between nucleotides −2326 and −2297 that conferred the transcriptional activation of the CYP3A7 gene. A 1-base pair mismatch within the corresponding region of the CYP3A4 gene was sufficient for a differential enhancer activity. A gel shift assay using nuclear extracts from HepG2 cells showed that Sp1 and Sp3 bound to the NF-κB-like element of the CYP3A7 but notCYP3A4 gene. Specific activation of the CYP3A7promoter by Sp1 and Sp3 was confirmed by a co-transfection of the p3A7NF-κB or p3A4NF-κB reporter gene with Sp1 or Sp3 expression plasmid into Drosophila cells, which lacked endogenous Sp family. Additionally, introduction of mutations into binding sites for hepatocyte nuclear factor 3β, upstream stimulatory factor 1, and a basic transcription element in the proximal promoter attenuated luciferase activity to 20% of the level seen with the intactCYP3A7 promoter. Thus, we conclude that the expression of the CYP3A7 gene in HepG2 cells is cooperatively regulated by Sp1, Sp3, hepatocyte nuclear factor 3β, and upstream stimulatory factor 1.