Characterization of the human cytochrome P4502D6 promoter - A potential role for antagonistic interactions between members of the nuclear receptor family
Characterization of the human cytochrome P4502D6 promoter - A potential role for antagonistic interactions between members of the nuclear receptor family
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DOI：
10.1074/jbc.271.41.25269
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发表时间：
1996-10-11
影响因子：
4.8
通讯作者：
Wolf, CR
中科院分区：
文献类型：
--
作者：
Cairns, W;Smith, CAD;Wolf, CR
人细胞色素P4502D6(CYP2D6)启动子在HepG2细胞中的功能图谱显示存在正负调控元件。在凝胶滞留分析中发现，这些调控元件中的一个与在大多数CYP2家族成员中高度保守的序列重叠，该元件由一个相隔1个碱基对(DR1)的简并AGGTCA直接重复序列组成，并被认为是类固醇受体超家族成员的靶标。利用HepG2核提取液，在DR1元件上形成了三个蛋白质-DNA复合体，其中一个依赖于HNF4的结合。其他DR1复合体被证明是由于孤儿受体鸡卵蛋白上游启动子转录因子I(COUP-TFI)的相互作用。在COS-7细胞中的实验表明，HNF4可以激活30倍的CYP2D6启动子。令人惊讶的是，DR1元件的突变导致了相对较小的23%的HepG2细胞活性下降。此外，COUP-TFI还能抑制HNF4对COS-7细胞中CYP2D6启动子的刺激作用，提示COUP-TFI可以减弱HNF4对HepG2细胞的抑制作用，但当HNF4在细胞中的表达水平通过共转染而增加时，则导致了CYP2D6启动子活性的增强，表明HNF4可以克服COUP-TFI的抑制作用。因此，DR1元件在控制CYP2D6基因转录中的作用取决于正负作用转录因子之间的平衡。
The functional mapping of the human cytochrome P4502D6 (CYP2D6) promoter in HepG2 cells revealed the presence of both positive and negative regulatory elements. One of these regulatory elements overlapped a sequence that is highly conserved in most members of the CYP2 family, This element, which consists of a degenerate AGGTCA direct repeat spaced by 1 base pair (DR1) and is known to be a target for members of the steroid receptor superfamily, was found to bind in vitro translated hepatocyte nuclear factor 4 (HNF4) in gel retardation analysis. Using HepG2 nuclear extracts, three protein-DNA complexes were formed on the DR1 element, one of which was confirmed to be dependent on the binding of HNF4. The other DR1 complexes were shown to be due to the interaction of the orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI). Experiments in COS-7 cells showed that HNF4 could activate the CYP2D6 promoter 30-fold. Surprisingly, mutation of the DR1 element produced a relatively minor 23% decrease in activity in HepG2 cells. Additionally, COUP-TFI was shown to inhibit HNF4 stimulation of the CYP2D6 promoter in COS-7 cells, suggesting that COUP-TFI could attenuate the effect of HNF4 in HepG2 cells, However, when HNF4 levels were increased in HepG2 cells by co-transfection, it resulted in the enhancement of CYP2D6 promoter activity, indicating that HNF4 could overcome the repressive effect of COUP-TFI. Therefore, the contribution of the DR1 element in controlling the transcription of the CYP2D6 gene depends on the balance between positively and negatively acting transcription factors.