A conserved nuclear receptor consensus sequence (DR-4) mediates transcriptional activation of the chicken CYP2H1 gene by phenobarbital in a hepatoma cell line
A conserved nuclear receptor consensus sequence (DR-4) mediates transcriptional activation of the chicken CYP2H1 gene by phenobarbital in a hepatoma cell line
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DOI：
10.1074/jbc.275.18.13362
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发表时间：
2000-05-05
影响因子：
4.8
通讯作者：
Meyer, UA
中科院分区：
文献类型：
--
作者：
Handschin, C;Meyer, UA
通过报告基因分析，在鸡肝癌细胞系（来亨鸡雄性肝癌（LMH））的CYP 2 H1基因5 '侧翼区发现了苯巴比妥反应性DNA元件，一个264 bp的增强子序列（PBRU）对苯巴比妥和多种苯巴比妥类诱导剂有反应。在264 bp的元件内的假定的转录因子结合位点的分析揭示了一个核受体半位点重复序列（DR-4）邻近一个假定的核因子-1位点。该基序类似于三个苯巴比妥诱导基因（大鼠CYP 2B 2、小鼠Cyp 2b 10和人CYP 2B 6）侧翼区域的苯巴比妥反应元件。DR-4六聚体半位点的定点诱变后，264-bp元件的激活被消除。该识别位点进化保守的证据是通过在LMH细胞中激活小鼠Cyp 2b 10苯巴比妥响应增强子来表明的，该增强子由激活CYP 2 H1 264-bp PBRU的相同诱导剂谱来激活。冈田酸对这种激活的抑制可以解释这种磷酸酶抑制剂对CYP 2B 1/2和Cyp 2b 10诱导的抑制作用。我们发现，这种抑制作用直接发生在264 bp PBRU，而近端启动子的CYP 2 H1诱导冈田酸在报告基因测定。这些实验利用肝癌衍生细胞系LMH的独特苯巴比妥诱导。
Phenobarbital-responsive DNA elements were identified in the 5'-flanking region of the chicken CYP2H1 gene by in reporter gene assays in a chicken hepatoma cell line (leghorn male hepatoma (LMH)), A 264-base pair (bp) enhancer sequence (phenobarbital-responsive unit (PBRU)) responded to phenobarbital and a variety of phenobarbital-type inducers. Analysis of putative transcription factor binding sites within the 264-bp element revealed a nuclear receptor half-site repeat (DR-4) neighboring a putative nuclear factor-1 site. This motif resembles phenobarbital response elements in the flanking regions of three phenobarbital-inducible genes, rat CYP2B2, mouse Cyp2b10 and human CYP2B6. Activation of the 264-bp element was eliminated after site-directed mutagenesis of the DR-4 hexamer half-sites. Evidence for evolutionary conservation of this recognition site was indicated by activation in LMH cells of a mouse Cyp2b10 phenobarbital-responsive enhancer by the same spectrum of inducers that activate the CYP2H1 264-bp PBRU. Inhibition of this activation by okadaic acid may explain the reported inhibitory effects on induction of CYP2B1/2 and Cyp2b10 by this phosphatase inhibitor. We show that this inhibition occurs directly on the 264-bp PBRU, whereas the proximal promoter of CYP2H1 is induced by okadaic acid in reporter gene assays. These experiments exploit the unique phenobarbital inducibility of the hepatoma-derived cell line LMH.