Hepatocyte Nuclear Factor 1 Regulates the Expression of the Organic Cation Transporter 1 via Binding to an Evolutionary Conserved Region in Intron 1 of the OCT1 Gene
Hepatocyte Nuclear Factor 1 Regulates the Expression of the Organic Cation Transporter 1 via Binding to an Evolutionary Conserved Region in Intron 1 of the OCT1 Gene
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DOI：
10.1124/jpet.113.206359
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发表时间：
2013-10-01
影响因子：
3.5
通讯作者：
Tzvetkov, Mladen V.
中科院分区：
文献类型：
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作者：
O'Brien, Valerie P.;Bokelmann, Kristin;Tzvetkov, Mladen V.
有机阳离子转运蛋白1 (OCT1)，也被称为溶质载体家族22成员1，在人类肝脏中有强烈的特异性表达。本研究表明，肝细胞核因子1 （HNF1）调节OCT1转录，并促进OCT1在肝脏的强烈特异性表达。生物信息学分析显示，在OCT1基因内含子1的进化保守区（ECR）中，HNF1结合基序具有很强的保守性。电泳迁移转移和染色质免疫沉淀试验证实了HNF1与内含子1 ECR的特异性结合。在HepG2细胞中进行的报告基因检测中，内含子1 ECR使SV40启动子活性提高了22倍，使OCT1启动子活性提高了13倍。当内含子1 ECR中的HNF1结合位点发生突变或内源性HNF1 α表达被小干扰RNA下调时，这种增加被逆转。在Huh7细胞中过表达HNF1 α后，内含子1 ECR使SV40启动子活性提高了11倍，使OCT1启动子活性提高了6倍。如果没有过表达HNF1 α，则分别仅增加3倍和2倍。最后，在人肝脏样本中，HNF1高表达与OCT1高表达显著相关（r = 0.48, P = 0.002, n - 40）。综上所述，HNF1是OCT1表达的强调节因子。影响HNF1活性的遗传变异、疾病状况或药物是否会影响oct1转运药物（如吗啡、托烷司琼、昂丹西琼、曲马多和二甲双胍）的药代动力学和疗效仍有待确定。除了OCT1，本研究还证明了物种间比较在发现功能相关基因组序列方面的有效性和实用性。
The organic cation transporter 1 (OCT1), also known as solute carrier family 22 member 1, is strongly and specifically expressed in the human liver. Here we show that the hepatocyte nuclear factor 1 (HNF1) regulates OCT1 transcription and contributes to the strong, liver-specific expression of OCT1. Bioinformatic analyses revealed strong conservation of HNF1 binding motifs in an evolutionary conserved region (ECR) in intron 1 of the OCT1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the specific binding of HNF1 to the intron 1 ECR. In reporter gene assays performed in HepG2 cells, the intron 1 ECR increased SV40 promoter activity by 22-fold and OCT1 promoter activity by 13-fold. The increase was reversed when the HNF1 binding sites in the intron 1 ECR were mutated or the endogenous HNF1 alpha expression was downregulated with small interfering RNA. Following HNF1 alpha overexpression in Huh7 cells, the intron 1 ECR increased SV40 promoter activity by 11-fold and OCT1 promoter activity by 6-fold. Without HNF1 alpha overexpression, the increases were only 3- and 2-fold, respectively. Finally, in human liver samples, high HNF1 expression was significantly correlated with high OCT1 expression (r = 0.48, P = 0.002, n - 40). In conclusion, HNF1 is a strong regulator of OCT1 expression. It remains to be determined whether genetic variants, disease conditions, or drugs that affect HNF1 activity may affect the pharmacokinetics and efficacy of OCT1-transported drugs such as morphine, tropisetron, ondansetron, tramadol, and metformin. Beyond OCT1, this study demonstrates the validity and usefulness of interspecies comparisons in the discovery of functionally relevant genomic sequences.