Regulation of the expression of human organic anion transporter 3 by hepatocyte nuclear factor 1 α/β and DNA methylation
Regulation of the expression of human organic anion transporter 3 by hepatocyte nuclear factor 1 α/β and DNA methylation
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DOI：
10.1124/mol.106.025494
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发表时间：
2006-09-01
影响因子：
3.6
通讯作者：
Sugiyama, Yuichi
中科院分区：
文献类型：
--
作者：
Kikuchi, Ryota;Kusuhara, Hiroyuki;Sugiyama, Yuichi
人有机阴离子转运蛋白3（hOAT 3/SLC 22 A8）主要在肾脏近端小管中表达，并在多种有机阴离子的尿排泄中起主要作用。对hOAT 3的启动子区进行了表征，以阐明hOAT 3组织特异性表达的机制。hOAT 3的最小启动子被鉴定为位于转录起始位点上游约300个碱基对处，其中存在典型的TATA和肝细胞核因子（HNF 1）结合基序，其在啮齿动物Oat 3基因中是保守的。反式激活试验显示，HNF 1 α和HNF 1 β显著增加hOAT 3启动子活性，其中HNF 1 β的反式激活效力低于HNF 1 α。HNF 1结合基序的突变阻止了反式激活。电泳迁移率变动分析证明HNF 1 α/HNF 1 α同源二聚体或HNF 1 α/HNF 1 β异源二聚体与hOAT 3启动子结合。还证明了hOAT 3的启动子活性受到DNA甲基化的抑制。此外，在缺乏内源性HNF 1 α和HNF 1 β表达的人胚肾293细胞中，通过HNF 1 α单独强制表达或HNF 1 α和HNF 1 β同时强制表达以及伴随的DNA去甲基化重新激活hOAT 3的表达，而单独强制表达HNF 1 β不能激活hOAT 3的表达。这表明HNF 1 α/HNF 1 β同源二聚体或HNF 1 α/HNF 1 β异源二聚体和DNA去甲基化对hOAT 3的组成型表达具有协同作用。这些结果表明，hOAT 3的组织特异性表达可能受到遗传（HNF 1 α和HNF 1 β）和表观遗传（DNA甲基化）因素的协同作用的调节。
Human organic anion transporter 3 (hOAT3/SLC22A8) is predominantly expressed in the proximal tubules of the kidney and plays a major role in the urinary excretion of a variety of organic anions. The promoter region of hOAT3 was characterized to elucidate the mechanism underlying the tissue-specific expression of hOAT3. The minimal promoter of hOAT3 was identified to be located approximately 300 base pairs upstream of the transcriptional start site, where there are canonical TATA and hepatocyte nuclear factor (HNF1) binding motifs, which are conserved in the rodent Oat3 genes. Transactivation assays revealed that HNF1 alpha and HNF1 beta markedly increased hOAT3 promoter activity, where the transactivation potency of HNF1 beta was lower than that of HNF1 alpha. Mutations in the HNF1 binding motif prevented the transactivation. Electrophoretic mobility shift assays demonstrated binding of the HNF1 alpha/HNF1 alpha homodimer or HNF1 alpha/HNF1 beta heterodimer to the hOAT3 promoter. It was also demonstrated that the promoter activity of hOAT3 is repressed by DNA methylation. Moreover, the expression of hOAT3 was activated de novo by forced expression of HNF1 alpha alone or both HNF1 alpha and HNF1 beta together with the concomitant DNA demethylation in human embryonic kidney 293 cells that lack expression of endogenous HNF1 alpha and HNF1 beta, whereas forced expression of HNF1 beta alone could not activate the expression of hOAT3. This suggests a synergistic action of the HNF1 alpha/HNF1 beta homodimer or HNF1 alpha/HNF1 beta heterodimer and DNA demethylation for the constitutive expression of hOAT3. These results indicate that the tissue-specific expression of hOAT3 might be regulated by the concerted effect of genetic (HNF1 alpha and HNF1 beta) and epigenetic (DNA methylation) factors.