Regulation of the expression of human organic anion transporter 3 by hepatocyte nuclear factor 1alpha/beta and DNA methylation.
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 HNF1alpha and HNF1beta markedly increased hOAT3 promoter activity, where the transactivation potency of HNF1beta was lower than that of HNF1alpha. Mutations in the HNF1 binding motif prevented the transactivation. Electrophoretic mobility shift assays demonstrated binding of the HNF1alpha/HNF1alpha homodimer or HNF1alpha/HNF1beta 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 HNF1alpha alone or both HNF1alpha and HNF1beta together with the concomitant DNA demethylation in human embryonic kidney 293 cells that lack expression of endogenous HNF1alpha and HNF1beta, whereas forced expression of HNF1beta alone could not activate the expression of hOAT3. This suggests a synergistic action of the HNF1alpha/HNF1alpha homodimer or HNF1alpha/HNF1beta 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 (HNF1alpha and HNF1beta) and epigenetic (DNA methylation) factors.
Kikuchi
Ryota
R
Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kusuhara
Hiroyuki
H
Hattori
Naka
N
Shiota
Kunio
K
Kim
Insook
I
Gonzalez
Frank J
FJ
Sugiyama
Yuichi
Y
D000595:Amino Acid Sequence; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D019295:Computational Biology; D019175:DNA Methylation / Q000187:drug effects; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000378:metabolism*; D051539:Hepatocyte Nuclear Factor 1-beta / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D029362:Organic Anion Transporters, Sodium-Independent / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*
D051538:Hepatocyte Nuclear Factor 1-alpha; D029362:Organic Anion Transporters, Sodium-Independent; D012097:Repressor Proteins; C114338:organic anion transport protein 3; D051539:Hepatocyte Nuclear Factor 1-beta; D001374:Azacitidine