Hepatocyte Nuclear Factor-1α Recruits the Transcriptional Co-Activator p300 on the GLUT2 Gene Promoter
- Nobuhiro Ban13,
- Yuichiro Yamada1,
- Yoshimichi Someya1,
- Kazumasa Miyawaki1,
- Yu Ihara1,
- Masaya Hosokawa1,
- Shinya Toyokuni2,
- Kinsuke Tsuda13 and
- Yutaka Seino1
- 1Department of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Japan
- 2Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Japan
- 3Graduate School of Human and Environmental Studies, Kyoto University, Japan
Mutations in the hepatocyte nuclear factor (HNF)-1α gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1α via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1α expression plasmid into a pancreatic β-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1α enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1α binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1α (amino acids 391–540) to interact with both the NH2-terminal region (amino acids 180–662) and the COOH-terminal region (amino acids 1,818–2,079) of p300. These findings demonstrated that HNF-1α binds to the 5′-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1α. In addition, these results provided new insight into the regulatory function of HNF-1α by suggesting a molecular basis for human GLUT2 gene expression.
Address correspondence and reprint requests to Yutaka Seino, M.D., Ph.D., Department of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan. E-mail:.
Received for publication 20 April 2001 and accepted in revised form 14 January 2002.
ABC, avidin-biotin complex; CBP, CREB-binding protein; CREB; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility shift assay; β-gal, β-galactosidase; GSIS, glucose-stimulated insulin secretion; GST, glutathione S-transferase; HAT, histone acetyltransferase; HNF, hepatocyte nuclear factor; MODY, maturity-onset diabetes of the young; P/CAF, p300/CBP-associated factor; PMSF, phenylmethylsulfonyl fluoride; SRC, steroid receptor co-activator; TBS, Tris-buffered saline; TBS-T, TBS containing 0.1% Tween-20; VP16, herpes virus acidic activation region.