Analysis of transcriptional network in pancreatic β-cells

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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 coactivator 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 NH_2-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 coactivator for HNF-1α. In addition, these results provided new insight into the regulatory function of HNF-1α or by suggesting a molecular basis for human GLUT2 gene expression.