Identification of transacting factors responsible for the tissue-specific expression of human glucose transporter type 2 isoform gene - Cooperative role of hepatocyte nuclear factors 1 alpha and 3 beta

Abstract

We investigated transacting factors binding to the cis-element important in tissue-specific expression of the human glucose transporter type 2 isoform (GLUT2) gene. By transient transfection assay, we determined that the 227-base pair fragment upstream of the ATG start site contained promoter activity and that the region from +87 to +132 (site C) was responsible for tissue-specific expression. DNase I footprinting and electrophoretic mobility shift assay indicated that site C contained one binding site for hepatocyte nuclear factor 1 (HNF1) and two binding sites for HNF3 The mutations at positions +101 and +103, which are considered to be critical in binding HNF1 and HNF3, resulted in a 53% decrease in promoter activity, whereas the mutation of the proximal HNF3 binding site (+115 and +117) reduced promoter activity by 28%. The mutations of these four sites resulted in marked decrease (70%) in promoter activity as well as diminished bindings of HNF1 and HNF3, A to G mutation, which causes conversion of the HNF1 and HNF3 binding sequence to the NF-Y binding site, resulted in a 22% decrease in promoter activity. We identified that both HNF1 and HNF3 function as transcriptional activators in GLUTS gene expression. Coexpression of the pGL+74 (+74 to +301) construct with the HNF1 alpha and HNF3 beta expression vectors in NIH 3T3 cells showed the synergistic effect on GLUTS promoter activity compared with the expression of HNF1 alpha, HNF3 beta, or a combination of HNF1 beta and HNF3 beta. These data suggest that HNF1 alpha and HNF3 beta may be the most important players in the tissue-specific expression of the human GLUTS gene.