Identification of transacting factors responsible for the tissue-specific expression of human glucose transporter type 2 isoform gene. Cooperative role of hepatocyte nuclear factors 1alpha and 3beta.
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 GLUT2 gene expression. Coexpression of the pGL+74 (+74 to +301) construct with the HNF1alpha and HNF3beta expression vectors in NIH 3T3 cells showed the synergistic effect on GLUT2 promoter activity compared with the expression of HNF1alpha, HNF3beta, or a combination of HNF1beta and HNF3beta. These data suggest that HNF1alpha and HNF3beta may be the most important players in the tissue-specific expression of the human GLUT2 gene.
Cha
J Y
JY
Department of Biochemistry and Molecular Biology and the Institute of Genetic Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea.
Kim
H
H
Kim
K S
KS
Hur
M W
MW
Ahn
Y
Y
D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics; D005786:Gene Expression Regulation; D051273:Glucose Transporter Type 2; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; D051273:Glucose Transporter Type 2; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009004:Monosaccharide Transport Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051542:Hepatocyte Nuclear Factor 3-beta; D051539:Hepatocyte Nuclear Factor 1-beta