Liver-specific Enhancer of the Glucokinase Gene

• Published in: The Journal of biological chemistry 1996-11, Vol.271 (46), p.29113-29120
• Main Authors: Iynedjian, P B, Marie, S, Wang, H, Gjinovci, A, Nazaryan, K
• Format: Article
• Language: English
• Subjects: Animals; Cells, Cultured; DNA Footprinting; Enhancer Elements, Genetic; Gene Expression Regulation, Enzymologic; Glucokinase - genetics; Humans; Liver - enzymology; Male; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Rats; Rats, Wistar; Sequence Deletion; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.46.29113

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between −1000 and −600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between −123 and −34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the β-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between −123 and −34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to −1000 inhibited luciferase expression. The gene fragment from −1003 to −707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The