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 beta-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 sequence between -732 and -578 upstream of the liver start of transcription in the human
glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver
glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.