LIVER-SPECIFIC ENHANCER OF THE GLUCOKINASE GENE

Glucokinase gene regions that are important for liver specific express ion of the enzyme have been functionally identified using transient tr ansfection of rat hepatocytes. Maximal luciferase activity was elicite d by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flankin g 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 bac kground level, occurred upon deletion of a 90-base pair sequence betwe en -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 in sulinoma cells, and conversely reporters with the beta-cell-specific p romoter were ineffective in primary hepatocytes. In FTO-2B hepatoma ce lls, a differentiated line expressing many liver-specific traits but n ot the endogenous glucokinase gene, the promoter proximal region betwe en -123 and -34 markedly stimulated the expression of transfected plas mids 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 TAT A 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 a cted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Bot h 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 thr ough 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 ab rogated the activity of the half enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upst ream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat en hancer. The human gene fragment ligated to the minimal rat liver gluco kinase promoter was shown to work as an enhancer in the hepatocyte tra nsfection system.