AN ARRAY OF BINDING-SITES FOR HEPATOCYTE NUCLEAR FACTOR-4 OF HIGH ANDLOW AFFINITIES MODULATES THE LIVER-SPECIFIC ENHANCER FOR THE HUMAN ALPHA(1)-MICROGLOBULIN BIKUNIN PRECURSOR/

alpha(1)-Microglobulin and bikunin are two plasma glycoproteins encode d by a gene for alpha(1)-microglobulin/bikunin precursor (AMBP). The s trict liver-specific transcription of the AMBP gene is controlled by a n elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enrich ed nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respect ively, Three further boxes, 7 to 9, have now been found in the enhance r area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respect ively. Electrophoretic mobility-shift assays with nuclear extracts fro m the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are bot h functional HNF-4-binding sites of high and low affinity respectively , whereas no binding capacity of box 9 was detected by this method. Tr ansfection of HepG2 and Chinese hamster ovary cells with chloramphenic ol acetyltransferase constructs harbouring the core or extended AMBP e nhancer with wild-type or mutated boxes and co-transfection with expre ssion plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The resp onse of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 e xhibits a repressor activity that can be detected when box 8 is ablate d. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce t he view that the AMBP enhancer is a quite elaborate and unusual exampl e of a modular enhancer whose activity is fine-tuned by the level of c ognate nuclear factors in the cell.