CHARACTERIZATION OF THE DISTAL ALPHA-FETOPROTEIN ENHANCER, A STRONG, LONG-DISTANCE, LIVER-SPECIFIC ACTIVATOR

High level expression of the alpha-fetoprotein (AFP) gene is controlle d by three upstream enhancers which function even in hepatic cell line s that repress the AFP gene promoter. The most distal (''Complex 3,'' at -6 kilobases) is the strongest in HepG2 cells. We mapped the main a ctivity of Complex 3 to a 170-base pair (BP) region from -6069 to -590 0; progressive deletion of the 5'- and 3'-ends identified an 84-bp seg ment which accounted for 90% of enhancer activity. Expression studies, which combined the deleted Complex 3 with an AFP or tk promoter chlor amphenicol acetyltransferase gene fusion, resolved five regions in the enhancer (Ia, Ib, II, III, and IV). Deletion of Regions Ia or II stro ngly reduced stimulation of the AFP promoter, while Regions Ia and Ib were essential for stimulation of the tk promoter. Footprinting indica ted multiple binding sites in regions Ia, Ib, and II. Gel shift and ol igonucleotide competition demonstrated that Regions Ia and II had high affinity HNF3- and C/EBP-binding sites, respectively, while additiona l unidentified factors bound throughout Regions I-III. Complex 3 is a powerful liver-specific transcriptional regulator and an important mod el of long distance gene activation.