Characterization of the distal alpha-fetoprotein enhancer, a strong, long distance, liver-specific activator.

High level expression of the alpha-fetoprotein (AFP) gene is controlled by three upstream enhancers which function even in hepatic cell lines that repress the AFP gene promoter. The most distal ("Complex 3," at -6 kilobases) is the strongest in HepG2 cells. We mapped the main activity of Complex 3 to a 170-base pair (BP) region from -6069 to -5900; progressive deletion of the 5'- and 3'-ends identified an 84-bp segment which accounted for 90% of enhancer activity. Expression studies, which combined the deleted Complex 3 with an AFP or tk promoter chloramphenicol acetyltransferase gene fusion, resolved five regions in the enhancer (Ia, Ib, II, III, and IV). Deletion of Regions Ia or II strongly reduced stimulation of the AFP promoter, while Regions Ia and Ib were essential for stimulation of the tk promoter. Footprinting indicated multiple binding sites in regions Ia, Ib, and II. Gel shift and oligonucleotide competition demonstrated that Regions Ia and II had high affinity HNF3- and C/EBP-binding sites, respectively, while additional unidentified factors bound throughout Regions I-III. Complex 3 is a powerful liver-specific transcriptional regulator and an important model of long distance gene activation.