Distant enhancers stimulate the albumin promoter through complex proximal binding sites

The albumin-alpha-fetoprotein locus epitomizes the main features of transcr iptional regulation of fetal and adult hepatocyte-specific genes: developme ntally regulated promoters and strong distant enhancers. Full enhancer acti vity required only a proximal albumin-promoter region containing the TATA b ox, hepatic nuclear factor 1 (HNF1), and nuclear factor Y (NF-Y) sites. Del etion of the HNF1 site abrogated enhancer and promoter activity, whereas me thylation of the site reduced all activity by about 3-fold. Deletion of the NF-Y site attenuated activity by about half, but much of the activity coul d be replaced by juxtaposition of an upstream region (designated distal ele ment TV). Gel shift and competition analysis demonstrated that binding of a rchitectural factors overlapped NF-Y binding. Moreover, a mutation that eli minated NF-Y binding but only minimally perturbed the surrounding region di d not affect enhancer function. In plasmids with a second promoter, the enh ancers simultaneously stimulated both albumin and Lu-fetoprotein promoters with minimal competition, but surprisingly some mutations in the albumin pr omoter attenuated expression from both promoters, whereas another uncoupled their expression. With single promoters, the function of the proximal prom oter region was controlled by three parameters in the following hierarchy: HNF1 binding > local architecture > NF-Y binding, but integrated two-promot er function had a much greater dependence on NF-Y.