Functional mapping of tissue-specific elements of the human alpha-fetoprotein gene enhancer

Serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC) patients and expression of the protein in cultured HCC cell lines are highly variable. These observations may arise from features correlated with tissue-specific expression of the gene. Extremely strong and potent liver-specific enhancer activity is confined from -4.1 to -3.3 kb upstream to the human AFP gene in contrast with that of the rodent which exists in three widely separated regions. To understand the tissue-specific expression of AFP, we examined cis-acting elements in the enhancer. Results revealed binding sites for selected liver-enriched transcription factors (LETFs) in both domains A (-4120 to -3756 bp) and B (-3492 to -3300 bp) of the gene. These sites included: one hepatocyte nuclear factor (HNF)-1 and HNF-4, two HNF-3, and two C/EBP binding sites in domain A. An adjacent domain B contained one HNF-3 site and three C/EBP sites plus a previously identified HNF-1 site. Each of these elements alone has the ability to stimulate heterogeneous promoter activity in a dose-dependent manner when transfected into AFP producing cells. A comparative study showed that the presence of two HNF-1 and one HNF-4 site is a characteristic feature of human but not rodent AFP enhancer. The mRNA levels of the liver-enriched transcription factors (LETFs) were variable in individual HCC cell lines and together with silencer activities may underlie differential expression of the AFP gene.