Functional analysis of the trans-acting factor binding sites of the mouse alpha-fetoprotein proximal promoter by site-directed mutagenesis.

The trans-acting factors of the mouse alpha-fetoprotein proximal promoter (-202 base pairs) are aligned as follows: regions Ia (HNF-1), Ib (C/EBP), II (NF-1 or C/EBP), II' (NF-1 or HNF-1), III (NP-III), IV (NP-IV), Va (NP-Va), and Vb (C/EBP). Site-specific mutation abolished protein binding to the corresponding mutated site with the exception of the NF-1 site, in which mutation causes partial protection. Transient expression analyses indicate that chloramphenicol acetyl-transferase (CAT) activity is reduced by mutations in regions Ia, II', Ib, II, and IV. Mutation of region III causes an increased activity and mutation of regions Va and Vb shows a slight inhibitory effect. Linking alpha-fetoprotein enhancer I to the wild type promoter resulted in a 12-fold stimulation of CAT activity. The activity of promoters with mutated C/EBP-binding sites (Ib, II, and Vb), was slightly above controls, indicating that enhancer I can reverse the effect of these mutations. Inhibition or stimulation of promoter activity resulting from mutations of the HNF-1 or NP-III binding sites, respectively, persisted when enhancer I was linked to the promoters, indicating that enhancer I cannot rescue these mutations. Mutation of both HNF-1-binding sites resulted in greater than 90% inhibition of CAT expression with and without enhancer I, indicating these sites are essential for promoter activity. The stimulation of promoter activity by mutation of the NP-III site suggests that this site may be essential for repression or attenuation of the alpha-fetoprotein gene. Our studies indicate that regulation of the alpha-fetoprotein gene requires the combinatorial effect of multiple cis- and trans-acting elements in the proximal promoter and that enhancer I may provide a factor(s) that specifically rescue the promoter from the inhibitory effect of mutation in the C/EBP-binding sites.