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

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