To identify the sequence of the negative regulatory element, gel retardation and DNase I footprint assays were performed using nuclear proteins from mouse liver and from a hepatoma cell line, H4IIE-C3.
DNase I footprinting and competition gel retardation assays showed that a sequence with an AT-rich core (AT motif) in the pre-S1 promoter region interacts with AFP1, a hepatoma nuclear factor that binds to the alpha-fetoprotein enhancer and promoter.
Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70.