FIG. 2.

SnoN, but not Ski, mediates repression of AFP expression. (A) SnoN-mediated transcription repression is compromised by disruption of p53 DNA binding. (Top) Hepa 1-6 hepatoma cells were transfected with AFP/lacZ (wild-type sequence for p53 binding) or DelA/lacZ (mutated for p53 binding) reporter constructs (500 ng/plate) along with the indicated expression vectors (p53 and SnoN; 500 ng/plate [each]). Each plate was also cotransfected with the pGL2 luciferase vector (50 ng/plate) to standardize and control for transfection efficiency. Expression levels relative to baseline are indicated for AFP/lacZ (gray) and DelA/lacZ (black). Cotransfected proteins exhibited equivalent levels of protein expression by Western blot analysis (data not shown). (Bottom) The p53/HNF-3 (FoxA) regulatory element centered at −850 in the AFP promoter (42) overlaps with consensus SBEs (89). The DelA/lacZ binding element has a 10-bp deletion within the 5′ p53 half site (shaded) and point mutations in the 3′ half site. (B) Ski activates AFP expression in the presence of p53. Identical transient transfection analyses were performed with the AFP/lacZ reporter and coexpressed p53, SnoN, and c-Ski constructs, as indicated. Expressed Ski protein repressed AFP only slightly and activated expression in the presence of p53. β-gal, β-galactosidase; luc, luciferase.