Fig. 7. Induction of involucrin expression by activated Notch1 and Notch2 expression and the conserved Notch1 ANK domain. (A) Suppression of involucrin promoter activity in differentiating keratinocytes by expression of anti-sense Notch1 cDNA but not dominant-negative RBP-Jκ. Keratinocytes were transfected with a reporter plasmid (Inv-luc) carrying the human involucrin promoter (Brissette et al., 1996) ± expression vectors for anti-sense mouse Notch1 cDNA or dominant-negative RBP-Jκ. Cells were kept under low calcium conditions or exposed to high calcium for 24 h. (B) Induction of involucrin promoter activity by activated Notch1 independently of RBP-Jκ activity. Keratinocytes were transfected with the Inv-luc reporter ± an expression vector for activated Notch1 (Notch1^ICD) and a vector for dominant-negative RBP-Jκ in increasing amounts. Empty plasmid vector was also added to ensure that all cells were transfected with the same amount of total DNA. (C) Identification of the minimal Notch responsive region of involucrin promoter. Primary keratinocytes were transfected with reporter plasmids carrying either 2473, 2116–2088 or 241 bases (Efimova et al., 1998) of the involucrin promoter region, ± an expression vector for activated Notch1 cDNA. (D) Differential induction of the RBP-Jκ responsive HES-AB promoter by activated Notch1 versus Notch2 expression. Keratinocytes were transfected with the HES-AB reporter with increasing amounts of expression vectors for either activated Notch1 or Notch2 proteins (Capobianco et al., 1997). An analogous dose–response experiment was also performed with the involucrin promoter (as in G), which, unlike the HES-AB promoter, showed similar induction by both activated Notch1 and Notch2 (not shown). (E–G) Selective induction of involucrin promoter activity by activated Notch2 and the conserved Notch1 ANK domain. Keratinocytes were transfected with reporter plasmids for the HES-AB (E), p21 (F) and involucrin (G) promoters with or without expression plasmids for the activated cytoplasmic region of Notch1 (Notch1^ICD), the same region with an internal deletion of the ANK domain (Notch1-ΔANK), the Notch1 ANK domain alone (ANK) and activated Notch2 (Notch2^ICD). The Notch1 ANK domain was also co-expressed with increasing amounts of the vector for dominant-negative RBP-Jκ as in (B); shown is the result with 1.5 µg of transfected plasmid DNA for the mutant RBP-Jκ.