Fig. 6. Induction of p21 expression by activated Notch1 through RBP-Jκ-dependent transcription. (A) Suppression of p21 promoter activity in differentiating keratinocytes by antisense Notch1 cDNA and dominant-negative RBP-Jκ. Keratinocytes were transfected with a reporter plasmid carrying the proximal 2.4 kb region of the human p21 promoter (p21-luc) ± expression vectors for anti-sense mouse Notch1 cDNA or dominant-negative RBP-Jκ (Kato et al., 1997). Cells were kept in low calcium medium or exposed to high calcium for the last 24 h of the experiment (72 h after transfection). (B) RBP-Jκ-dependent induction of p21 promoter activity by activated Notch1. Keratinocytes were transfected with the p21-luc reporter ± an expression vector for activated Notch1 (Notch1ICD) and a vector for the dominant-negative RBP-Jκ mutant in increasing amounts. Empty plasmid vector was also added to ensure that all cells were transfected with the same amount of total DNA. (C) Calcium and Notch1 responsiveness of a minimal p21 promoter region containing the fully conserved RBP-Jκ binding site. Keratinocytes were transfected with the reporters p21-luc, carrying the 2.4 kb p21 promoter, or p21-(557/382)-luc, containing 175 bp of the p21 promoter containing the RBP-Jκ binding site (–557 to –382 position) fused to a minimal promoter (pluc-MCS vector; Stratagene). Cells were either kept under low calcium conditions, exposed to high calcium for 24 h or co-transfected with an expression vector for activated Notch1. (D) Binding of the RBP-Jκ protein to the endogenous p21 promoter as assessed by chromatin immunoprecipitation. Primary keratinocytes under low calcium conditions were processed for chromatin immunoprecipitation with antibodies against the RBP-Jκ or SP1 proteins and affinity-purified IgGs. The immunoprecipitates were analyzed by PCR with oligonucleotide primers specific for the indicated regions of the mouse p21WAF1/Cip1 promoter and for a region of the mouse c-jun promoter containing a canonical RBP-Jκ binding site. Preliminary PCR reactions were carried out with naked DNA to determine optimal conditions for amplification of each DNA.