Fig. 2.

The murine Myc 3′ enhancer contains a SPS that drives NTC dimer-dependent transcription. (A) Schematic of the RBPJ–Notch1–MAML NTC complex on DNA containing an SPS site. The two RBPJ motifs are designated A and B, and the critical R1984 interankyrin repeat domain contact is highlighted. See ref. 16 for details. RBPJ, gold; Notch1 RAM-ANK, purple; MAML1, green. (B) Assembly of dimeric NTCs on the 3′ Myc enhancer element. EMSA was performed with oligonucleotide probes containing the putative SPS and the indicated combinations of recombinant RBPJ, Notch1 RAM-ANKs (RA1), and N-terminal MAML1 (MAML). RA1-R1984A corresponds to the dimerization-defective Notch1 mutant. (C) Schematic of the luciferase reporter gene constructs including the NDME WT and mutant sequences. Note: RBPJ is denoted by its alternative name, CSL. (D) T6E cells were transfected with Renilla luciferase control (pRLTK), the indicated Myc firefly luciferase reporter genes, and either empty vector or vectors driving expression of WT NICD1 or NICD1 with the R1984A mutation. (E) Murine T-ALL T6E cells transfected with pRL-TK and one of the reporter constructs shown in C. Cells were cultured with DMSO or 1 μM compound E for 6 h before harvest. (F) Activity of the murine Myc enhancer reporter construct (MycProNDME) in various cell lines with and without overexpression of NICD1. In D–F, cells were harvested 48 h after transfection. Data were obtained in triplicate in independent experiments; error bars correspond to the SEM. **P < 0.001.