Fig. 6. ß-catenin/TCF complex directly regulates the Ngn1 promoter. (A) A schematic representation of the mouse neurogenin 1 (Ngn1) promoter (WT), and its mutant within the putative TCF binding site (-1167 to -1160) located in the Ngn1 promoter (Mut). NPCs were transfected with a vector containing the Ngn1 promoter (2.7 kb: wild type or mutant) driving luciferase expression. (B) Relative luciferase activity was measured after 13 hours of incubation. Mutation of the TCF binding site in the Ngn1 promoter reduced endogenous transcriptional activity. (C) Chromatin complex was immunoprecipitated with anti-ß-catenin (Santa Cruz Biotechnology and Sigma) or control IgG, and was subjected to PCR analysis to amplify Ngn1 genomic sequence. (D) RT-PCR analysis of Ngn1 expression. NPCs were infected with a retrovirus encoding either GFP alone (control) or both GFP and S33Y ß-catenin, and the expression level of Ngn1 mRNA was analyzed. Gapdh was used for standardization of the samples. No genomic amplification was observed from the RNA treated without reverse transcriptase (-RT).