Fig. 4. Smad2-P–Smad4–LEF1 directly bind to the promoter of the E-cadherin gene. (A) Promoter analysis of the murine E-cadherin gene revealed one unique LEF1-binding site (independent of the standard TCF and/or LEF site located in the E-pal) and a Smad-binding element (SBE), both within close proximity to the E-pal LEF1-binding region. (B) Binding of the Smad2-P–Smad4–LEF1 protein complex to the endogenous loci was confirmed by chromatin immunoprecipitation. Chromatin was immunoprecipitated with antibodies against IgG (negative control), LEF1, Smad4, Smad2-P or -catenin. PCR analysis using precipitated DNA showed binding of LEF1, Smad4 and Smad2-P to LEF1 (non-E-pal), LEF1 (E-pal) and SBE regions of the E-cadherin promoter. Whereas LEF1 and Smad4 interact with their respective binding regions, all three proteins are observed because they form a transcription complex. No signal was observed from unfused MEE cells whereas a moderate signal was detected from fused MEE cells that was heavily increased upon treatment with exogenous TGF3. -catenin was not detected for interaction with these binding sites.