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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Dev Growth Differ. 2012 Jul 8;54(6):633–648. doi: 10.1111/j.1440-169X.2012.01364.x

Fig. 6. Smad4-Snail-SIP1 binds to the promoter of the E-cadherin gene.

Fig. 6

(A) A schematic diagram of the proposed interaction between the Snail-SIP1-Smad4 transcription complex and the E-Cadherin gene promoter is shown to demonstrate our hypothesis of transcriptional repression. The transcription initiation site (+1) is indicated by arrows. Positions of regulatory E-boxes in the E-cadherin promoter (E1–E3) (bound by E-cadherin repressors) are indicated by grey boxes. E1 and E2 boxes are adjacent in the mouse promoter, forming the E-pal element (Moreno-Bueno et al., 2009) (B) Binding of the Snail-SIP1-Smad4 complex to the endogenous loci was confirmed by chromatin immunoprecipitation. Chromatin was immunoprecipitated with antibodies against IgG (negative control), Smad4, Snail, SIP1 and β-actin (non-specific control). PCR analysis using precipitated DNA showed binding of Snail-SIP1-Smad4 and SIP1-Smad4 to E-pal and E-box regions respectively, of the E-Cadherin promoter, in response to TGFβ3 (5ng/mL) treatment for 24 h. No signal was observed from untreated cells and β-Actin was not detected to interact with these binding sites. (C) Using a wild type (−650- to +95) pGL3-Lux-E-Cadherin reporter plasmid and three mutant plasmids (mutations on E-Box, E-pal and both E-box and E-pal), we were able to demonstrate that these binding sites are critical for E-Cadherin promoter activity. Luciferase assays, performed in MES cells treated with 5 and 10ng/mL TGFβ3 with wild type and mutant plasmids, showed a high level of E-Cadherin promoter activity in all groups of untreated cells. TGFβ3 (5ng/mL) treatment significantly reduced E-Cadherin activity in the wild type transfected cells. E-pal and E-box mutations reversed TGFβ3’s inhibitory effect on promoter activity, with the double mutant demonstrating the most significant effect. Repression of E-Cadherin gene activity by TGFβ3 can be restored and reversed by blocking both Smad dependent and Smad independent pathways (MEK1/2 blocked with U0126, p38MAPK blocked with SB202190), except for the PI3 kinase pathway (blocked with LY294002). Both TGFβ treatment groups differed significantly from the negative control (untreated and empty vector treated cells) groups, (p ≤ 0001, as indicated by *).