Abstract
T cell factor 4 (TCF4) interacts with beta-catenin in the WNT signaling pathway and transactivates downstream target genes involved in cancer progression. To identify proteins that regulate TCF4-mediated biological responses, we performed a yeast two-hybrid screen to search for a TCF4-binding protein(s) and found that MAD2B interacts with TCF4. We confirmed that MAD2B is a TCF4-binding protein by co-immunoprecipitation. Using the TOPFLASH reporter assay, we found that MAD2B blocks TCF4-mediated transactivation. The MAD2B binding regions of TCF4 were identified by TCF4 deletion mapping and electrophoretic mobility shift assay analysis. TCF4 and MAD2B interactions abolished the DNA binding ability of TCF4. Knockdown of MAD2B in SW480 colorectal cancer cells led to the conversion of epithelial cells to a mesenchymal fibroblastoid phenotype (epithelial-mesenchymal transdifferentiation). An E-cadherin promoter reporter analysis showed that MAD2B modulates TCF4-mediated E-cadherin expression. MAD2B knockdown blocked E-cadherin expression and significantly induced mesenchymal markers, such as N-cadherin and vimentin. Mesenchymal induction was accompanied by F-actin redistribution and the appearance of a fibroblastoid phenotype. MAD2B knockdown also increased both mRNA and protein levels of Slug, a known TCF4-induced E-cadherin transcriptional repressor. A chromatin immunoprecipitation assay showed that MAD2B silencing enhances the ability of TCF4 to bind the Slug promoter. Thus, MAD2B is a novel TCF4-interacting protein. This study provides the first evidence for the involvement of MAD2B in TCF4-mediated epithelial-mesenchymal transdifferentiation.
Highlights
The WNT signaling cascade is a crucial cell development and growth regulatory pathway [1,2,3]
This study provides the first evidence for the involvement of MAD2B in T cell factor 4 (TCF4)-mediated epithelial-mesenchymal transdifferentiation
We found that FLAG-MAD2B, but not FLAGMAD2, co-precipitated with HA-TCF4, suggesting that the interaction between TCF4 and MAD2B is specific (Fig. 1B)
Summary
Plasmid Construction—For the yeast two-hybrid assay, TCF4 and its derivatives were cloned by inserting PCR-amplified gene fragments into the EcoRI and BamHI sites of pAS2–1 (Clontech). The TCF4 gene was cloned into pcDNA3.0-HA (Invitrogen) between the BamHI and XhoI sites. The human MAD2B gene was cloned into pRK5-FLAG (BD Biosciences) between the EcoRI and XbaI sites. The EGFPMAD2B plasmid was constructed by inserting the MAD2B gene into the EcoRI and SmaI sites of EGFP-N3 (Clontech). The Escherichia coli expression plasmid pET29b-MAD2B was constructed by inserting the MAD2B gene into the EcoRI and XhoI sites of the pET29b plasmid (Novagen). The pCDNA3-HA-TCF4 and pRK5FLAG-MAD2B plasmids were transfected into the cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were mounted with Prolong Gold antifade reagent (Invitrogen) and imaged with a confocal microscope
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call