GRG5/AES represses TCF-β-catenin mediated transcription and co-operates with both dnTCF4 and Grg1-L for transcriptional repression.

<p>HEK293 cells were transfected with either the reporter construct TCF-Luc (containing three TCF consensus binding sites upstream of the firefly luciferase cDNA), or the control reporter TCF*-Luc (with mutations in the TCF-binding sites) and the indicated combinations of expression plasmids. A) Addition of GRG5/AES plasmid decreases the luciferase activity induced by transfected βcat and endogenous TCFs in a dose dependent manner. At the highest dose (1.2 μg/μl), GRG5/AES-mediated repression is comparable to the effect of dominant negative TCF4 (dnTCF4), a previous established repressor of the canonical Wnt pathway [13]. B) Repression of TCF-β-catenin mediated transcription by various Groucho-family proteins. In contrast to “long” Groucho Grg1 (Grg1-L), GRG5/AES, GRG4 and Grg1-S (a short GRG1 isoform [63]) effectively repressed TCF-dependent luciferase activity. 1.2 µg were used for transfection of each Groucho expression plasmid. C) Co-transfection of GRG5/AES with either dnTCF4 or Grg1L (0.4µg of each plasmid) results in higher repression of TCF-β-catenin dependent luciferase activity then transfections with each effector plasmid individually. β-galactosidase assays were performed as an internal control for transfection efficiency. All the assays were done in triplicate in three independent experiments.