We further substantiated the positive role of Daxx in regulating TCF4-mediated transactivation and target gene expression by RNA interference approach. 293
Paclitaxel transfected with the pSUPER-Daxx, a construct reported previously for Daxx depletion [13], were subjected to reporter gene analysis. As expected, transfection of pSUPER-Daxx decreased endogenous Daxx level (Fig. 3A, left panel). Under such condition, the LiCl-induced TOPFLASH reporter activity was significantly attenuated as compared to that of cells transfected with pSUPER empty vector (right panel, lanes 3 and 4). Similar observation was made in HCT116 cell line ( Fig. 3B). Accordingly, depletion of Daxx expression in 293 cells reduced the Wnt3a-induced expression levels of c-Myc and cyclin D1 while TCF4 and β-catenin remained similar expression level ( Fig. 3C, lanes 3 and 4). Likewise, the levels of c-Myc and cyclin D1 were diminished in HCT116
cells transfected with pSUPER-Daxx ( Fig. 3D). To avoid the off-target effect by one Daxx siRNA, we also used lentivirus-mediated delivery of short-hairpin RNA (shRNA) against distinct mRNA region of Daxx. In accordance with the results of pSUPER-Daxx, the lentiviral infection of Daxx shRNA caused a reduction of Daxx level and concomitantly rendered lower levels of c-Myc and cyclin D1 in HCT116 cells ( Fig. 3E). These results further support the notion that Daxx acts as a positive coregulator for activating β-catenin/TCF4 transcriptional potential.
