Rabbit Anti-Human TCF3 (Phospho-Thr355) polyclonal antibody

Protein N-glycosylation and the Wnt/beta-catenin signaling pathways play critical roles in development and cancer. Although N-glycosylation has been shown to influence Wnt signaling through its effects on Wnt ligands, it is unclear whether the Wnt/beta-catenin pathway impacts protein N-glycosylation. In this study, we show that promoters of the first N-glycosylation gene, DPAGT1, from Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human epidermoid carcinoma (A253) cells contain the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus sequence. Treatment of cells with a Wnt activator, lithium chloride, up-regulated DPAGT1 transcript levels that correlated with an increase in the beta-catenin abundance. Furthermore, exposure of cells to a Wnt receptor ligand, Wnt3a, resulted in an increase in the DPAGT1 transcript levels that was abrogated by the Wnt inhibitor, Dick-kopf-1. DNA mobility shift assays revealed specific protein complexes at the DPAGT1 TCF/LEF binding region that were competed off with antibodies to either Tcf3/4 or beta-catenin. Chromatin immunoprecipitation analysis confirmed the presence of beta-catenin at the DPAGT1 promoter in vivo. In addition, the DPAGT1 TCF/LEF sequence drove the expression of the luciferase reporter gene. Furthermore, up-regulation of DPAGT1 transcripts by Wnt3a led to altered N-glycosylation of E-cadherin. Interestingly, the DPAGT1 TCF/LEF sequence also interacted with gamma-catenin, a close homologue of beta-catenin, although not in a lithium chloride-dependent manner. Our results provide the first evidence that the Wnt/beta-catenin signaling pathway regulates the metabolic pathway of protein N-glycosylation by targeting DPAGT1 expression. Moreover, they suggest the existence of another regulatory mechanism involving the interaction of Tcf with gamma-catenin at the DPAGT1 promoter.

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naive pluripotency by holding back differentiation. The canonical Wnt/beta-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/beta-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15(Ink4b), p16(Ink4a) and p19(Arf), which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of beta-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.