Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus

Abstract

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 naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-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 β-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.

Author summary

Studying how to safely expand stem cells in culture is essential for regenerative medicine applications. Hence there is a clear need to decode how the cell cycle of mouse embryonic stem cells (mESCs) is regulated. Tcf3 and Tcf1 belong to the Tcf family of proteins. Tcf/Lef are effectors of the Wnt/β-catenin pathway and Tcf3 controls mESC pluripotency. Here we identified a recruitment site for Tcf1 embedded into a number of cell cycle repressor genes such as p15 ^Ink4b , p16 ^Ink4a and p19 ^Arf . Tcf1-mediated activation of these genes drastically slows down proliferation of mESCs. In conclusion, here we showed that the Wnt pathway, besides controlling mESC pluripotency via Tcf3, also regulates mESC cell cycle through the recruitment of Tcf1 to the regulatory sites of key cell cycle genes.