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 p15Ink4b, p16Ink4a and p19Arf, 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.

We are grateful for the support from ERC grant (242630-RERE) (MPC), the Ministerio de Economia y Competitividad y FEDER (SAF2011-28580, and BFU2014-54717-P, BFU2015-71984-ERC to MPC), an AGAUR grant from Secretaria d´Universitats i Investigació del Departament d´Economia i Coneixement de la Generalitat de Catalunya (2014SGR1137 to MPC), Ministerio de Ciencia e Innovación FPI (to FA), the European Union's Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to MPC), the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017, the CERCA Programme/Generalitat de Catalunya (to MPC); KU Leuven starting grant (STG) and KU Leuven C1 funds (C14/16/078) (to FL), AFR Postdoctoral Grant from the Luxembourg National Research Fund (FNR); ANEMO project N. 4001584/PDR 2012-1 and Dutch Province of Limburg (to GE), Short Term Mobility Award, CNR (to AC).