Wnt/β-catenin signaling pathway safeguards epigenetic stability and homeostasis of mouse embryonic stem cells.

Wnt/β-catenin signaling regulates both mouse embryonic stem cell (mESC) self-renewal and differentiation. Here we show that the activity of the Wnt/β-catenin signaling pathway safeguards normal DNA methylation of mESCs. Indeed, loss of Wnt activity correlated with loss of DNA methylation, especially at the imprinting control regions (ICRs) in prolonged in vitro mESC cultures. To address the role of Wnt signaling on ICR methylation we performed Reduced Representation Bisulfite Sequencing (RRBS) on both “Young” and “Old” mESCs, being at early and late passages, respectively. We found that several ICRs (both maternal and paternal) showed loss of methylation in “Old” cells that correlates with lower Wnt downstream target expression and lower β-catenin protein level, when compared to “Young” mESCs. In parallel, we analyzed the ICR methylation level also in two Wnt mutant clones after prolonged in vitro culture (O-S33Y #1 and #2). The mutant clones maintained high Wnt activity with passages, similar to “Young” mESCs and ICR methylation level at around 50%, further supporting the beneficial role of Wnt/β-catenin pathway on mESC epigenetic stability and homeostasis. Methylation levels of E14 mESCs displaying different levels of Wnt/β-catenin activity and β-catenin protein, analyzed by RRBS, in duplicate.