β-catenin mediates endodermal commitment of human ES cells via distinct transactivation functions

β-catenin, acting as the core effector of canonical Wnt signaling pathway, plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models, the β-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood, partly due to the rapid and complex cell fate transitions during early differentiation.

In this study, we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of β-catenin. Our analysis showed that a truncated β-catenin lacking both N- and C-terminal domains (ΔN¹⁴⁸C) could robustly rescue the DE formation, whereas hyperactive β-catenin mutants with S33Y mutation or N-terminal deletion (ΔN⁹⁰) had limited ability to induce DE lineage. Notably, the ΔN¹⁴⁸C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak β-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes, whereas the hyperactive β-catenin mutants activated mesoderm genes.

Our study uncovered an unconventional regulatory function of β-catenin through weak transactivation, indicating that the levels of β-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.