Distinct regulation of Wnt signaling and EPZ5676 governs human UiPSM reprogramming via the CDX2/T/TBX6 core axis.

An ideal in vitro system to study somitogenesis is induced presomitic mesoderm (UiPSM); the regulatory pathways of this system are not well understood. Here, integrated multi-omic and functional analyses were performed to elucidate the roles of Wnt signaling and EPZ5676 in UiPSM reprogramming. The findings showed that the Wnt/β-catenin signaling activation by CHIR is essential to induce UiPSM, which facilitates chromatin remodeling and the activation of major presomitic mesoderm (PSM) transcription factors (TFs). Conversely, EPZ5676 controls when reprogramming occurs and increases the efficiency of reprogramming by controlling chromatin dynamics without dictating cell fate. The TFs CDX2, T (Brachyury), and TBX6 were found to be a central regulatory axis whose functions were partially redundant. Mechanistically, CDX2 directly interacts with the regulatory regions of WNT3A, T, and TBX6, maintaining their chromatin accessibility and transcriptional activity and creating a positive feedback loop of Wnt/β-catenin-dependence. Moreover, the interaction between CDX2, T, and TBX6 with Wnt signaling is reciprocal to establish a stable regulatory network that preserves UiPSM identity and stemness. Taken together, this work shows that Wnt/β-catenin signaling coordinates UiPSM reprogramming and maintenance via the CDX2/T/TBX6 axis, which offers new mechanistic understanding of human mesoderm lineage specification and somatic cell reprogramming.