ALKBH5 modulates endoderm fate diversion from hESCs through the ALKBH5-GATA6-WNT/ß-catenin regulatory axis [CUT&Tag]
N6-methyladenonsine (m6A) modification locates ubiquitously in mammalian mRNA, and profoundly impacts various physiological processes and pathogenesis. However, the precise involvement of m6A in early endoderm development has yet to be fully elucidated. Here, we reported that depletion of the m6A demethylase ALKBH5 in human embryonic stem cells (hESCs) severely impaired definitive endoderm (DE) differentiation. Within this process, ALKBH5-/- hESCs failed to undergo the primitive streak (PS) intermediate transition, which is considered as a prelude to endoderm specification. Mechanistically, we demonstrated that ALKBH5 deficiency induced m6A hypermethylation around the 3' untranslated region (3'UTR) of GATA6 transcripts and destabilized GATA6 mRNA in a YTHDF2-dependent manner. Moreover, dysregulation of GATA6 expression ablated its occupancy with critical regulators of Wnt/ß-catenin signaling pathway, thereby disrupting the signaling logic underlying DE formation. Overall, our findings unveil a mechanism whereby the ALKBH5-GATA6-WNT/ß-catenin axis modulates human in vitro DE induction, and present novel insights on m6A modification in early embryonic development. Overall design: We analyzed the endogenous GATA6 protein occupancy during primitive streak specification from hESCs