biostudies-literatureLiang ZKey Research and Development Program of Zhejiang ProvinceNational Natural Science Foundation of ChinaScientific foundation of Zhejiang Province10879-10896https://www.ebi.ac.uk/biostudies/studies/S-EPMC11472173biostudies-literatureUnknown52(18)N6-methyladenonsine (m6A) is ubiquitously distributed in mammalian mRNA. However, the precise involvement of m6A in early development has yet to be fully elucidated. Here, we report that deletion of the m6A demethylase ALKBH5 in human embryonic stem cells (hESCs) severely impairs definitive endoderm (DE) differentiation. ALKBH5-/- hESCs fail to undergo the primitive streak (PS) intermediate transition that precedes endoderm specification. Mechanistically, we show that ALKBH5 deficiency induces m6A hypermethylation around the 3' untranslated region (3'UTR) of GATA6 transcripts and destabilizes GATA6 mRNA in a YTHDF2-dependent manner. Moreover, GATA6 binds to the promoters of critical regulatory genes involved in Wnt/β-catenin signaling transduction, including the canonical Wnt antagonist DKNucleic acids researchALKBH5 governs human endoderm fate by regulating the DKK1/4-mediated Wnt/β-catenin activation.PMC114721732024SSYS00309206820132300639Fan YZhao CWang BLiang ZFu YQin YMa ZZhai ZWang KPei DLi WHuang TWang XKuang JfalseALKBH5 governs human endoderm fate by regulating the DKK1/4-mediated Wnt/β-catenin activation.N6-methyladenonsine (m6A) is ubiquitously distributed in mammalian mRNA. However, the precise involvement of m6A in early development has yet to be fully elucidated. Here, we report that deletion of the m6A demethylase ALKBH5 in human embryonic stem cells (hESCs) severely impairs definitive endoderm (DE) differentiation. ALKBH5-/- hESCs fail to undergo the primitive streak (PS) intermediate transition that precedes endoderm specification. Mechanistically, we show that ALKBH5 deficiency induces m6A hypermethylation around the 3' untranslated region (3'UTR) of GATA6 transcripts and destabilizes GATA6 mRNA in a YTHDF2-dependent manner. Moreover, GATA6 binds to the promoters of critical regulatory genes involved in Wnt/β-catenin signaling transduction, including the canonical Wnt antagonist DK2024-01-01T00:00:00Z2024 Oct2026-06-23T03:11:26.103Z2025-04-04T13:54:16.546ZS-EPMC114721733916649210.1093/nar/gkae707