BMAL1 regulates human embryonic stem cell neurodifferentiation through YTHDF2-dependent m6A modification

BMAL1 is a core circadian transcription factor that also regulates stem cell fate, yet its role in human embryonic stem cell neurodifferentiation remains incompletely understood. Circadian disruption is associated with adverse neurodevelopmental outcomes, while N6 methyladenosine (m6A) RNA modification is a key regulator of early development. How BMAL1 interfaces with m6A dependent regulation during neural differentiation is unclear. Using CRISPR-Cas9 mediated genome editing, we generate BMAL1-deficient human embryonic stem cells and characterize their differentiation toward neural stem cells. Loss of BMAL1 does not compromise pluripotency but markedly impairs neurodifferentiation, resulting in defective neural rosette formation and suppression of neural lineage gene programs. Transcriptomic and CUT&Tag analyses reveal that BMAL1 directly regulates genes involved in embryonic development and neurogenesis. Integrated m6A profiling demonstrates altered m6A distribution in BMAL1-deficient cells, particularly reduced m6A enrichment near transcription end sites of downregulated genes. Notably, BMAL1 loss leads to a pronounced reduction in the m6A reader YTHDF2 at the protein level. Mechanistically, BMAL1 binds to an E-box motif in the YTHDF2 promoter and regulates alternative splicing of YTHDF2 transcripts, favoring a variant with a highly structured 5′ untranslated region and reduced translational efficiency. Genetic disruption of this E-box phenocopies BMAL1 deficiency. Functional rescue experiments show that YTHDF2 overexpression partially restores neural differentiation defects caused by BMAL1 loss. These findings identify a BMAL1-m6A-YTHDF2 regulatory axis that integrates transcriptional and post-transcriptional control to govern early human neural differentiation, providing mechanistic insight into how circadian disruption may contribute to neurodevelopmental disorders.