Fto-mediated m6A modification is essential for cerebellar development through regulating epigenetic reprogramming.

Background: Growing evidence highlights the importance of epitranscriptomic regulation in cerebellar development and function, especially through m⁶A methylation. Nevertheless, the precise function of the RNA demethylase Fto in the cerebellum is still uncertain.

Methods: An Fto knockout (Fto^KO) mouse model was generated to investigate the role of Fto in cerebellar development. Cerebellar function was assessed using the behavioral tests and Nissl staining. Immunofluorescence was performed to detect molecular expression levels and subcellular localization. Dot blot, m⁶A-RIP-seq, ATAC-seq and CUT&Tag-seq were used to confirm m⁶A levels and chromatin accessibility. Co-IP was employed to test molecular interactions.

Results: Fto^KO mice exhibited cerebellar ataxia, including tremors and abnormal gait patterns. Reduced FTO expression at embryonic day 13.5 (E13.5) and postnatal day 3 (P3) stages resulted in increased TUJ1 expression, as well as reductions in neuronal functional genes (Map2) and self-renewal genes (Sox2, Sox9, Nestin and Pax6). Mechanistically, Kat8 upregulation was linked to the high m⁶A levels regulated by Fto loss. Furthermore, IGF2BP3 specifically recruited acetyltransferase KAT8 to control gene transcription during early cerebellar development by regulating H4K16ac modification, which alters chromatin accessibility in neural developmental pathways.

Conclusions: In summary, Fto^KO-induced Kat8 upregulation in an m⁶A-dependent manner resulted in enhanced KAT8 recruitment by IGF2BP3, which improved chromatin accessibility and H4K16ac modification, thereby promoting cerebellar developmental dysfunction.