Nat10-mediated ac4C epitranscriptomics orchestrates hematopoietic stem/progenitor cell fate determination via translation control.

Epigenetic regulation is crucial for balancing hematopoietic stem cell (HSC) self-renewal and differentiation, thereby maintaining hematopoietic homeostasis. Although Nat10-mediated RNA ac4C modification has been implicated in malignant hematopoiesis, its role in normal hematopoiesis remains unexplored. Here, we developed ULAC-seq to map ac4C in rare hematopoietic stem/progenitor cells (HSPCs) and revealed dynamic, cell-type-specific ac4C patterns, peaking in megakaryocyte-erythroid progenitors (MEPs), correlating with elevated Nat10 expression. Nat10 knockout disrupts HSC self-renewal and arrests MEP differentiation, leading to fetal and postnatal hematopoietic failure. Mechanistically, Nat10 deposits ac4C on mRNAs encoding key hematopoietic transcription regulators (e.g., Nfix), thereby enhancing their translation. Nat10 loss reduces Nfix protein levels and suppresses expression of its target genes (e.g., Mpl) that govern HSPC fate, while Nfix reconstitution rescues colony-forming defects in Nat10-null HSPCs. Our findings reveal that Nat10 orchestrates hematopoiesis through ac4C-dependent translational control of transcriptional factors, establishing an epitranscriptome-transcriptome regulatory axis essential for HSC maintenance and function.