Data availability. All of the sequencing data is available via Gene Expression Omnibus (GEO) under the accession number GSE117826.

Mutations in genes involved in DNA methylation (DNAme; e.g., TET2, DNMT3A) , are frequently observed in hematological malignancies 1 – 3 and clonal hematopoiesis 4 , 5 . Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid vs. myelo-monocytic progenitors upon Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells (HSCs). To reconcile genome-wide DNAme changes with specific erythroid vs. myelo-monocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A -mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the hematopoietic differentiation topography, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in transcription factor binding-motif CpG enrichment.

SUMMARY Stem cells balance cellular fates through asymmetric and symmetric divisions in order to self-renew or to generate downstream progenitors. Symmetric commitment divisions in stem cells are required for rapid regeneration during tissue damage and stress. The control of symmetric commitment remains poorly defined. Using single-cell RNA sequencing (scRNA-seq) in combination with transcriptomic profiling of HSPCs (hematopoietic stem and progenitor cells) from control and m6A methyltransferase Mettl3 conditional knockout mice, we found that m6A-deficient hematopoietic stem cells (HSCs) fail to symmetrically differentiate. Dividing HSCs are expanded and are blocked in an intermediate state that molecularly and functionally resembles multipotent progenitors. Mechanistically, RNA methylation controls Myc mRNA abundance in differentiating HSCs. We identified MYC as a marker for HSC asymmetric and symmetric commitment. Overall, our results indicate that RNA methylation controls symmetric commitment and cell identity of HSCs and may provide a general mechanism for how stem cells regulate differentiation fate choice.