PRC2-regulated glycolysis-to-oxphos metabolic switch coordinates the onset of terminal erythropoiesis

Abstract Erythropoiesis is a complex, tightly regulated process involving sequential changes in epigenetics, metabolism, and proliferation. While conventional immunophenotyping combined with transcriptomic and epigenomic profiling has provided valuable insights, these approaches lack the resolution to fully capture dynamic transitions. A comprehensive understanding of how these programs are coordinated remains incomplete. To address this, we integrated single-cell RNA sequencing (scRNA-seq), cellular indexing of transcriptomes and epitopes (CITE-seq), histone modification profiling, and chromatin accessibility analysis to dissect murine erythropoiesis at high resolution. scRNA-seq identified nine transcriptional clusters (C1–C9) along a continuous trajectory. Conventional R3 cells (CD71⁺Ter119high) were further resolved into three subclusters (C5–C7), exhibiting heterogeneity in ribosome biogenesis, heme metabolism, hemoglobin gene expression, chromatin condensation, and enucleation priming. Notably, C4 displayed the highest unique molecular identifier (UMI) counts and total RNA content, co-expressing markers from neighboring clusters alongside its own signature genes. Differential expression analysis of C4 versus neighboring clusters revealed three coordinated changes: (1) initiation of heme biosynthesis and hemoglobin gene expression, (2) a decline in ribosome biogenesis compared to C3, and (3) a metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS), marked by a 98% reduction in Pkm (pyruvate kinase M) expression. To probe this metabolic shift, we FACS-sorted R2 (CD71⁺Ter119⁻), R2–3 (CD71⁺Ter119med, corresponding to C4), and R3 populations to assess intracellular pyruvate and ATP levels and chromatin accessibility. Consistent with reduced Pkm, R2–3 cells showed a sharp decline in pyruvate and diminished ATAC-seq signals at the Pkm locus, suggesting epigenetic repression. CUT&Tag data revealed increased H3K27me3 at Pkm in R2–3, implicating Polycomb Repressive Complex 2 (PRC2) in the regulation of this metabolic switch. To test PRC2's functional role, we generated Ezh2 conditional knockout mice using Vav-Cre. These mice exhibited defective erythropoiesis, with R2–3 cell accumulation and greatly reduced downstream R3/R4 populations, resulting in anemia. scRNA-seq of Ezh2 KO bone marrow precursors revealed premature chromatin opening at hemoglobin and heme synthesis gene loci (e.g., Alas2, Fech), even though these genes were not direct PRC2 targets in wild-type cells. Hb gene expression in KO R4 cells remained comparable to wild-type, but heme synthesis gene expression was suboptimal, suggesting impaired heme availability. Strikingly, Pkm expression increased ~160-fold in KO C4 cells, and glycolysis gene scores were elevated across KO clusters, indicating enhanced pyruvate-generating capacity. FACS-sorted KO R2–3 and R3 cells confirmed pyruvate accumulation, exhibited reduced ATP levels, and showed impaired proliferation and increased apoptosis. To test the effect of pyruvate accumulation directly, we treated G1ER cells (arrested at the R2–R2–3 transition) with increasing pyruvate (10–50 mM). Intracellular pyruvate increased with dose, while 40–50 mM pyruvate led to ATP reduction, impaired proliferation, and increased cell death. Similarly, in vitro–differentiated Lin⁻ bone marrow cells treated with 50 mM pyruvate exhibited reduced frequencies of R2–3 and R3 cells and elevated cell death. In contrast, Lin⁺ cells cultured under the same conditions were less affected, suggesting lineage-stage-specific sensitivity to pyruvate overload. In summary, we identify a metabolically distinct transitional population (C4) in which erythroid precursors undergo a PRC2-regulated shift from glycolysis to OXPHOS, coinciding with the onset of heme and hemoglobin production. PRC2 suppresses Pkm expression via H3K27me3 deposition, thereby restraining pyruvate production and coordinating energy metabolism with terminal differentiation. Loss of PRC2 disrupts this metabolic control, leading to pyruvate accumulation, ATP deficiency, and defective erythropoiesis culminating in anemia.