Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

Transcriptional reprogramming drives differentiation and coordinates cellular responses. While mRNA expression in distinct cell types has been extensively analyzed, the mechanisms that control RNA synthesis upon lineage specifications remain unclear. Here, we induce erythroid differentiation in human cells, track transcription and its regulation at nucleotide-resolution, and identify molecular mechanisms that orchestrate gene and enhancer activity during erythroid specification. We uncover waves of transcription and reveal that a brief differentiation signal launches sustained and propagating changes in RNA synthesis and mRNA expression over cell divisions. NRF2, a strong trans-activator upon oxidative stress, drives erythroid differentiation without a detectable increase in reactive oxygen species. In erythroid precursors, NRF2 induces architecturally primed, differentiation-linked enhancers, and genes encoding globin and antioxidant proteins. Projecting signal-induced transcription to DNA accessibility and mRNA expression in single human bone marrow cells, reveals ordered activation of myeloid (GABPA) and erythroid (GATA1, TAL1 and HEMGN) factors in lineage-specification, followed by NRF2-triggered antioxidant response in the late erythroid cells. This study establishes molecular mechanisms that prime, execute, and temporally coordinate RNA synthesis during erythroid differentiation. Furthermore, we show that master regulators of differentiation and stress co-orchestrate erythropoiesis and produce the antioxidant machinery before erythroid cells mature to oxygen transporting enucleated erythrocytes.