RNA Polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation [NET-seq]

The controlled release of promoter-proximal RNA polymerase II (Pol II) pausing into productive elongation is a major step in gene regulation. Yet, functionally analyzing Pol II pausing is difficult because the factors that regulate pause release are generally essential. Here, we identified heterozygous loss-of-function mutations in SUPT5H, encoding SPT5, in individuals with β-thalassemia trait unlinked to the β-globin locus. Recapitulating the pathogenic mutations in human erythroid cells or acute treatment with transcription inhibitors replicates the patient phenotype of altered globin gene expression. Furthermore, in SUPT5H-edited cells, Pol II pause release was globally disrupted. We observed dynamic patterns of pausing at cell cycle and erythroid differentiation genes at the transition from progenitors to precursors during erythropoiesis. At this same transition, in SUPT5H-edited cells, we observed delayed differentiation and altered cell cycle kinetics, as well as a delay in the onset of gene expression programs. Therefore, hindering pause release perturbs cell cycle and delays differentiation at key stages of erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation. SUPT5H editing of hematopoietic stem and progenitor cells (HSPCs) to recapitualte los-of-function patient mutations and follow imapct on globins and erythropooesis using NET-seq, RNA-seq, Nanostring, and FACS