Loss of Runx1 triggers inactivation of a Pu.1 gene enhancer and terminal erythroid differentiation

Pu.1 is an ETS family transcription factor (TF) that plays a critical role in early erythropoiesis by promoting limited self-renewal of erythroid progenitors and directly antagonizing terminal erythroid differentiation. We add to Pu.1’s role in erythropoiesis by showing that loss of chromatin sites containing Pu.1 and Runx1 DNA-binding motifs are the dominant chromatin change in hematopoietic cells as they commit to the erythroid cell fate and begin terminal differentiation. Despite the critical role of Pu.1 in erythropoiesis it is not known how Pu.1 is regulated in these cells. We show that during early erythropoiesis the Pu.1 locus is dramatically remodeled leading to loss of chromatin accessibility upstream of the Pu.1 promoter including loss of a well-known strong Pu.1 enhancer at -14kb, the “URE”. Consistent with genome-wide changes, the URE contains Pu.1 and Runx1 DNA-binding motifs. Loss of enhancer activity is associated with downregulation of both factors and scRNAseq data shows that Runx1 is one of the TFs most closely correlated with Pu.1 expression in uncommitted and immature erythroid progenitors. Forced expression of either factor prevents endogenous Pu.1 downregulation and blocks terminal erythroid differentiation leading to the production of two novel erythroid lines which expand ex vivo for at least 4 months and maintain the ability to terminally differentiate. Forced expression of Runx1 in mice lacking the Pu.1 URE does not block terminal erythroid differentiation. The molecular mechanism of graded URE inactivation in erythroid cells represents a distinct pattern of Pu.1 repression from T-cells which actively repress Pu.1 through the URE.