RUNX1B expression distinguishes megakaryocytic and erythroid lineage fate in adult hematopoiesis

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny, which also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter model, we demonstrate here that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast, the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid specification. Accordingly, the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that the level of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will provide the opportunity to further investigate and define the molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Moreover, comparison of a RUNX1C null (KO) PreMegE to its WT counterpart demonstrated considerably enhanced erythroid specification at the expense of megakaryopoiesis in the absence of P1-specified RUNX1C expression. Overall design: mRNA profiles of wild type (WT), Runx1 P2-hCD4+ (P2+), Runx1 P2-hCD4- (P2-) and RUNX1C knockout (KO) bone marrow Pre-Megakaryocyte/Erythroid (PreMegE) progenitors were generated from young adult (12-16 weeks) mice by deep sequencing, in triplicate, using Illumina NextSeq 500.

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Draper JE, Sroczynska P, Tsoulaki O, Leong HS, Fadlullah MZ, Miller C, Kouskoff V, Lacaud G