Dynamic regulation of runx1 at the onset of definitive hematopoiesis

Hematopoietic stem cells (HSCs) have been used in medicine for several decades. However, the difficulty of generating, maintaining or expanding them in culture has made it difficult to advance HSC-based therapies further. In the embryo, HSCs first arise from a subset of the dorsal aorta endothelium, the so-called hemogenic endothelium (HE), through a process known as endothelial-to-hematopoietic transition (EHT). The transcription factor Runx1 is critically required for EHT. To begin to elucidate the regulatory pathways converging on Runx1, and thus orchestrating hematopoietic specification and EHT, we have identified and characterized several hematopoietic Runx1 enhancers. Analysis of enhancer-reporter transgenic mouse lines showed that one of these, the +23 enhancer, is sufficient to mediate reporter gene expression to all cells undergoing EHT, including HE and hematopoietic stem and progenitor cells. Two others, in contrast, show a more restricted pattern, with one marking HE but not hematopoietic stem or progenitor cells, while the other marks hematopoietic progenitors but not the HSC lineage or HE. Mutation of candidate transcription factor motifs in these enhancers implicated distinct upstream factors that may contribute to their tissue specificity. Deletion of individual Runx1 enhancers from the endogenous locus in mESCs resulted in a moderate reduction of Runx1 expression in cell populations undergoing EHT. Our results are consistent with a shadow enhancer model where each element is acting in an additive manner. Ongoing work using next generation chromosome conformation capture (NG Capture-C) further dissects Runx1 cis-interactions and supports a model of dynamic enhancer-promoter interactions at the onset of hematopoiesis. Ultimately, a better understanding of factors and signals involved in establishing the HSC lineage in mouse ontogeny is expected to inform the development of culture protocols to generate these cells in vitro.