An intronic RUNXI Enhancer marks Hematopoietic stem cells

Abstract

RUNX1 is essential for definitive hematopoeisis and is frequently mutated in human leukemias. In addition, RUNX1 haploinsufficiency causes familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). However, the precise transcriptional regulation of Runx1 remains largely unknown due to the extremely large size of the mammalian Runx gene loci which spans approximately 1.0 Mb in human RUNX genes. Here we report the identification, through an innovative, combinatorial in silico approach involving comparative genomics and retroviral integration sites (RIS) mapping; and characterization of a highly evolutionarily conserved Runx1 intronic enhancer. This enhancer initially observed to be active in hematopoietic sites in the early zebrafish embryo, was found, by quantitative chromatin immunoprecipitation (qChIP), to be within b openb chromatin regions only in mouse cell lines expressing Runx1. To further characterize this element, +24.1 mCNE-GFP transgenic mouse lines were generated. Analyses of transgenic mouse embryos show that this Runx1 +24.1 enhancer targets reporter gene expression to hemogenic sites where the de novo generation of HSC/progenitor cells occur. Significantly, this enhancer is preferentially active in hemogenic endothelial cells (EC) but not in non-hemogenic EC. Furthermore, transplantation assays reveal that long-term hematopoietic stem cells (LT-HSC) are enriched in Runx1 +24.1 enhancer-targeted adult bone marrow cells, even within the HSC-containing, c-Kit+Sca-1+Lin- (KSL) fraction. Taken together, our results support the role of +24.1 mCNE as an enhancer which targets HSC. We anticipate that our Runx1 +24.1 stem cell enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic EC/HSC behavior in vivo and consequently become an invaluable tool for research on stem cell and cancer biology.