A “Master Regulatory Cis-element” Governs the Hematopoietic Stem/Progenitor Cell Compartment, Vascular Integrity, and Cardiovascular Development

Abstract 1304

The expression and activities of master regulatory proteins are exquisitely controlled to ensure the proper development of complex organisms. As a master regulator can orchestrate multiple developmental processes, presumably mechanisms involving a large ensemble of cis-elements and trans-acting factors are deployed to establish this control. GATA-2, a master regulator of hematopoiesis expressed in hematopoietic, endothelial, neuronal, and fat cells is likely subject to such complex regulation. GATA-2 occupies dispersed sites of the Gata2 locus, highlighting a positive autoregulatory mechanism. As GATA-1 levels rise during erythropoiesis, GATA-1 displaces GATA-2 from the Gata2 locus, instigating repression. “GATA switches” occur at five dispersed Gata2 sites (−77, −3.9, −2.8, −1.8 and +9.5 kb). Targeted deletion of GATA factor switch sites −2.8 and −1.8 revealed that these sites individually contribute to maximal Gata2 expression, but are otherwise dispensable for embryogenesis and steady-state hematopoiesis. Herein, we describe the targeted deletion of an intronic GATA switch site (+9.5) comprised of an E-box GATA factor composite cis-element. Strikingly, whereas the Gata2 gene knockout results in lethal anemia by E10.5, the +9.5 mutation yielded embryonic lethality around E13.5. The +9.5 mutation greatly reduced Gata2 expression in fetal livers, heart and endothelial cells, but not in the brain. Consequently, by E12.5 mutant mice exhibited severely reduced numbers of hematopoietic stem/progenitor cells (HSPCs) in fetal livers as assayed by cell sorting and colony assays. However, mutant mice were not overtly anemic and Ter119+ cells were abundant in the mutant fetal livers, accounting for the delay in lethality compared to the Gata2 knockout. Additionally, +9.5 mutant embryos displayed vascular/cardiovascular malformations and severe hemorrhaging. This work reveals that, unlike the −2.8 and −1.8 GATA switch sites, the +9.5 composite element functions as a “master regulatory cis-element” required for HSPC development, vascular integrity, and cardiovascular development. Genomic analyses identified a large cohort of loci containing conserved intronic composite elements resembling the +9.5 element, with heavy representation at genes critical for HSPC and vascular development/function, as well as novel genes of unknown function.