Cis-element mutated in GATA2-dependent immunodeficiency governs hematopoiesis and vascular integrity

Abstract

Haploinsufficiency for GATA2 causes human immunodeficiency syndromes characterized by mycobacterial infection, myelodysplasia, lymphedema, or aplastic anemia that progress to myeloid leukemia. GATA2 encodes a master regulator of hematopoiesis that is also linked to endothelial biology. Though the disease-causing mutations commonly occur in the GATA-2 DNA binding domain, we identified a patient with mycobacterial infection and myelodysplasia who had an uncharacterized heterozygous deletion in a GATA2 cis-element consisting of an E-box and a GATA motif. Targeted deletion of the equivalent murine element to yield homozygous mutant mice revealed embryonic lethality later than occurred with global Gata2 knockout, hematopoietic stem/progenitor cell depletion, and impaired vascular integrity. Heterozygous mutant mice were viable, but embryos exhibited deficits in definitive, but not primitive, hematopoietic stem/progenitor activity and reduced expression of Gata2 and its target genes. Mechanistic analysis revealed disruption of the endothelial cell transcriptome and loss of vascular integrity. Thus, the composite element disrupted in a human immunodeficiency is essential for establishment of the murine hematopoietic stem/progenitor cell compartment in the fetal liver and for essential vascular processes.

Mechanism governing a stem cell-generating cis -regulatory element

“…+9.5 element uniquely endows hemogenic endothelium of the AGM with the capacity to generate long-term repopulating HSCs (LT-HSCs) that populate the fetal liver (5,25). In addition, the small number of HSCs generated from +9.5 −/− AGM undergo apoptosis and lack LT activity, indicating that the +9.5 element also confers LT-HSC survival (25); a similar conclusion emerged from analysis of a conditional Gata2 KO mouse (52).…”

“…In addition, the small number of HSCs generated from +9.5 −/− AGM undergo apoptosis and lack LT activity, indicating that the +9.5 element also confers LT-HSC survival (25); a similar conclusion emerged from analysis of a conditional Gata2 KO mouse (52). The +9.5 site confers maximal Gata2 expression in the AGM and definitive hematopoietic precursors in the fetal liver (5,25). A critical question is what molecular attributes underlie the uniquely important +9.5 site activity.…”

“…In contrast to the −1.8 and −2.8 site deletions, targeted deletion of the +9.5 intronic site is embryonically lethal at E13.5-E14.5 (5). The +9.5 site is essential for GATA-2 expression in hematopoietic stem and progenitor cells (HSPCs) and in endothelium during embryogenesis (5,9,25,26). Definitive hematopoiesis is severely impaired in +9.5 −/− mice due to defective HSC production, as demonstrated by competitive transplants and imaging of HSC genesis from hemogenic endothelium in the dorsal aorta (25).…”

“…Following NeoR excision, the targeted allele has a 126-bp Xba I-to-Not I fragment containing a single LoxP site substituted for the GATA motifs and intervening sequence. or near the +9.5 site (5,45). Thus, mutations of the +9.5 element, an essential mediator of definitive hematopoiesis in the mouse, underlie human hematopoietic pathology.…”

“…Although a single Gata2 allele is sufficient to confer viability in Gata2 +/− and +9.5 +/− mouse strains, heterozygosity in these mice reduces Gata2 expression, as well as HSC generation and function (5,19,20,25). To assess the influence of the −3.9 mutation on HSC genesis, we conducted whole-mount 3D embryo imaging to visualize c-Kit + hematopoietic clusters containing HSCs in E10.5 aorta gonad mesonephros (AGM) of −3.9 +/+ and −3.9 −/− littermates.…”