Cis-element mutated in GATA2-dependent immunodeficiency governs hematopoiesis and vascular integrity
Kirby D. Johnson, … , Steven M. Holland, Emery H. Bresnick
Kirby D. Johnson, … , Steven M. Holland, Emery H. Bresnick
Published September 10, 2012
Citation Information: J Clin Invest. 2012;122(10):3692-3704. https://doi.org/10.1172/JCI61623.
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Research Article

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 GATA2cis-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.

Authors

Kirby D. Johnson, Amy P. Hsu, Myung-Jeom Ryu, Jinyong Wang, Xin Gao, Meghan E. Boyer, Yangang Liu, Youngsook Lee, Katherine R. Calvo, Sunduz Keles, Jing Zhang, Steven M. Holland, Emery H. Bresnick

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Figure 8

Mechanisms underlying the vascular integrity defect of +9.5–/– embryos.

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Mechanisms underlying the vascular integrity defect of +9.5–/– embryos. ...
(A) Microarray-based comparison of gene expression in PECAM-1–positive cells purified from E12.5 +9.5+/+ and +9.5–/– embryos. A single-cell suspension was generated from the embryo proper lacking the fetal liver, and PECAM-1–positive cells were isolated by adsorption to magnetic beads loaded with anti–PECAM-1 antibody. Microarray analysis was conducted with four +9.5+/+ and four +9.5–/– embryos, and statistically significant downregulated and upregulated genes are shown in the heat map. (B) Gene ontology analysis of genes downregulated in +9.5–/– embryos using the DAVID Bioinformatics Program ( http://david.abcc.ncifcrf.gov/). A P value of 0.05 was used as the standard cutoff level. org., organization. (C) Quantitative RT-PCR validation of gene expression changes in PECAM-1+ cells. (D) ChIP-seq profiles of endogenous GATA-2 occupancy of ITGB3 and FERMT3 loci in HUVECs. (E) Itgb3 downregulation in MAE cells upon siRNA-mediated knockdown of GATA-2. Left: Representative Western blot showing the extent of GATA-2 knockdown. Right: Dot plot of Itgb3 mRNA levels in MAE cells treated with control or Gata2 siRNA, respectively. Each dot represents data from a single experiment, and data from 13 independent experiments are shown. Mean and SEM are indicated by long and short horizontal bars, respectively (P < 0.0001).