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 1

Disruption of the GATA2 E-box–GATA composite cis-element in the MonoMAC patient.

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Disruption of the GATA2 E-box–GATA composite cis-element in the MonoMAC ...
(A) ChIP-seq analysis of GATA-2 occupancy at GATA2 in human erythroid, endothelial, and neural cells. Datasets for K562 erythroleukemia and HUVECs were described (27, 43). The ChIP-seq data set from SH-SY5Y neuroblastomas (from R. Kumar, P.J. Farnham, and E.H. Bresnick, unpublished observations) was performed identically to the other analyses. (B) +9.5 kb site deletion in MonoMAC. Sequencing identified a 28-bp deletion from 1 allele of the MonoMAC patient (c.572+512del28 allele) versus control. Human and mouse sequence comparison illustrating conserved composite element. Red text: GATA motifs and E-box (CATCTG). Human transcript variant 1 (NM_001145661.1) includes two upstream noncoding exons. The murine intron 4 +9.5 region corresponds to human intron 5. (C) Co-inheritance of SNP-containing G allele and +9.5 kb site deletion (intron 5 del28) by one son of the MonoMAC patient determined by sequencing, demonstrating linkage of GATA2 +9.5 kb site deletion and exon 4 SNP rs34799090 (c.481C>G). (D) Differential allele expression in the +9.5 kb site deletion MonoMAC patient. SNP probes distinguished between GATA2 alleles in monocyte and T cell cDNAs (mean ± SD, 2 RT reactions per sample analyzed in quadruplicate; *P < 0.0001). (E) Bone marrow megakaryocytic dysplasia from the +9.5 kb site deletion MonoMAC patient versus normal human marrow megakaryocytes. Normal human megakaryocytes from H&E-stained core marrow biopsy and Wright-Giemsa–stained aspirate smear demonstrating normal maturation with multiple connected nuclear lobes. Dysplastic megakaryocytes from +9.5 kb site deletion patient with thrombocytopenia showing atypical separated or detached nuclear lobes (core biopsy, H&E; aspirate smear, Wright-Giemsa) and atypical mononuclear megakaryocyte; dysplastic megakaryocyte detected by Factor VIIIvw immunohistochemistry. Original magnification, ×1000.