Pathogenic Mutation that Dislocates GATA2 Zinc Fingers Establishes a Hematopoiesis-Disrupting Signaling Network

While certain human genetic variants are conspicuously loss-of-function, decoding the functional impact of many variants is challenging. Previously, we described a leukemia predisposition syndrome (GATA2-deficiency) patient with a germline GATA2 variant that inserts nine amino acids between the two zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies in Gata2 enhancer-mutant hematopoietic progenitor cells to reveal how the insertion impacts GATA2 function genome-wide. Despite being nuclear-localized, 9aa-Ins was severely defective, with activation more impaired than repression. Variation of the inter-zinc finger spacer length revealed that repression tolerated insertions that were detrimental to activation. GATA2 deficiency generated a hematopoiesis-disrupting signaling network in progenitor cells with reduced Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) signaling and elevated Interleukin-6 (IL-6) signaling. As insufficient GM-CSF signaling causes pulmonary alveolar proteinosis and excessive IL-6 signaling causes bone marrow failure, hallmark phenotypes of GATA2-deficiency patients, these results establish molecular mechanisms underlying GATA2-linked pathologies. Overall design: 25000 Hoxb8-immortalized wild type hematopoietic progenitors (hi-77+/+) infected with control (empty) retrovirus, Gata2 -77 enhancer deleted mutant progenitors (hi-77-/-) infected with empty retrovirus or GATA2- or 9aa-Ins-expressing retrovirus were FACS isolated for GFP+