Single-Nucleotide Human Disease Mutation Inactivates a Blood-Regenerative Enhancer

Coding and regulatory GATA2 mutations that deregulate protein expression and/or function cause MDS/AML (McReynolds et al., 2018). In the mouse, decreased GATA-2 expression impairs hematopoietic stem/progenitor cell (HSPC) genesis and function. While prior studies demonstrated the requirement for Gata2 +9.5 and -77 enhancers to regulate GATA-2 and HSPC transitions in vivo (Gao et al., 2013; Johnson et al., 2012; Johnson et al., 2015; Mehta et al., 2017; Sanalkumar et al., 2014), nothing is known about the contribution of the individual motifs within these enhancers for GATA-2 expression, hematopoiesis and hematopoietic regeneration.

To elucidate mechanisms conferring +9.5 enhancer activity that regulate HSPC genesis and function, we used CRISPR/Cas9 technology to generate cis-element-mutant mouse strains. The first strain recapitulates human disease mutations and involves corruption of the E-box and a C>T transition within an Ets site (+9.5(E-box;Ets)), while retaining a GATA motif. The second strain contains the Ets 1017+572C>T transition (+9.5(Ets)), representing the most frequent noncoding GATA2 mutation in human patients (Ganapathi et al., 2015; Hsu et al., 2013). We compared the phenotypes of these strains with our previously generated +9.5(E-box;GATA)^-/- strain (+9.5^-/-) (Gao et al., 2013; Johnson et al., 2012) to ascertain how the +9.5 regulates hematopoietic processes. +9.5^-/- embryos have nearly complete ablation of HSC emergence and die by E14. +9.5(E-box;Ets^-/- embryos recapitulate +9.5 defects, indicating that neither the GATA nor Ets motifs suffice for +9.5 developmental functions. In contrast, +9.5(Ets)^-/- mutant animals are born at normal Mendelian ratios - the sole example of a +9.5 mutation in the mouse that is not embryonic lethal. Mutant E15.5 embryos have minor decreases in fetal liver cell number (20%, P=0.023), no hemorrhaging or edema, and Gata2 mRNA is reduced 25-50% (P=0.006).

+9.5(Ets)^-/- adult mice exhibited normal steady-state hematopoiesis. To test if stress-dependent hematopoietic regeneration revealed Ets motif activity, 5-fluorouracil (5FU) was used to kill cycling cells and promote quiescent HSC proliferation. Two doses of 5FU (250 mg/kg) were administered at an interval of eleven days to stimulate maximal HSPC expansion (Xu et al., 2017). +9.5(Ets)^-/- mice had a significantly reduced (P<0.0001) median survival of 14.5 days, versus 22 and 21 days for wild type (WT) and +9.5(Ets)^+/-, respectively. We hypothesized that the hypersensitivity of +9.5(Ets)^-/- mice to myeloablative stress was due to impaired HSC mobilization and/or recovery. To identify cell populations altered in +9.5(Ets)^-/- bone marrow, we quantified immunophenotypic HSPCs in untreated and treated (9 and 11 days post-5FU) mice. In untreated mice, +9.5(Ets)^-/- and WT HSC (Lin⁻Sca1⁺Kit⁺CD48^-CD150⁺) numbers were similar. MPPs (Lin^-Sca1⁺Kit⁺CD48^-CD150^-) were 9-fold lower in mutants. Nine and eleven days post-5FU treatment, HSCs increased 17- and 10-fold in WT (P<0.001 and P=0.003, respectively), but not +9.5(Ets)^-/-, mice. To assess HSPC function, bone marrow was competitively transplanted into lethally-irradiated mice. Four months post-transplant, +9.5(Ets)^-/- marrow was defective in multi-lineage repopulation (3-fold decrease in donor-derived cells from peripheral blood (P=0.0005), with significant reductions of HSPCs in bone marrow). These results demonstrate that the Ets motif confers Gata2 function as a key step in HSC regeneration.

In summary, multiple cis-elements confer the developmental function of an essential enhancer, while a single cis-element mutated in disease confers regeneration in response to injury. Our results establish a paradigm in which the cis-element predisposition mutation, combined with a secondary insult, underlies pathogenesis and illustrates the power of micro-dissecting enhancers to unveil physiological and pathological mechanisms. Studies are underway to rigorously analyze the functional consequences of diverse secondary insults to identify conditions that cause bone marrow failure and hematologic malignancy. Comparing and contrasting developmental and regenerative genetic networks, including those operational in single cells from our unique models, will reveal broad principles and strategies to manipulate normal and malignant hematopoiesis, for example in the context of GATA-2-dependent pathologies.