Abstract
GATA-2 levels must be stringently regulated to ensure normal hematopoiesis, and human GATA-2 mutations cause hematologic disorders. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells and are essential for hematopoiesis and embryonic development. Mechanisms underlying how the enhancers control Gata2 expression and GATA-2 instigated genetic networks in a cell-specific manner are not completely understood. Targeted deletion of an intronic Gata2 enhancer 9.5 kb downstream of the transcription start site (+9.5) abrogates HSC genesis in the aorta-gonad-mesonephros (AGM) region (Gao et al., JEM, 2013). By contrast, the -77 kb enhancer (-77) activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation (Johnson et al., Science Advances, 2015). To dissect relationships between the enhancers, we developed a compound heterozygous (CH) mouse model bearing +9.5 and -77 enhancer mutations on different Gata2 alleles. While the CH embryos were alive at E13.5, nearly all died by E14.5 (p = 3.58 x 10-5). Flow cytometric analyses and embryo confocal imaging demonstrated that CH embryos have modestly reduced HSC numbers in the fetal liver (2.9-fold) and the AGM (41%, p = 7.8 x 10-5), which was comparable to +9.5+/- embryos. Thus, -77 does not genetically interact with +9.5 to control HSC emergence. Flow cytometric analysis revealed that Lin-Sca1-Kit+ myelo-erythroid progenitors were 6.6-fold lower in CH vs. WT embryos (p = 1.8 x 10-11), with the difference involving disproportionate losses of GMP (8.6-fold; p = 3.7 x 10-6) and MEP (379-fold; p = 3.2 x 10-9). By contrast, +9.5+/- fetal livers had 2-fold fewer myeloid progenitors, which involved similar reductions of CMP (2.1-fold; p = 1 x 10-6), GMP (2.6-fold; p = 0.0007) and MEP (1.9-fold; p = 0.002). Consistent with the myelo-erythroid progenitor reductions and MEP depletion, CH fetal livers lacked BFU-E (p < 0.001) and CFU-GEMM (p < 0.001) in a colony assay. These results illustrate a genetic interaction between +9.5 and -77 in progenitors, but not HSCs, and a new paradigm in which both enhancers must reside on a single allele to generate MEPs.
As erythroid precursor cells express GATA-2, we tested whether the -77 deletion impairs erythroid maturation due to a reduction in myelo-erythroid progenitors or due to a cell-autonomous requirement of the enhancer in erythroid precursors. -77-/- E14.5 fetal livers were pale and smaller than WT counterparts, and -77-/- fetal liver cellularity was reduced 7.2-fold (5.3 x 10-4). When liver size was taken into account, there was little difference in the number of E14.5 R1 cells in -77-/- liver vs. WT littermates (p = 0.31). However, -77-/- R2-R5 cells declined sharply (R2, 8.2-fold, p = 0.004; R3, 14-fold, p < 10-5; R4, 9.7-fold, p = 0.002; R5, 14-fold, p = 0.087). The mutant R1 cells were defective in forming BFU-Es and CFU-Es. Analysis of transcriptomes of purified 77-/- and WT R1 cells from E14.5 fetal livers revealed 2805 and 2519 upregulated and downregulated (p < 0.05) genes, respectively, in -77-/- R1 cells. The -77 enhancer conferred GATA-2 expression, which strongly upregulated GATA-1 and therefore a large GATA-1 target gene cohort. A comparison of WT and -77-/- R1 cell transcriptomes with those of early (Tgbfr3low) and late (Tgbfr3high) BFU-Es (Gao et al., Blood, 2016) revealed a -77-/- R1 signature that correlated with the early BFU-E signature (r = 0.73, p < 10-4) and negatively correlated with the late BFU-E signature (r = -0.42, p = 4 x 10-4) differing from WT cells. In addition to GATA-1 target gene alterations, 253 of the -77-activated genes were not GATA-1-regulated in the G1E-ER-GATA-1 system. These genes included Ryk, which encodes a non-canonical Wnt receptor, and had not been studied in erythroid cells. Two Ryk shRNAs significantly decreased BFU-Es and CFU-GMs in lineage-depleted fetal liver cells. Ongoing studies are integrating Ryk function into signaling circuits that control erythroid maturation and analyzing other -77-regulated targets predicted to constitute new regulators of erythroid cell maturation/function. Thus, loss of the -77 enhancer creates multi-faceted defects in erythroid precursors, involving deficiencies of constituents of signaling and transcriptional circuitry required to enable and drive erythroid maturation.
No relevant conflicts of interest to declare.
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