Our efforts to forge principles for how GATA factors control development and function of the hematopoietic system led to the discovery of conserved Gata2 enhancers (+9.5 and -77) essential for hematopoiesis and other blood-regulatory enhancers. While genomic studies commonly predict enhancers, there were no reports of enhancers essential for stem cell genesis or progenitor fate decisions. Strikingly, +9.5 mutations resemble GATA2 coding mutations in causing human GATA2-deficiency syndrome involving immunodeficiency, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). -77 disruption also causes MDS/AML. This foundation led clinical centers to analyze -77 and +9.5 genetic variation. We discovered: 1) Whereas +9.5 and -77 confer progenitor cell fate, only +9.5 triggers hematopoietic stem cell genesis. 2) GATA2 loss with EVI1 upregulation is leukemogenic. 3) An ensemble of “+9.5-like” enhancers exist, including one regulating a c-Kit facilitator that promotes erythrocyte regeneration and survival in severe anemia. 4) GATA2-regulated G-protein- coupled receptor circuits control hematopoiesis. 5) We described gain-of-function (GOF) GATA2 mutants, suggesting a paradigm-shift. 6) +9.5 single-nucleotide disease mutation dissociates developmental and regenerative activities, creating a bone marrow failure predisposition. The disease mutations, innovative mouse models, rescue assay and multiomics will enable the following aims. Aim 1 will determine how a blood disease-causing enhancer establishes progenitor cell fate. -77-/- progenitors are defective in erythroid and granulocytic differentiation, yet competent to generate macrophages. Our proteomic and single-cell transcriptome data revealed that skewed differentiation involves upregulated interferon (IFN) response proteins. We hypothesize that convergence of GATA2 and IFN mechanisms is critical in progenitor biology. We will establish the -77-regulated transcriptome/proteome that controls progenitor fate and address key mechanistic issues. Aim 2 will elucidate genetic determinants for the function of essential GATA2 enhancers. We generated compound heterozygous (CH) enhancer mutant mice (-77+/-;+9.5+/-) and discovered that both enhancers must reside on the same allele to regulate progenitor function. We are unaware of any example of a dual enhancer requirement for progenitor function, the mechanisms may inform human +9.5 and -77 enhanceropathies, we will elucidate the mechanisms. Aim 3 will test models for how GATA2 human disease mutants exert gain-of-function activity. We innovated a rigorous rescue assay in which restoring GATA2 in - 77-/- progenitors normalizes skewed differentiation and transcription. Mutants assumed to be loss-of-function (LOF) surprisingly retained activity to rescue granulopoiesis and select target genes. We will test if all disease mutants exhibit GOF activity, elucidate the mechanisms and determine if activities parse mutants into groups that may ultimately inform cli...