Pathogenic GATA2 genetic variants utilize an obligate enhancer mechanism to distort a multilineage differentiation program

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2- deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2- dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA- binding C- terminal zinc finger, T354M disproportionately required the N- terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-epsilon (C/EBP epsilon) enhancer, creating a feedforward loop operating with the T - cell Acute Lymphocyte Leukemia - 1 (TAL1) transcription factor. Elevating C/EBP epsilon partially normalized hematopoietic defects of GATA2- deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.