PROJECT SUMMARY/ABSTRACT The objective of this proposal is to determine how GATA2 deficiency elevates innate immune signaling, including Interferon-gamma (IFNγ) and Toll-like receptor (TLR) signaling, in fetal progenitor cells and the functional consequences of hypersensitive innate immune signaling. GATA2 is a master transcription factor essential for hematopoiesis in embryos and adults. Gata2 expression is regulated by the activity of two enhancers, -77 kb upstream and +9.5 kb downstream of the Gata2 transcription start site. Mutations in the coding and non-coding regions of GATA2 cause a hereditary syndrome with a wide variety of hematologic manifestations, including immunodeficiency, acute myeloid leukemia, myelodysplastic syndrome, and bone marrow failure. Studies in our group demonstrated that GATA2 regulates genes encoding important cellular signaling components to establish and maintain hematopoietic stem and progenitor cell function. Genetic loss of an enhancer 77 kb upstream of Gata2 start site is embryonically lethal after E15.5, downregulates Gata2 expression in myeloid-erythroid progenitors, upregulates components of IFNγ, TLR, and Interleukin-6 signaling, and impairs hematopoiesis with the loss of erythroid and granulocytic differentiation and the retention of monocytic differentiation. My results demonstrate that GATA2-deficient primary (-77-/-) lineage-depleted progenitors isolated from E14.5 fetal livers and ER-HOXB8-immortalized (hi-77-/-) fetal progenitors exhibit elevated TLR signaling, and GATA2 deficiency creates signaling crosstalk between IFNγ and TLR1/2 and TLR2/6 to elevate cytokine/chemokine gene expression and protein production in myeloid cell progeny. Furthermore, GATA2 deficiency de-represses the activity of the myeloid/lymphoid transcription factor PU.1 to elevate myeloid and B cell-lineage gene expression. Since PU.1 cooperates with signal-dependent transcription factors such as STATs, IRFs, NF-κB, and AP-1 to regulate gene expression, and reducing PU.1 level suppressed TLR1/2-mediated gene regulation and dysregulated a subset of IFNγ-activated genes in hi-77-/- progenitors, I hypothesize that GATA2 deficiency in fetal progenitors increases PU.1 activity, and through cooperation with IFNγ- and TLR-regulated transcription factors, GATA2-deficient fetal progenitor cells are rendered hypersensitive to innate immune signaling. I will use multidisciplinary approaches to test hypotheses in the following Specific Aims. Aim 1: To determine the molecular mechanisms by which PU.1 elevates IFNγ- and TLR1/2-dependent transcriptional responses in GATA2-deficient fetal progenitors; Aim 2: To determine the functional consequences of elevated TLR signaling in GATA2-deficient fetal progenitors. Using multidisciplinary approaches to address innovative questions, this study will advance our understanding of how GATA2 dysregulation alters critical cell biological processes to generate hematologic pathologies and will provide immense ...