Molecular Mechanism of Severe Congenital Neutropenia

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DESCRIPTION (provided by applicant): Severe Congenital Neutropenia (SCN) predisposes affected humans to opportunistic infections because of a profound deficiency in the production of neutrophils. The majority of SCN patients express mutant forms of neutrophil elastase (a serine protease encoded by ELA2). To date, mouse models of SCN using ELA2 mutants from SCN patients have not reproduced the disease; limiting dissection of the molecular mechanisms underlying SCN. Growth Factor Independence-1 (GFI1) is a transcriptional repressor oncoprotein with a classic C2-H2 zinc-finger DNA-binding domain. We have recently shown that the affected individuals in a family of SCN patients are heterozygous for mutations in GFI1. Importantly, GFI1-/- mice and GFI1-mutant SCN humans exhibit a strikingly similar disease and dramatically lack any mature neutrophils. Granulopoiesis is controlled by transcription factors. GFI1 is a transcription factor. Thus, GFI1 mutations may be more clearly associated with blocks in both human and murine granulopoiesis than other SCN-associated mutations. An exploration of GFI1 in neutrophil differentiation provides a unique opportunity to identify biochemical pathways disrupted in SCN patients, and to develop an animal model of this inherited bone marrow failure syndrome. Our preliminary evidence indicates that GFI1 targets ELA2. We hypothesize that GFI1-mutant proteins found in SCN patients cause neutropenia because they act as dominant negative proteins; interfering with GFI1 transcriptional control over ELA2 and myeloid specific genes. We propose to utilize retroviral vector transduction to validate SCN-mutant-GFI1 proteins as disease-causing entities in murine and human hematopoietic stem cells. Moreover, the role of wild-type GFI1 gene dosage in controlling mutant GFI1 molecules and their mechanism of action will be discerned, then correlated to functional and molecular analyses of patient blood. Finally, we will molecularly characterize ELA2 as a GFI1-target gene in myelopoiesis, and we will biologically test suggested genetic interactions. These studies will provide an animal model of SCN, and are designed to dissect the molecular mechanism underlying this bone marrow failure syndrome. More generally, this work should impact our understanding of hematopoiesis, as well as engender greater understanding of GFI1 function.