The zinc finger protein Growth factor independent-1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cell (HSC) maintenance and granulocytic lineage differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 (encoding GFI1N382S) which generate dominant negative acting proteins. GFI1N382S proteins sequester limiting cofactors to deregulate a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs and suggest that that transcriptional control of microRNA genes is critical for GFI1N382S-associated SCN phenotypes. First, the expression of Gfi1 and miR21 and miR-196 is reciprocal: 1) in wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), 3) during treatment-induced differentiation of human myeloid leukemia cell lines, and 4) upon conditional deletion of Gfi1 in primary sorted murine CMP and GMP. Biochemical analyses reveal that miR21 and miR-196 are direct transcriptional targets of Gfi1. Subsequently, forced expression of wild type Gfi1 rescues expression of microRNA in Gfi1−/− Lin- bone marrow cells, while forced expression of Gfi1N382S in wild type Lin- bone marrow cells significantly deregulates miR-21 and miR-196 expression. Similarly, we demonstrate elevated miR21 and miR196b levels in CD34+ cells from a GFI1N382S SCN patient. Flow cytometric analysis and colony assays reveal that the overexpression or knockdown of either miR induces changes in myeloid development, but that co-expression of both miR (as seen in Gfi1−/− mice and GFI1N382S SCN patients) completely blocks G-CSF-induced granulopoiesis. These data provide a molecular understanding of SCN disease pathogenesis.

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