A Microrna Feed-Forward Loop Amplifies GFI1N382S Transcriptional Reprogramming In Severe Congenital Neutropenia

Abstract 2239

The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell self renewal and myeloid differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 that generate dominant negative acting proteins. Moreover, GFI1-mutant SCN patients and Gfi1-/- mice display a unique accumulation of myeloid progenitors. Recently we showed that Gfi1 regulation of HoxA9, Pbx1 and Meis1 underlies these phenomena, in that the Gfi1-Hox transcriptional circuit controls the accumulation of myeloid progenitors in vivo. We have also shown that Gfi1 regulates miR-21 during myelopoiesis, and that miR-21 is deregulated by Gfi1N382S expression. Our new data link these concepts by demonstrating that forced expression of miR-21 in bone marrow cells results in the accumulation of myeloid progenitors in transplant recipients. Moreover, miR-21 directly targets the Ski oncoprotein, and Ski-/- bone marrow cells show an accumulation of myeloid progenitors. Thus, Gfi1-/-, miR-21 overexpressing-, and Ski-/- myeloid progenitors accumulate in the marrow. Strikingly, Ski is dramatically reduced in miR-21 overexpressing Lin- bone marrow cells. Nearly undetectable Ski expression in Gfi1-/- bone marrow cells can be completely rescued by antagonizing miR-21 activity. Since Ski is a corepressor and Gfi1 is a transcriptional repressor, we next tested whether the two proteins physically interact. Indeed, endogenous Ski and Gfi1 can be coimmunoprecipitated. Synthetic Ski and Gfi1 proteins reveal that the interaction is mediated through Ski carboxy-terminal and Gfi1 zinc-finger domains. Chromatin immunoprecipitation reveals Ski and Gfi1 co-occupy several Gfi1 target genes (including HoxA9), which are derepressed upon Gfi1 or Ski knockdown. However, while Gfi1 binds and regulates the miR-21 gene, Ski is not bound to the miR-21 gene, and Ski knockdown has no effect upon miR-21 levels. Thus, the data point to a novel feed-forward transcriptional circuit. Gfi1N382S deregulation of miR-21 amplifies the dominant-negative effect of Gfi1N382S through miR-21 targeting of Ski, leading to further derepression of Gfi1-Ski target genes.