Gfi1-Mediated Repression of C-Fos, Egr1 and Egr2, and Inhibition of ERK1/2 Signaling Contribute to the Role of Gfi1 in Granulopoiesis

Gfi1 is a transcriptional repressor that plays an important role in hematopoiesis. In the myeloid system, Gfi1 promotes granulopoiesis and antagonizes the alternative monocyte development in part through repressing PU.1 and CSF1 , which promote monopoiesis. However, the molecular mechanism by which Gfi1 favors neutrophil over monocyte development remains incompletely understood. G-CSF is a lineage-specific cytokine that plays a dominant role in granulopoiesis. We recently showed that a G-CSFR mutant in which tyrosine 729 was mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of ERK1/2 and augmented activation of downstream targets c-Fos and Egr1. To address whether Gfi1 may promote granulopoiesis independent of its repression of Csf1 , we established murine myeloid 32D and multipotent FDCP-mix cells that constitutively expressed G-CSFR Y729F and inducibly expressed Gfi1 in response to doxycycline (Dox; 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1). 32D and FDCP-mix cells showed no response to CSF1. In the absence of Dox, 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1 differentiated into monocytes when cultured in G-CSF. Interestingly, addition of Dox to induce Gfi1 expression rescued neutrophil development in both cell lines. Induction of Gfi1 expression with Dox also markedly attenuated the induction of c-Fos, Egr1 and Egr2 expression in response to G-CSF. In contrast, the mRNA levels of c-Fos, Egr1 and Egr2 were dramatically elevated in lineage marker negative (Lin^-) bone marrow (BM) cells from Gfi1^-/- mice as compared to BM cells from Gfi1^+/+ mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that Gfi1 bound to and repressed the activities of c-Fos , Egr1 and Egr2 promoters. c-Fos , Egr1 and Egr2 are immediate early genes that are activated by the ERK1/2 pathway. Notably, G-CSF-stimulated activation of ERK1/2 was stronger in Lin^- BM cells from Gfi1^-/- mice than in cells from Gfi1^+/+ mice. It has been shown that BM cells from Gfi1^-/- mice are unable to differentiate into mature neutrophils in vitro , but instead give rise to atypical monocytes. Interestingly, the MEK1/2 inhibitor U0126 or PD0325901 reduced the mRNA levels of c-Fos, Egr1 and Egr2, and partially rescued G-CSF-induced neutrophil development in Gfi1^-/- BM cells. Together, our data indicate that Gfi1 inhibits the expression of c-Fos, Egr1 and Egr2 through direct transcriptional repression of their genes and indirect inhibition of ERK1/2 signaling pathway, and that Gfi1-mediated downregulation of c-Fos, Egr1 and Egr2 may contribute to the role of Gfi1 in granulopoiesis.