Growth factor independence-1 (Gfi-1) is a zinc-finger transcriptional repressor that plays a critical role in hematopoiesis. Gfi-1 regulates the development of myeloid and lymphoid cells, and controls hematopoietic stem cell self-renewal. Gfi-1 is weakly oncogenic but strongly cooperates with oncoprotein Myc in lymphomagenesis. How Gfi-1 functions in hematopoiesis remains poorly understood. Data presented here demonstrate that Gfi-1 represses p21Cip1 and MBP through two distinct mechanisms.
Gfi-1 interacts with Myc-interacting zinc-finger protein (Miz-1), a transcriptional activator regulating cell cycle progression and apoptosis, and is recruited by Miz-1 to the promoter of Miz-1 target gene p21Cip1 which encodes a potent cell cycle inhibitor, leading to transcriptional repression. Repression of p21Cip1 by Gfi-1 is independent of direct DNA-binding. Knockdown or deficiency of Gfi-1 results in augmented p21Cip1 expression. Interestingly, Gfi-1 forms a ternary Gfi-1/Miz-1/Myc complex on the p21Cip1 promoter and collaborates with Myc in the repression of p21Cip1. This Miz-1-dependent transcriptional repression by Gfi-1 also applies to other Miz-1 target genes encoding cell cycle inhibitors p15Ink4b and p27Kip1. Consistent with the mechanism of Miz-1-dependent transcriptional repression, Gfi-1 also represses growth inhibitory cytokine TGF-β-activated p21Cip1 independent of DNA-binding. Interestingly, Gfi-1 expression is downregulated by TGF-β, suggesting a role of Gfi-1 in TGF-β-mediated growth inhibition.
MBP encodes a cytotoxic granule protein expressed in eosinophils and basophils. Our data identify MBP as a new target of Gfi-1-mediated transcriptional repression. Unlike p21Cip1, however, the repression of MBP by Gfi-1 requires Gfi-1 direct DNA-binding as evidenced by the fact that the Gfi-1 dominant negative mutant N382S, which is defective for DNA-binding, relieves the transcriptional repression of MBP by Gfi-1. Indeed, knockdown of Gfi-1 results in enhanced expression of MBP. Expression of the N382S mutant has been shown to cause premature apoptosis of myeloid cells induced to differentiate by G-CSF. Interestingly, overexpression of MBP also results in increased apoptosis during G-CSF-stimulated terminal neutrophilic differentiation, indicating that elevated MBP expression may contribute to the N382S-associated apoptosis of differentiating myeloid cells. These data suggest that the transcriptional repression of MBP by Gfi-1 may contribute to the role of Gfi-1 in regulating granulocyte development.
Taken together, our study demonstrates Gfi-1-mediated transcriptional repression of p21Cip1 and MBP by two different mechanisms. Gfi-1, via binding to Miz-1, is recruited to p21Cip1 and other Miz-1 target genes leading to transcriptional repression, and Gfi-1 represses MBP, however, through direct DNA-binding. These findings provide new insights into the transcriptional regulation by Gfi-1 and may have broad implications for better understanding the role of Gfi-1 in normal hematopoiesis and tumorigenesis.