Gfi1 Is Repressed by p53 and Inhibits DNA Damage-Induced Apoptosis

Abstract 1228

Gfi1 is a transcriptional repressor that plays a critical role in hematopoiesis. Gfi1 has also been implicated in lymphomagenesis when aberrantly activated as a result of deregulated expression. It is still poorly understood how Gfi1 expression is regulated and how it acts in the hematopoietic system. We show here that Gfi1 transcription was repressed by the tumor suppressor p53 in hematopoietic cells. Treatment of cells with doxorubicin (Doxo), which induces topoisomerase II-mediated DNA double strand breaks (DSB), led to a steady increase in p53 protein level, which was accompanied by a gradual decline in Gfi1 expression at both protein and mRNA levels. Knockdown of p53 resulted in increased Gfi1 expression and also abolished Doxo-induced Gfi1 downregulation. In contrast, Gfi1 expression was reduced and its downregulation in response to DNA damage was restored in p53-deficient cells transfected with the p53/estrogen receptor ligand binding domain fusion protein (p53ER^TAM) in the presence of 4-hydroxytamoxifen (4-OHT). In luciferase reporter assays, Doxo treatment inhibited the activity of a ∼2.4-kb Gfi1 promoter fragment in p53^+/+ cells, but not in p53^−/− cells. Consistent with this, the wild type p53, but not a DNA binding-defective p53 mutant, repressed the Gfi1 promoter fragment. Chromatin immunoprecipitation (ChIP) assays demonstrated that p53 bound to the proximal region of the Gfi1 promoter. Detailed mapping of the Gfi1 promoter indicated that the core promoter region of Gfi1 spanning −33 to +6 bp is sufficient for p53-mediated repression. This core promoter region contains a putative p53 repressive response element (RRE) and, when the RRE was mutated, p53 failed to bind to and repress the Gfi1 promoter. Significantly, apoptosis induction by Doxo treatment was inhibited upon Gfi1 overexpression, but augmented following Gfi1 knockdown. Together, these data establish for the first time that Gfi1 is repressed by p53 and indicate that Gfi1 acts to protect hematopoietic cells from DNA damage-induced apoptosis. Our findings have important implications for understanding the roles of Gfi-1 in normal hematopoiesis and lymphomagenesis.