Identification of Gfi1 as a Direct Target of the Leukemogenic Fusion Protein AML1-ETO9a

AML1-ETO9a (AE9a), a C-terminal splice variant of t(8;21), is highly leukemogenic in mice. Using combined gene expression and promoter occupancy (ChIP-on-chip) profiling of an AE9a induced murine AML model, we identified Gfi1, a transcriptional repressor important for neutrophil differentiation, as a direct target of AE9a. The level of Gfi1 mRNA was down-regulated following leukemia development. We further show that t(8;21) AML-M2 patient samples have lower Gfi1 mRNA levels compared to non-t(8;21) AML-M2 samples. In addition, Gfi1 protein was down-regulated in the human hematopoietic cell line U937 upon AE or AE9a expression. Two consensus AML1 sites (TGTGGT) located at positions −1750 and +405 from the Gfi1 transcriptional initiation site, respectively, are found in the proximity of the peaks of ChIP-on-chip profiling. We confirmed direct DNA binding of AE9a to these regions by ChIP assays. Furthermore, we showed that the activity of the Gfi1 promoter region −1880 to +481 is repressed by AE and AE9a in luciferase reporter assays. To address the effects of down-regulation of Gfi1 on proliferation, we overexpressed it in a primary AML1-ETO C-terminal mutant (AML1-ETOtr) leukemia cell line. Its overexpression slowed down growth and promoted apoptosis and differentiation of this cell line. In this report, we provide evidence that AE9a directly down-regulates Gfi1, thereby inhibiting hematopoietic differentiation, which may contribute to leukemia development.