IFN-γ up-regulates PU.1 expression in erythroblasts through IRF-1. (A) Quantitative PCR analysis of the expression of IRF-1 mRNA in CD71⁺ erythroblasts from WT, CD70TG, IFN-γ^−/−, and CD70TG*IFN-γ^−/− mice. Data are mean ± SD for 3 mice per group. *Significant difference (P < .05) between CD70TG mice and all other groups (1-way ANOVA with Bonferroni correction). (B) Quantitative PCR analysis of IRF-1 mRNA in CD71⁺ erythroblasts or MEPs from WT mice cultured overnight with or without IFN-γ. Data are the fold induction of IRF-1 expression with IFN-γ compared with the medium control for 7 (CD71⁺) or 6 (MEPs) mice per group, pooled from 2 independently performed experiments. *P < .001 (CD71⁺) or P < .05 (MEPs; paired Student t test). (C) Sequence analysis of the promoter region of the human SPI1 (PU.1) gene, displaying a conserved putative IRF-1/2 binding site approximately 15 kb upstream of the transcription start site (based on the UCSC Genome Browser). (D) Effect of IFN-γ on the in vitro BFU-e and CFU-e colony-forming potential of human erythroid precursor cells cultured overnight with or without IFN-γ. Data are mean ± SD from triplicate analysis. Results are representative from 2 independently performed experiments. *P < .05 (nonpaired Student t test). (E) ChIP analysis of the binding of IRF-1 to the putative IRF-1/2 binding site in the SPI1 (PU.1) promoter of human erythroid precursor cells cultured overnight with or without IFN-γ. RFE indicates relative fold enrichment. Data are mean ± SD from 3 independent experiments. *P < .05 (nonpaired Student t test). (F) Effect of IFN-γ on the BFU-e and CFU-e potential of human erythroid precursor cells transduced with shRNA directed against IRF-1 or PU.1. Data are mean ± SD from 3 independent experiments. *P < .05 (1-way ANOVA with Bonferroni correction).