Eomesodermin promotes interferon-[gamma] expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines

The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-[gamma] (IFN-[gamma]). However, in contrast to T-bet, the role of Eomes in the expression of IFN-[gamma] remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-[gamma] in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-[gamma] in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners.