Ets-1 is involved in the regulation of the T-bet enhancer in vivo and in vitro. (A) B cells were purified from the spleens of wild-type C57Bl/6 mice and either stimulated for 1 hour with 25 μg/mL of LPS and 50 ng/mL of IFN-γ (dark histograms) or left untreated (open histograms). ChIP analysis was performed using normal rabbit serum (Ig), anti-STAT1 (Stat1), or anti–Ets-1 (Ets-1) Abs. Quantification of immunoprecipitated DNA fragments was performed by real-time PCR using primers for the promoter region (CNS I), the enhancer (CNS IV), and the irrelevant Foxp3 gene (Foxp3). Values were normalized to corresponding input control and are expressed as the fold enrichment relative to normal rabbit serum for each experiment. Results are representative of 3 individual experiments. (B) The ETS-binding sites in CNS IV are essential for T-bet enhancer activity. Luciferase activity of pGL3 empty vector (pGL3), wild-type T-bet CNS IV (Wt-CNS IV), or CNS IV carrying mutated ETS-binding sites (Mut-CNS IV). Constructs were transfected into the WEHI B-cell line and normalized luciferase activities were measured in nonactivated and overnight IFN-γ (50 ng/mL)–stimulated cells. Bar graphs show the average fold increase in luciferase activity after activation of 4 independent experiments. Errors bars indicate SDs.