Fig. 2.

T-bet recruitment to the endogenous IFN-γ promoter and enhancement of histone modifications. (A) T-bet transactivation of the endogenous IFN-γ gene. Shown are representative results of RT-PCR analyses of IFN-γ and GAPDH mRNAs in Jurkat cells. Cells were nontransfected (first two lanes) or subjected to short-term transfection with T-bet expression vector, followed by stimulation with nothing or PMA and ionomycin overnight, as indicated. RT-PCR of IFN-γ and GAPDH mRNAs were performed within the linear range of amplification, and products were detected by Southern blot hybridizations using internal oligonucleotide probes. (B and C) Jurkat T cells, stably infected with either an empty retrovector or one directing constitutive T-bet expression, were divided equally and stimulated with nothing or the combination of PMA and ionomycin as indicated. These cell populations were then used for ChIPs with antibodies specific for T-bet or IgG negative control or PCR from preimmunoprecipitation chromatin (input) (B) or anti-acetyl-histone H4, antidimethyl-histone H3(K4), or nonspecific IgG (C), as indicated. Western blots documented activation-induced expression of low levels of T-bet in the empty vector-infected cells and constitutive expression of T-bet in the transduced population (data not shown). In each case, similar results were obtained in short-term transfections with IRES-Thy1.1 expression vector plasmids followed by MACS selection of positive cells and ChIPs (data not shown).