Figure 4

IRF2 is required for chromatin accessibility and active histone modifications at the TLR3 promoter. A IRF2 and BAF47 are required for the chromatin accessibility at the TLR3 promoter. HeLa cells transfected with the control or small interference RNA constructs as indicated above the panel were selected with puromycin for three days. Following treatment with IFN-α for 12 hours, HeLa nuclei were isolated and briefly digested with Hind III for 10 minutes. The purified genomic DNA was digested to completion with Nhe I and analyzed by LM-PCR using primers specific for the TLR3 promoter. The data were quantified using phosphoimager analysis. The intensity of the Hind III bands were normalized by that of the Nhe I bands and indicated below the panel. The positions of Hind III and Nhe I sites were indicated in Figure 1C. B IRF2 is required for H3K4me3 modification at the TLR3 promoter in both unstimulated and stimulated states. HeLa cells transfected with the control or small interference RNA construct targeting IRF2 were selected with puromycin for three days. Following treatment with IFN-α for 12 hours, chromatin fractions were prepared and immunoprecipitated with H3K4me3 antibodies. The resulting DNA samples were analyzed using q-PCR with primers for either the TLR3 promoter or exon II regions. C IRF2 is required for H3K9ac/K14ac modification at the TLR3 promoter in both unstimulated and stimulated states. HeLa cells transfected with the control or small interference RNA construct targeting IRF2 were selected with puromycin for three days. Following treatment with IFN-α for 12 hours, chromatin fractions were prepared and immunoprecipitated with H3K9ac/K14ac antibodies. The resulting DNA samples were analyzed using q-PCR with primers for either the TLR3 promoter or exon II regions.