IRF-binding site mediates the BAF complex activity at the TLR3 promoter. A The TLR3 promoter is regulated by BAF complex in a chromatin-dependent manner. SW-13 cells were transfected with the pREP4 or pGL3 reporter vectors in the absence or presence of a BRG1 expression vector for 48 hours. The luciferase activity was analyzed using a dual-luciferase assay kit from Promega. Error bars indicate the range of three independent experiments. B The BAF complex is associated constitutively with the TLR3 promoter. Chromatin was prepared by sonication from HeLa cells treated with IFN-α for 12 hours prior to formaldehyde cross-linking. DNA purified from immunoprecipitate with antibody against BRG1 or pre-immune serum was analyzed with primers covering the TLR3 promoter using quantitative-PCR. C Deletion analysis of the TLR3 promoter. The TLR3 promoter was deleted from 5′ end and analyzed similarly as in panel A. IFN indicates the cells were treated with 500 units/ml if IFN-α for 12 hours before harvesting for analyzing the luciferase activity. The potential transcription factor binding sites in the promoter region indicated below the panel. The numbers indicate that the position of deletion and are relative to the transcription initiation site. The Hind III and Nhe I sites used in the restriction enzyme accessibility assays (Figure 4A) are also indicated. D The IRF-binding site is essential for the TLR3 activity. The Sp1 and IRF-E sequences in the TLR3 promoter were point-mutated respectively and analyzed as in panel C. wt-TLR3pr: the wild type TLR3 promoter in pREP4-luc reporter vector; MutSp1: the Sp1 binding site in the TLR3 promoter was point-mutated; mutIRF-E: the IRF binding site in the TLR3 promoter was point-mutated.