FIG. 1.

Histone H2B mediates dsDNA-induced cellular activation. (A) A human bone marrow expression cDNA library was screened based on the ability to mediate dsDNA-induced IFN-β promoter activation. (B to D) HEK293 cells were transfected with control siRNA or siRNA targeting H2B. (B) The cells were treated with 0, 0.1, 0.5, or 2.5 μg/ml of dsDNA [poly(dA·dT)], and the supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) for IFN-β (PBL). (C) The cells were treated with 0.5 μg/ml of dsDNA [poly(dA·dT)] for 0, 3, 6, 12, 24, and 48 h, and the levels of phosphorylated IRF3 (p-IRF3) in the nucleus and IRF3 (normalization control) in the whole-cell lysates were examined by immunoblotting analysis. (D) The cells were further transfected with expression plasmids for TBK1 and IKKi, and the supernatants were subjected to ELISA for IFN-β (PBL). (E) HEK293 cells were transfected with histone H2B-FLAG and treated with biotin-poly(dA·dT) or biotin-poly(dG·dC). The cell lysates were collected, and a pull-down assay was performed with streptavidin-agarose. The complex was analyzed by immunoblotting using anti-FLAG Ab. (F) HEK293 cells were transfected with HA-GFP, HA-H2B-GFP, HA-H2B N′-tail-GFP, or HA-H2B αH-GFP. Twenty-four hours after the first transfection, the cells were further transfected with the HPV18 genome. Forty-eight hours after the second transfection, the cell lysates were collected and immunoprecipitation was performed with anti-HA Ab. The standard PCR targeting the HPV18 E6 gene was conducted with each cell lysate and immunoprecipitated complex. (G) HEK293 cells were transfected with H2B siRNA and then further transfected with pGL3 IFN-β and pTK-RL plus control, H2B FL, the H2B N′-tail, or the H2B αH plasmid. After treatment with 0, 0.1, 0.5, or 2.5 μg/ml of dsDNA [poly(dA·dT)] for 24 h, a luciferase assay was performed. (H) HEK293 cells were transfected with control siRNA or siRNA targeting either H2B, H1, H2A, H3, or H4. The cells were further transfected with pGL3 IFN-β and pTK-RL and treated with or without 0.5 μg/ml of dsDNA [poly(dA·dT)], dsRNA [poly(I:C)], or TNF-α (50 ng/ml) for 24 h, and then a luciferase assay was performed. The luciferase activity is depicted as the IFN-β promoter activity relative to samples obtained from the cells treated with control siRNA in each stimulation (relative IFN-β promoter activity). All data except for those in panel represent means ± standard deviations (SD) of six to eight samples. *, P < 0.05.