Transactivation capacity of the regulatory sites in the CCR5 promoter. Transient transfection of promoter constructs in Tera-2 cells. (A) Transient transfection of the full-length CCR5 upstream (CCR5-pA1) and downstream (CCR5-pB1) promoter constructs. Cells were left untreated or treated with TNF-α (10 ng/mL) for 24 hours, leading to activation of only the β2m promoter. (B) Transient transfection of full-length and truncated upstream (CCR5-pA1 and CCR5-pA4) and downstream (CCR5-pB1 and CCR5-pB4) promoter constructs and the HLA-B250 promoter construct, as a positive control. Cells were left untreated or treated with IFN-γ (500 U/mL) for 24 hours, leading to HLA-B250 promoter activity only. (C) Transient transfection of CCR5 promoter constructs and a β2m promoter construct as a positive control with an IRF-1 expression vector or empty control vector, showing lack of CCR5 promoter transactivation by IRF-1. (D) Transient transfection of CCR5 promoter constructs with a CREB-1 expression vector, showing transactivation of the downstream promoter constructs by CREB-1. (E) Cotransfection of the CCR5 downstream promoter construct CCR5-pB3 with CREB-1 and ATF-1 expression vectors, revealing low transactivation activity of ATF-1 and absent enhancement of CREB-1 induced promoter activity. (F) Cotransfection of the CCR5 downstream promoter construct CCR5-pB3 with CREB-1 and CBP, p300, and P/CAF expression vectors, revealing lack of coinduction of the CCR5 promoter by CBP, p300, and P/CAF. Depicted are relative light units (RLU) per second obtained for luciferase activity and normalized with Renilla luciferase activity. Shown are means plus or minus SEM of 3 independent experiments (A,C,E,F) or a representative of 3 independent experiments (B,D) performed in triplicate.