B cell activating factor (BAFF) gene promoter activity depends upon co-activator, p300

B-cell activating factor (BAFF) plays a critical role for mature B cell generation and maintenance. We have previously described that mouse BAFF (mBAFF) transcript expression was increased by toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS), through reactive oxygen species (ROS) production and NF-κB activation. Here, we investigated whether mBAFF expression could be regulated by promoter activation through the cooperation of NF-κB and p300, co-activator to various transcription factors. We cloned mBAFF promoter into luciferase-expressing pGL3-basic vector and computer-analyzed its NF-κB binding motif. Due to the existence of NF-κB binding motifs, activity in 2.0 kb mBAFF promoter was higher than that in 1.0 or 0.5 kb mBAFF promoter. When Raw 264.7 murine macrophages were stimulated with LPS, 2.0 kb mBAFF promoter activity was increased time dependently. Serum deprivation (0.5% FBS) producing ROS and exogenous H2O2 treatment also enhanced mBAFF promoter activity, which was reduced by N-acetyl-l-cysteine (NAC), a well-known ROS scavenger. LPS and serum-starved ROS production increased NF-κB activation. mBAFF promoter activity was augmented by co-transfection with p65 and/or co-activator, p300. It was inhibited by dominant negative (DN) p300. Binding of p300 to BAFF promoter was detected by chromatin immunoprecipitation (ChIP) assay. Data suggest that mBAFF expression could be regulated by promoter activation through NF-κB activation, which might be dependent on the cooperation with co-activator, p300.