CREB/ATF proteins enhance the basal and CD154 and IL-4-induced transcriptional activity of the human I gamma 1 proximal promoter

To understand the underlying basis for the strong IL-4- and CD154-mediated I gamma1 promoter activity in Ramos 2G6 B cells, we carried out transient t ransfection assays with luciferase-based constructs containing approximatel y 2.2 kb and 500 bp of the human I gamma1 proximal promoter region. As a co mparison, the corresponding regions of the human I gamma3 promoter were tes ted under identical conditions. We found that both I gamma1 and I gamma3 pr omoter constructs were activated upon transfection into Ramos B cells and t hat activity was significantly upregulated by CD154 and IL-4 signals. Howev er, the I gamma1 promoter was measurably stronger than the I gamma3 promote r with respect to both basal and induced responses. Sequence comparison rev ealed a divergent 36-bp region containing multiple putative transcription f actor binding sites in the I gamma1 but not the I gamma3 promoter. A mutati onal "swap" of this sequence resulted in a marked decrease and increase in I gamma1 and I gamma3 basal and induced promoter activity, respectively. Ge l retardation assays with I gamma1-specific probes revealed CREB-containing complexes that were not observed with the corresponding I gamma3 probes. M utation of a single nucleotide in overlapping CREB sites in the I gamma1 se quence resulted in a significant decrease in basal activity with a correspo nding reduction in the level of IL-4- and CD154-mediated transcription.