CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human lγ1 proximal promoter

To understand the underlying basis for the strong IL-4- and CD154-mediated Iγ1 promoter activity in Ramos 2G6 B cells, we carried out transient transfection assays with luciferasebased constructs containing approximately 2.2 kb and 500 bp of the human Iγ1 proximal promoter region. As a comparison, the corresponding regions of the human Iγ3 promoter were tested under identical conditions. We found that both lγ1 and Iγ3 promoter constructs were activated upon transfection into Ramos B cells and that activity was significantly up-regulated by CD154 and IL-4 signals. However, the lγ1 promoter was measurably stronger than the Iγ3 promoter with respect to both basal and induced responses. Sequence comparison revealed a divergent 36-bp region containing multiple putative transcription factor binding sites in the Iγ1 but not the Iγ3 promoter. A mutational "swap" of this sequence resulted in a marked decrease and increase in Iγ1 and lγ3 basal and induced promoter activity, respectively. Gel retardation assays with Iγ1-specific probes revealed CREB-containing complexes that were not observed with the corresponding lγ3 probes. Mutation of a single nucleotide in overlapping CREB sites in the Iγ1 sequence resulted in a significant decrease in basal activity with a corresponding reduction in the level of IL-4- and CD154-mediated transcription.