CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human Igamma1 proximal promoter.
To understand the underlying basis for the strong IL-4- and CD154-mediated Igamma1 promoter activity in Ramos 2G6 B cells, we carried out transient transfection assays with luciferase-based constructs containing approximately 2.2 kb and 500 bp of the human Igamma1 proximal promoter region. As a comparison, the corresponding regions of the human Igamma3 promoter were tested under identical conditions. We found that both Igamma1 and Igamma3 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 Igamma1 promoter was measurably stronger than the Igamma3 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 Igamma1 but not the Igamma3 promoter. A mutational "swap" of this sequence resulted in a marked decrease and increase in Igamma1 and Igamma3 basal and induced promoter activity, respectively. Gel retardation assays with Igamma1-specific probes revealed CREB-containing complexes that were not observed with the corresponding Igamma3 probes. Mutation of a single nucleotide in overlapping CREB sites in the Igamma1 sequence resulted in a significant decrease in basal activity with a corresponding reduction in the level of IL-4- and CD154-mediated transcription.
Bhushan
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Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, USA.
Covey
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D051696:Activating Transcription Factors; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000502:physiology*; D023201:CD40 Ligand / Q000502:physiology*; D002460:Cell Line; D017124:Conserved Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D015847:Interleukin-4 / Q000502:physiology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D051696:Activating Transcription Factors; D001798:Blood Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D007074:Immunoglobulin G; D014157:Transcription Factors; D023201:CD40 Ligand; D015847:Interleukin-4