c-Rel is a selective activator of a novel IL-4/CD40 responsive element in the human Ig gamma4 germline promoter.
Induction of isotype switching to a specific C(H) gene correlates with the transcriptional activation of the same gene in germline (GL) configuration. Expression of correctly spliced GL transcripts is necessary to target a switch region for recombination. In human B cells, the IgE and IgG4 isotypes are both induced by IL-4 through sequential switching, but are functionally antagonistic because IgG4 appears to have IgE-blocking activity. In order to understand the molecular mechanisms that regulate IgG4 production, we undertook a systematic analysis of the gamma4 GL promoter. A HindIII/NaeI region (-421/+474) highly conserved in the human gamma locus mediated the synergistic activation of a reporter gene in response to IL-4 and CD40 cross-linking. STAT6 binding to the proximal gamma4 GL promoter was essential for both IL-4-induced activation and CD40-dependent enhancement of transcription. Of note, a 45bp region (-76/-32) centered around the STAT6 binding motif drove robust synergistic activation of a heterologous fos promoter upon stimulation with IL-4 and CD40 cross-linking. This finding suggested that the (-76/-32) region may contain a novel IL-4/CD40 responsive element (RE). Electrophoretic mobility shift assays (EMSA) analysis using BL-2 nuclear extracts and in vitro translated NF-kappaB/Rel family proteins revealed the presence of a motif that overlaps the 5' end of the STAT6 element and binds selectively c-Rel. A mutation that abrogated c-Rel, but not STAT6, binding strongly impaired the CD40-induced enhancement of IL-4-dependent gamma4 GL transcription in reporter assays. These results indicate that c-Rel is selectively involved in the CD40-dependent activation of the IL-4/CD40 RE in the proximal gamma4 GL promoter.
Agresti
Alessandra
A
Molecular Immunoregulation Unit, San Raffaele Scientific Institute, 20132 Milan, Italy.
Vercelli
Donata
D
D001483:Base Sequence; D019013:CD40 Antigens / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D007074:Immunoglobulin G / Q000145:classification / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D015847:Interleukin-4 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051560:Proto-Oncogene Proteins c-bcl-6; D020569:Proto-Oncogene Proteins c-rel / Q000502:physiology*; D020218:Response Elements; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology
D019013:CD40 Antigens; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D051560:Proto-Oncogene Proteins c-bcl-6; D020569:Proto-Oncogene Proteins c-rel; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D015847:Interleukin-4