Regulation of the germline immunoglobulin C γ1 promoter by CD40 ligand and IL-4: dual role for tandemNF- κB binding sites

Abstract

Transcription of germline Ig constant region genes and associated switch regions is anearly and essential step in heavy chain class switch recombination. Transcription of the germlineCγ1 and Cε Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independentlyinduce transcription of these genes and act in synergy with IL-4 to increase expression. In thepresent study, we investigated the role of three tandem NF-κB sites (site 1, −95; site 2, −71; site3, −53) in the regulation of the germline Cγ1 Ig promoter by CD40 Ligand (CD40L) and IL-4 inthe mouse B lymphoma cell line, BCL 1-3B3. Germline γ1 transcripts areinduced by CD40L and by IL-4 in BCL 1-3B3 and the combination of signals issynergistic, as in normal B cells. EMSA with crude nuclear extracts demonstrated that stimulationwith CD40L results in the induction of NF- κB complexes that bind to each of the threeNF- κB sites and are composed mainly of p50 and RelB, but also include c-Rel and p65.Surprisingly, site-specific mutagenesis of the NF- κB sites did not reduceCD40-responsiveness of germline γ1 promoter-luciferase reporter constructs transientlytransfected into BCL 1-3B3. Mutation in any one NF- κB site, however,significantly reduced overall transcriptional activity of the promoter, both basal and induced,suggesting a role in basal promoter function. In addition, activation of the promoter by IL-4 wasblocked by mutation of all three NF- κB sites and similarly reduced by mutation of site 1,suggesting that NF- κB-STAT6 interactions may be necessary for STAT6-mediatedtransactivation of the germline γ1 promoter. The results suggest that the three NF- κB sites may serve as a focus for formation of a higher-order transcription complex includingSTAT6, NF- κB and components of the basal transcription apparatus. © 1999 ElsevierScience Ltd. All rights reserved.