Regulation of the germline immunoglobulin Cγ1 promoter by CD40 ligand and IL-4: Dual role for tandem NF-κB binding sites

Transcription of germline Ig constant region genes and associated switch regions is an early and essential step in heavy chain class switch recombination. Transcription of the germline Cγ1 and Ca Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independently induce transcription of these genes and act in synergy with IL-4 to increase expression. In the present study, we investigated the role of three tandem NF-κcB sites (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germline Cγ1 Ig promoter by CD40 Ligand (CD40L) and IL-4 in the mouse B lymphoma cell line, BCL₁-3B3. Germline γ1 transcripts are induced by CD40L and by IL-4 in BCL₁-3B3 and the combination of signals is synergistic, as in normal B cells. EMSA with crude nuclear extracts demonstrated that stimulation with CD40L results in the induction of NF-κB complexes that bind to each of the three NFκB 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 reduce CD40-responsiveness of germline γ1 promoter- luciferase reporter constructs transiently transfected into BCL₁-3B3. 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 was blocked 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-mediated transactivation 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 including STAT6, NF-κB and components of the basal transcription apparatus.