Regulation of the germline immunoglobulin C gamma 1 promoter by CD40 ligand and IL-4: dual role for tandem NF-kappa B binding sites

Transcription of germline Ig constant region genes and associated switch re gions is an early and essential step in heavy chain class switch recombinat ion. Transcription of the germline C gamma 1 and CE Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independently induce transcri ption of these genes and act in synergy with IL-4 to increase expression. I n the present study, we investigated the role of three tandem NF-kappa B si tes (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germli ne C gamma 1 Ig promoter by CD40 Ligand (CD40L) and IL,-4 in the mouse B ly mphoma cell line, BCL1-3B3. Germline yl transcripts are induced by CD40L an d 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 stimulat ion with CD40L results in the induction of NF-kappa B complexes that bind t o each of the three NF-kappa B sites and are composed mainly of p50 and Rel B, but also include c-Rel and p65. Surprisingly, site-specific mutagenesis of the NF-KB sites did not reduce CD40-responsiveness of germline yl promot er-luciferase reporter constructs transiently transfected into BCL1-3B3. Mu tation in any one NF-kappa B site, however, significantly reduced overall t ranscriptional 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-kappa B sites and similarly reduced by mutation of site 1, suggesting that NF-kappa B-STAT6 interactio ns may be necessary for STAT6-mediated transactivation of the germline yl p romoter. The results suggest that the three NF-KB sites may serve as a focu s for formation of a higher-order transcription complex including STAT6, NF -kappa B and components of the basal transcription apparatus. (C) 1999 Else vier Science Ltd. All rights reserved.