title: Regulation of the germline immunoglobulin Cgamma1 promoter by CD40 ligand and IL-4: dual role for tandem NF-kappaB binding sites.
abstract: 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 Cgamma1 and C epsilon 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-kappaB sites (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germline Cgamma1 Ig promoter by CD40 Ligand (CD40L) and IL-4 in the mouse B lymphoma cell line, BCL1-3B3. Germline gamma1 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-kappaB complexes that bind to each of the three NF-kappaB sites and are composed mainly of p50 and RelB, but also include c-Rel and p65. Surprisingly, site-specific mutagenesis of the NF-kappaB sites did not reduce CD40-responsiveness of germline gamma1 promoter-luciferase reporter constructs transiently transfected into BCL1-3B3. Mutation in any one NF-kappaB 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-kappaB sites and similarly reduced by mutation of site 1, suggesting that NF-kappaB-STAT6 interactions may be necessary for STAT6-mediated transactivation of the germline gamma1 promoter. The results suggest that the three NF-kappaB sites may serve as a focus for formation of a higher-order transcription complex including STAT6, NF-kappaB and components of the basal transcription apparatus.
authors.affiliation: Department of Microbiology, University of Texas Health Science Center at San Antonio, 78284-7758, USA.
authors.forename: W D
authors.initials: WD
authors.lastname: Warren
authors.forename: K L
authors.initials: KL
authors.lastname: Roberts
authors.forename: L A
authors.initials: LA
authors.lastname: Linehan
authors.forename: M T
authors.initials: MT
authors.lastname: Berton
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D023201:CD40 Ligand; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000276:immunology*; D005803:Genes, Immunoglobulin; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D015847:Interleukin-4 / Q000276:immunology / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000276:immunology / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000276:immunology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000276:immunology
chemical_list: D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D050800:STAT6 Transcription Factor; C494100:Stat6 protein, mouse; D015534:Trans-Activators; D023201:CD40 Ligand; D015847:Interleukin-4