NFE, A NEW TRANSCRIPTIONAL ACTIVATOR THAT FACILITATES P50 AND C-REL-DEPENDENT IGH 3'-ENHANCER ACTIVITY

The induction of immunoglobulin heavy chain (IgH) 3' enhancer activity has been coupled to ligand/receptor-dependent activation of resting B cells. To search for transcriptional target sites that account for th is induction, extracts from lipopolysaccharide (LPS)-stimulated B cell s and cell lines were used. Here we describe, by gel-retardation analy sis, the identification of an NF-kappa B site and an adjacent nuclear factor ets-like (NFE) site in the 3' enhancer. The NFE motif binds fou r protein complexes in resting B cell extracts, of which two are down- regulated upon LPS stimulation. Gel shift-shift experiments of the NF- kappa B complexes with specific antibodies identified p50 and c-Rel pr oteins to be the predominant factors in primary LPS-stimulated cell ex tracts. Site-directed mutagenesis of these motifs demonstrates that th ey contribute to part of the enhancer activity in plasma cells. One co py of the NF kappa B/NFE motifs, linked to a heterologous reporter con struct, displays lymphoid-restricted reporter gene activity in transie nt transfection assays. Mutation of either site abrogates all promoter activity. Complementation experiments demonstrate that although p50 a nd c-Rel expression vectors reconstitute transcription of an intact NF -kappa B/NFE reporter construct in a dose-dependent manner, mutation o f the NFE site or the NF-kappa B site abrogates essentially all transc riptional activity in both plasma cells and in COS cells. Taken togeth er, we provide evidence for the existence of an activator, NFE, which in combination with the p50 and c-Rel proteins, are part of the transc ription factor machinery that regulates 3' enhancer activity, and thus the control of the IgH locus in late B lymphocyte development.