Introduction Antigenic stimulation of T cells is induced by T-cell-receptor ligation and results in the release of immune mediators such as cytokines and chemokines. This activation initiates a nuclear program that results in activation of early growth response factors (EGRs), NF-κB, NF-AT, AP-1 and STAT proteins, and de novo transcription of the corresponding genes. In general terms, these immediate-early genes encode DNA-binding transcription factors. Following transcription, protein expression and nuclear translocation, these effector proteins bind to their cognate promoters and initiate transcription of cytokine, chemokine and inflammatory genes. The precise mechanism of how, in lymphocytes, individual transcription factors interact with coactivators and basal transcription factors, and how these DNA-binding proteins control transcription of inflammatory genes is currently unclear. The four EGR zinc-finger transcription factors (EGR-1 to EGR-4) are transiently and coordinately induced in T cells upon antigenic stimulation and the individual proteins regulate expression of genes encoding immune effectors like interleukin 2 (IL-2) (Skerka et al., 1995), tumor necrosis factor α (TNF- α) (Krämer et al., 1994), the β chain of the IL-2 receptor (Lin and Leonard, 1997), Fas (Dinkel et al., 1997) and the Fas ligand (Li-Weber et al., 1999). In addition, EGR proteins are expressed in distinct cell types and regulate transcription of a wide range of genes, including genes involved in the control of cell growth and apoptosis (Gashler and Sukhatme, 1995). There is a large panel of EGR-regulated genes and these target genes show tissue-specific expression (Gashler and Sukhatme, 1995). The restricted expression pattern of the target genes contrasts with the ubiquitous expression of EGR proteins and it is hypothesized that the tissue-specific transcription is mediated by additional nuclear proteins that cooperate with EGR proteins. We have previously shown that, in T cells, EGR-1 and EGR-4 form stable complexes with the nuclear factor of activated T cells (NFAT) (Decker et al., 2003). NF-κB is a transcription factor that plays a key role in inflammation and the immune response. The NF-κB family has five members, including RelA (p65), RelB, c-Rel, NF-κB1 (p105-p50) and NF-κB2 (p100-p52) (Hayden and Gosh, 2004). 3203 Here, we characterize the basis for the T-cell-specific activity of the human zinc-finger protein early growth response factor 4 (EGR-4). A yeast two-hybrid screen showed interaction of EGR-4 with NF-κB p50. Using recombinant proteins, stable physical complex formation was confirmed for EGR-4 and EGR-3 with p50 and with p65 using glutathione-S-transferase pull-down assays and surface-plasmon-resonance and peptide-spot analyses. In vivo interaction of EGR-4 and EGR-3 with NF-κB p65 was demonstrated by immunoprecipitation experiments and fluorescence-resonance-energy transfer (FRET) analysis showing interaction in the nucleus of transfected Jurkat T cells. In transfection assays, EGR-p50 complexes were transcriptionally inactive and EGR-p65 complexes strongly activated transcription of the promoters of the human genes encoding the cytokines interleukin 2, tissue necrosis factor α and ICAM-1. The EGR-p65 complexes increased reporter-gene activity about 100-fold and thus exceeded the transcriptional activities of the p65 homodimer and the p65/p50 heterodimers. The major interaction domain for p65 was localized within the third zinc finger of EGR-4 using deletion mutants for pull-down assays and peptide- spot assays. By computer modeling, this interaction domain was localized to an α-helical region and shown to have the central amino acids surface exposed and thus accessible for interaction. In summary, in T cells, the two zinc-finger proteins EGR-4 and EGR-3 interact with the specific nuclear mediator NF-κB and control transcription of genes encoding inflammatory cytokines. Key words: EGR, NF-κB, IL-2, TNF-α, ICAM-1 Summary Early growth response proteins EGR-4 and EGR-3 interact with immune inflammatory mediators NF-κB p50 and p65 Gerhard D. Wieland 1, *, Nina Nehmann 1, *, Doreen Müller 1 , Hermann Eibel 2 , Ulrich Siebenlist 3 , Jürgen Sühnel 4 , Peter F. Zipfel 1 and Christine Skerka 1,‡ 1 Department of Infection Biology, Leibniz-Institute for Natural Products, Research and Infection Biology, Hans-Knoell-Institute, Butenbergstrasse 11a, 07745 Jena, Germany 2 Research Group for Rheumatology, Albert-Ludwig-University, Hugstetter Strasse 55, 79106 Freiburg, Germany 3 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD 20892, USA 4 Biocomputing Group, Institute of Molecular Biotechnology, Beutenbergstrasse 11, 07745 Jena, Germany *These authors contributed equally to this work ‡ Author for correspondence (e-mail: christine.skerka@hki-jena.de) Accepted 19 April 2005 Journal of Cell Science 118, 3203-3212 Published by The Company of Biologists 2005 doi:10.1242/jcs.02445 Research Article Journal฀of฀Cell฀Science