Characterization of constitutive and inducible transcription factors binding to the P2 NF-AT site in the human interleukin-4 promoter.
Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites--the P2 site--located between -173 and -150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes--a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at -173 to -150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site.
Li-Weber
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Tumor Immunology Program, German Cancer Research Center, Heidelberg. m.li-weber@dkfz-heidelberg.de
Salgame
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Hu
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Krammer
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D002478:Cells, Cultured; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D051863:Host Cell Factor C1; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics*; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D050809:POU Domain Factors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D050809:POU Domain Factors; C494165:POU2F1 protein, human; C058435:POU2F2 protein, human; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D015847:Interleukin-4