Th2-specific protein/DNA interactions at the proximal nuclear factor-AT site contribute to the functional activity of the human IL-4 promoter.
IL-4 is a pleiotropic immunoregulatory cytokine secreted by activated Th2, but not Th1, cells. The proximal IL-4 promoter contains MARE, C/EBP, P0, octamer-like, P1, and activating protein-1 elements. The half c-Maf binding site (MARE), P0, and P1 sites were previously shown to be involved in Th2-specific transcriptional activity. Except the MARE and P1 site, the molecular basis for Th2 specificity of the P0 site has not been analyzed. Here, we provide the first detailed analysis of the P0 binding factors and show that in Th2, but not in Th1, cells, NF-AT and proteins of the activating protein-1 family are involved in cooperative binding to the P0 and the adjacent octamer-like site. In the mouse Th2 D10 cells, Oct-1/Oct-2 are also found to participate in formation of the P0-binding complexes. Mutation, deletion, and methylation interference analysis demonstrate that both the P0 and the octamer-like sequence are required for inducible binding. Furthermore, we provide the first report of the functional relevance of each site in the human IL-4 promoter by mutagenesis/transfection analysis and demonstrate that the octamer-like, P0 and P1 sites are important for the biologic function of the IL-4 promoter. The MARE site, although it was shown to be critical for the function of the murine IL-4 promoter, does not appear essential for human IL-4 promoter activity in Jurkat T cells. These findings suggest that besides c-Maf, another Th2-specific factor(s) may be involved in tissue-specific expression of the IL-4 gene.
Tumor Immunology Program, German Cancer Research Center, Heidelberg. m.li-weber@dkfz-heidelberg.de
M
M
Li-Weber
P
P
Salgame
C
C
Hu
I V
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Davydov
O
O
Laur
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S
Klevenz
P H
PH
Krammer
D000818:Animals; D001483:Base Sequence; D002999:Clone Cells; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051863:Host Cell Factor C1; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics* / Q000502:physiology; D015759:Ionomycin / Q000494:pharmacology; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000276:immunology*; D011485:Protein Binding / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018418:Th2 Cells / Q000276:immunology / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; C494165:POU2F1 protein, human; C058435:POU2F2 protein, human; C494166:Pou2f1 protein, mouse; C489206:Pou2f2 protein, mouse; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D015847:Interleukin-4; D015759:Ionomycin; D004247:DNA; D013755:Tetradecanoylphorbol Acetate