Coordinate expression and distinct DNA-binding characteristics of the four EGR-zinc finger proteins in Jurkat T lymphocytes.
The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of proteins that are composed of three homologous consecutive zinc fingers of the Cys2-His2 type and different flanking sequence. Upon growth stimulation of resting cells the four EGR-genes are simultaneously transcribed. We have analyzed the expression of the four EGR-proteins in Jurkat T cells and show by Western blot analysis that the four EGR-proteins are coordinately induced upon treatment with a combination of PHA and PMA. As the individual proteins are reported to bind to identical target sequences, we have analyzed the DNA-binding of the native proteins. Using nuclear extract in which we have demonstrated expression of all four EGR-proteins, only EGR-1, but no other member of this protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to oligonucleotides that include the EGR-consensus sites. The distinct binding affinities suggest that in vivo EGR-proteins bind to different target sequences and that each EGR-protein regulates distinct target genes. This is underlined by demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter element with the sequence GGG GTG GGG. This G-rich sequence serves as an overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As similar overlapping binding sites for EGR-1 and Sp1 have been identified in several human and mouse gene promoters, we raise the question whether the Sp1 binding sites described in a large number of eukaryotic gene promoters also represent binding sites for EGR-1.
Skerka
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Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Decker
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Zipfel
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D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D051767:Early Growth Response Protein 2; D051776:Early Growth Response Protein 3; D051765:Early Growth Response Transcription Factors; D005807:Genes, Overlapping; D006801:Humans; D017874:Immediate-Early Proteins; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D008934:Mitogens / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; C497326:EGR2 protein, human; C111028:EGR4 protein, human; D051766:Early Growth Response Protein 1; D051767:Early Growth Response Protein 2; D051765:Early Growth Response Transcription Factors; C486109:Egr1 protein, mouse; C486392:Egr4 protein, mouse; D017874:Immediate-Early Proteins; D007376:Interleukin-2; D008934:Mitogens; D009838:Oligodeoxyribonucleotides; D010835:Phytohemagglutinins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051776:Early Growth Response Protein 3; D013755:Tetradecanoylphorbol Acetate