Detection and characterization of cellular EGR-1 binding to its recognition site.
Most of what is known of the Egr-1 DNA binding site GCGGGGGCG was originally identified by experiments using DNA sequences and bacterially expressed or in vitro translated EGR-1 protein. Here we report the binding of cellular EGR-1 protein derived from HeLa, mouse and human fibroblasts to its consensus sequence. Binding is strongly but transiently stimulated in these cells by serum, phorbol ester, or by okadaic acid, an inhibitor of protein serine/threonine phosphatases 1 and 2A, suggesting the regulation of this gene expression and its DNA binding activity to be under the control of protein kinase(s) and phosphatase(s). When EGR-1 synthesis is stimulated under the above conditions, binding of the transcription factor Sp1 to its recognition site in the Egr-1 promoter is reduced with a concomitant appearance of EGR-1 DNA binding. This is likely a result of competition between Sp1 and the newly synthesized EGR-1, since there is a partial overlap in the binding sequences recognized by these proteins. In cotransfection experiments EGR-1 activated transcription through multiple copies of GCGGGGGCG 5' to a minimum promoter of c-fos. Interestingly, EGR-1 is shown to down-regulate the transcription of its own gene expression, whereas Sp1 activated Egr-1 gene expression. The detection of cellular EGR-1 binding to the Egr-1 consensus sequence in the different cell types provides a model for studying the mechanism by which an immediate-early gene is regulated by various ligands.
Cao
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Signal Transduction Laboratory Institute of Molecular and Cell Biology, National University of Singapore.
Mahendran
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R
Guy
G R
GR
Tan
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YH
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001769:Blood; D016384:Consensus Sequence; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000494:pharmacology; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015032:Zinc / Q000494:pharmacology; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D010703:Phorbol Esters; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D003513:Cycloheximide; D015032:Zinc