Abstract
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.
Highlights
Quiescent cells in culture can be activated to reenter the cell cycle by treating the cells with serum or growth factors
Deletion/mutation of a putative suppressor gene wtl [16,17,18]. This gene encodes a protein with four zinc fingers, shares at least 65% amino acid sequence similarity with EGR-1, and bindstothesameconsensus sequence asEGR-1(15).In contrasttoEGR-1 which activatestranscription, wtl repressestranscriptionthroughthe recognition element [19, 20]
We report the binding and characterizationof cellular EGR-1 to its consensussequence and haveinvestigated how other transcription factorscould affect EGR-1 binding anhdow EGR-1 controls itsown gene expression
Summary
Clear extractsderived from control (lane 2 ) , serum-induced (lanes 35),or TPA-induced (lanes6-8) HeLa cells. Complex CIII was not affected in translated EGR-1 to the EBS[11,12] They were designated the presence of a 200-fold excess of the consensus sequences as low affinity binding sites since they did not compete as for AP-1 [29] or nuclear factor kB [30] These positions of CII and CIII were determined by incubating a results suggest that CIII consists of transcription factor Spl small amount of nuclear extract with "P-labeled P4 (Fig. 5, bound tothe core and flanking GGG sequence of EBS, lane 1 ) In relation to these complexes Spl bound strongly, whereas CII consistsof the EGR-1binding complex. 5' deletion mutation (p425) of the Egr-1 promoter [21, 28]
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