A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1.
Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, DNA-binding, and nuclear localization activities. Deletion analysis, as well as fusions to the DNA-binding domain of GAL4, indicated that the activation potential of Egr-1 is distributed over an extensive serine/threonine-rich N-terminal domain. In addition, a novel negative regulatory function has been precisely mapped 5' of the zinc fingers: amino acids 281 to 314 are sufficient to confer the ability to repress transcription on a heterologous DNA-binding domain. Specific DNA-binding activity was shown to reside in the three zinc fingers of Egr-1, as predicted by homology to other known DNA-binding proteins. Finally, nuclear localization of Egr-1 is specified by signals in the DNA-binding domain and basic flanking sequences, as determined by subcellular fractionation and indirect immunofluorescence. Basic residues 315 to 330 confer partial nuclear localization on the bacterial protein beta-galactosidase. A bipartite signal consisting of this basic region in conjunction with either the second or third zinc finger, but not the first, suffices to target beta-galactosidase exclusively to the nucleus. Our work shows that Egr-1 is a functionally complex protein and suggests that it may play different roles in the diverse settings in which it is induced.
Gashler
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AL
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637-1963.
Swaminathan
S
S
Sukhatme
V P
VP
D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry*; D051766:Early Growth Response Protein 1; D005786:Gene Expression Regulation; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*; D015533:Transcriptional Activation; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors