Role of zinc-finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human synaptobrevin II gene.
Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1. Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background. Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation.
Petersohn
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Institute for Genetics, University of Cologne, Germany.
Thiel
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D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D017874:Immediate-Early Proteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D050683:R-SNARE Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D016704:Synapsins / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D016329:Sp1 Transcription Factor; D016704:Synapsins; D014157:Transcription Factors