Open AccessRole of Zinc‐Finger Proteins Sp1 and Zif268/egr‐1 in Transcriptional Regulation of the Human Synaptobrevin II Gene
Author(s) -
Petersohn Dirk,
Thiel Gerald
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.0827u.x
Subject(s) - biology , reporter gene , microbiology and biotechnology , zinc finger , promoter , gene , gene expression , transcription factor , genetics
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 fusion 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.