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Synaptobrevin 11 is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. Th 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 hind 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 tor 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 11 gene upstream ot a minimal promoter and the Escherichia coli chloramphenicol acetyItransferase (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 transativated 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. [ABSTRACT FROM AUTHOR]

The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc-finger motifs which belongs to the Krüpple family of zinc-finger proteins. The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin. These binding activities are characteristic of zinc-finger proteins. Immunochemical analysis using antibodies produced against the recombinat protein detected its expression in human mammary epithelial cells but not in stroma cells, which is consistent with the pattern of expression observed at the RNA level in cell cultures. Western blot analysis demonstrated the expression of 1 33-kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional transp-acting domain. These data establish the unique structure of the OZF protein which is distinct from previously identified zinc-finger proteins. In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis. [ABSTRACT FROM AUTHOR]