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J Biol Chem, Vol. 273, Issue 6, 3588-3597, February 6, 1998

Transcriptional Repression by v-Ski and c-Ski Mediated by a Specific DNA Binding Site

Rebekka NicolDagger § and Ed Stavnezer§

From the Dagger  Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 and the § Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

The Ski oncoprotein has been shown to bind DNA and activate transcription in conjunction with other cellular factors. Because tumor cells or myogenic cells were used for those studies, it is not clear that those activities of Ski are related to its transforming ability. In this study, we use a nuclear extract of c-ski-transformed cells to identify a specific DNA binding site for Ski with the consensus sequence GTCTAGAC. We demonstrate that both c-Ski and v-Ski in nuclear extracts are components of complexes that bind specifically to this site. By evaluating the features of the sequence that are critical for binding, we show that binding is cooperative. Although Ski cannot bind to this sequence on its own, we use cross-linking with ultraviolet light to show that Ski binds to this site along with several unidentified cellular proteins. Furthermore, we find that Ski represses transcription either through upstream copies of this element or when brought to the promoter by a heterologous DNA binding domain. This is the first demonstration that Ski acts as a repressor rather than an activator and could provide new insights into regulation of gene expression by Ski.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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