DNA-BINDING AND TRANSCRIPTIONAL ACTIVATION BY THE SKI ONCOPROTEIN MEDIATED BY INTERACTION WITH NFI

The Ski oncoprotein has been found to bind nonspecifically to DNA in a ssociation with unindentified nuclear factors. In addition, Ski has be en shown to activate transcription of muscle-specific and viral promot ers/enhancers. The present study was undertaken to identify Ski's DNA binding and transcriptional activation partners by identifying specifi c DNA binding sites. We used nuclear extracts from a v-Ski-transduced mouse L-cell line and selected Ski-bound sequences from a pool of dege nerate oligonucleotides with anti-Ski monoclonal antibodies. Two seque nces were identified by this technique. The first (TGGC/ANNNNNT/GCCAA) is the previously identified binding site of the nuclear factor I (NF I) family of transcription factors. The second (TCCCNNGGGA) is the bin ding site of Olf-1/EBF. By electrophoretic mobility shift assays we fi nd that Ski is a component of one or more NFI complexes but we fail to detect Ski in Olf-1/EBF complexes. We show that Ski binds NFI protein s and activates transcription of NFI reporters, but only in the presen ce of NFI. We also find that homodimerization of Ski is essential for co-activation with NFI. However, the C-terminal dimerization domain of c-Ski, which is missing in v-Ski, can be substituted by the leucine z ipper domain of GCN4.