DNA binding and transcriptional activation by the Ski oncoprotein mediated by interaction with NFI.
The Ski oncoprotein has been found to bind non-specifically to DNA in association with unindentified nuclear factors. In addition, Ski has been shown to activate transcription of muscle-specific and viral promoters/enhancers. The present study was undertaken to identify Ski's DNA binding and transcriptional activation partners by identifying specific 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 degenerate oligonucleotides with anti-Ski monoclonal antibodies. Two sequences were identified by this technique. The first (TGGC/ANNNNNT/GCCAA) is the previously identified binding site of the nuclear factor I (NFI) family of transcription factors. The second (TCCCNNGGGA) is the binding site of Olf-1/EBF. By electophoretic mobility shift assays we find 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 proteins and activates transcription of NFI reporters, but only in the presence 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 zipper domain of GCN4.
Tarapore
P
P
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
Richmond
C
C
Zheng
G
G
Cohen
S B
SB
Kelder
B
B
Kopchick
J
J
Kruse
U
U
Sippel
A E
AE
Colmenares
C
C
Stavnezer
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007739:L Cells; D016350:Leucine Zippers; D051379:Mice; D051839:NFI Transcription Factors; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; C080333:transcription factor nuclear factor 1; C070358:v-ski protein, Avian retrovirus; D004247:DNA