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J. Biol. Chem., Vol. 265, Issue 36, 22243-22254, 12, 1990

Rous sarcoma virus enhancer factor I is a ubiquitous CCAAT transcription factor highly related to CBF and NF-Y

M Faber and L Sealy
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.

We have characterized enhancer factor I (EFI), a trans-acting factor present in avian nuclear extracts which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter. Through deletion and point mutagenesis, we show that EFI is a member of the CCAAT family of transcription factors. Although the CCAAT motif is essential for protein-DNA recognition, EFI shows surprising latitude in the nucleotide sequences flanking the CCAAT motif to which it will bind with high affinity. EFI will cross-bind to the binding sites of a number of previously described CCAAT factors, including CBF, NF-Y, CP2, CP1, CTF/NF-1, and c/EBP, with a range of affinities that is at most 10- fold lower than the high affinity binding site for EFI in the Rous sarcoma virus long terminal repeat. We present evidence, however, that EFI is probably identical or very closely related to CBF and NF-Y. This is based on the fact that EFI in avian nuclear extracts binds with equal or 2-fold greater affinity to the binding sites of NF-Y and CBF, despite less than 50% homology (outside the CCAAT motif) between the EFI, NF-Y, and CBF recognition sequences. Moreover, radiolabeled EFI, NF-Y, or CBF DNAs give rise to identical gel retardation patterns in extracts from a variety of different cell types. EFI, CBF, and NF-Y appear to fractionate identically upon ion exchange chromatography, separating into two heterologous components (A and B) which must be recombined to recover substantial DNA binding activity. Molecular weight estimates for the two heterologous components of EFI, CBF, and a Y-box binding protein (Celada, A., and Maki, R.A. (1989) Mol. Cell. Biol. 9, 3097-3100) are very similar. EFI DNA binding activity has recently been shown to be induced by serum and the oncogene v-src (Dutta, A., Stoeckle, M.Y., and Hanafusa, H. (1990) Genes & Dev. 4, 243- 254). The close relationship or identity between EFI, CBF, and NF-Y, thus has important implications regarding the mechanisms by which serum or the oncogene v-src may affect changes in gene expression.
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