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J Biol Chem, Vol. 274, Issue 33, 23043-23051, August 13, 1999

Transactivation of the Human cdc2 Promoter by Adenovirus E1A
E1A INDUCES THE EXPRESSION AND ASSEMBLY OF A HETEROMERIC COMPLEX CONSISTING OF THE CCAAT BOX BINDING FACTOR, CBF/NF-Y, AND A 110-kDa DNA-BINDING PROTEIN

Chien-Yuan Kao, Akihide Tanimoto, Nobuyuki Arima, Yasuyuki Sasaguri, and R. Padmanabhan

From the  Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421 and the  Department of Pathology and Cell Biology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi, Kitakyushu 807, Japan

Cyclin-dependent kinases (CDKs) play an important role in the eukaryotic cell cycle progression. Cdc2 (CDK1) is expressed in late G₁/S phase and required for G₂ to M phase transition in higher eukaryotes. The oncoproteins, SV40 large T antigen and adenovirus E1A, induce a 110-kDa protein which specifically recognizes the two inverted CCAAT motifs of the cdc2 promoter in cycling cells and plays an essential role in transactivation of the human cdc2 promoter. Since these CCAAT motifs also conform to the consensus binding sites for the ubiquitous heterotrimeric transcription factor, CBF/NF-Y, the role of CBF/NF-Y in the transactivation of the cdc2 promoter was examined in this study. Our results indicate that CBF/NF-Y and the 110-kDa protein interact with the CCAAT box motif to form a heteromeric complex. However, mutagenesis of the pentanucleotide CCAAT motif or in the presence of urea greater than 2.5 M, no heteromeric complex was formed. In contrast, the 110-kDa protein could still bind the mutant CCAAT motif or with the wild type motif in the presence of 2.5 M urea. Furthermore, E1A.12S induced the gene expression of all three subunits of CBF/NF-Y. Coexpression of E1A and a dominant negative mutant NF-YA subunit significantly reduced the E1A-mediated transactivation of the cdc2 promoter in a dose-dependent manner. These results support the conclusion that E1A protein mediates optimal transactivation of the human cdc2 promoter by inducing the expression and assembly of a heteromeric complex consisting of the 110-kDa protein and the CBF/NF-Y which interacts with the two CCAAT motifs of the cdc2 promoter.

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