THE ADENOVIRUS E4-6 7 PROTEIN TRANSACTIVATES THE E2 PROMOTER BY INDUCING DIMERIZATION OF A HETEROMERIC E2F COMPLEX/

Binding of the mammalian transcription factor E2F to the adenovirus E2 a early promoter is modulated through interaction with the viral E4-6/ 7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E 2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 pro duct. The C-terminal 70 amino acids of E4-6/7 are necessary and suffic ient for induction of E2F binding and for transactivation. To assess t he mechanism(s) of E2a transactivation and the induction of cooperativ e E2F binding by the E4-6/7 protein, we have analyzed a series of poin t mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally ; an E4-6/7 mutant with a phenylalanine-to-proline substitution at ami no acid 125 (F-125-P) efficiently interacts with E2F but does not indu ce E2F binding to the E2a promoter and is defective for transactivatio n. Induction of E2F stable complex formation at the E2a promoter by th e F-125-P mutant protein is restored by divalent E4-6/7-specific monoc lonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant prot ein. These and other data support the involvement of E4-6/7 dimerizati on in the induction of cooperative and stable E2F binding and transact ivation of the E2a promoter. We present evidence that at least two cel lular components are involved in E2F DNA binding activity and that bot h are required for E2F induction by the E4-6/7 product. The recently c loned E2F-related activities E2F-1 and DP-1 individually bind to an E2 F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP- 1, and E4-6/7 are sufficient to form the induced E2F complex at the E2 a promoter.