The C-terminal 70 amino acids of the adenovirus E4-ORF6/7 protein are essential and sufficient for E2F complex formation

Abstract

E2F is a cellular transcription factor that binds to the adenovirus (Ad) E1A enhancer and E2aE promoter regions, to the cellular c-myc P2 and dihydrofolate reductase promoters, and to other viral and cellular regulatory regions. The binding activity of E2F to the Ad E2aE promoter is dramatically increased during an adenovirus infection (termed E2F induction). E2F induction is dependent on the expression of the 150 amino acid E4-ORF6/7 protein which forms a direct, physical complex with E2F to mediate the cooperative and stable binding of E2F to inverted sites in the E2aE promoter. Using in vitro DNA binding assays to measure the formation of the infection-specific complexes, we have defined the minimal domain of the E4-ORF6/7 protein, the C-terminal 70 amino acids, required to complex with E2F and stabilize its binding at the E2aE promoter. The ability of mutant E4-ORF6/7 proteins to form the stable E2F-E2aE promoter complex in vitro correlated well with their ability to trans-activate E2 transcription in vivo. These observations support a model in which the E4-ORF6/7 protein binds to E2F to induce the cooperative binding of two E2F molecules to the E2aE promoter thereby activating E2 transcription.

Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

“…The results presented represent the average of five experiments. The E2a-CAT vector was previously described (42). The E2F-1-CAT vector was constructed from a plasmid provided by Peggy Farnham (University of Wisconsin) and contains positions Ϫ176 to ϩ36 of the murine E2F-1 promoter region (25) linked to the CAT gene.…”

“…1A) (16,17,26,36,48). The induction of E2F binding to the Ad5 E2a promoter in vitro is directly correlated with transcriptional activation of the E2a promoter in vivo (38,39,41,42,49). The dissociation of Rb family members from E2Fs by the E1A proteins also results in activation of cellular genes containing E2F response elements and the induction of cell cycle progression (8).…”

The E4-6/7 Protein Functionally Compensates for the Loss of E1A Expression in Adenovirus Infection

“…Nuclear extracts were prepared according to the method of Dignam et al (5). Nuclear fractions were dialyzed against dialysis buffer (DB; 20 mM HEPES [pH 7.5], 100 mM KCl, 10% glycerol, 5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride), and the dialysate was cleared by centrifugation at 25,000 ϫ g. In vitro DNA binding assays were essentially performed as described previously (26,27). Briefly, binding reaction mixtures (20 l) contained 5 to 10 g of nuclear extract, 2 g of sonicated salmon sperm DNA, and 20,000 cpm of 32 P-labeled Ad5 E2a promoter region probe DNA (1 to 2 fmol of DNA) in DB supplemented with Nonidet P-40 (final concentration, 0.1%).…”

“…Free E2F is then bound by the Ad E4-6/7 protein, which forms a complex with E2F and induces the cooperative and stable binding of E2F to the inverted binding sites in the Ad5 E2a promoter (12,13,15,22,30). The induction of E2F binding to the Ad5 E2a promoter in vitro is directly correlated with transcriptional activation of the E2a promoter as assayed using transient-expression assays and virus infection assays in vivo (24,25,26,27,32).…”

Differential Roles of Two Tandem E2F Sites in Repression of the Human p107 Promoter by Retinoblastoma and p107 Proteins

“…A nuclear extract of HeLa cells was prepared as described by Dignam et al (11). Band shift and competition assays (21) were performed as described by O'Connor and Hearing (59). DNA-protein complexes were separated from free DNA by electrophoresis in a 4% 30:1 (acrylamide-bisacrylamide) polyacrylamide gel run in 0.25ϫ Tris-borate-EDTA at 4ЊC.…”