The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

Abstract

Binding of the mammalian transcription factor E2F to the adenovirus E2a 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 E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point 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 amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F 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 E2a promoter.

Separation of Lipid Transport Functions by Mutations in the Extracellular Domain of Scavenger Receptor Class B, Type I

“…Materials-The following antibodies were used: anti-M45 epitope hybridoma medium (a generous gift from Dr. Patrick Hearing, State University of New York, Stony Brook; 1:100 for immunofluorescence, 1:250 for flow cytometry, and 1:500 for immunoblotting) (40), anti-His 6 monoclonal IgG (Roche Applied Science; 1:500 for immunoblotting), anti-SR-BI extracellular domain polyclonal antibody 356 (directed against residues 174 -356; 1:250 for flow cytometry) (41), anti-SR-BI C-terminal domain polyclonal antibody (Novus Biologicals, Inc.; 1:5000 for immunoblotting), anti-SR-BI extracellular domain polyclonal antibody (Novus Biologicals, Inc.; 1:500 for immunofluorescence), peroxidase-conjugated goat anti-mouse or anti-rabbit secondary IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:10,000 for immunoblotting), fluorescein (Sigma)-or phycoerythrin (Molecular Probes, Inc.)-conjugated anti-rabbit antibody (1:100 for flow cytometry), and Alexa 488-conjugated goat anti-mouse or anti-rabbit secondary IgG (Molecular Probes, Inc.; 1:1000 for immunofluorescence).…”

A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria

“…An 18 aa epitope of adenovirus protein E4-6͞7, which is recognized by the monoclonal antibody M45 (18), was fused to the C terminus of AvrA as follows. A 925 nt fragment containing avrA was amplified by PCR using two degenerate primers (5Ј-CGGAGAATTCAGATGATATTTTCGGTG-CAGG-3Ј and 5Ј-CCGATCCATGGGTTTAAGTAAA-GACTTATATTCAGC-3Ј) and subsequently cloned into the tagging vector pSB504 (14).…”

Adenovirus early E4 genes in viral oncogenesis

“…In contrast, the E4 ORF6/7 gene product binds free E2F, which then dimerizes (Obert et al, 1994) and binds with increased stability to the two inverted E2F-binding sites in the Ad5 E2 early promoter Huang and Hearing, 1989b;Marton et al, 1990;Raychaudhuri et al, 1989) and the cellular E2F-1 promoter (Schaley et al, 2000). The induction of stabilized E2F-binding in vitro correlates with transcriptional activation of both promoters in vivo (Reichel et al, 1989;Neill et al, 1990;Neill and Nevins, 1991;O'Connor and Hearing, 1991;Obert et al, 1994;Schaley et al, 2000). A recent study suggests that E4 ORF6/7 alone is su cient to displace pRb and p107 from E2F complexes thereby activating expression of E2 via induction of E2F-binding to the E2 early promoter, and resulting in signi®cantly enhanced replication of an E1A-defective Ad in HeLa cells .…”

“…Binding of the E1A proteins to Rb family members dissociates them from E2Fs (Kovesdi et al, 1986a(Kovesdi et al, ,b, 1987Cress and Nevins, 1996a), resulting in activation of cellular genes containing E2F-binding sites, and induction of cell cycle progression (Cress and Nevins, 1996b). In contrast, the E4 ORF6/7 gene product binds free E2F, which then dimerizes (Obert et al, 1994) and binds with increased stability to the two inverted E2F-binding sites in the Ad5 E2 early promoter Huang and Hearing, 1989b;Marton et al, 1990;Raychaudhuri et al, 1989) and the cellular E2F-1 promoter (Schaley et al, 2000). The induction of stabilized E2F-binding in vitro correlates with transcriptional activation of both promoters in vivo (Reichel et al, 1989;Neill et al, 1990;Neill and Nevins, 1991;O'Connor and Hearing, 1991;Obert et al, 1994;Schaley et al, 2000).…”