Multiple, distinct trans-activation functions are encoded by the simian virus 40 large T and small t antigens, only some of which require the 82-residue amino-terminal common domain.
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Simian virus 40 (SV40) small t and large T antigens can each trans activate the adenovirus (Ad) E2A and the Ad VA-I promoters. The first 82 amino acids of large T and small t are identical. However, this large T-small t common domain between residues 1 and 82 does not trans activate, suggesting that large T and small t each encode separate trans-activation functions. To determine whether the large T or small t unique domains, which are required for trans activation of the E2A promoter, are sufficient for this activity, we have employed expression plasmids separately encoding the common and unique domains of large T and small t. Cotransfection of a large T unique domain expression plasmid efficiently trans activated the E2A promoter. Optimal trans activation by large T required the motif that binds cellular proteins such as the retinoblastoma gene product, which is located in the large T unique domain, and additional large T structures outside this motif. In contrast, the small t unique domain did not trans activate the E2A promoter. Experiments utilizing E2A promoter mutants containing only the ATF- or EIIF-binding sites demonstrated that trans activation by small t involves only the EIIF transcription factor and that this function requires both the common (residues 1 to 82) and the small t unique domains expressed as a colinear protein. trans activation by large T, in contrast, involves at least three mechanisms. There appear to be at least two mechanisms that involve the EIIF transcription factor, at least one of which does not require the common domain (residues 1 to 82) and one mechanism that involves the ATF factor and does require both the common and the large T unique domains. (+info)
Trans-activation of the adenovirus E2 promoter by human papillomavirus type 16 E7 is mediated by retinoblastoma-dependent and -independent pathways.
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In common with the adenovirus E1A and simian virus 40 large T oncoproteins, the E7 protein of human papillomavirus (HPV) type 16 interacts with the retinoblastoma (Rb) tumour suppressor protein (pRb). The functional importance of this interaction for HPV-16 E7 protein was investigated by analysis of the transactivating function of E7 at the adenovirus E2 promoter in a set of breast tumour cell lines. Trans-activation by HPV-16 E7 in two pRb-deficient cell lines demonstrated that pRb is not essential for E7-mediated trans-activation, but reconstitution of Rb expression indicated the existence of an Rb-mediated pathway of E7 trans-activation. This pathway results from suppression by E7 of a trans-repressing function encoded by the Rb gene. The E7 protein is shown to be capable of interacting in vivo with the Rb-related protein p107. Furthermore, analysis of a fusion construct between the amino terminus of Rb and the carboxy terminus of p107 suggests that, in common with pRb, the p107 protein trans-represses the adenovirus E2 early promoter. Therefore it is proposed that the pRb-independent pathway of E7 trans-activation is a consequence of the suppression of trans-repression by p107. (+info)
Helix-destabilizing properties of the adenovirus DNA-binding protein.
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The adenovirus DNA-binding protein (DBP) is a multifunctional protein that is essential for viral DNA replication. DBP binds both single-stranded and double-stranded DNA as well as RNA in a sequence-independent manner. Previous studies showed that DBP does not promote melting of duplex poly(dA-dT) in contrast to prokaryotic single-strand-binding proteins. However, here we show that DBP can displace oligonucleotides annealed to single-stranded M13 DNA. Depending upon the DBP concentration, strands of at least 200 nucleotides can be unwound. Although unwinding of short (17-bp), fully duplex DNA is facilitated by DBP, unwinding of larger (28-bp) duplexes is only possible if single-stranded protruding ends are present. These protruding ends must be at least 4 nucleotides long for optimal unwinding, and both 5' and 3' single-stranded overhangs suffice. DBP-promoted strand displacement is sensitive to MgCl2 and NaCl and not dependent upon ATP. Our results suggest that DBP, through formation of a protein chain on the displaced strand, may destabilize duplex DNA ahead of the replication fork, thereby assisting in strand displacement during replication. (+info)
The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.
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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, and 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. (+info)
Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein.
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The cellular protein p107 shares many structural and biochemical features with the retinoblastoma gene product, pRB. We have isolated a full-length cDNA for human p107 and have used this clone to study the function of p107. We show that, like pRB, p107 is a potent inhibitor of E2F-mediated trans-activation, and overexpression of p107 can inhibit proliferation in certain cell types, arresting sensitive cells in G1. Several experiments, however, showed that growth inhibition by pRB and p107 did not occur through the same mechanism. First, in the cervical carcinoma cell line C33A, p107 was able to block cell proliferation, whereas pRB could not, even though both proteins were potent inhibitors of E2F-mediated transcription in this cell line. Second, growth arrest by pRB and p107 was rescued differentially by various cell cycle regulators. Third, some mutants of p107 that cannot associate with adenovirus E1A were still able to inhibit cell proliferation, whereas analogous mutants in pRB are known to be unable to block cell growth. Together, these results suggest a biological role of p107 that is related, but not identical, to that of pRB. (+info)
The E2A gene product contains two separable and functionally distinct transcription activation domains.
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The E2A gene encodes transcription factors of the helix-loop-helix (HLH) family which are implicated in cell-specific transcriptional control in several cell lineages, including pancreatic beta cells. In the present work, we show by deletion mapping of both the E2A protein itself and the Gal4-E2A fusion protein that the protein contains at least two distinct activation domains. One domain (located between amino acids 1 and 153) functions efficiently in a variety of mammalian cell lines. The second domain (located between amino acids 369 and 485) functions preferentially in pancreatic beta cell lines. The latter domain shows a pattern of heptad repeats of leucine residues characteristics of "leucine zipper" transcription factors; site-directed mutagenesis of leucines within this repeat led to substantial reductions in activity. The selective properties of this activation domain may contribute to cell-specific transcription directed by the E2A gene. (+info)
Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation.
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The E2F transcription factor has been implicated in the regulation of genes whose products are involved in cell proliferation. Two proteins have recently been identified with E2F-like properties. One of these proteins, E2F-1, has been shown to mediate E2F-dependent trans-activation and to bind the hypophosphorylated form of the retinoblastoma protein (pRB). The other protein, murine DP-1, was purified from an E2F DNA-affinity column, and it was subsequently shown to bind the consensus E2F DNA-binding site. To study a possible interaction between E2F-1 and DP-1, we have now isolated a cDNA for the human homolog of DP-1. Human DP-1 and E2F-1 associate both in vivo and in vitro, and this interaction leads to enhanced binding to E2F DNA-binding sites. The association of E2F-1 and DP-1 leads to cooperative activation of an E2F-responsive promoter. Finally, we demonstrate that E2F-1 and DP-1 association is required for stable interaction with pRB in vivo and that trans-activation by E2F-1/DP-1 heterodimers is inhibited by pRB. We suggest that "E2F" is the activity that is formed when an E2F-1-related protein and a DP-1-related protein dimerize. (+info)
Regions of the retinoblastoma gene product required for its interaction with the E2F transcription factor are necessary for E2 promoter repression and pRb-mediated growth suppression.
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Studies of naturally occurring mutations of the RB1 tumor suppressor gene have indicated that the E1A/T antigen-binding domain is important for pRb function. Mutations engineered within the C-terminal 135 amino acids of pRb also abrogate its growth-suppressive function during the G1 interval of the cell cycle. Both the pRb E1A/T antigen-binding domain and the C-terminal domain are required for interaction with the E2F transcription factor. A series of mutated pRb proteins has been used to define the C-terminal sequences which determine E2F binding, adenovirus E2 promoter inhibition, and negative growth control. Deletion of the C terminus to residue 870 allowed full pRb function, while further deletion to residue 841 inactivated pRb in each assay. Amino acid sequences immediately C-terminal to the E1A/T antigen-binding domain were absolutely required for pRb activity. Mutations which prevented pRb from interacting with E2F also eliminated pRb-mediated E2 promoter repression and inactivated the ability of pRb to suppress cell growth. (+info)