Cellular E2F Research Articles

Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes.

Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of E1A mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.

The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA

E2F is a cellular DNA-binding factor. Its binding activity is changed within adenovirus-infected cells so that it binds cooperatively to pairs of properly spaced and oriented E2F recognition sites. In the work described in this report, the conversion to cooperative binding was shown to require the adenovirus E4 17-kilodalton (kDa) polypeptide. Mutant viruses carrying alterations within the E4 17-kDa coding region failed to generate the infection-specific, cooperatively binding form of E2F. It was possible to alter E2F from uninfected cells so that it bound cooperatively by incubation with a partially purified fraction obtained from infected cells. The E4 17-kDa protein copurified with this activity and was also found to be present in a complex containing E2F. Consistent with its ability to alter the binding of E2F to its recognition sites within the E2 promoter, the E4 17-kDa polypeptide contributed to maximal expression of E2 mRNAs in some cell types. Its ability to enhance E2 transcription did not require expression of the E1A transactivator protein. These results are consistent with a model which proposes that the E4 17-kDa polypeptide binds to the cellular E2F factor, altering its binding behavior and thereby enhancing its ability to stimulate transcription.

Cooperative binding of two E2F molecules to an Ela-responsive promoter is triggered by the adenovirus Ela, but not by a cellular Ela-like activity.

The binding of the cellular E2F transcription factor to the central EIa-responsive element of the adenovirus EIIa early promoter (EIIaE) was compared in extracts of HeLa cells which had been infected with either wild-type adenovirus or the EIa-deficient mutant dl312. No quantitative differences in the E2F-binding activity were detected as a function of EIa gene expression. However, complexes formed by the E2F factor in the presence of EIa were qualitatively different from those formed on the same sequence element in the absence of EIa. Specifically, the formation of complexes containing two E2F molecules is favoured by EIa, probably through the induction of protein-protein interactions. Protein binding to EIIaE promoter in extracts from non-infected F9 embryonal carcinoma cells, prepared before and after in vitro differentiation of these cells was also analysed. The higher expression of EIIaE in undifferentiated cells, which was originally attributed to a cellular EIa-like function, may be correlated with the increased binding activity of a murine E2F-like protein which does not, however, result in the simultaneous occupation of both E2F sites on the EIIaE promoter, suggesting that the viral EIa and the presumptive cellular EIa-like functions trans-activate the EIIaE promoter through different pathways.

An adenovirus early region 4 gene product is required for induction of the infection-specific form of cellular E2F activity.

E2F is a cellular, sequence-specific DNA-binding factor that binds to pairs of sites that occur upstream of the E1A and E2 early mRNA cap sites. During adenovirus infection, there is induction of a form of E2F that binds cooperatively to the pair of sites in the E2 control region. Production of the infection-specific E2F activity is dependent on early region 4 (E4), as extracts of cells infected with a mutant that lacks E4 did not contain this activity. Instead, two new forms of E2F were seen with the E4 mutant. Infection with mutant viruses unable to make E1A gene products produced the wild-type infection-specific E2F activity after a delay. Mutations in the E1B-55 kD-, E1B-21 kD-, E2-72 kD-, and E3-coding regions had no effect on production of infection-specific E2F. Analysis of cell lines confirmed the results obtained with mutant viruses. Cells that expressed E1A but not E4 genes (e.g., 293 cells) did not contain infection-specific E2F. Cell lines that expressed the E4 gene contained the activity. These observations demonstrate that E4 participates in the infection-induced change in E2F-binding activity. The data are consistent with E1A playing an indirect role in the process by mediating the efficient expression of E4 gene products which, in turn, induce the alteration in E2F activity.

The cellular transcription factor E2f requires viral E1A and E4 gene products for increased DNA-binding activity and functions to stimulate adenovirus E2A gene expression

Whereas a wide variety of cellular proteins interact with the cis-regulatory elements of the adenovirus E1A and E2A genes, only the DNA-binding activity of the cellular E2f factor is modulated by viral early-gene expression. An analysis of cellular E2f protein levels and adenovirus early-gene expression in a panel of independently cloned virus-transformed rodent cell lines and in virus-infected rodent cells has established that both the E1A 289-amino-acid (289R) protein and a yet-to-be-defined E4 gene product are required for maximal E2f DNA-binding activity. To distinguish between the multiple roles the E1A protein could serve in this process, the E2f DNA-binding activity was determined in a virus-transformed cell line which contains a conditional-lethal mutation affecting the 289R protein. Since E4 gene expression was not altered by the incubation conditions, the observation of reduced cellular E2f activity at the nonpermissive temperature suggests a direct role for the E1A 289R protein in E2f activation. When a virus containing a deletion in the E4 gene was introduced into cell lines which can complement the E4 gene defect, a correlation between high cellular E2f levels and increased rates of E2A gene transcription was observed. A time course analysis of the viral infection revealed that E2f functions catalytically to stimulate viral E2A gene transcription. These observations have led to several hypotheses concerning possible mechanisms by which elevated E2A gene expression, which leads to cytotoxicity, might be avoided in the transformed cell.