Apoptosis induced in mammalian cells by small peptides that functionally antagonize the Rb-regulated E2F transcription factor.

A variety of studies implicate the E2F transcription factor as a critical regulator of the mammalian cell cycle. The E2F pathway is aberrant in most, if not all, human tumor cells; therefore, therapeutic regimes that modulate E2F activity may provide an approach for reinstating growth control in situations where normal physiological control is lost. To elucidate the role of E2F in the cell cycle and assess its value as a therapeutic target, we have introduced peptides that functionally antagonize E2F DNA binding activity into mammalian cells. Introduction of these peptides into mammalian tumor cells caused the rapid onset of apoptosis, an outcome that correlates with the inactivation of physiological E2F.

Functional interaction between DP-1 and p53.

The cellular transcription factor DRTF1/E2F and the tumor suppressor protein p53 play important roles in controlling early cell cycle events. DRTF1/E2F is believed to coordinate and integrate the transcription of cell cycle-regulating genes, for example, those involved in DNA synthesis, with the activity of regulatory proteins, such as the retinoblastoma tumor suppressor gene product (pRb), which modulate its transcriptional activity. In contrast, p53 is thought to monitor the integrity of chromosomal DNA and when appropriate interfere with cell cycle progression, for example, in response to DNA damage. Generic DRTF1/E2F DNA binding activity and transcriptional activation arise when members of two distinct families of proteins, such as DP-1 and E2F-1, interact as DP/E2F heterodimers. In many cell types, DP-1 is a widespread component of DRTF1/E2F DNA binding activity which when expressed at high levels oncogenically transforms embryonic fibroblasts. Here, we document an association between DP-1 and p53 and demonstrate its presence in mammalian cell extracts. In vitro p53 interacts with an immunochemically distinct form of DP-1 and in vivo can regulate transcription driven by the DP-1/E2F-1 heterodimer. At the biochemical level, p53 competes with E2F-1 for DP-1, with a consequent reduction in DNA binding activity. Mutational analysis defines within DP-1 a C-terminal region required for the interaction with p53 and within p53 an N-terminal region distinct from that required to bind to MDM2. Our results establish DRTF1/E2F as a common cellular target in growth control mediated through the activities of pRb and p53 and suggest an alternative mechanism through which p53 may regulate cellular proliferation.

Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster.

The cellular transcription factor DRTF1/E2F is implicated in the control of early cell cycle progression due to its interaction with important regulators of cellular proliferation, such as pocket proteins (for example, the retinoblastoma tumour suppressor gene product), cyclins and cyclin-dependent kinase subunits. In mammalian cells DRTF1/E2F is a heterodimeric DNA binding activity which arises when a DP protein interacts with an E2F protein. Here, we report an analysis of DRTF1/E2F in Drosophila cells, and show that many features of the pathway which regulate its transcriptional activity are conserved in mammalian cells, such as the interaction with pocket proteins, binding to cyclin A and cdk2, and its modulation by viral oncoproteins. We show that a Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila DRTF1/E2F. An analysis of the expression patterns of a Drosophila DP and E2F protein indicated that DmDP is developmentally regulated and in later embryonic stages preferentially expressed in proliferating cells. In contrast, the expression of DmE2F-1 in late stage embryos occurs in a restricted group of neural cells, whereas in early embryos it is widely expressed, but in a segmentally restricted fashion. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of DmDP and DmE2F-1 suggest that the formation of DP/E2F heterodimers, and hence DRTF1/E2F, is subject to complex regulatory cues.

Molecular characterization of Xenopus laevis DP proteins.

It is widely believed that in mammalian cells the cellular transcription factor (DRTF1/E2F integrates cell-cycle events with the transcription apparatus by interacting with important regulators of the cell cycle, such as the retinoblastoma gene product (pRb) and related proteins, cyclins, and cyclin-dependent kinases. Here, we have defined DRTF1/E2F in Xenopus laevis that, like its mammalian counterpart, specifically binds to the E2F site, is regulated during development, and interacts with pRb and related proteins. We have isolated cDNAs that encode the functional homologue of mammalian DP-1, X1 DP-1, together with a close relative, X1 DP-2. X1 DP-1, which is highly conserved with murine DP-1, is a major DNA binding component of X1 DRTF1/E2F. Both DP-1 and DP-2 synergistically interact with members of the E2F family of proteins, E2F-1, E2F-2, and E2F-3, to generate DNA binding complexes that specifically recognize the E2F site and functionally interact with E2F-1 in E2F site-dependent transcriptional activation of cellular genes. DP-1 and DP-2 encode maternally stored transcripts that are expressed during early development. In the adult however, the expression of DP-1 and DP-2 is tissue restricted. This study therefore defines a new family of transcription factors, the DP proteins, members of which can interact combinatorially with E2F proteins to generate an array of DNA binding complexes that integrate cell-cycle progression with the transcription apparatus through the E2F binding site. The tissue-specific expression of DP family members suggests that the combination of DP/E2F heterodimers that constitute DRTF1/E2F is influenced by the phenotype of the cell.

DP-1: a cell cycle-regulated and phosphorylated component of transcription factor DRTF1/E2F which is functionally important for recognition by pRb and the adenovirus E4 orf 6/7 protein.

The cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus through its cyclical interactions with important regulators of cellular proliferation. Two sequence-specific DNA binding proteins, DP-1 and E2F-1, are components of DRTF1/E2F which synergistically interact in a DP-1/E2F-1 heterodimer. Here, we show that DP-1 is a very frequent, possibly universal, component of DRTF1/E2F in 3T3 cells since it is present in all forms of the DNA binding activity that occur during cell cycle progression. Furthermore, the DP-1 polypeptide, which is phosphorylated, undergoes a phosphorylation-dependent mobility shift during the cell cycle suggesting that its level of phosphorylation is regulated during cell cycle progression. A C-terminal region in DP-1 can interact with pRb which, in the context of the DP-1/E2F-1 heterodimer, contributes to the efficiency of pRb binding. The DP-1/E2F-1 heterodimer specifically interacts with the adenovirus type 5 E4 orf 6/7 protein, to produce a DNA binding activity which binds co-operatively to, and transcriptionally activates through, two appropriately positioned E2F sites in a manner which resembles the regulation of DRTF1/E2F by E4 orf 6/7 during adenovirus infection. We conclude that DP-1 is a frequent and cell cycle-regulated component of DRTF1/E2F, and that in the DP-1/E2F-1 heterodimer it is functionally important for recognition by pRb and the E4 orf 6/7 protein.

Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation.

Human papillomavirus type 16 E7 regulates E2F and contributes to mitogenic signalling.

We have produced human papillomavirus type 16 E7 protein in a bacterial expression system and examined the mitogenic activity of this protein in Swiss 3T3 cells after scrape loading. The ability of E7 to induce cellular DNA synthesis in quiescent mouse fibroblasts is strongly enhanced by the presence of a single growth factor such as insulin. Although only weakly mitogenic, introduction of E7 alone resulted in the rapid induction of the transcriptionally active form of E2F, which was not enhanced further by the addition of insulin. Mutant E7 proteins defective for RB binding failed to induce the active form of E2F or act synergistically with insulin to stimulate DNA synthesis. The ability of E7 to regulate E2F may therefore be necessary, but is not sufficient, for full induction of DNA synthesis.

A new component of the transcription factor DRTF1/E2F.

Transcription factor DRTF1/E2F coordinates events in the cell cycle with transcription by its cyclical interactions with important regulators of cellular proliferation like the retinoblastoma tumour-suppressor gene product (Rb) and the Rb-related protein, p107 (refs 1-8). DRTF1/E2F binding sites occur in the control regions of genes involved in proliferation, and both Rb and p107 repress the capacity of DRTF1/E2F to activate transcription (refs 11, 12; M. Zamanian and N.B.L.T., manuscript submitted). Mutant Rb proteins isolated from tumour cells are unable to bind DRTF1/E2F (refs 11-13), and certain viral oncoproteins, such as adenovirus E1A, sequester Rb and p107 in order to free active DRTF1/E2F (refs 5, 11, 12, 14, 15). Here we report the isolation of a complementary DNA encoding DRTF1-polypeptide-1 (DP-1), a major sequence-specific binding protein that is present in DRTF1/E2F, including Rb- and p107-associated DRTF1/E2F. The DNA-binding domain of DP-1 contains a region that resembles that of E2F-1 (refs 16, 17), and recognizes the same sequence. DRTF1/E2F thus appears to contain at least two sequence-specific DNA-binding proteins.

Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

Cyclin A and the retinoblastoma gene product complex with a common transcription factor.

The retinoblastoma gene (Rb) product is a negative regulator of cellular proliferation, an effect that could be mediated in part at the transcriptional level through its ability to complex with the sequence-specific transcription factor DRTF1. This interaction is modulated by adenovirus E1a, which sequesters the Rb protein and several other cellular proteins, including cyclin A, a molecule that undergoes cyclical accumulation and destruction during each cell cycle and which is required for cell cycle progression. Cyclin A, which also complexes with DRTF1, facilitates the efficient assembly of the Rb protein into the complex. This suggests a role for cyclin A in regulating transcription and defines a transcription factor through which molecules that regulate the cell cycle in a negative fashion, such as Rb, and in a positive fashion, such as cyclin A, interact. Mutant loss-of-function Rb alleles, which occur in a variety of tumour cells, also fail to complex with E1a and large T antigen. Here we report on a naturally occurring loss-of-function Rb allele encoding a protein that fails to complex with DRTF1. This might explain how mutation in the Rb gene prevents negative growth control.

Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor.

The transforming proteins of several DNA tumour viruses, including adenovirus E1a and simian virus 40 large T antigen, complex with the retinoblastoma (Rb) tumour-suppressor gene product. This requires regions in these viral proteins necessary for transformation and is thought to inactivate the growth-suppressing properties of the Rb protein by disrupting its interaction with cellular targets. Indeed, regions of Rb required to form a complex with E1a and large T antigen are often mutated in transformed cells. The level at which the Rb protein regulates proliferation is unknown, although one possibility is transcription. We have previously characterized a sequence-specific transcription factor, DRTF1, the activity of which is downregulated as embryonal carcinoma stem cells differentiate. DRTF1 is found in several discrete protein complexes (a, b and c) which are of different sizes but have the same DNA specificity. We now show that one of these also contains the Rb protein and, further, that the adenovirus E1a protein causes the dissociation of the Rb protein from this complex. This requires conserved regions 1 and 2 of E1a that are known to be required for efficient transformation. These results demonstrate that the Rb protein forms a complex with a DNA-bound transcription factor, and suggests that the Rb protein might act by regulating transcription.