The MDM2 proto-oncogene is found amplified in a variety of tumours. The oncogenic capacity of the MDM2 protein is attributed to its ability to bind the p53 tumour-suppressor protein and mask its transcriptional activation potential. Here we show that MDM2 makes a functional contact with two cooperating transcription factors, E2F1 and DP1 (refs 4,5), which are involved in S-phase progression. MDM2 contacts the activation domain of E2F1 using residues conserved in the activation domain of p53. However, in contrast to its repression of p53 activity, MDM2 stimulates the activation capacity of E2F1/DP1. These results indicate that MDM2 not only releases a proliferative block by silencing the tumour suppressor p53, it also positively augments proliferation by stimulating the S-phase inducing transcription factors E2F1/DP1.

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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.

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.

The E2F family of transcription factors are thought to play an important role in the control of cell cycle progression. There is now also increasing evidence that some family members may act as oncogenes or tumour suppressor genes. The characterization of these proteins in human primary haemopoietic cells and acute myeloid leukaemia (AML) blasts may thus give an insight to the molecular mechanisms governing proliferation and leukaemogenesis in these cells. Therefore we analysed the expression of E2F‐DNA binding activity and the constituent proteins found in the complexes in human primary haemopoietic cells of various lineages. We also studied blasts from 18 patients with acute myeloid leukaemia (AML). On electromobility shift assays (EMSA) a single E2F‐DNA binding complex was detected in T cells, B cells and monocytes which was shown to contain E2F‐4, DP‐1 and p130, indicating that all quiescent haemopoietic cells have the same complex. Examination of 18 AML samples by EMSA revealed the presence of E2F binding and no gross abnormalities were detected. An E2F‐4/p130 complex was detected in representative samples of all FAB types analysed. Thus abnormalities of E2F function are unlikely to play a primary pathogenic role in AML.

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