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.

E2F1 overexpression has been shown to induce apoptosis in cooperation with p53. Using Saos-2 cells, which are null for p53 and lack functional Rb, we have demonstrated that E2F1 overexpression can also induce apoptosis in the absence of p53 and retinoblastoma protein (Rb). E2Fl-induced apoptosis can be specifically inhibited by Rb but not mdm2, which is known for its ability to inhibit p53-induced apoptosis. Through the study of the apoptotic function of a set of E2F1 mutants, it was clear that the transactivation and the apoptotic function of E2F1 are uncoupled. The transactivation-defective E2F1 mutants E2Fl(1-374), E2Fl(390-1)DF(Amdm2), and E2Fl(406-415)(ARb) can induce apoptosis as effectively as wild-type E2F1. In contrast to E2F1 transactivation, the DNA-binding activity of E2F1 was proven to be essential for its apoptotic function, as the DNA-binding-defective mutants E2Fl(132) and E2Fl(132)(1-374) failed to induce apoptosis. Therefore Rb may inhibit E2Fl-induced apoptosis by mechanisms other than the suppression of the transactivation of E2F1. This hypothesis was supported by our observation that although Rb overexpression can specifically repress the apoptosis induced by wild-type E2F1 and a Rb-binding-competent E2F1 mutant E2Fl(390-1)DF(Amdm2), it failed to inhibit the apoptosis induced by mutants E2Fl(1-374) and E2Fl(~406-415)(~Rb), which are defective or reduced in Rb binding and transactivation. All of these points argue for a novel function for E2F1 and Rb in controlling apoptosis. The results also indicate that transcriptional repression rather than the transactivation function of E2F1 may be involved in its apoptotic function. The results presented here may provide us some physiological implication of the repression function of the Rb-E2F1 complex.

contributed equally to this workThe CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins such as the transcription factors TFIIE and TFIIF. In addition, CBP associates with two other TSs, P/CAF and SRC1. We set out to establish whether the intrinsic AT activity of CBP contributes to transcriptional activation. We show that a region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter. The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity. Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo. We also find that the HAT domains of CBP and P/CAF share sequence similarity. Four conserved motifs are identified, three of which are analogous to motifs A, B and D, found in other N-acetyltransferases. The fourth motif, termed E, is unique to CBP and P/CAF. Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo. These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription. The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family.

Transcription by RNA polymerase (pol) III is under cell-cycle control, being higher in S and G2 than in G0 and early G1 phases. Many transformed cell types have elevated pol III activity, presumably to sustain sufficient protein synthesis for unrestrained growth. The retinoblastoma tumour-suppressor protein (Rb) restricts cellular proliferation, and is often found mutated in transformed cells. Here we demonstrate that Rb can repress the level of transcription from pol III templates both in vitro and vivo. Analysis of Rb-deficient SAOS2 cells and primary fibroblasts from Rb-/- mice demonstrates elevated levels of pol III activity in the absence of functional Rb protein. Rb-induced repression of pol III activity is alleviated by mutations in the Rb pocket domain that occur naturally in tumours, and by viral transforming proteins that bind and inactivate Rb. These results implicate repression of pol III transcription as a mechanism for Rb-induced growth arrest, and suggest that restraining protein biosynthesis may be important in the prevention of tumour development.