INHIBITION OF E2F-1 TRANSACTIVATION BY DIRECT BINDING OF THE RETINOBLASTOMA PROTEIN

Loss of a functional retinoblastoma tumor suppressor gene product, pRB , is a key step in the development of many human tumors. pRB is a nega tive regulator of cell proliferation and appears to participate in con trol of entry into the S phase of the cell cycle. The recent demonstra tion that pRB binds to transcription factor E2F has provided a model f or the mechanism of pRB-mediated growth regulation. Since adenovirus E IA proteins dissociate the pRB-E2F complexes and stimulate E2F-depende nt transcription, it has been suggested that pRB inhibits E2F transact ivation. Although some evidence for this hypothesis has been provided, it has not been possible to determine the mechanism of pRB-mediated i nhibition of E2F transactivation. In this study, we constructed mutant s of E2F-1 that do not bind to pRB yet retain the ability to transacti vate the adenovirus E2 promoter through E2F DNA-binding sites. We demo nstrated that transactivation mediated by the wild-type E2F-1 protein was inhibited by overexpression of wild-type pRB but not by a naturall y occurring mutant of pRB. Transactivation mediated by mutants of E2F- 1 which do not bind to pRB was not affected by overexpression of wild- type pRB. Furthermore, when the E2F-1 transactivation domain was fused to the GAL4 DNA-binding domain, pRB inhibited GAL4-E2F-1 transactivat ion through GAL4 sites. Expression of pRB did not inhibit transactivat ion mediated by GAL4-E2F-1 mutant constructs which were devoid of pRB binding. In conclusion, these data demonstrate that pRB inhibits E2F-d ependent transactivation by direct protein-protein interaction.