THE TRANSCRIPTION FACTOR E2F-1 IS A DOWNSTREAM TARGET OF RE ACTION

Reintroduction of RE into SAOS2 (RB(-/-)) cells causes a G(1) arrest a nd characteristic cellular swelling. Coexpression of the cellular tran scription factor E2F-1 could overcome these effects. The ability of E2 F-1 to bind to RE was neither necessary nor sufficient for this effect , and S phase entry was not accompanied by RE hyperphosphorylation und er these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RE mutant. These data, toge ther with the fact that RE binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RE action. Mutational analysis showed that t he ability of E2F-1 to bind to DNA was necessary and sufficient to blo ck the formation of large cells by RE, whereas the ability to induce S -phase entry required a functional transactivation domain as well. Thu s, the induction of a G(1) arrest and the formation of large cells by RE in these cells can be genetically dissociated. Furthermore, the abi lity of the E2F-1 DNA-binding domain alone to block one manifestation of RE action is consistent with the notion that RB-E2F complexes activ ely repress transcription upon binding to certain E2F-responsive promo ters. In keeping with this view, we shea here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated transcriptional repression.