INDEPENDENT REGIONS OF ADENOVIRUS E1A ARE REQUIRED FOR BINDING TO ANDDISSOCIATION OF E2F-PROTEIN COMPLEXES

The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a rela ted gene product, p107, in association with the cyclin A-cdk2 or the c yclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a funct ional pRB leads to the repression of E2F activity. The products of the adenovirus EIA gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of EIA require d for this function are also essential for binding to a number of cell ular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regio ns of EIA, as well as in vivo expression of EIA proteins containing de letions of either conserved region 1 (CRI) or CR2, we find that CR2 of EIA can form stable complexes with E2F. EIA proteins containing both CRI and CR2 also associate with E2F, although the presence of these pr oteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that EIA-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Compl exes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that EIA may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR 2 domain of EIA for binding to pRB. These results indicate that EIA CR I and E2F-1 may bind to the same or overlapping sites on pRB and that EIA CR2 binds to an independent region. On the basis of our results, w e propose a two-step model for the release of E2F from pRB and p107 ce llular proteins.