Induction of the cellular E2F-1 promoter by the adenovirus E4-6/7 protein

The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with differen t members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. T his induction of E2F DNA binding activity strongly correlates with increase d E2a transcription when analyzed using virus infection and transient expre ssion assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and sta ble DNA binding to the Ad5 E2a promoter region, not all of these viruses ca rry the inverted E2F binding site targets in their E2a promoter regions. Th e Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. Ho wever, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single bindi ng site. A possible explanation of these results is that the Ad E4-6/7 prot ein evolved to induce cellular gene expression. Consistent,vith this notion , we show that infection with different adenovirus isolates induces the bin ding of E2F to an inverted configuration of binding sites present in the ce llular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promo ter linked to chloramphenicol acetyltransferase or green fluorescent protei n reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induc tion of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms t o induce the expression of the E2F-1 gene. The E1A proteins displace repres sors of E2F activity (the Rb family members) and thus relieve E2F-1 promote r repression; the E4-6/7 protein complements this function by stably recrui ting active E2F to the E2F-1 promoter to transactivate expression.