A SILENCER ELEMENT IN THE RETINOBLASTOMA TUMOR-SUPPRESSOR GENE

We have previously identified two positive regulatory elements in the retinoblastoma gene (RB) promoter. One is an ATF site and the other we have called a retinoblastoma binding factor 1 site. In addition, a co nsensus E2F site is located directly 3' to the ATF site. Here we demon strate with gel shift assays that E2F and E2F-dependent complexes can bind in vitro to the E2F site of the RB promoter (RB-E2F site). Moreov er, we demonstrate that deletion of the RB-E2F site results in stimula tion of RB promoter activity in transient transfection assays in sever al cell lines. Specific point mutations in the E2F site that inhibit t he binding of E2F and its complexes also stimulate the RB promoter act ivity, suggesting that a factor(s) that can bind to the RB-E2F site co uld be a silencer. RB promoter activity was also stimulated two- to fo ur-fold by mutation of the E2F site in the RB-negative cell lines, J82 and HTB9. Taken together, our results show that the E2F site in the p romoter of the RB tumor-suppressor gene acts as a silencer element and that the silencer effect of the E2F site is not necessarily dependent on the presence of the RB protein.