SUBUNIT COMPOSITION DETERMINES E2F DNA-BINDING SITE-SPECIFICITY

The product of the retinoblastoma (Rb) susceptibility gene, Rb-l, regu lates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transc ription factor composed of two subunits each encoded by one of two rel ated gene families, denoted E2F and DP. Five E2F genes, E2F-1 through E2F-5, and two DP genes, DP-1 and DP-2, have been isolated from mammal s, and heterodimeric complexes of these proteins are expressed in most , if not all, vertebrate cells. It is not yet clear whether E2F/DP com plexes regulate overlapping and/or specific cellular genes. Moreover, little is known about whether Rb regulates all or a subset of E2F-depe ndent genes. Using recombinant E2F, DP, and Rb proteins prepared in ba culovirus-infected cells and a repetitive immunoprecipitation-PCR proc edure (CASTing), we have identified consensus DNA-binding sites for E2 F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. Our data indicate that (i) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (i i) E2F sites differ with respect to their intrinsic DNA-bending proper ties; (iii) E2F/DP complexes induce distinct degrees of DNA bending; a nd (iv) complex-specific E2F sites selected in vitro function distinct ly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complex es may regulate subsets of MF-dependent cellular genes.