CONSTITUTIVE PROTECTION OF E2F RECOGNITION SEQUENCES IN THE HUMAN THYMIDINE KINASE PROMOTER DURING CELL-CYCLE PROGRESSION

The sequences responsible for S phase-specific induction of the human thymidine kinase (TK) gene have been mapped to a small region that con tains putative E2F binding sites, We have analyzed protein-DNA interac tions at the TK promoter during cell cycle progression in human fibrob lasts using an in vivo footprinting approach, We found 14 protein bind ing sites that were occupied in vivo, All of the sites (among them two inverted CCAAT boxes and several Spl sites) bound transcription facto rs constitutively throughout the cell cycle, i.e. none of the factor b inding was cell cycle-dependent, An E2F-like site located between nucl eotides -97 and -89 relative to the major transcription start site was protected in G(0), G(1), S, and G(2) phases, This cell cycle-independ ent protection of E2F sequences in the TK promoter differs from the G( 0)/G(1)-restricted binding of E2F complexes observed for genes in whic h the E2F sites function as repressor elements (Tommasi, S., and Pfeif er, G. P. (1995) Mol. Cell. Biol. 15, 6901-6913; Zwicker, J., Liu, N., Engeland, K., Lucibello, F. C., and Muller, R. (1996) Science 271, 15 95-1597). A comparison of several genes containing E2F motifs indicate s that E2F sites located in proximity to the transcription initiation site (-50 to +20) in TATA-less promoters predominantly function as rep ressor elements, while in other genes constitutively bound E2F complex es located further upstream mediate activation presumably in conjuncti on with a functional TATA box.