THE G(1) S BOUNDARY-SPECIFIC ENHANCER OF THE RAT CDC2 PROMOTER/

Multiple species of G(1) cyclins and cyclin-dependent kinases are indu ced sequentially during G, phase, and the expression of cyclin A and c dc2 genes is subsequently induced at the G(1)/S boundary. To analyze t he mechanism of cdc2 promoter activation, the 5'-flanking region of th e rat cdc2 gene was isolated and its structural features were characte rized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, w hich contains the E box, Sp1, and E2F motifs. The expression of 5' seq uential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The pr esumed enhancer region was further analyzed by the introduction of bas e substitutions and by the formation of DNA-protein complexes with cel l extracts prepared at various times during the G(1)-to-S-phase progre ssion. These analyses revealed that the enhancer sequence, AAGTTACAAAT A, located from -276 to -265, confers strong inducibility on the basal promoter at the G(1)/S boundary. The base substitutions introduced in to the moths of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter a t the G(1)/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G(1) phase.