The G₁/S Boundary-Specific Enhancer of the Rat cdc2 Promoter

Abstract

Multiple species of G₁ cyclins and cyclin-dependent kinases are induced sequentially during G₁ phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G₁/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5′-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions ‒183 to ‒122, which contains the E box, Sp1, and E2F motifs. The expression of 5′ sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from ‒276 to ‒265, confers strong inducibility on the basal promoter at the G₁/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G₁/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G₁ phase.