Enhancer and silencer binding proteins involved in the rat cdc2 promoter activation at the G1/S boundary.

Abstract

Expression of the rat cdc2 gene during G1-S phase progression is negatively and positively regulated by the silencer and enhancer elements located upstream of the basal promoter. The silencer and enhancer sequences resemble each other, but the silencer contains extra internal AG residues. The cDNA clones encoding the enhancer binding proteins cdc2E1 and cdc2E2 were isolated by South-Western blotting. cdc2E1 and cdc2E2 comprise 436 and 256 amino acids and have two RNA binding domains which contain an RNP1 octamer and an RNP 2 hexamer. Both cdc2E1 and cdc2E2 bind to the double-stranded and single-stranded silencer and enhancer sequences, but their binding affinity to the enhancer was stronger than that to the silencer. Transfection of quiescent 3Y1 cells with the cdc2 promoter-luciferase constructs, followed by serum stimulation, showed that the promoter activation at the G1-S phase boundary was reduced greatly by base substitutions within the enhancer, but not within the silencer. Gel shift assays with oligonucleotides containing both the silencer and enhancer showed that formation of the large complex was greatly reduced if base-substitutions were introduced into the enhancer, but not within the silencer. The complex was supershifted completely by anti-cdc2E1 antibody and partially by anti-cdc2E2 antibody. These results suggest that cdc2E1 and cdc2E2 preferentially form the multimeric complex at the enhancer site after the late G1 phase for activation of the cdc2 promoter.