Negative regulation of the rat cdc2 promoter in G1 by the silencer element.
Expression of the cdc2 gene is induced steeply at the G1-S-phase boundary. The previous analysis of promoter elements that confer inducibility revealed the enhancer at positions -276 to -265. Enhancer activity is suppressed by the upstream sequence that seems to contain the silencer. The silencer element was analyzed by fusing several oligonucleotides covering the silencer region upstream of the enhancer in the cdc2 promoter-luciferase construct. Oligonucleotide IV, which suppressed enhancer activity, was further dissected by the introduction of base substitutions and by forming the DNA-protein complexes with quiescent rat cell extract. The silencer element, AAGTAGTAAAAATA, was finally identified at positions -374 to -360, which resembles the enhancer sequencer but contains extra internal AG residues. Silencer complexes were formed with the quiescent cell extract, whereas the amounts of the complexes decreased with the progression of the cell cycle, and nearly no complexes were formed with the late G1 cell extracts. Conversely, the enhancer complex begins to be formed after late G1. Among the three silencer complexes, the formation of the slowest-migrating complex (complex III) was inhibited by the enhancer sequence, suggesting that a common factor interacts with both the silencer and enhancer. These results suggest that the conversion of complex formation from the silencer to the enhancer site regulates the induction of cdc2 promoter activity at the G1-S-phase boundary.
Tsubota
Y
Y
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Takeuchi
A
A
Shimizu
M
M
Nakajima
T
T
Nojima
H
H
Oda
K
K
D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000235:genetics*; D002457:Cell Extracts; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005347:Fibroblasts; D016193:G1 Phase / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*
D002457:Cell Extracts; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D004247:DNA; D016203:CDC2 Protein Kinase