A specific CCAAT-binding protein, CBP/tk, may be involved in the regulation of thymidine kinase gene expression in human IMR-90 diploid fibroblasts during senescence.
The aging of IMR-90 human diploid fibroblasts in culture is accompanied by a 5-7 fold decrease in the level of thymidine kinase (TK) mRNA and TK activity (Chang, Z. F., and Chen, K. Y. (1988) J. Biol. Chem. 263, 11431-11435). We have employed a gel mobility shift analysis to investigate the molecular basis of the age-dependent attenuation of TK gene expression. Several cis-elements including two inverted CCAAT boxes, located at base pairs (bp) -36 and -67, and GC-rich Sp1 binding sites have been identified in the TK promoter. A 28-bp (-91 to -64) fragment containing the distal inverted CCAAT element was excised from the TK promoter to examine possible differences in nuclear protein binding between young and old IMR-90 cells. A prominent DNA-protein complex was identified in serum-stimulated young cells by a gel mobility shift assay. Competition analysis indicated that the binding was highly specific. The nuclear protein responsible for the complex formation was named CBP/tk (CCAAT Binding Protein for TK gene) since methylation interference assay showed that the inverted CCAAT box was involved in binding. The appearance of the CBP/tk-28-bp complex in IMR-90 cells was (i) serum-dependent, becoming prominent 12-24 h after serum stimulation, and (ii) age-dependent, prominent only in young but not in old IMR-90 cells. Similar serum- and age-dependent complex formations were also observed using a 67-bp fragment (-63 to +4) containing the proximal CCAAT element and a TATA box. In contrast, the binding activities for the Sp1 sequence were the same in young and old cells and appeared to be serum-independent. CBP/tk binding activity in nuclear extracts was abolished by heat (60 degrees C, 5 min) or treatment with proteinase K (0.1 microgram/ml) and sodium dodecyl sulfate (0.005%), but not by Nonidet P-40 or Triton X-100. Treatment of nuclear extracts with alkaline phosphatase or lectins (concanavalin A and wheat germ agglutinin) did not affect the binding activity. Metal chelators such as 1,10-ortho-phenanthroline (0.5 mM) inhibited the CBP/tk binding activity. Cycloheximide added to the serum-stimulated cultures at an early or mid-G1 phase inhibited the CBP/tk binding activity. The half-life of the serum-induced CBP/tk binding activity was estimated to be less than 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)
Pang
J H
JH
Department of Chemistry, Rutgers University, Piscataway, New Jersey 08855-0939.
Chen
K Y
KY
D001483:Base Sequence; D001667:Binding, Competitive; D022762:CCAAT-Enhancer-Binding Proteins; D002453:Cell Cycle; D002478:Cells, Cultured; D016922:Cellular Senescence; D002614:Chelating Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000502:physiology*; D003902:Detergents; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013937:Thymidine Kinase / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D002614:Chelating Agents; D004268:DNA-Binding Proteins; D003902:Detergents; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D003513:Cycloheximide; D013937:Thymidine Kinase