The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIα gene by heat shock

Manabu Furukawa, Takeshi Uchiumi, Minoru Nomoto, Hiroshi Takano, Richard I. Morimoto, Seijo Naito, Michihiko Kuwano, Kimitoshi Kohno

Research output: Contribution to journalArticle

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Abstract

Expression of the DNA topoisomerase IIα (topoIIα) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIα mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 °C for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIα gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) -295 to +85) and of three deletion constructs (nt -197 to +85, -154 to +85, and -74 to +85) was increased ~3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt -20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt -74 and - 21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3-24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIα gene promoter. Finally, in vivo footprint analysis of the topoIIα gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIα gene by heat shock requires the release of a negative regulatory factor from ICE1.

Original languageEnglish
Pages (from-to)10550-10555
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number17
DOIs
Publication statusPublished - Apr 24 1998

Fingerprint

Type II DNA Topoisomerase
Transcriptional Activation
Shock
Hot Temperature
Genes
Chemical activation
Nucleotides
Oligonucleotides
Electrophoretic mobility
Mutation
Electrophoretic Mobility Shift Assay
Luciferases
Urinary Bladder Neoplasms
Human Activities
Transfection
Cultured Cells
Assays
Plasmids

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIα gene by heat shock. / Furukawa, Manabu; Uchiumi, Takeshi; Nomoto, Minoru; Takano, Hiroshi; Morimoto, Richard I.; Naito, Seijo; Kuwano, Michihiko; Kohno, Kimitoshi.

In: Journal of Biological Chemistry, Vol. 273, No. 17, 24.04.1998, p. 10550-10555.

Research output: Contribution to journalArticle

Furukawa, Manabu ; Uchiumi, Takeshi ; Nomoto, Minoru ; Takano, Hiroshi ; Morimoto, Richard I. ; Naito, Seijo ; Kuwano, Michihiko ; Kohno, Kimitoshi. / The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIα gene by heat shock. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 17. pp. 10550-10555.
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AU - Morimoto, Richard I.

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