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J Biol Chem, Vol. 273, Issue 17, 10550-10555, April 24, 1998
From the Departments of Expression of the DNA topoisomerase II
The Role of an Inverted CCAAT Element in Transcriptional
Activation of the Human DNA Topoisomerase II
Gene by Heat
Shock
,,
,, and Biochemistry and ** Urology,
Kyushu University School of Medicine, Maidashi, Higashi-ku, Fukuoka
812-8582, Japan, the ¶ Department of Molecular Biology, School of
Medicine, University of Occupational and Environmental Health,
Yahatanishi-ku, Kitakyushu 807, Japan, and the Department of
Biochemistry, Molecular Biology, and Cell Biology, Northwestern
University, Evanston, Illinois 60208
(topoII) gene is highly sensitive to various environmental stimuli
including heat shock. The amount of topoII 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 topoII 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 topoII gene
promoter. Finally, in vivo footprint analysis of the
topoII 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 topoII gene by heat shock requires the release of a negative regulatory factor from ICE1.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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