The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIalpha gene by heat shock.
Expression of the DNA topoisomerase IIalpha (topoIIalpha) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIalpha 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 degreesC for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIalpha 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 approximately 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 topoIIalpha gene promoter. Finally, in vivo footprint analysis of the topoIIalpha 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 topoIIalpha gene by heat shock requires the release of a negative regulatory factor from ICE1.
Furukawa
M
M
Department of Biochemistry, Kyushu University School of Medicine, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. mana@biochem1.med.kyushu-u.ac.jp
Uchiumi
T
T
Nomoto
M
M
Takano
H
H
Morimoto
R I
RI
Naito
S
S
Kuwano
M
M
Kohno
K
K
D000951:Antigens, Neoplasm; D001483:Base Sequence; D004247:DNA; D018983:DNA Footprinting; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D018869:Heat-Shock Response; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D015533:Transcriptional Activation
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II