THE ROLE OF AN INVERTED CCAAT ELEMENT IN TRANSCRIPTIONAL ACTIVATION OF THE HUMAN DNA TOPOISOMERASE II-ALPHA GENE BY HEAT-SHOCK

Expression of the DNA topoisomerase II alpha (topoII alpha) gene is hi ghly sensitive to various environmental stimuli including heat shock. The amount of topoII alpha mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stres s at 43 degrees C for 1 h. The effect of heat shock on the transcripti onal activity of the human topoII alpha gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasm ids containing various lengths of the promoter sequence. The transcrip tional activity of the full-length promoter (nucleotides (nt) -295 to +85) and of three deletion constructs (nt -197 to +85, -154 to +85, an d -74 to +85) was increased similar to 3-fold 24 h after heat shock st ress. In contrast, the transcriptional activity of the minimal promote r (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 -2 1, was not increased by heat shock. Furthermore, the transcriptional a ctivity of promoter constructs containing mutations in the GC box or h eat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobi lity shift assays revealed reduced binding of a nuclear factor to an o ligonucleotide 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 alpha gene promoter. Finally, in vivo footprint analysis of the topoII alpha gene promoter revealed that two G residu es of ICE1 that were protected in control cells became sensitive to di methyl sulfate modification after heat shock. These results suggest th at transcriptional activation of the topoII alpha gene by heat shock r equires the release of a negative regulatory factor from ICE1.