Evidence for repressional role of an inverted CCAAT box in cell cycle-dependent transcription of the human DNA topoisomerase II alpha gene

Expression of DNA topoisomerase II alpha (topo II alpha) is cell cycle-regu lated at both the transcriptional and the post- transcriptional levels. In order to identify cis-acting elements responsible for transcriptional regul ation during the cell cycle, we investigated NIH/3T3 cells stably transfect ed with luciferase reporter plasmids containing various lengths of the huma n topo II alpha gene promoter. Serum-deprived cells expressed low levels of luciferase, and following serum-induced cell cycle reentry luciferase leve ls were gradually elevated 2-fold. During S phase, a steep S-fold increase in luciferase activity was seen, reaching its maximum approximately 22 h af ter serum addition. This pattern was observed with both a full-length (nucl eotides (nt) -295 to +90] and a deletion (nt -90 to +90) promoter construct . In contrast, when testing a deletion construct (nt -51 to +90) lacking th e first inverted CCAAT box (ICB1) the S phase-specific induction was absent . Mutation of ICB1 revealed that it had a repressive character, since lucif erase levels rose rapidly to maximal levels immediately following serum add ition. Furthermore, electrophoretic mobility shift assays demonstrated a ma rked decrease in ICB1 binding activity following serum addition. Together, this suggests a role of ICB1 in cell cycle-dependent repression of topo II alpha transcription.