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J Biol Chem, Vol. 274, Issue 26, 18753-18758, June 25, 1999

Evidence for Repressional Role of an Inverted CCAAT Box in Cell Cycle-dependent Transcription of the Human DNA Topoisomerase II Gene

Jacob Falck^§, Peter B. Jensen^§, and Maxwell Sehested

From the  Department of Pathology, Laboratory Center and ^§ Department of Oncology, Finsen Center, Rigshospitalet, DK-2100 Copenhagen, Denmark

Expression of DNA topoisomerase II (topo II) is cell cycle-regulated at both the transcriptional and the post-transcriptional levels. In order to identify cis-acting elements responsible for transcriptional regulation during the cell cycle, we investigated NIH/3T3 cells stably transfected with luciferase reporter plasmids containing various lengths of the human topo II gene promoter. Serum-deprived cells expressed low levels of luciferase, and following serum-induced cell cycle re-entry luciferase levels were gradually elevated 2-fold. During S phase, a steep 3-fold increase in luciferase activity was seen, reaching its maximum approximately 22 h after serum addition. This pattern was observed with both a full-length (nucleotides (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 the first inverted CCAAT box (ICB1) the S phase-specific induction was absent. Mutation of ICB1 revealed that it had a repressive character, since luciferase levels rose rapidly to maximal levels immediately following serum addition. Furthermore, electrophoretic mobility shift assays demonstrated a marked decrease in ICB1 binding activity following serum addition. Together, this suggests a role of ICB1 in cell cycle-dependent repression of topo II transcription.

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