Abstract
Expression of DNA topoisomerase IIalpha (topo IIalpha) 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 IIalpha 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 IIalpha transcription.
Highlights
Type II DNA topoisomerases are essential nuclear enzymes that regulate DNA topology by creating a transient double strand break through which a second intact double helix is passed
Promoter Activity in Stable Cell Lines Exhibits Wild Type Behavior—The topoisomerase II (topo II)␣ transcription pattern is characterized by an increase during S phase of the cell cycle
Upon serum addition, when the cells treated with Me2SO re-entered the cell cycle, an immediate elevation of luciferase activity was detected reaching a plateau after approximately 4 – 6 h (Fig. 1A)
Summary
Type II DNA topoisomerases are essential nuclear enzymes that regulate DNA topology by creating a transient double strand break through which a second intact double helix is passed (reviewed in Refs. 1 and 2). Levels of topo II␣ enzyme changes within a single cell cycle, with low levels during G0/G1 and accumulation during S and G2 to reach maximal levels during mitosis [11] This accumulation is obtained by enhancement of both transcriptional activity and mRNA stability during S phase [12, 13]. Reduction in the amount of topo II enzyme is one mechanism by which cancer cells acquire drug resistance (reviewed in Ref. 15) For this reason, considerable effort has been made to determine the mechanisms regulating topo II expression. When Swiss 3T3 cells are confluence-arrested topo II␣ mRNA is rapidly down-regulated in response to reduced binding of the transcription factor NF-Y to an ICB [16] Another ICB has been associated with both transcriptional activation and repression of the topo II␣ promoter in response to heat shock and p53 expression, respectively [17, 18]. The transcription factors c-MYB, B-MYB, NF-M, and Sp3 have been identified as trans-activators of the topo II␣ gene promoter in various cellular backgrounds (20 –22)
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