RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate

Abstract

DNA topoisomerase II (Topo II) is a major component of mitotic chromosomes and an important drug target in cancer chemotherapy, however, its role in chromosome structure and dynamics remains controversial. We have used RNAi to deplete Topo II in Drosophila S2 cells in order to carry out a detailed functional analysis of the role of the protein during mitosis. We find that Topo II is not required for the assembly of a functional kinetochore or the targeting of chromosomal passenger proteins, nonetheless, it is essential for anaphase sister chromatid separation. In response to a long-running controversy, we show that Topo II does have some role in mitotic chromatin condensation. Chromosomes formed in its absence have a 2.5-fold decrease in the level of chromatin compaction, and are morphologically abnormal. However, it is clear that the overall programme of mitotic chromosome condensation can proceed without Topo II. Surprisingly, in metaphase cells depleted of Topo II, one or more chromosome arms frequently stretch out from the metaphase plate to the vicinity of the spindle pole. This is not kinetochore-based movement, as the centromere of the affected chromosome is located on the plate. This observation raises the possibility that further unexpected functions for Topo II may remain to be discovered.

Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase IIα Mutant Human Cell Line

“…Our results exclude any active role for ICRF-193-inhibited topoII␣ (e.g., DNA breakage via topoII␣ poisoning, or structural interference) in the effect of ICRF-193 on condensation, because topoII␣ depletion does not affect this ( Figure 4C). Our conclusion that topoII␣ is not essential for chromosome condensation, although consistent with some studies (Andreassen et al, 1997;Chang et al, 2003;Sakaguchi and Kikuchi, 2004), seems at odds with reports that topoII inhibitors prevent condensation (see Introduction) which, combined with the fact that TOPOII␤Ϫ/Ϫ cells divide normally (Yang et al, 2000), imply that topoII␣ is essential for condensation. These discrepancies may reflect differences in interpretation, however, because there clearly is an effect on the timing of condensation, and this may be more or less pronounced depending on cell type and whether microtubule inhibitors are used to allow further time for condensation to occur.…”

“…In vitro studies of chromosome condensation in mitotic extracts (Newport and Spann, 1987;Adachi et al, 1991;Hirano and Mitchison, 1991, 1993), in which topoII is immunodepleted or inactivated by inhibitors, showed varying requirements for topoII. In the absence of suitable topoII mutants, in vivo studies in higher eukaryotes have made use of topoII inhibitors (Andoh et al, 1993;Downes et al, 1994;Gorbsky, 1994;Ishida et al, 1994;Gimenez-Abian et al, 1995;Andreassen et al, 1997;Gimenez-Abian et al, 2000) or RNA interference (Chang et al, 2003;Sakaguchi and Kikuchi, 2004). Such studies mostly support a role for topoII in chromosome condensation, but again, condensation was impaired to varying degrees, and in some cases (Andreassen et al, 1997;Chang et al, 2003;Sakaguchi and Kikuchi, 2004) impairment was surprisingly mild or absent.…”

“…In the absence of suitable topoII mutants, in vivo studies in higher eukaryotes have made use of topoII inhibitors (Andoh et al, 1993;Downes et al, 1994;Gorbsky, 1994;Ishida et al, 1994;Gimenez-Abian et al, 1995;Andreassen et al, 1997;Gimenez-Abian et al, 2000) or RNA interference (Chang et al, 2003;Sakaguchi and Kikuchi, 2004). Such studies mostly support a role for topoII in chromosome condensation, but again, condensation was impaired to varying degrees, and in some cases (Andreassen et al, 1997;Chang et al, 2003;Sakaguchi and Kikuchi, 2004) impairment was surprisingly mild or absent. Thus, although both structural (Mirkovitch et al, 1988;Hart and Laemmli, 1998) and catalytic (Koshland and Strunnikov, 1996;Hirano, 2000) models for how topoII may be involved in chromosome condensation have been proposed, the issue of whether topoII is required for condensation remains controversial.…”

Effects of Scaffold/Matrix Alteration on Centromeric Function and Gene Expression

“…This impaired function provides an explanation for the observed increase in the level of the mitotic checkpoint protein BubR1 on the total centromere population following DST treatment. Other studies have also shown that the disruption of chromosomal scaffold/matrix proteins decreases mitotic chromosome condensation and sister chromatid resolution (62)(63)(64).…”

A role for Drosophila Polo protein in chromosome resolution and segregation during mitosis

“…The chromosome missegregation phenotype of Polo-depleted cells resemble the ones described for lack of SMC4 (structural maintenance chromosome 4) and Topoisomerase II, proteins required for chromosome resolution. [11][12][13] Interestingly, Plk1-dependent phosphorylation of Topoisomerase II is essential for its function in chromosome segregation during mitosis. 14 We therefore examined prometaphase/ metaphase chromosome resolution in Polo-depleted cells.…”