The Analysis of Mutant Alleles of Different Strength Reveals Multiple Functions of Topoisomerase 2 in Regulation of Drosophila Chromosome Structure

Abstract

Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.

Author Summary

Type II topoisomerases (Topo II) are enzymes that disentangle DNA molecules during essential cellular processes such as DNA replication, chromosome condensation and mitotic cell division. Topo II is a major component of mitotic chromosomes and it is a well known target for cancer chemotherapy. Topo II inhibitors block the Topo II enzymatic activity leading to extensive DNA damage, which ultimately kills the cancer cell. Thus, investigating the role of Topo II in the assembly and structural maintenance of chromosomes is not only relevant to understand chromosome biology but might also have a translational impact on cancer therapy. Here we used Drosophila as model system to analyze the effect of Topo II depletion on chromosome stability. We show that the chromosomal phenotypes of mutant flies vary with the amount of residual Topo II, ranging from site-specific chromosome breaks, variations in chromosome number (aneuploidy and poliploidy) and dramatic defects in chromosome morphology. The chromosomal phenotypes observed in flies recapitulate all phenotypes seen in Topo II-depleted vertebrate chromosomes, reconciling the phenotypic discrepancies reported in previous studies. In addition, our finding that the Topo II dependent phenotypes vary with the residual amount of the enzyme provides useful information on the possible outcome of cancer therapy with Topo II inhibitors.