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.

Topoisomerases modulate the timing of meiotic DNA breakage and chromosome morphogenesis inSaccharomyces cerevisiae

“…The meiotic functions of topoisomerases have been primarily elucidated in the context of the meiotic divisions where, similar to mitosis, topo II has a major role in disentangling DNA to facilitate chromosome segregation (Gomez et al, 2014;Hartsuiker et al, 1998;Hughes & Hawley, 2014;Jaramillo-Lambert et al, 2016;Kallio & Lahdetie, 1996;Mengoli et al, 2014;Tateno & Kamiguchi, 2001). However, both topoisomerases are also present and active in meiotic prophase (Borde et al, 1999;Cobb et al, 1997;Stern & Hotta, 1983).…”

LhrandHmrare required for sister chromatid detachment during anaphase but not for centromere function

“…Furthermore, we found that the mutants exhibit mitotic indexes and frequencies of well-aligned metaphase plates comparable to those seen in wild type controls. However, we discovered that the mutants have ~6% chromosome aberrations, most of which were incomplete isochromatid breaks that are likely to be generated during anaphase (Mengoli et al, 2014). Consistent with this idea, 15% of anaphases showed chromatin bridges, broken bridges, and/or acentric fragments caused by breaks in heterochromatin or at the euchromatin/heterochromatin junction.…”

“…However, they displayed relatively high frequencies of incomplete chromosome breaks, namely broken chromosomes without the corresponding fragment or complete chromosome complements plus an extra acentric fragment. These two types of chromosome aberrations are likely generated during anaphase (Mengoli et al, 2014). Notably, we did not observe aneuploid cells with unbroken chromosomes in either Hmr or Lhr mutant brains or failure of the centromeres to separate and move towards the poles.…”

“…The proportion of breaks in the Y chromosome in males is also higher for Hmr and Lhr than any previous condition analyzed. It is likely that the high frequency of heterochromatic breaks observed in Hmr and Lhr mutants reflects a specific fragility of heterochromatin and euchromatin-heterochromatin junctions during anaphase, similar to that observed in Top2 mutants (Mengoli et al, 2014). However, while in Top2 mutants most incomplete isochromatid breaks involve the Y chromosome and a distal heterochromatic region in the 3L arm (region h47), in Hmr and Lhr mutants isochromatid breaks involve the Y and both the second and the third chromosome heterochromatin (Tables 1 and 2).…”

“…These incomplete isochromatid breaks are rather rare in other mutants that exhibit chromosome aberrations (mei-9, mei-41 (ATR), mus-102, tim2,dPdxk,tws), where they ranged from 2 to 5% of total isochromatid breaks (Gatti, 1979;Benna et al, 2010;Marzio et al 2014;Merigliano et al, 2017). Such incomplete isochromatid breaks are in contrast very frequent in Topoisomerase2 (Top2) mutants, where they are caused by the rupture of chromosome bridges generated during anaphase due to failure of sister chromatid decatenation (Mengoli et al 2014). Although incomplete isochromatid breaks and at least some complete isochromatid breaks are likely to result from breaks generated during anaphase (Figure 3), chromatid deletions (breaks involving only one of the two sister chromatids; see Figure 2 and Table 1) cannot be the outcome of anaphase defects but rather must result from lesions produced during S or G2 phase.…”