Academic journal article Genetics

Disruption of Topoisomerase II Perturbs Pairing in Drosophila Cell Culture

Academic journal article Genetics

Disruption of Topoisomerase II Perturbs Pairing in Drosophila Cell Culture

Article excerpt

ABSTRACT

Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc^sub 167^ cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc^sub 167^ cells into G^sub 1^, S, and G^sub 2^ subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing.

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ONE way in which the genome can be organized is through the physical pairing of homologous chromosomes. Such pairing occurs during meiosis, when it aligns chromosomes in preparation for recombination and segregation, as well as in nonmeiotic nuclei. Although most extensively studied in the Dipteran insect Drosophila melanogaster, nonmeiotic association of homologous chromosomal regions has also been observed elsewhere, where it participates in gene expression and other vital processes (reviewed by WU and MORRIS 1999; DUNCAN 2002; GRANT-DOWNTON and DICKINSON 2004; MCKEE 2004; ZICKLER 2006). Importantly, nonmeiotic pairing can influence gene regulation and DNA repair through the processes of transvection (reviewed by PIRROTTA 1999; WU and MORRIS 1999; DUNCAN 2002; KASSIS 2002; KENNISON and SOUTHWORTH 2002) and recombination (reviewed by GLOOR 2002; WYMAN et al. 2004; also see RONG and GOLIC 2003), respectively. Pairing of homologous chromosomal regions has also been implicated in mammalian X-inactivation (MARAHRENS 1999; BACHER ET AL. 2006; DIAZ-PEREZ et al. 2006; XU et al. 2006) and imprinting (LASALLE and LALANDE 1996; RIESSELMANN and HAAF 1999). Intriguingly, a reduced level of pairing at imprinted regions may be associated with syndromes such as autism and the Prader-Willi and Angelman syndromes (LASALLE and LALANDE 1996; THATCHER et al. 2005). Despite its role in multiple phenomena, however, the mechanism(s) of nonmeiotic homolog pairing remains largely unknown.

To better understand nonmeiotic pairing, we have focused on Drosophila, where homologous chromosomes are essentially paired in all somatic cells throughout development (STEVENS 1907, 1908; METZ 1916; reviewed by DUNCAN 2002; MCKEE 2004). FISH analyses of embryos and tissues such as from the larval brain or imaginal discs indicate that, typically, a given chromosome region is paired in 60-100% of nuclei, but that this level of pairing can vary depending on the time of development, region being assayed, and phase of the cell cycle (KOPCZYNSKI and MUSKAVITCH 1992; CSINK and HENIKOFF 1998; FUNG et al. 1998; GEMKOW ET AL. 1998; SASS and HENIKOFF 1999; VAZQUEZ ET AL. 2002; RONSHAUGEN and LEVINE 2004; FRITSCH et al. 2006). For example, analysis of multiple chromosomal locations during early embryogenesis indicates that some regions initiate pairing earlier than do others, but that all regions eventually achieve high levels of pairing (FUNG et al. 1998). Other studies have examined the impact of the cell cycle on pairing. In one case, BrdU labeling of the larval central nervous system (CNS) revealed that the percentage of nuclei in which a euchromatic region, 59E, and a pericentromeric repeat, AACAC, were paired decreased during S phase from . …

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