Topoisomerase IIα Is Required for Embryonic Development and Liver Regeneration in Zebrafish

Abstract

Topoisomerases solve the topological problems encountered by DNA throughout the lifetime of a cell. Topoisomerase II␣, which is highly conserved among eukaryotes, untangles replicated chromosomes during mitosis and is absolutely required for cell viability. A homozygous lethal mutant, can4, was identified in a screen to identify genes important for cell proliferation in zebrafish by utilizing an antibody against a mitosis-specific marker, phospho-histone H3. Mutant embryos have a decrease in the number of proliferating cells and display increases in DNA content and apoptosis, as well as mitotic spindle defects. Positional cloning revealed that the genetic defect underlying these phenotypes was the result of a mutation in the zebrafish topoisomerase II␣ (top2a) gene. top2a was found to be required for decatenation but not for condensation in embryonic mitoses. In addition to being required for development, top2a was found to be a haploinsufficient regulator of adult liver regrowth in zebrafish. Regeneration analysis of other adult tissues, including fins, revealed no heterozygous phenotype. Our results confirm a conserved role for TOP2A in vertebrates as well as a dose-sensitive requirement for top2a in adults.The accurate and complete replication and separation of chromosomes during mitosis is vital for the viability of cells. One potential complication encountered during cell division is the topological and tensional pressures put on DNA during these processes. Topoisomerases are responsible for enzymatically winding, unwinding, knotting, and unknotting reactions that are necessary for solving the topological problems of DNA. Genetic and biochemical studies in bacteria and yeast have revealed two classes of topoisomerases, type I and type II (38). Type I topoisomerases act on DNA by creating a temporary single-strand nick in DNA, passing the intact strand through the broken strand and then religating the nick. Type II topoisomerases, alternatively, function by binding to two double-stranded DNA molecules, generating a double-stranded break in one of the strands, passing the intact strand through the broken strand, and religating the broken strand.During DNA replication, sister chromatids become tangled with each other as a by-product of their duplication. The primary cellular function of type II topoisomerases during mitosis is the decatenation (untangling) of sister chromatids. Consistent with this essential role, genetic studies Saccharomyces cerevisiae have revealed that temperature-sensitive topoisomerase II mutants (TOP2) are unviable at nonpermissive temperatures (10,16,36). These mutants arrest during M phase due to their incompletely decatenated chromatids.Humans have two TOP2 homologues, topoisomerase II alpha (TOP2A) and topoisomerase II beta (TOP2B). Although they are both functionally redundant with the yeast homologue, TOP2A and TOP2B are genetically unique. As the products of different loci, TOP2A and TOP2B have different expression patterns. Specifically, TOP2A expression peaks during M phase, wh...

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

“…However, DNA damage typically blocks replication to prevent propagation of mutated DNA and to allow DNA repair. Indeed, topoisomerase 2a mutants have DNA damage and reduced cell proliferation (Dovey et al, 2009), and we found these mutants had very little BrdU incorporation (not shown), which is unlike the increase in DNA replication in uhrf1 and dnmt1 mutants. Other studies have reported that DNMT1 depletion from cancer cells causes an acute cell cycle arrest in S-phase (Milutinovic et al, 2003;Unterberger et al, 2006) and a recent study using Xenopus egg extracts shows a requirement for UHRF1 in origin licensing and processivity during DNA replication (Taylor et al, 2013).…”

Topoisomerase IIβ is required for lamina-specific targeting of retinal ganglion cell axons and dendrites

“…Both gene products are broadly expressed at 1 dpf; top2a RNA is detectable in the head and neural tube (Dovey et al, 2009), whereas top2b probe labels the whole embryo. After this time point, top2a expression persists in proliferative zones (Dovey et al, 2009), whereas top2b becomes restricted to postmitotic neurons.…”

“…Both gene products are broadly expressed at 1 dpf; top2a RNA is detectable in the head and neural tube (Dovey et al, 2009), whereas top2b probe labels the whole embryo. After this time point, top2a expression persists in proliferative zones (Dovey et al, 2009), whereas top2b becomes restricted to postmitotic neurons. The zebrafish data corroborate observations from mammals; top2b has been shown in developing rat cerebellum to be expressed just as top2a diminishes, as neurons exit the cell cycle and begin to differentiate morphologically (Tsutsui et al, 2001).…”

“…Because of the length of chromosomes, and the association of genomic DNA with multiple proteins, local supercoiling cannot be resolved or altered by twisting the full chromosome; topoisomerases make local changes to create desirable topology. Top2a appears to be particularly important for the untangling of chromosomes prior to segregation during mitosis (Dovey et al, 2009), and perhaps has some function in transcription also (Mondal and Parvin, 2001;Mondal et al, 2003). Top2b, by contrast, appears dispensable for proliferation but involved in several facets of transcription.…”

Transcriptome profile of human neuroblastoma cells in the hypomagnetic field

“…The dynamic expression changes of CCAR1 during the early incubation period (12-18 h) ( Figure 6) suggest that the HMF might affect hormone-induced transcriptional regulation processes [54,55], as this is the primary function of CCAR1. Expression changes were also verified in genes that are involved in genetic material modification and epigenetic control of gene expression, such as chromatin condensation (TOP2A) [56,57], histone acetylation [58] (SAP30 and SAP18 [59,60]), and alternative splicing (SRSF2IP [61,62]). Illustrating the relationship among these genes, namely the molecular network leading to changes in cell proliferation (Figure 7), as well as tracing the changes in gene expression during exposure to the HMF, will be helpful for our understanding of their roles.…”