Identification of Genes That Promote or Antagonize Somatic Homolog Pairing Using a High-Throughput FISH-Based Screen

Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS Genetics (Impact Factor: 7.53). 05/2012; 8(5):e1002667. DOI: 10.1371/journal.pgen.1002667
Source: PubMed


The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate "pairing promoting genes" and candidate "anti-pairing genes," providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing.

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Available from: Eric F Joyce, Oct 02, 2015
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    • "Brain cell preparations from 72 to 96-hour third-instar larvae with empty guts showed mitotic pro-metaphases, metaphases and anaphases. Somatic pairing was observed in metaphase plates as reported for other dipterans (Stevens 1908, Metz 1916, Joyce et al. 2012). We distinguished two types of chromosome numbers within the studied samples: 2n=10 and 2n=11. "
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    ABSTRACT: The horn fly, Haematobia irritans is an obligate haematophagous cosmopolitan insect pest. The first reports of attacks on livestock by H. irritans in Argentina and Uruguay occurred in 1991, and since 1993 it is considered an economically important pest. Knowledge on the genetic characteristics of the horn fly increases our understanding of the phenotypes resistant to insecticides that repeatedly develop in these insects. The karyotype of H. irritans, as previously described using flies from an inbred colony, shows a chromosome complement of 2n=10 without heterochromosomes (sex chromosomes). In this study, we analyze for the first time the chromosome structure and variation of four wild populations of H. irritans recently established in the Southern Cone of South America, collected in Argentina and Uruguay. In these wild type populations, we confirmed and characterized the previously published " standard " karyotype of 2n=10 without sex chromosomes; however, surprisingly a supernumerary element, called B-chromosome, was found in about half of mitotic preparations. The existence of statistically significant karyotypic diversity was demonstrated through the application of orcein staining, C-banding and H-banding. This study represents the first discovery and characterization of horn fly karyotypes with 2n=11 (2n=10+B). All spermatocytes analyzed showed 5 chromosome bivalents, and therefore, 2n=10 without an extra chro-CompCytogen 9(1): 31–50 (2015)
    Comparative cytogenetics 02/2015; 9(1):31-50. DOI:10.3897/CompCytogen.v9i1.8535 · 1.21 Impact Factor
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    • "Multiple attempts have been made to understand molecular mechanisms responsible for this phenomenon. Two independent screens have been done, and many candidate genes have been identified, but the mechanism of this phenomenon still remains elusive [23], [24], [25]. It has been proposed that the process of zygotic transcription per se is what establishes somatic pairing [22], [24]. "
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    ABSTRACT: Three-dimensional organization of the genome is important for regulation of gene expression and maintenance of genomic stability. It also defines, and is defined by, contacts between different chromosomal loci. Interactions between loci positioned on different chromosomes, i.e. "trans" interactions are one type of such contacts. Here, we describe a case of inducible trans interaction in chromosomes of the budding yeast S. cerevisiae. Special DNA sequences, inserted in two ectopic chromosomal loci positioned in trans, pair with one another in an inducible manner. The spatial proximity diagnostic of pairing is observable by both chromosome capture analysis (3C) and epifluorescence microscopy in whole cells. Protein synthesis de novo appears to be required for this process. The three-dimensional organization of the yeast nucleus imposes a constraint on such pairing, presumably by dictating the probability with which the two sequences collide with one another.
    PLoS ONE 09/2013; 8(9):e75895. DOI:10.1371/journal.pone.0075895 · 3.23 Impact Factor
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    • "In addition to its well-known functions in chromatin condensation along the chromosome axis during nuclear division and in interphase, condensin complexes inhibit associations between homologues (reviewed by Wood et al. 2010). Drosophila condensin II antagonizes the pairing of homologues (Joyce et al. 2012) and reduces transvection by limiting interactions between homologues during interphase. Furthermore, the condensin II subunits SMC4, Cap-H2 and Cap-D3 are necessary to disassemble aligned polytene chromosomes (Hartl et al. 2008a). "
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    ABSTRACT: In plants as in other eukaryotes, the structural maintenance of chromosome (SMC) protein complexes cohesin, condensin and SMC5/6 are essential for sister chromatid cohesion, chromosome condensation, DNA repair and recombination. The presence of paralogous genes for various components of the different SMC complexes suggests the diversification of their biological functions during the evolution of higher plants. In Arabidopsis thaliana, we identified two candidate genes (Cap-D2 and Cap-D3) which may express conserved proteins presumably associated with condensin. In silico analyses using public databases suggest that both genes are controlled by factors acting in a cell cycle-dependent manner. Cap-D2 is essential because homozygous T-DNA insertion mutants were not viable. The heterozygous mutant showed wild-type growth habit but reduced fertility. For Cap-D3, we selected two homozygous mutants expressing truncated transcripts which are obviously not fully functional. Both mutants show reduced pollen fertility and seed set (one of them also reduced plant vigour), a lower chromatin density and frequent (peri)centromere association in interphase nuclei. Sister chromatid cohesion was impaired compared to wild-type in the cap-D3 mutants but not in the heterozygous cap-D2 mutant. At superresolution (Structured Illumination Microscopy), we found no alteration of chromatin substructure for both cap-D mutants. Chromosome-associated polypeptide (CAP)-D3 and the cohesin subunit SMC3 form similar but positionally non-overlapping reticulate structures in 2C-16C nuclei, suggesting their importance for interphase chromatin architecture in differentiated nuclei. Thus, we presume that CAP-D proteins are required for fertility, growth, chromatin organisation, sister chromatid cohesion and in a process preventing the association of centromeric repeats.
    Chromosoma 08/2013; 122(6). DOI:10.1007/s00412-013-0424-y · 4.60 Impact Factor
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