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

Joyce, Eric F.; Williams, Benjamin R.; Xie, Tiao; Wu, Chuan

doi:10.1371/journal.pgen.1002667

Cited by 153 publications

(267 citation statements)

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“…S10); while simultaneous visualization of both genomic regions and transcripts has been achieved previously (43), visualization of both DNA and RNA typically requires sequential hybridizations to avoid cross-talk between the probe sets targeting DNA and those targeting RNA (44). Oligopaints are also suitable for conducting high-throughput FISH in 384-well plates (29) (Fig. 4C; SI Appendix, Fig.…”

Section: Oligopaints Robustly Label Interphase and Metaphase Chromosomentioning

confidence: 99%

“…Also, as these probes are typically short (∼20-50 bases) (24)(25)(26) and single stranded, they diffuse efficiently into fixed cells and tissues and are unhindered by competitive hybridization with complementary probe fragments. Oligo probes have allowed the visualization of single-copy viral DNA as well as individual mRNA molecules using branched DNA signal amplification (27) or a handful to a few dozen short oligo probes (26,28), and, by targeting blocks of repetitive sequences as a strategy to amplify signal, enabled the first FISHbased genome-wide RNAi screen (29). Oligo probes have also been generated directly from genomic DNA using parallel PCR reactions (30,31).…”

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confidence: 99%

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Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

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Joyce

Apostolopoulos

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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410

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A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

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Section: Oligopaints Robustly Label Interphase and Metaphase Chromosomentioning

confidence: 99%

mentioning

confidence: 99%

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Beliveau

Joyce

Apostolopoulos

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

410

472

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“…These methods represent a true discovery approach and have the advantage of not making any assumptions about involved pathways. Screening strategies to identify genepositioning factors have recently become feasible due to the development of high-throughput FISH methods using high-content imaging (Joyce et al, 2012;Shachar et al, 2015a,b) in which the position of a large number of genes or large number of conditions, such as RNAi knockdowns, can be probed in a single experiment. These approaches have yielded the most comprehensive sets of gene-positioning factors to date and have led to the characterization of several novel positioning pathways.…”

Section: Box 1 Basic Organization Of Chromatin In the Nucleusmentioning

confidence: 99%

Causes and consequences of nuclear gene positioning

Shachar

Misteli

2017

Journal of Cell Science

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The eukaryotic genome is organized in a manner that allows folding of the genetic material in the confined space of the cell nucleus, while at the same time enabling its physiological function. A major principle of spatial genome organization is the non-random position of genomic loci relative to other loci and to nuclear bodies. The mechanisms that determine the spatial position of a locus, and how position affects function, are just beginning to be characterized. Initial results suggest that there are multiple, gene-specific mechanisms and the involvement of a wide range of cellular machineries. In this Commentary, we review recent findings from candidate approaches and unbiased screening methods that provide initial insight into the cellular mechanisms of positioning and their functional consequences. We highlight several specific mechanisms, including tethering of genome regions to the nuclear periphery, passage through S-phase and histone modifications, that contribute to gene positioning in yeast, plants and mammals.

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“…transvection, have been identified, and may function differently in different cell types (Hartl et al 2008;Bateman et al 2012b;Joyce et al 2012). Finally, data based on DNA-FISH show high levels of somatic homolog pairing in third instar larvae, but lower levels during embryogenesis, which would likely negatively impact transvection in early tissues (Fung et al 1998).…”

Section: Factors Other Than Enhancer Strength Influence Transvectionmentioning

confidence: 99%

The Capacity to Act in Trans Varies Among Drosophila Enhancers

Blick

Mayer-Hirshfeld

Malibiran³

et al. 2016

Genetics

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The interphase nucleus is organized such that genomic segments interact in cis, on the same chromosome, and in trans, between different chromosomes. In Drosophila and other Dipterans, extensive interactions are observed between homologous chromosomes, which can permit enhancers and promoters to communicate in trans. Enhancer action in trans has been observed for a handful of genes in Drosophila, but it is as yet unclear whether this is a general property of all enhancers or specific to a few. Here, we test a collection of well-characterized enhancers for the capacity to act in trans. Specifically, we tested 18 enhancers that are active in either the eye or wing disc of third instar Drosophila larvae and, using two different assays, found evidence that each enhancer can act in trans. However, the degree to which trans-action was supported varied greatly between enhancers. Quantitative analysis of enhancer activity supports a model wherein an enhancer's strength of transcriptional activation is a major determinant of its ability to act in trans, but that additional factors may also contribute to an enhancer's trans-activity. In sum, our data suggest that a capacity to activate a promoter on a paired chromosome is common among Drosophila enhancers.KEYWORDS RMCE; interchromosomal interactions; long-range enhancer; somatic homolog pairing; transvection T HE spatial organization of the interphase eukaryotic genome is characterized by extensive long-distance interactions between distal chromosome regions (Sanyal et al. 2012). Interactions have been identified between sequences on the same chromosome (in cis) or on different chromosomes (in trans) (Lieberman-Aiden et al. 2009;Duan et al. 2010;Sexton et al. 2012;van de Werken et al. 2012;Nagano et al. 2013;Zhang et al. 2013). Many long-distance interactions in cis underlie the activation of specific genes, and in some cases, sequences have been identified that facilitate interactions between a distal enhancer and a specific promoter target (Zhou and Levine 1999;Calhoun et al. 2002;Calhoun and Levine 2003;Lin 2003;Akbari et al. 2008;Fujioka et al. 2009;Majumder et al. 2015). In contrast, the genetic impacts of trans-interactions between chromosomes are less clearly understood. Examples of gene regulation involving interchromosomal associations have been described (Spilianakis et al. 2005;Bacher et al. 2006;Xu et al. 2006;Apostolou and Thanos 2008;Sandhu et al. 2009;Markenscoff-Papadimitriou et al. 2014;Patel et al. 2014), but it remains unclear whether it is common for sequences that regulate gene expression to communicate between different chromosomes when they are physically juxtaposed.In Drosophila melanogaster, extensive trans-interactions are observed between homologous chromosomes in virtually all somatic tissues, a phenomenon known as somatic homolog pairing (reviewed by McKee 2004;Bosco 2012). The close proximity of homologous chromosomes in Drosophila can permit an enhancer to act in trans on a promoter on the paired homolog, a form of pairing-dependent ...

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

Cited by 153 publications

References 104 publications

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Causes and consequences of nuclear gene positioning

The Capacity to Act in Trans Varies Among Drosophila Enhancers

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