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      A multiplexed DNA FISH strategy for assessing genome architecture in Caenorhabditis elegans

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Eukaryotic DNA is highly organized within nuclei and this organization is important for genome function. Fluorescent in situ hybridization (FISH) approaches allow 3D architectures of genomes to be visualized. Scalable FISH technologies, which can be applied to whole animals, are needed to help unravel how genomic architecture regulates, or is regulated by, gene expression during development, growth, reproduction, and aging. Here, we describe a multiplexed DNA FISH Oligopaint library that targets the entire Caenorhabditis elegans genome at chromosome, three megabase, and 500 kb scales. We describe a hybridization strategy that provides flexibility to DNA FISH experiments by coupling a single primary probe synthesis reaction to dye conjugated detection oligos via bridge oligos, eliminating the time and cost typically associated with labeling probe sets for individual experiments. The approach allows visualization of genome organization at varying scales in all/most cells across all stages of development in an intact animal model system.

          eLife digest

          DNA contains the instructions needed to build and maintain a living organism. How DNA is physically arranged inside a cell is not random, and DNA organization is important because it can affect, for example, which genes are active, and which are not.

          Researchers often use a technique called “fluorescence in situ hybridization” (or FISH for short) to study how DNA is organized in cells. FISH tethers fluorescent molecules to defined sections of DNA, making those sections glow under the right wavelength of light. It is possible to collect images of the fluorescent DNA regions under a microscope to see where they are in relation to each other and to the rest of the cell.

          Fields, Nguyen et al. have now created a new library of FISH molecules that can be used to analyze the DNA of a microscopic worm known as Caenorhabditis elegans – a model organism that is widely used to study genetics, animal development, and cell biology. The library can be used to visualize the worm’s whole genome at different scales. The library enables accurate and reliable investigations of how DNA is organized inside C. elegans, including in intact worms, meaning it also offers the first chance to study DNA organization in a whole organism through all stages of its life cycle.

          This new resource could help to reveal the relationships between DNA organization, cell specialization and gene activity in different cells at different stages of development. This could help to clarify the relationships between physical DNA organization and biological change. This design strategy behind this whole genome library should also be adaptable for similar studies in other animal species.

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          Most cited references 44

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          Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

          We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.
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            Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions

            The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories (CT), and numerous models have been proposed for how chromosomes fold within CTs 1 . These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid revolutions in the study of 3D genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide 2 . In the present study, we investigated the 3D organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term “topological domains”, as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, suggesting that topological domains are an inherent property of mammalian genomes. Lastly, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, tRNAs, and SINE retrotransposons, suggesting that these factors may play a role in establishing the topological domain structure of the genome.
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              A guided tour into subcellular colocalization analysis in light microscopy.

              It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                14 May 2019
                2019
                : 8
                Affiliations
                [1 ]deptLaboratory of Genetics University of Wisconsin-Madison MadisonUnited States
                [2 ]deptDepartment of Genetics Harvard Medical School BostonUnited States
                Stowers Institute for Medical Research United States
                Weill Cornell Medicine United States
                Stowers Institute for Medical Research United States
                University of California, Santa Cruz United States
                University of California, San Francisco United States
                Stowers Institute for Medical Research United States
                Author notes
                [‡]

                Department of Genetics, Penn Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

                [†]

                These authors contributed equally to this work.

                Article
                42823
                10.7554/eLife.42823
                6516958
                © 2019, Fields et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Product
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: RO1GM088289
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: 5DP1GM106412
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01HD091797
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01GM123289
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Tools and Resources
                Chromosomes and Gene Expression
                Custom metadata
                The design and validation of an oligo-based DNA FISH library that targets the entire Caenorhabditis elegans genome at chromosome, three megabase, and 500 kb scales.

                Life sciences

                c. elegans, oligopaint, genome architecture, dna fish

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