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

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          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.

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          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 references22

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          Genome sequence of the nematode C. elegans: a platform for investigating biology.

          (1999)
          The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.
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            daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans.

            A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the long-lived dauer diapause stage. daf-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor. Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism, diapause, and longevity.
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              Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

              Introduction Somatic cells within an organism possess genomes that are, with only a few minor exceptions, identical. However, various cell types may possess different epigenomes including the variation of DNA methylation and histone modification patterns. Epigenome variability accounts for cell-type-specific gene expression and silencing patterns in multicellular organisms. The impact of higher-order nuclear architecture on these patterns is not yet known [1]. Studies of higher-order chromatin arrangements in numerous cell types from different species form an indispensable part of a comprehensive approach to understanding epigenome evolution and cell-type-specific variability. Numerous research groups have attempted to map the large-scale organization and distribution of chromatin in cycling and postmitotic cell types (for reviews see [2,3,4,5,6,7,8]). Reliable topological maps, however, for the three-dimensional (3D) and 4D (3D plus spatiotemporal) arrangements of the two haploid chromosome complements in a diploid somatic cell nucleus have been lacking so far. Such 3D and 4D maps would provide the necessary foundation for studying the effect of higher-order chromatin distribution on nuclear functions, and are needed for different cell types at various stages of the cell cycle and at various stages of terminal differentiation. In addition to their importance for epigenome research, these maps should also help to understand karyotype evolution [9,10,11,12] and the formation of chromosomal rearrangements in irradiated or cancer cells [13,14,15,16,17]. In a 2D analysis of human fibroblast prometaphase rosettes, Nagele et al. [18,19] measured distances and angular separations for a number of chromosomes. These authors concluded that the maternal and paternal chromosome sets were separate, and that the heterologous chromosomes in each set showed highly nonrandom distributions. Subsequent studies further emphasized a highly ordered chromosome territory (CT) pattern for the nuclei of polarized human bronchial epithelial cells [20] and for nuclei of quiescent (G0) diploid (46, XY) human fibroblasts in culture [21]. Koss [20] reported that angles between the center of the nucleus and homologous pairs of Chromosome 1, 7, and X CTs were nearly identical in about two-thirds of bronchial epithelial cell nuclei to the angles reported by Nagele et al. for the same chromosome pairs in fibroblast prometaphase rosettes [18]. In contrast, Allison and Nestor [22] found a relatively random array of chromosomes on the mitotic ring of prometaphase and anaphase cells in cultured human diploid fibroblasts, diploid cells from human lung tissue, and human lymphocytes. The causes of these discrepancies have so far remained elusive. For nuclei of human lymphocytes, phytohemagglutinin-stimulated lymphoblasts, and lymphoblastoid cell lines, several groups have consistently reported a preferential positioning of gene-rich CTs (e.g., Homo sapiens chromosome [HSA] 19) towards the center of the nucleus, and of gene-poor CTs (e.g., HSAs 18 and Y) towards the nuclear periphery [23,24,25,26]. We recently confirmed this gene-density-correlated radial CT positioning for several other normal and malignant human cell types [26]. Bickmore and colleagues [23,27] also reported gene-density-correlated CT arrangements for cycling human fibroblasts. In contrast, Sun et al. [28] and our group [23,24,25,26] provided support for chromosome-size-correlated radial arrangements in quiescent fibroblasts. Although Sun et al. refer to nuclei studied in the G1-phase of the cell cycle, we believe that most of the cells included in their analysis were in a quiescent state (G0), since fibroblasts were grown on coverslips to 90%–95% confluence. Bridger et al. [27] reported that Chromosome 18 CTs were significantly closer to the nuclear periphery in S-phase fibroblasts than in quiescent fibroblasts. These findings suggest that cycling and noncycling fibroblasts differ in higher-order chromatin organization. We tested this hypothesis further in the present study. To overcome some of the technical limitations of previous studies, and to explore some of their inconsistencies, we employed 3D fluorescence in situ hybridization (FISH) protocols that allowed the differential coloring of all 24 chromosome types (22 autosomes plus X and Y) simultaneously within a population of human male fibroblasts (46, XY) under conditions preserving the 3D nuclear shape and structure to the highest possible degree [29,30]. In addition, we performed a series of two-color 3D FISH experiments in semi-confluent cultures, and determined the radial 3D positions of a subset of CTs (HSAs 1, 17–20, and Y) in quiescent (G0) and cycling (early S-phase) fibroblasts. Our data demonstrate unequivocally that the 3D arrangements of chromosomes in quiescent and cycling human fibroblasts follow probabilistic rules, and suggest that nuclear functions in human fibroblasts do not require a deterministic neighborhood pattern of homologous and heterologous chromosomes. Throughout, when we use the term “probabilistic chromosome order,” we mean an order that cannot be explained simply as a consequence of geometrical constraints that affect the distribution of chromosomes in mitotic rosettes or of CTs in cell nuclei. Constraints may enforce an arrangement of large and small chromosomes or CTs that deviates significantly from the prediction of a random order of points without any functional implications. Our long-term goal is to contribute to the elucidation of the set of rules (most likely a combination of probabilistic and deterministic) that generate cell-type-specific, functionally relevant higher-order chromatin arrangements. Results Differential Coloring of All 24 Chromosome Types in Nuclei of Human Male Diploid Fibroblasts Early-passage human fibroblast cultures (46, XY) were grown to confluence and maintained at this stage for several days before being fixed with buffered 4% paraformaldehyde. Under these conditions, the overwhelming majority (>99%) of cells were postmitotic (G0), as demonstrated by a lack of both pKi67 staining and incorporation of thymidine analogs (data not shown). Two 3D multiplex FISH (M-FISH) protocols were used for the differential coloring of all 24 human chromosome types (22 autosomes plus X and Y). The first approach was based on 3D M-FISH with 24 chromosome paint probes. Probes were differentially labeled using a combinatorial labeling scheme with seven different haptens/fluorochromes [31]. DAPI was used to stain nuclear DNA. Light-optical serial sections were separately recorded for each fluorochrome using digital wide-field epifluorescence microscopy (Figure 1). A second approach, called ReFISH [32], achieved differential staining of all 24 human chromosome types in two sequential FISH experiments with triple-labeled probe subsets. Light-optical serial sectioning of the same nuclei with laser confocal microscopy was performed after both the first and the second hybridization. Both approaches provided stringent accuracy for color classification of all CTs, and yielded the same results. Therefore, we combined data from 31 nuclei studied with the first approach and from 23 nuclei studied with the second approach (54 nuclei in total). Following careful correction for chromatic shifts, and image deconvolution in the case of wide-field microscopy (Figure S1), we performed overlays of the corresponding light-optical sections from all channels with voxel accuracy. CT classification was carried out on these overlays by the computer program goldFISH [33] (Figures 1B and S1C). This program classifies chromosomes by virtue of differences in the combinatorial fluorescent labeling schemes. Figure 1C shows the 3D reconstruction of a nucleus with all CTs viewed from different angles. Although the present experiments were not designed to address the issue of chromatin intermingling from neighboring CTs, it is obvious that goldFISH should have led to numerous misclassifications if there were excessive, widespread intermingling (for further discussion of CT boundaries, see [34]). For each individual CT the classification achieved by goldFISH was confirmed or rejected by careful visual inspection of light-optical sections. Any CT that could not be classified with certainty was omitted from further consideration. We were thus able to identify 2,030 CTs (82%) from a total of 2,484 CTs present in the 54 diploid fibroblast nuclei. As reference points for all distance and angle measurements reported below, we determined the 3D location of the fluorescence intensity gravity centers (IGCs) of individual painted CTs and the IGC of the nucleus (CN). Unless stated otherwise, when we describe below the position of a CT or prometaphase chromosome (PC) and report distance and angle measurements, we are referring to the 3D position of the CT's or PC's IGC. As a control for the reliability of the CT localizations, we subjected nuclei first studied by 24-color 3D FISH to a sequential five-color FISH experiment with individually labeled paint probes for Chromosomes 1 (Cy5), 3 (Cy3), 10 (FITC), 12 (Cy3.5), and 20 (Cy5.5). We were able to retrieve 11 of the 31 originally studied nuclei and to determine whether 3D positions of CTs first classified in the 24-color 3D FISH experiment could be confirmed after the second hybridization. In 96% of the re-hybridized CTs, the 3D position of the IGC differed by less than 1 μm, the range being between 0.01 and 1.3 μm. Size-Correlated Radial CT Positions in Nuclei of Quiescent (G0) Fibroblasts For every identified CT we measured the 3D radial CN–CT distance (from the CN to the CT's IGC). For a graphic overview of the location of each CT in 2D nuclear projections, the 3D positions of all IGCs obtained for a given CT were normalized and drawn into an ellipse representing the nuclear rim (Figure 2). As representative examples, Figure 2A shows nuclear projections of the normalized 3D IGC locations of CTs of HSAs 1, 7, 11, 18, 19, and Y, while Figure 2B shows cumulative 3D CN–CT graphs for the same CTs. Figures S2 and S4 provide the respective data for the entire chromosome complement. Notably, 3D radial CN–CT distance measurements did not reveal a significant difference between the positions of the gene-poor HSA 18 and the gene-rich HSA 19, although distinctly peripheral and interior locations, respectively, have been found for these two chromosomes in the spherical nuclei of lymphocytes and several other cell types (see Introduction). In summary, our data (Figures 2B, S2, S4, and S7 [left panel]) demonstrate that the territories of all small chromosomes—independent of their gene density—were preferentially found close to the center of the nucleus, while the territories of large chromosomes were preferentially located towards the nuclear rim. Figure 3 displays the positive correlation obtained in quiescent human fibroblasts for the mean normalized radial CN–CT distances and the DNA content of the chromosomes. The broad variability of radial CT positions seen in the set of 54 G0 nuclei indicates that radial CT arrangements in quiescent fibroblasts follow probabilistic, not deterministic, rules. To visualize the relative average positions of the IGCs of all heterologous CTs, we generated multidimensional scaling (MDS) plots [35,36] based on the mean of all normalized 3D CT–CT distances (Figure 4). Consistent with the data shown in Figure 3A, we found CTs from small chromosomes preferentially clustering towards the center of the nucleus, while CTs from large chromosomes were preferentially located towards the periphery. The acrocentric chromosomes (13–15, 21, and 22) carry nucleolar organizer regions (NORs) on their short arms, and active NORs are associated with the nucleoli. Since nucleoli are generally located away from the nuclear envelope in the inner nuclear space, we expected that normalized 3D CN–CT distances for all acrocentric chromosomes should be significantly shorter on average than 3D CN–CT distances for the largest chromosomes. Figure 5 confirms this expectation in the sample of 54 3D evaluated nuclei, emphasizing the sensitivity of the IGC approach. We also found a highly significant difference (p 0.05; Mann-Whitney U-test [U-test]). In contrast, the gene-poor Y territory was slightly more shifted towards the nuclear interior than the gene-rich HSA 17 CTs (Figure 6B and 6E). This shift was significant for cycling fibroblasts (p 0.05; U-test), but located significantly closer to the nuclear center than expected in the case of a uniform radial distribution (p 0.05; one-tailed K-S test of goodness of fit). With few exceptions pairwise comparisons of the mean angular separation between a pair of homologous CTs with the respective mean angle distribution in 60 random point distribution model nuclei did not show a significant difference (p > 0.05; two-tailed K-S test). Significant differences (p 0.05; two-tailed K-S test). (426 KB JPG). Click here for additional data file. Figure S11 Significance Levels for Pairwise Comparisons between Heterologous 3D CT–CN–CT Angles in 54 G0 Fibroblast Nuclei Significance levels were determined by the two-tailed K-S test. Green, not significant, p > 0.05; yellow, p 0.05; two-tailed K-S test). (328 KB JPG). Click here for additional data file. Video S1 Model Nucleus: CT Simulation The video shows the simulation of CT expansion in a fibroblast model nucleus according to the SCD model (compare with Figure 1). (567 KB MPG). Click here for additional data file.
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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
                : e42823
                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.

                Author information
                http://orcid.org/0000-0002-8410-9374
                http://orcid.org/0000-0001-9268-6596
                http://orcid.org/0000-0002-7974-8155
                Article
                42823
                10.7554/eLife.42823
                6516958
                31084706
                eece8460-d3e6-44ce-a7f1-1baf5ca63cbe
                © 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.

                History
                : 14 October 2018
                : 02 April 2019
                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
                dna fish,genome architecture,oligopaint,c. elegans
                Life sciences
                dna fish, genome architecture, oligopaint, c. elegans

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