Abstract
A comprehensive analysis of the tridimensional (3D) organization of the genome is crucial to understand gene regulation. Three-dimensional DNA fluorescent in situ hybridization (3D-FISH) is a method of choice to study nuclear organization at the single-cell level. The labeling of DNA loci of interest provides information on their spatial arrangement, such as their location within the nucleus or their relative positioning. The single-cell information of spatial positioning of genomic loci can thus be integrated with functional genomic and epigenomic features, such as gene activity, epigenetic states, or cell population averaged chromatin interaction profiles obtained using chromosome conformation capture methods. Moreover, the development of a diversity of super-resolution (SR) microscopy techniques now allows the study of structural chromatin properties at subdiffraction resolution, making a finer characterization of shapes and volumes possible, as well as allowing the analysis of quantitative intermingling of genomic regions of interest. Here, we present and describe a 3D-FISH protocol adapted for both conventional and SR microscopy such as 3D structured illumination microscopy (3D-SIM), which can be used for the measurement of 3D distances between loci and the analysis of higher-order chromatin structures in cultured Drosophila and mammalian cells.
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Acknowledgements
We thank Julio Mateos-Langerak and Thierry Cheutin for critical reading of the manuscript. We thank the Montpellier Resources Imagerie facility (BioCampus Montpellier, CNRS, INSERM, Université de Montpellier). Q.S. is supported by the French Ministry of Higher Education and Research and La Ligue Nationale Contre le Cancer. F.B. and G.C. are supported by the CNRS. Research at the G.C. laboratory is supported by grants from the CNRS, the European Research Council (2017-AdG No 788972, 3DEpi) the European Union’s Horizon 2020 research and innovation program under grant agreement no 676556 (MuG), the Agence Nationale de la Recherche (ANR-15-CE12-0006 EpiDevoMath), the Fondation pour la Recherche Médicale (DEI20151234396), the INSERM, the French National Cancer Institute (INCa), the MSDAVENIR Reseach fund (MSDAVENIR DS-2018-0041 GENE-IGH) and the Laboratory of Excellence EpiGenMed.
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Szabo, Q., Cavalli, G., Bantignies, F. (2021). Higher-Order Chromatin Organization Using 3D DNA Fluorescent In Situ Hybridization. In: Bodega, B., Lanzuolo, C. (eds) Capturing Chromosome Conformation. Methods in Molecular Biology, vol 2157. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0664-3_13
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DOI: https://doi.org/10.1007/978-1-0716-0664-3_13
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