Skip to main content
SpringerLink
Log in

Higher-Order Chromatin Organization Using 3D DNA Fluorescent In Situ Hybridization

  • Protocol
  • First Online:
Capturing Chromosome Conformation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2157))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bickmore WA (2013) The spatial organization of the human genome. Annu Rev Genomics Hum Genet 14:67–84. https://doi.org/10.1146/annurev-genom-091212-153515

    Article  CAS  PubMed  Google Scholar 

  2. Cremer T, Cremer M (2010) Chromosome territories. Cold Spring Harb Perspect Biol 2(3):a003889. https://doi.org/10.1101/cshperspect.a003889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326(5950):289–293. https://doi.org/10.1126/science.1181369

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485(7398):376–380. https://doi.org/10.1038/nature11082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hou C, Li L, Qin ZS, Corces VG (2012) Gene density, transcription, and insulators contribute to the partition of the Drosophila genome into physical domains. Mol Cell 48(3):471–484. https://doi.org/10.1016/j.molcel.2012.08.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot T, van Berkum NL, Meisig J, Sedat J, Gribnau J, Barillot E, Bluthgen N, Dekker J, Heard E (2012) Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485(7398):381–385. https://doi.org/10.1038/nature11049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sexton T, Yaffe E, Kenigsberg E, Bantignies F, Leblanc B, Hoichman M, Parrinello H, Tanay A, Cavalli G (2012) Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148:458–472. https://doi.org/10.1016/j.cell.2012.01.010

    Article  CAS  PubMed  Google Scholar 

  8. Bintu B, Mateo LJ, Su JH, Sinnott-Armstrong NA, Parker M, Kinrot S, Yamaya K, Boettiger AN, Zhuang X (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362(6413). https://doi.org/10.1126/science.aau1783

  9. Boettiger AN, Bintu B, Moffitt JR, Wang S, Beliveau BJ, Fudenberg G, Imakaev M, Mirny LA, Wu CT, Zhuang X (2016) Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529(7586):418–422. https://doi.org/10.1038/nature16496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cattoni DI, Cardozo Gizzi AM, Georgieva M, Di Stefano M, Valeri A, Chamousset D, Houbron C, Dejardin S, Fiche JB, Gonzalez I, Chang JM, Sexton T, Marti-Renom MA, Bantignies F, Cavalli G, Nollmann M (2017) Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions. Nat Commun 8(1):1753. https://doi.org/10.1038/s41467-017-01962-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fudenberg G, Imakaev M (2017) FISH-ing for captured contacts: towards reconciling FISH and 3C. Nat Methods 14(7):673–678. https://doi.org/10.1038/nmeth.4329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Szabo Q, Jost D, Chang JM, Cattoni DI, Papadopoulos GL, Bonev B, Sexton T, Gurgo J, Jacquier C, Nollmann M, Bantignies F, Cavalli G (2018) TADs are 3D structural units of higher-order chromosome organization in Drosophila. Sci Adv 4(2):eaar8082. https://doi.org/10.1126/sciadv.aar8082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang S, Su JH, Beliveau BJ, Bintu B, Moffitt JR, Wu CT, Zhuang X (2016) Spatial organization of chromatin domains and compartments in single chromosomes. Science 353(6299):598–602. https://doi.org/10.1126/science.aaf8084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Williamson I, Berlivet S, Eskeland R, Boyle S, Illingworth RS, Paquette D, Dostie J, Bickmore WA (2014) Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization. Genes Dev 28(24):2778–2791. https://doi.org/10.1101/gad.251694.114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nir G, Farabella I, Perez Estrada C, Ebeling CG, Beliveau BJ, Sasaki HM, Lee SD, Nguyen SC, McCole RB, Chattoraj S, Erceg J, AlHaj Abed J, Martins NMC, Nguyen HQ, Hannan MA, Russell S, Durand NC, Rao SSP, Kishi JY, Soler-Vila P, Di Pierro M, Onuchic JN, Callahan SP, Schreiner JM, Stuckey JA, Yin P, Aiden EL, Marti-Renom MA, Wu CT (2018) Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling. PLoS Genet 14(12):e1007872. https://doi.org/10.1371/journal.pgen.1007872

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Beliveau BJ, Boettiger AN, Avendano MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu CT (2015) Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 6:7147. https://doi.org/10.1038/ncomms8147

    Article  CAS  PubMed  Google Scholar 

  17. Beliveau BJ, Kishi JY, Nir G, Sasaki HM, Saka SK, Nguyen SC, Wu CT, Yin P (2018) OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes. Proc Natl Acad Sci U S A 115(10):E2183–E2192. https://doi.org/10.1073/pnas.1714530115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Demmerle J, Innocent C, North AJ, Ball G, Muller M, Miron E, Matsuda A, Dobbie IM, Markaki Y, Schermelleh L (2017) Strategic and practical guidelines for successful structured illumination microscopy. Nat Protoc 12(5):988–1010. https://doi.org/10.1038/nprot.2017.019

    Article  CAS  PubMed  Google Scholar 

  19. Ball G, Demmerle J, Kaufmann R, Davis I, Dobbie IM, Schermelleh L (2015) SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci Rep 5:15915. https://doi.org/10.1038/srep15915

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  21. Matsuda A, Schermelleh L, Hirano Y, Haraguchi T, Hiraoka Y (2018) Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy. Sci Rep 8(1):7583. https://doi.org/10.1038/s41598-018-25922-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Durand NC, Robinson JT, Shamim MS, Machol I, Mesirov JP, Lander ES, Aiden EL (2016) Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Syst 3(1):99–101. https://doi.org/10.1016/j.cels.2015.07.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Institute of Human Genetics, CNRS and University of Montpellier, Montpellier Cedex 5, France

    Quentin Szabo, Giacomo Cavalli & Frédéric Bantignies

Authors
  1. Quentin Szabo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Giacomo Cavalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frédéric Bantignies
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frédéric Bantignies .

Editor information

Editors and Affiliations

  1. Istituto Nazionale di Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy

    Beatrice Bodega

  2. Istituto Nazionale di Genetica Molecolare, Milan, Italy

    Chiara Lanzuolo

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

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

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0664-3_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0663-6

  • Online ISBN: 978-1-0716-0664-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation