Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Methods for mapping 3D chromosome architecture

Abstract

Determining how chromosomes are positioned and folded within the nucleus is critical to understanding the role of chromatin topology in gene regulation. Several methods are available for studying chromosome architecture, each with different strengths and limitations. Established imaging approaches and proximity ligation-based chromosome conformation capture (3C) techniques (such as DNA-FISH and Hi-C, respectively) have revealed the existence of chromosome territories, functional nuclear landmarks (such as splicing speckles and the nuclear lamina) and topologically associating domains. Improvements to these methods and the recent development of ligation-free approaches, including GAM, SPRITE and ChIA-Drop, are now helping to uncover new aspects of 3D genome topology that confirm the nucleus to be a complex, highly organized organelle.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Methods for studying the major features of 3D chromatin folding across different genomic scales.
Fig. 2: Imaging-based approaches to visualize chromatin contacts.
Fig. 3: 3C and its derivatives.
Fig. 4: Ligation-free methods to map chromatin contacts genome-wide.
Fig. 5: Comparison of long-range chromatin contacts across methods.

References

  1. Pombo, A. & Dillon, N. Three-dimensional genome architecture: players and mechanisms. Nat. Rev. Mol. Cell Biol. 16, 245–257 (2015).

    CAS  PubMed  Article  Google Scholar 

  2. Dekker, J. et al. The 4D nucleome project. Nature 549, 219–226 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Spielmann, M., Lupiáñez, D. G. & Mundlos, S. Structural variation in the 3D genome. Nat. Rev. Genet. 19, 453–467 (2018). This review explains the influence of disrupted chromatin folding on gene regulation in disease with examples from developmental disorders.

    CAS  PubMed  Article  Google Scholar 

  4. Krijger, P. H. & de Laat, W. Regulation of disease-associated gene expression in the 3D genome. Nat. Rev. Mol. Cell Biol. 17, 771–782 (2016).

    CAS  PubMed  Article  Google Scholar 

  5. Gall, J. G. & Pardue, M. L. Formation and detection of RNA–DNA hybrid molecules in cytological preparations. Proc. Natl Acad. Sci. USA 63, 378–383 (1969).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. Speicher, M. R., Gwyn Ballard, S. & Ward, D. C. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12, 368–375 (1996).

    CAS  PubMed  Article  Google Scholar 

  7. Wang, S. et al. Spatial organization of chromatin domains and compartments in single chromosomes. Science 353, 598–602 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Ma, H., Reyes-Gutierrez, P. & Pederson, T. Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors. Proc. Natl Acad. Sci. USA 110, 21048–21053 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. Ma, H. et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 34, 528–530 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Beagrie, R. A. et al. Complex multi-enhancer contacts captured by genome architecture mapping. Nature 543, 519–524 (2017). This study introduces GAM, a ligation-free technique to map chromatin contacts genome-wide. GAM confirms the presence of TADs and reveals multiway contacts between super-enhancers spanning tens of megabases.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Quinodoz, S. A. et al. Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell 174, 744–757.e24 (2018). This paper describes SPRITE, a ligation-free approach to map chromatin interactions and DNA–RNA contacts across the entire genome. SPRITE detects chromosomal hubs at nuclear bodies that bring together regions from different chromosomes.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Zheng, M. et al. Multiplex chromatin interactions with single-molecule precision. Nature 566, 558–562 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Cutter, A. R. & Hayes, J. J. A brief review of nucleosome structure. FEBS Lett. 589, 2914–2922 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Robinett, C. C. et al. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J. Cell Biol. 135, 1685–1700 (1996).

    CAS  PubMed  Article  Google Scholar 

  15. Belmont, A. S. & Straight, A. F. In vivo visualization of chromosomes using lac operator-repressor binding. Trends Cell Biol. 8, 121–124 (1998).

    CAS  PubMed  Article  Google Scholar 

  16. Lucas, J. S., Zhang, Y., Dudko, O. K. & Murre, C. 3D trajectories adopted by coding and regulatory DNA elements: first-passage times for genomic interactions. Cell 158, 339–352 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Germier, T., Sylvain, A., Silvia, K., David, L. & Kerstin, B. Real-time imaging of specific genomic loci in eukaryotic cells using the ANCHOR DNA labelling system. Methods 142, 16–23 (2018).

    PubMed  Article  CAS  Google Scholar 

  18. Barutcu, A. R., Maass, P. G., Lewandowski, J. P., Weiner, C. L. & Rinn, J. L. A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus. Nat. Commun. 9, 1444–1455 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  19. Barbieri, M. et al. Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 24, 515–524 (2017).

    CAS  PubMed  Article  Google Scholar 

  20. Maass, P. G., Barutcu, A. R., Weiner, C. L. & Rinn, J. L. Inter-chromosomal contact properties in live-cell imaging and in Hi-C. Mol. Cell 69, 1039–1045.e3 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Finn, E. H. et al. Extensive heterogeneity and intrinsic variation in spatial genome organization. Cell 176, 1502–1515.e10 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Boettiger, A. N. et al. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Luperchio, T. R. et al. Chromosome conformation paints reveal the role of lamina association in genome organization and regulation. Preprint at bioRxiv https://doi.org/10.1101/122226 (2017).

  24. Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385 (2012). This study uses 5C to identify the compartmentalization of chromosomes into TADs.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Branco, M. R. & Pombo, A. Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations. PLOS Biol. 4, e1380780–e1380788 (2006). This microscopy study of contacts between chromosomes uses cryo-FISH to show the previously underestimated extent of chromosome intermingling between chromosome territories.

    Article  CAS  Google Scholar 

  26. Ferrai, C. et al. Poised transcription factories prime silent uPA gene prior to activation. PLOS Biol. 8, e1000270 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. Simonis, M. et al. Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture–on-chip (4C). Nat. Genet. 38, 1348–1354 (2006).

    CAS  PubMed  Article  Google Scholar 

  28. Beliveau, B. J. et al. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl Acad. Sci. USA 109, 21301–21306 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A. & Bickmore, W. A. Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis. Chromosome Res. 19, 901–909 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Beliveau, B. J. et al. Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat. Commun. 6, 7147–7160 (2015).

    CAS  PubMed  Article  Google Scholar 

  31. Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Bintu, B. et al. Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362, eaau1783 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. Ni, Y. et al. Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes. eLife 6, 21660 (2017). This elegant study uses super-resolution microscopy and DNA-FISH to fine-map enhancer–promoter contacts.

    Article  Google Scholar 

  34. Stevens, T. J. et al. 3D structures of individual mammalian genomes studied by single-cell Hi-C. Nature 544, 59–64 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Gibcus, J. H. et al. A pathway for mitotic chromosome formation. Science 359, eaao6135 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  36. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Shao, S. et al. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res. 44, e86–e98 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. Fu, Y. et al. CRISPR–dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. Nat. Commun. 7, 11707–11714 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Wang, S., Su, J. H., Zhang, F. & Zhuang, X. An RNA-aptamer-based two-color CRISPR labeling system. Sci. Rep. 6, 26857–26863 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Gu, B. et al. Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements. Science 359, 1050–1055 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).

    CAS  Article  PubMed  Google Scholar 

  42. Zhao, Z. et al. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat. Genet. 38, 1341–1347 (2006).

    CAS  PubMed  Article  Google Scholar 

  43. Dostie, J. et al. Chromosome conformation capture carbon copy (5C): a massively parallel solution for mapping interactions between genomic elements. Genome Res. 16, 1299–1309 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009). This article describes Hi-C, a 3C-based approach to map chromatin contacts genome-wide using selection of ligated DNA fragments.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Denker, A. & de Laat, W. The second decade of 3C technologies: detailed insights into nuclear organization. Genes Dev. 30, 1357–1382 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. van de Werken, H. J. et al. Robust 4C-seq data analysis to screen for regulatory DNA interactions. Nat. Methods 9, 969–972 (2012).

    PubMed  Article  CAS  Google Scholar 

  47. Franke, M. et al. Formation of new chromatin domains determines pathogenicity of genomic duplications. Nature 538, 265–269 (2016).

    CAS  PubMed  Article  Google Scholar 

  48. Symmons, O. et al. The Shh topological domain facilitates the action of remote enhancers by reducing the effects of genomic distances. Dev. Cell 39, 529–543 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Loviglio, M. N. et al. Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes. Mol. Psychiatry 22, 836–849 (2017).

    CAS  PubMed  Article  Google Scholar 

  50. Kundu, S. et al. Polycomb repressive complex 1 generates discrete compacted domains that change during differentiation. Mol. Cell 65, 432–446.e5 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Hughes, J. R. et al. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment. Nat. Genet. 46, 205–212 (2014).

    CAS  PubMed  Article  Google Scholar 

  52. Mifsud, B. et al. Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat. Genet. 47, 598–606 (2015).

    CAS  PubMed  Article  Google Scholar 

  53. Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043.e19 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Andrey, G. et al. Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding. Genome Res. 27, 223–233 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Schoenfelder, S., Javierre, B. M., Furlan-Magaril, M., Wingett, S. W. & Fraser, P. Promoter capture Hi-C: high-resolution, genome-wide profiling of promoter interactions. J. Vis. Exp. 136, e57320 (2018).

    Google Scholar 

  56. Belton, J. M. et al. Hi-C: a comprehensive technique to capture the conformation of genomes. Methods 58, 268–276 (2012).

    CAS  PubMed  Article  Google Scholar 

  57. Kalhor, R., Tjong, H., Jayathilaka, N., Alber, F. & Chen, L. Genome architectures revealed by tethered chromosome conformation capture and population-based modeling. Nat. Biotechnol. 30, 90–98 (2011).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  58. Rodley, C. D. M., Bertels, F., Jones, B. & O’Sullivan, J. M. Global identification of yeast chromosome interactions using genome conformation capture. Fungal Genet. Biol. 46, 879–886 (2009). This paper describes GCC, a 3C-based approach to map chromatin contacts genome-wide without selection of ligated DNA fragments.

    CAS  PubMed  Article  Google Scholar 

  59. Servant, N., Varoquaux, N., Heard, E., Barillot, E. & Vert, J.-P. Effective normalization for copy number variation in Hi-C data. BMC Bioinformatics 19, 313–313 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  60. Dixon, J. R. et al. Integrative detection and analysis of structural variation in cancer genomes. Nat. Genet. 50, 1388–1398 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. Vidal, E. et al. OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes. Nucleic Acids Res. 46, e49–e58 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  62. Ma, W. et al. Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes. Nat. Methods 12, 71–78 (2015).

    PubMed  Article  CAS  Google Scholar 

  63. Ma, W. et al. Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution. Methods 142, 59–73 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). This article describes a high-resolution contact map of the human genome, which reveals the presence of loop domains and introduces the concept of loop formation between divergent CTCF sites.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Hsieh, T.-Han S. et al. Mapping nucleosome resolution chromosome folding in yeast by micro-C. Cell 162, 108–119 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. Hsieh, T. S., Fudenberg, G., Goloborodko, A. & Rando, O. J. Micro-C XL: assaying chromosome conformation from the nucleosome to the entire genome. Nat. Methods 13, 1009–1011 (2016).

    CAS  PubMed  Article  Google Scholar 

  67. Hsieh, T.-H. S. et al. Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Preprint at bioRxiv https://doi.org/10.1101/638775 (2019).

  68. Li, T., Jia, L., Cao, Y., Chen, Q. & Li, C. OCEAN-C: mapping hubs of open chromatin interactions across the genome reveals gene regulatory networks. Genome Biol. 19, 54–68 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  69. Gavrilov, A. A. et al. Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub. Nucleic Acids Res. 41, 3563–3575 (2013). This paper presents electron microscopy and confocal microscopy images showing the changes that occur in chromatin ultrastructure during a proximity-ligation assay.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. Lu, L., Liu, X., Peng, J., Li, Y. & Jin, F. Easy Hi-C: a simple efficient protocol for 3D genome mapping in small cell populations. Preprint at bioRxiv https://doi.org/10.1101/245688 (2018).

  71. Nagano, T. et al. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell. Nat. Protoc. 10, 1986–2003 (2015).

    CAS  PubMed  Article  Google Scholar 

  72. Nagano, T. et al. Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature 502, 59–64 (2013).

    CAS  PubMed  Article  Google Scholar 

  73. Flyamer, I. M. et al. Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition. Nature 544, 110–114 (2017). This study uses single-cell Hi-C and DNA-FISH to study differences in 3D genome folding between oocytes and zygotes, and reveals the absence of chromatin compartments in the oocyte, as well as single-cell heterogeneity in TAD organization.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. Nagano, T. et al. Cell-cycle dynamics of chromosomal organization at single-cell resolution. Nature 547, 61–67 (2017). This paper describes the use of single-cell Hi-C to map chromatin contacts throughout the cell cycle in thousands of individual cells, which shows the emergence of chromatin structures after mitosis and the temporal dynamics of different chromatin topologies.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  75. Ramani, V. et al. Massively multiplex single-cell Hi-C. Nat. Methods 14, 263–266 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  76. Tan, L., Xing, D., Chang, C.-H., Li, H. & Xie, X. S. Three-dimensional genome structures of single diploid human cells. Science 361, 924–928 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  77. Horike, S., Cai, S., Miyano, M., Cheng, J. F. & Kohwi-Shigematsu, T. Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat. Genet. 37, 31–40 (2005).

    CAS  PubMed  Article  Google Scholar 

  78. Schoenfelder, S. et al. Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat. Genet. 42, 53–61 (2010).

    CAS  PubMed  Article  Google Scholar 

  79. Fullwood, M. J. et al. An oestrogen-receptor-α-bound human chromatin interactome. Nature 462, 58–64 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  80. Mumbach, M. R. et al. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat. Methods 13, 919–922 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  81. Fang, R. et al. Mapping of long-range chromatin interactions by proximity ligation-assisted ChIP-seq. Cell Res. 26, 1345–1348 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  82. Davies, J. O., Oudelaar, A. M., Higgs, D. R. & Hughes, J. R. How best to identify chromosomal interactions: a comparison of approaches. Nat. Methods 14, 125–134 (2017).

    CAS  PubMed  Article  Google Scholar 

  83. Zhang, Y. et al. Enhancing Hi-C data resolution with deep convolutional neural network HiCPlus. Nat. Commun. 9, 750–758 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  84. O’Sullivan, J. M., Hendy, M. D., Pichugina, T., Wake, G. C. & Langowski, J. The statistical-mechanics of chromosome conformation capture. Nucleus 4, 390–398 (2013).

    PubMed  PubMed Central  Article  Google Scholar 

  85. Chen, H. et al. Dynamic interplay between enhancer–promoter topology and gene activity. Nat. Genet. 50, 1296–1303 (2018). In this study, live-cell imaging shows that transcription directly depends on contact between enhancers and promoters.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  86. van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase. Nat. Biotechnol. 18, 424–428 (2000).

    PubMed  Article  CAS  Google Scholar 

  87. Vogel, M. J., Peric-Hupkes, D. & van Steensel, B. Detection of in vivo protein–DNA interactions using DamID in mammalian cells. Nat. Protoc. 2, 1467–1478 (2007).

    CAS  PubMed  Article  Google Scholar 

  88. Marshall, O. J., Southall, T. D., Cheetham, S. W. & Brand, A. H. Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing. Nat. Protoc. 11, 1586–1598 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  89. Peric-Hupkes, D. et al. Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol. Cell 38, 603–613 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. Guelen, L. et al. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453, 948–951 (2008). This article describes DamID, an elegant approach to map chromatin contacts at the nuclear lamina.

    CAS  PubMed  Article  Google Scholar 

  91. Spector, D. L. & Lamond, A. I. Nuclear speckles. Cold Spring Harb. Perspect. Biol. 3, a000646 (2011).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  92. Chen, Y. et al. Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler. J. Cell Biol. 270, 4025–4048 (2018). This article introduces TSA-seq, a genome-wide sequencing technique, to map spatial distances between DNA and nuclear bodies, such as splicing speckles.

    Article  CAS  Google Scholar 

  93. Redolfi, J. et al. DamC reveals principles of chromatin folding in vivo without crosslinking and ligation. Nat. Struct. Mol. Biol. 26, 471–480 (2019). This article introduces DamC, an approach that allows mapping of chromatin contacts in vivo and without crosslinking and ligation.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  94. Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012). In this article, Hi-C experiments reveal the compartmentalization of chromosomes into TADs.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  95. Pombo, A. & Branco, M. R. Functional organisation of the genome during interphase. Curr. Opin. Genet. Dev. 17, 451–455 (2007).

    CAS  PubMed  Article  Google Scholar 

  96. Li, Y. et al. The effects of chemical fixation on the cellular nanostructure. Exp. Cell Res. 358, 253–259 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  97. Oudelaar, A. M., Davies, J. O. J., Downes, D. J., Higgs, D. R. & Hughes, J. R. Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C. Nucleic Acids Res. 45, e184–e192 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  98. Guillot, P. V., Xie, S. Q., Hollinshead, M. & Pombo, A. Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells. Exp. Cell Res. 295, 460–468 (2004).

    CAS  PubMed  Article  Google Scholar 

  99. Solovei, I. et al. Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH). Exp. Cell Res. 276, 10–23 (2002).

    CAS  PubMed  Article  Google Scholar 

  100. Markaki, Y. et al. The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture. Bioessays 34, 412–426 (2012).

    PubMed  Article  Google Scholar 

  101. Xie, S. Q., Lavitas, L. M. & Pombo, A. CryoFISH: fluorescence in situ hybridization on ultrathin cryosections. Methods Mol. Biol. 659, 219–230 (2010).

    CAS  PubMed  Article  Google Scholar 

  102. Brown, J. M. et al. A tissue-specific self-interacting chromatin domain forms independently of enhancer–promoter interactions. Nat. Commun. 9, 3849–3863 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  103. Allahyar, A. et al. Enhancer hubs and loop collisions identified from single-allele topologies. Nat. Genet. 50, 1151–1160 (2018).

    CAS  PubMed  Article  Google Scholar 

  104. Olivares-Chauvet, P. et al. Capturing pairwise and multi-way chromosomal conformations using chromosomal walks. Nature 540, 296–300 (2016).

    CAS  PubMed  Article  Google Scholar 

  105. Darrow, E. M. et al. Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture. Proc. Natl Acad. Sci. USA 113, E4504–E4512 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  106. Oudelaar, A. M. et al. Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains. Nat. Genet. 50, 1744–1751 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  107. Branco, M. R., Branco, T., Ramirez, F. & Pombo, A. Changes in chromosome organization during PHA-activation of resting human lymphocytes measured by cryo-FISH. Chromosome Res. 16, 413–426 (2008).

    CAS  PubMed  Article  Google Scholar 

  108. Rosin, L. F., Nguyen, S. C. & Joyce, E. F. Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei. PLOS Genet. 14, e1007393 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  109. Loviglio, M. N. et al. Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes. Mol. Psychiatry 22, 836–849 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  110. Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R. & Flavell, R. A. Interchromosomal associations between alternatively expressed loci. Nature 435, 637–645 (2005).

    CAS  PubMed  Article  Google Scholar 

  111. Monahan, K., Horta, A. & Lomvardas, S. LHX2- and LDB1-mediated trans interactions regulate olfactory receptor choice. Nature 565, 448–453 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  112. Hakim, O. et al. Diverse gene reprogramming events occur in the same spatial clusters of distal regulatory elements. Genome Res. 21, 697–706 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  113. Giorgetti, L. & Heard, E. Closing the loop: 3C versus DNA FISH. Genome Biol. 17, 215–223 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  114. Tang, Z. et al. CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription. Cell 163, 1611–1627 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  115. Wang, X. T., Dong, P. F., Zhang, H. Y. & Peng, C. Structural heterogeneity and functional diversity of topologically associating domains in mammalian genomes. Nucleic Acids Res. 43, 7237–7246 (2015). This paper describes a chromosome-wide effort to map TADs using DNA-FISH. In addition to revealing the single-cell behaviour of TADs, the paper shows that the imaging data have a high correlation with Hi-C data.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  116. Fudenberg, G. & Imakaev, M. FISH-ing for captured contacts: towards reconciling FISH and 3C. Nat. Methods 14, 673–678 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  117. Rao, S. S. P. et al. Cohesin loss eliminates all loop domains. Cell 171, 305–320.e24 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  118. Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972 (2016).

    CAS  PubMed  Article  Google Scholar 

  119. Aitchison, J. D. & Rout, M. P. The interactome challenge. J. Cell Biol. 211, 729 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  120. Cremer, T. & Cremer, M. Chromosome territories. Cold Spring Harb. Perspect. Biol. 2, a003889 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  121. Parada, L. & Misteli, T. Chromosome positioning in the interphase nucleus. Trends Cell Biol. 12, 425–432 (2002).

    CAS  PubMed  Article  Google Scholar 

  122. Hacisuleyman, E. et al. Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat. Struct. Mol. Biol. 21, 198–206 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  123. Maass, P. G. et al. Reorganization of inter-chromosomal interactions in the 2q37-deletion syndrome. EMBO J. 37, e96257 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  124. Maharana, S. et al. Chromosome intermingling — the physical basis of chromosome organization in differentiated cells. Nucleic Acids Res. 44, 5148–5160 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  125. Zhang, Y. et al. Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell 148, 908–921 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  126. Lomvardas, S. et al. Interchromosomal interactions and olfactory receptor choice. Cell 126, 403–413 (2006).

    CAS  PubMed  Article  Google Scholar 

  127. Cairns, J. et al. CHiCAGO: robust detection of DNA looping interactions in capture Hi-C data. Genome Biol. 17, 127–143 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  128. Fanucchi, S., Shibayama, Y., Burd, S., Weinberg, M. S. & Mhlanga, M. M. Chromosomal contact permits transcription between coregulated genes. Cell 155, 606–620 (2013).

    CAS  PubMed  Article  Google Scholar 

  129. Jackson, D. A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  130. Zink, D., Bornfleth, H., Visser, A., Cremer, C. & Cremer, T. Organization of early and late replicating DNA in human chromosome territories. Exp. Cell Res. 247, 176–188 (1999).

    CAS  PubMed  Article  Google Scholar 

  131. Visser, A. E. et al. Spatial distributions of early and late replicating chromatin in interphase chromosome territories. Exp. Cell Res. 243, 398–407 (1998).

    CAS  PubMed  Article  Google Scholar 

  132. Ferreira, J., Paolella, G., Ramos, C. & Lamond, A. I. Spatial organization of large-scale chromatin domains in the nucleus: a magnified view of single chromosome territories. J. Cell Biol. 139, 1597–1610 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  133. Sadoni, N. et al. Nuclear organization of mammalian genomes. J. Cell Biol. 146, 1211–1226 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  134. Hiratani, I. et al. Global reorganization of replication domains during embryonic stem cell differentiation. PLOS Biol. 6, e245 (2008).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  135. Schwaiger, M. et al. Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome. Genes. Dev. 23, 589–601 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  136. Pope, B. D. et al. Topologically associating domains are stable units of replication-timing regulation. Nature 515, 402–405 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  137. Monneron, A. & Bernhard, W. Fine structural organization of the interphase nucleus in some mammalian cells. J. Ultrastruct. Res. 27, 266–288 (1969).

    CAS  PubMed  Article  Google Scholar 

  138. Verschure, P. J., van der Kraan, I., Manders, E. M. M. & van Driel, R. Spatial relationship between transcription sites and chromosome territories. J. Cell Biol. 147, 13–24 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  139. Dundr, M. & Misteli, T. Biogenesis of nuclear bodies. Cold Spring Harb. Perspect. Biol. 2, a000711 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  140. Mao, Y. S., Zhang, B. & Spector, D. L. Biogenesis and function of nuclear bodies. Trends Genet. 27, 295–306 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  141. Pederson, T. The nucleolus. Cold Spring Harb. Perspect. Biol. 3, a000638 (2011).

    PubMed  PubMed Central  Google Scholar 

  142. Shopland, L. S., Johnson, C. V., Byron, M., McNeil, J. & Lawrence, J. B. Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighborhoods. J. Cell Biol. 162, 981–990 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  143. Brown, J. M. et al. Association between active genes occurs at nuclear speckles and is modulated by chromatin environment. J. Cell Biol. 182, 1083–1097 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  144. Iborra, F. J., Pombo, A., Jackson, D. A. & Cook, P. R. Active RNA polymerases are localized within discrete transcription “factories’ in human nuclei. J. Cell Sci. 109, 1427–1436 (1996).

    CAS  PubMed  Google Scholar 

  145. Pombo, A. et al. Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III. EMBO J. 18, 2241–2253 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  146. Xie, S. Q., Martin, S., Guillot, P. V., Bentley, D. L. & Pombo, A. Splicing speckles are not reservoirs of RNA polymerase II, but contain an inactive form, phosphorylated on serine2 residues of the C-terminal domain. Mol. Biol. Cell 17, 1723–1733 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  147. Osborne, C. S. et al. Active genes dynamically colocalize to shared sites of ongoing transcription. Nat. Genet. 36, 1065–1071 (2004).

    CAS  PubMed  Article  Google Scholar 

  148. Osborne, C. S. & Eskiw, C. H. Where shall we meet? A role for genome organisation and nuclear sub-compartments in mediating interchromosomal interactions. J. Cell Biochem. 104, 1553–1561 (2008).

    CAS  PubMed  Article  Google Scholar 

  149. Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25, 833–840 (2018).

    CAS  PubMed  Article  Google Scholar 

  150. Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, aar3958 (2018).

    Article  CAS  Google Scholar 

  151. Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  152. Bantignies, F. et al. Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila. Cell 144, 214–226 (2011).

    CAS  PubMed  Article  Google Scholar 

  153. Tiwari, V. K., Cope, L., McGarvey, K. M., Ohm, J. E. & Baylin, S. B. A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations. Genome Res. 18, 1171–1179 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  154. Fraser, J. et al. Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation. Mol. Syst. Biol. 11, 852–865 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  155. Nemeth, A. et al. Initial genomics of the human nucleolus. PLOS Genet. 6, e1000889 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  156. Kumaran, R. I. & Spector, D. L. A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence. J. Cell Biol. 180, 51–65 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  157. Sabbattini, P., Georgiou, A., Sinclair, C. & Dillon, N. Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the λ5–VpreB1 locus control region. Mol. Cell Biol. 19, 671–679 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  158. Finlan, L. E. et al. Recruitment to the nuclear periphery can alter expression of genes in human cells. PLOS Genet. 4, e1000039 (2008). This article demonstrates that the nuclear environment influences gene expression; bringing genes into the repressive context of the nuclear lamina can lead to downregulation of their expression.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  159. Wang, H. et al. CRISPR-mediated programmable 3D genome positioning and nuclear organization. Cell 175, 1405–1417.e14 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  160. Grewal, S. I. & Elgin, S. C. Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399–406 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  161. Sexton, T. et al. Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148, 458–472 (2012).

    CAS  PubMed  Article  Google Scholar 

  162. Lupianez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  163. Hnisz, D. et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351, 1454–1458 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  164. Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  165. Symmons, O. et al. Functional and topological characteristics of mammalian regulatory domains. Genome Res. 24, 390–400 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  166. Jackson, D. A., Bartlett, J. & Cook, P. R. Sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in human cells: the role of transcription units. Nucleic Acids Res. 24, 1212–1219 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  167. de Wit, E. et al. CTCF binding polarity determines chromatin looping. Mol. Cell 60, 676–684 (2015).

    PubMed  Article  CAS  Google Scholar 

  168. Gomez-Marin, C. et al. Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders. Proc. Natl Acad. Sci. USA 112, 7542–7547 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  169. Vietri Rudan, M. et al. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep. 10, 1297–1309 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  170. Weinreb, C. & Raphael, B. J. Identification of hierarchical chromatin domains. Bioinformatics 32, 1601–1609 (2016).

    CAS  PubMed  Article  Google Scholar 

  171. Fudenberg, G. et al. Formation of chromosomal domains by loop extrusion. Cell Rep. 15, 2038–2049 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  172. Tolhuis, B., Palstra, R. J., Splinter, E., Grosveld, F. & de Laat, W. Looping and interaction between hypersensitive sites in the active β-globin locus. Mol. Cell 10, 1453–1465 (2002).

    CAS  PubMed  Article  Google Scholar 

  173. Lettice, L. A. A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum. Mol. Genet. 12, 1725–1735 (2003).

    CAS  PubMed  Article  Google Scholar 

  174. Nobrega, M. A., Ovcharenko, I., Afzal, V. & Rubin, E. M. Scanning human gene deserts for long-range enhancers. Science 302, 413–413 (2003).

    CAS  PubMed  Article  Google Scholar 

  175. Qin, Y. et al. Long-range activation of Sox9 in Odd Sex (Ods) mice. Hum. Mol. Genet. 13, 1213–1218 (2004).

    CAS  PubMed  Article  Google Scholar 

  176. Javierre, B. M. et al. Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167, 1369–1384.e19 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  177. Zullo, J. M. et al. DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina. Cell 149, 1474–1487 (2012).

    CAS  PubMed  Article  Google Scholar 

  178. Reddy, K. L., Zullo, J. M., Bertolino, E. & Singh, H. Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 452, 243–247 (2008).

    CAS  PubMed  Article  Google Scholar 

  179. Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  180. Strom, A. R. et al. Phase separation drives heterochromatin domain formation. Nature 547, 241–245 (2017). This article introduces phase separation as a model for heterochromatin formation in the nucleus by showing liquid–liquid phase separation of the heterochromatic protein HPa1 in vitro.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  181. Kim, S. et al. The dynamic three-dimensional organization of the diploid yeast genome. eLife 6, 23623–23645 (2017).

    Article  Google Scholar 

  182. Chetverina, D., Aoki, T., Erokhin, M., Georgiev, P. & Schedl, P. Making connections: insulators organize eukaryotic chromosomes into independent cis-regulatory networks. Bioessays 36, 163–172 (2014).

    CAS  PubMed  Article  Google Scholar 

  183. Schoenfelder, S. et al. The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements. Genome Res. 25, 582–597 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  184. Deng, W. et al. Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158, 849–860 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  185. Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  186. Engreitz, J. M. et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539, 452–455 (2016). This study shows that regulation of genes can occur though the promoters of nearby genes, highlighting the functional importance of transcription factories.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  187. Mayer, R. et al. Common themes and cell type specific variations of higher order chromatin arrangements in the mouse. BMC Cell Biol. 6, 44–65 (2005).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  188. Ahmed, K. et al. Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo. PLOS ONE 5, e10531 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  189. Li, Y. et al. CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells. PLOS ONE 9, e114485 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  190. Kerpedjiev, P. et al. HiGlass: web-based visual exploration and analysis of genome interaction maps. Genome Biol. 19, 125–136 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  191. Bonev, B. et al. Multiscale 3D genome rewiring during mouse neural development. Cell 171, 557–572.e24 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  192. Naumova, N., Smith, E. M., Zhan, Y. & Dekker, J. Analysis of long-range chromatin interactions using chromosome conformation capture. Methods 58, 192–203 (2012).

    CAS  PubMed  Article  Google Scholar 

  193. van de Werken, H. J. et al. 4C technology: protocols and data analysis. Methods Enzymol. 513, 89–112 (2012).

    PubMed  Article  CAS  Google Scholar 

  194. Schwartzman, O. et al. UMI-4C for quantitative and targeted chromosomal contact profiling. Nat. Methods 13, 685–691 (2016).

    CAS  PubMed  Article  Google Scholar 

  195. Dostie, J. & Dekker, J. Mapping networks of physical interactions between genomic elements using 5C technology. Nat. Protoc. 2, 988–1002 (2007).

    CAS  PubMed  Article  Google Scholar 

  196. Kim, J. H. et al. 5C-ID: increased resolution chromosome-conformation-capture-carbon-copy with in situ 3C and double alternating primer design. Methods 142, 39–46 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  197. Belaghzal, H., Dekker, J. & Gibcus, J. H. Hi-C 2.0: an optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation. Methods 123, 56–65 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  198. Li, X. et al. Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions. Nat. Protoc. 12, 899–915 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  199. Davies, J. O. et al. Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat. Methods 13, 74–80 (2016).

    CAS  PubMed  Article  Google Scholar 

  200. Croft, J. A. et al. Differences in the localization and morphology of chromosomes in the human nucleus. J. Cell Biol. 145, 1119–1131 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  201. Cremer, M. et al. Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol. Biol. 463, 205–239 (2008).

    CAS  PubMed  Article  Google Scholar 

  202. Chen, B. et al. Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci. Nucleic Acids Res. 44, e75–e87 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  203. Takei, Y., Shah, S., Harvey, S., Qi, L. S. & Cai, L. Multiplexed dynamic imaging of genomic loci by combined CRISPR imaging and DNA sequential FISH. Biophys. J. 112, 1773–1776 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  204. Shimizu, N., Maekawa, M., Asai, S. & Shimizu, Y. Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes. Chromosome Res. 23, 649–662 (2015).

    CAS  PubMed  Article  Google Scholar 

  205. Müller, S., Neusser, M. & Wienberg, J. Towards unlimited colors for fluorescence in-situhybridization (FISH). Chromosome Res. 10, 223–232 (2002).

    PubMed  Article  Google Scholar 

  206. Hepperger, C., Otten, S., von Hase, J. & Dietzel, S. Preservation of large-scale chromatin structure in FISH experiments. Chromosoma 116, 117–133 (2007).

    CAS  PubMed  Article  Google Scholar 

  207. Volpi, E. V. et al. Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. J. Cell Sci. 113, 1565–1576 (2000).

    CAS  PubMed  Google Scholar 

  208. Williams, R. R., Broad, S., Sheer, D. & Ragoussis, J. Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei. Exp. Cell Res. 272, 163–175 (2002).

    CAS  PubMed  Article  Google Scholar 

  209. Chambeyron, S. & Bickmore, W. A. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription. Genes Dev. 18, 1119–1130 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  210. Raap, A. K., Marijnen, J. G., Vrolijk, J. & van der Ploeg, M. Denaturation, renaturation, and loss of DNA during in situ hybridization procedures. Cytometry 7, 235–242 (1986).

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

The authors thank the Helmholtz Association (Germany) for support, C. Thieme (our laboratory) for help plotting GAM and SPRITE contact maps in Figs 1 and 5a,b, and S. Quinodoz (M. Guttman laboratory) for sharing SPRITE contact cluster data (Figs 1 and 5b). A.P. acknowledges support from the National Institutes of Health Common Fund 4D Nucleome Program grant U54DK107977. The authors apologize to the many scientists whose studies were not discussed in our review due to length constraints.

Author information

Authors and Affiliations

Authors

Contributions

All authors contributed to all aspects of the article.

Corresponding authors

Correspondence to Rieke Kempfer or Ana Pombo.

Ethics declarations

Competing interests

A.P. has filed a patent application on GAM: Pombo, A., Edwards, P. A. W., Nicodemi, M., Beagrie, R. A. & Scialdone, A. Patent application on ‘Genome Architecture Mapping’. Patent PCT/EP2015/079413 (2015).

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Chromosome territories

The nuclear volumes occupied by each specific chromosome. Chromosomes tend to interact predominantly within themselves and occupy distinct regions within the interphase nucleus.

Chromosomal compartments

Chromosomes fold into distinct subcompartments, which correlate with transcriptional activity (A compartment) or repression (B compartment). The A and B compartments are defined by Hi-C contact frequencies.

Topologically associating domains

(TADs). Chromosomal regions that fold into self-associating domains, with high internal interaction frequencies, demarcated by a clear drop of local interactions with neighbouring regions at their boundaries.

Chromatin loops

Local regions of high interaction frequency between two genomic loci indicate that these regions form the basis of a DNA loop. Loops often form between regions with divergent CCCTC-binding factor (CTCF) sites, or between enhancers and their target promoters.

CCCTC-binding factor

(CTCF). A transcription factor with 11 conserved zinc-finger (ZF) domains. This nuclear protein is able to use different combinations of the ZF domains to bind different DNA target sequences and proteins. CTCF is enriched at topologically associating domain (TAD) borders, where its binding can be important to specify TAD border definition.

Chromatin

The combination of DNA, RNA and protein that constitutes the chromosomes in eukaryotic cells. Broadly, heterochromatin is associated with transcriptional repression and euchromatin is associated with transcriptional activity.

Nuclear lamina

A protein mesh, consisting of lamins and other membrane-associated proteins, at the inner nuclear membrane that contributes to nuclear structure and function. Chromatin in the proximity of the lamina tends to be heterochromatic and transcriptionally repressed.

Fluorescence in situ hybridization

A technique that can be used to visualize the location of nucleic acid sequences within the nucleus using sequence-specific fluorescent probes that hybridize to the regions of interest, combined with microscopy.

Chromosome conformation capture

(3C). A technique used to detect the frequency of interactions between any specified two loci in the genome. Interactions between loci are captured by formaldehyde fixation, followed by restriction enzyme digestion and ligation. The frequencies of interactions between loci are determined by quantitative real-time PCR.

Hi-C

(High-throughput chromosome conformation capture). A genome-wide version of chromosome conformation capture that allows all chromatin interactions in the genome to be mapped simultaneously. The frequencies of interactions between loci are determined by paired end sequencing.

Proximity ligation

Fixation of cells, followed by fragmentation of chromatin and ligation of nearby, crosslinked DNA fragments.

Genome architecture mapping

(GAM). A genome-wide approach to detect chromatin contacts based on their physical distances within the nucleus. DNA loci are detected in thin nuclear slices by DNA extraction and sequencing. Chromatin contacts are inferred from co-segregation frequencies of pairs of DNA loci across a large (400–1,000) collection of nuclear slices.

Split-pool recognition of interactions by tag extension

(SPRITE). A ligation-free approach to detect chromatin interactions by tagging crosslinked chromatin complexes. The DNA (and RNA) molecules within an individual chromatin complex are identified after sequencing by their unique combination of barcodes that have been sequentially added using a split-pool strategy.

Chromatin immunoprecipitation

(ChIP). A method used to determine whether a given protein binds to, or is localized to, specific chromatin loci in vivo, detected after (native or crosslinked) chromatin purification and immunoprecipitation, followed by DNA detection by PCR, microarray hybridization or sequencing.

Genomic resolution

The size of the window (often in the range of kilobases) when, for most assays, reads after sequencing are mapped to the genome and then binned into equally sized genomic windows (bins).

Sequencing depth

The average number of reads representing a given nucleotide in the reconstructed sequence. A 10× sequence depth means that each nucleotide of the transcript was sequenced, on average, 10 times.

Nuclear bodies

Membrane-less compartments in the nucleus with high concentrations of DNA binding proteins, chromatin modifiers or RNAs that can be involved in shaping chromatin structure and modulating gene regulation. Nuclear bodies include the nucleolus, splicing speckles and Polycomb bodies.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kempfer, R., Pombo, A. Methods for mapping 3D chromosome architecture. Nat Rev Genet 21, 207–226 (2020). https://doi.org/10.1038/s41576-019-0195-2

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41576-019-0195-2

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing