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.

  • Protocol
  • Published:

Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies

Nature Protocols volume 15, pages 364–397 (2020)Cite this article

Subjects

Abstract

We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromatin conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. By contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long-molecule sequencing on an Oxford Nanopore or Pacific Biosciences platform. Each concatemer of proximity ligation products represents a snapshot topology of a different individual allele, revealing its multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox is able to generate background models and three-way interaction profiles from the same dataset. These profiles can be used to distinguish whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously occurring at chromatin hubs. The entire procedure can be completed in 2 w, and requires standard molecular biology and data analysis skills and equipment, plus access to a third-generation sequencing platform.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic overview.
Fig. 2: Schematic overview of the MC-4C pipeline and its modules.
Fig. 3: Intermediate controls in the MC-4C protocol.
Fig. 4: Read-size distribution of an MC-4C experiment.
Fig. 5: Chromosome coverage of an MC-4C experiment.
Fig. 6: Sequence saturation plots.
Fig. 7: Read-size distribution of two MC-4C datasets.
Fig. 8: Chromosome coverage of two example MC-4C experiments.
Fig. 9: Read-size distribution of two MC-4C datasets.
Fig. 10: Overall profile of an MC-4C dataset.
Fig. 11: Quality check for sequencing saturation and UMI diversity.
Fig. 12: Viewpoint-SOI analysis for the Hbb-b1 gene and HS2 as an SOI.
Fig. 13: Association matrix of an MC-4C dataset.
Fig. 14: Association analysis across an ROI.

Similar content being viewed by others

Data availability

All raw sequencing data used in this study are available through the European Nucleotide Archive (https://www.ebi.ac.uk/ena/data/view/PRJEB23327). The processed data can be downloaded from the Mendeley Data repository (https://doi.org/10.17632/wbk8hk87r2.3). The test data used in the Walkthrough section of this protocol can be downloaded from github (https://github.com/deLaatLab/mc4c_test-data/archive/master.zip) and are included as Supplementary Data 2.

Code availability

The MC-4C pipeline (version 1.02, at the moment of protocol preparation), including all scripts used to produce figures in this protocol, is available under MIT License at github in mc4c_py repository (https://github.com/deLaatLab/mc4c_py) and is available directly as Supplementary Data 1. The data and results presented in this protocol were prepared using version 1.02 of the MC-4C pipeline.

References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Brant, L. et al. Exploiting native forces to capture chromosome conformation in mammalian cell nuclei. Mol. Syst. Biol. 12, 891 (2016).

    Article  Google Scholar 

  4. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).

    Article  CAS  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. Splinter, E., de Wit, E., van de Werken, H. J., Klous, P. & de Laat, W. Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation. Methods 58, 221–230 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Li, G. et al. ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol. 11, R22 (2010).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Fullwood, M. J. & Ruan, Y. ChIP-based methods for the identification of long-range chromatin interactions. J. Cell. Biochem. 107, 30–39 (2009).

    Article  CAS  Google Scholar 

  12. Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

    Article  CAS  Google Scholar 

  13. Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Bianco, S., Chiariello, A. M., Annunziatella, C., Esposito, A. & Nicodemi, M. Predicting chromatin architecture from models of polymer physics. Chromosome Res. 25, 25–34 (2017).

    Article  CAS  Google Scholar 

  17. Serra, F. et al. Automatic analysis and 3D-modelling of Hi-C data using TADbit reveals structural features of the fly chromatin colors. PLoS Comput. Biol. 13, e1005665 (2017).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. 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, E4504–12 (2016).

    Article  CAS  Google Scholar 

  20. Quinodoz, S. A. et al. Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell 174, 744–757.e24 (2018).

    Article  CAS  Google Scholar 

  21. Beagrie, R. A. et al. Complex multi-enhancer contacts captured by genome architecture mapping. Nature 543, 519–524 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  24. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  25. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  26. Koranne, S. Hierarchical data format 5: HDF5. in Handbook of Open Source Tools 191–200 (Springer, 2011).

  27. Walt, S., van der, van der Walt, S., Chris Colbert, S. & Varoquaux, G. The NumPy array: a structure for efficient numerical computation. Comput. Sci. Eng. 13, 22–30 (2011).

    Article  Google Scholar 

  28. McKinney, W. Python for Data Analysis: Data Wrangling with Pandas, NumPy, and IPython (‘O’Reilly Media, Inc.’, 2017).

  29. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

  30. Nakayama, T. et al. Cas9-based genome editing in Xenopus tropicalis. Methods Enzymol. 546, 355–375 (2014).

    Article  CAS  Google Scholar 

  31. Koh, C. M. Isolation of genomic DNA from mammalian cells. Methods Enzymol. 529, 161–169 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank E. Vos and E. deWit and other laboratory members for their input at various stages of the development of MC-4C. This work was supported by an NWO VIDI grant (639.072.715) to J.d.R and an NWO/CW TOP grant (714.012.002) and NWO VICI grant (724.012.003) to W.d.L., and by the NIH Common Fund Program, grant U01CA200147, as a Transformative Collaborative Project Award (TCPA; TCPA-2017-DE-LAAT).

Author information

Author notes

  1. These authors contributed equally: Carlo Vermeulen, Amin Allahyar.

Authors and Affiliations

  1. Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands

    Carlo Vermeulen, Amin Allahyar, Britta A. M. Bouwman, Peter H. L. Krijger, Marjon J. A. M. Verstegen, Geert Geeven, Christian Valdes-Quezada & Wouter de Laat

  2. Oncode Institute, Center for Molecular Medicine, University Medical Center, Utrecht University, Utrecht, the Netherlands

    Ivo Renkens, Roy Straver, Wigard P. Kloosterman & Jeroen de Ridder

Authors
  1. Carlo Vermeulen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amin Allahyar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Britta A. M. Bouwman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter H. L. Krijger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marjon J. A. M. Verstegen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Geert Geeven
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christian Valdes-Quezada
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ivo Renkens
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Roy Straver
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wigard P. Kloosterman
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jeroen de Ridder
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Wouter de Laat
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.V. and B.A.M.B. designed and performed experiments. C.V. and A.A. wrote manuscript and designed the figures. A.A. designed and performed the computational analysis, designed the figures and wrote the manuscript. P.H.L.K., M.J.A.M.V. and C.V.-Q. performed ‘C’ methods experiments. R.S. and A.A. implemented the pipeline in Python. I.J.R. performed and W.P.K. designed and supervised MinION sequencing experiments. G.G. helped with computational analysis. J.d.R. designed and supervised the computational analyses and pipelines and co-wrote the manuscript. W.d.L. conceived and supervised the study and wrote the manuscript.

Corresponding authors

Correspondence to Jeroen de Ridder or Wouter de Laat.

Ethics declarations

Competing interests

C.V., B.A.M.B., P.H.L.K., M.J.A.M.V. and G.G. are shareholders of Cergentis. W.d.L. is founder and shareholder of Cergentis. W.P.K. and J.d.R. are co-founders and shareholders of Cyclomics.

Additional information

Peer review information Nature Protocols thanks Jian Ma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Related link

Key references using this protocol

Allahyar, A. et al. Nat. Genet. 50, 1151–1160 (2018): https://doi.org/10.1038/s41588-018-0161-5

Integrated supplementary information

Supplementary Fig. 1 gRNA template.

5 μl of gRNA template PCR was loaded on a 2% agarose gel stained with ethidium bromide.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Manual

Reporting Summary

Supplementary Data 1

MC-4C pipeline

Supplementary Data 2

MC-4C test dataset

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vermeulen, C., Allahyar, A., Bouwman, B.A.M. et al. Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies. Nat Protoc 15, 364–397 (2020). https://doi.org/10.1038/s41596-019-0242-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0242-7

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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