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:

Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions

Nature Protocols volume 8, pages 509–524 (2013)Cite this article

Subjects

Abstract

Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1–8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

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

Figure 1: Overview of the multiplexed 3C-seq procedure.
Figure 2: Flowchart of multiplexed 3C-seq data generation and processing.
Figure 3: 3C-seq primer design and positioning.
Figure 4: Examples of successful digestion and ligation efficiencies.
Figure 5: Typical interaction profiles obtained from a multiplexed 3C-seq experiment.
Figure 6: Comparison of interactions detected for the same 3C-seq sample after single or multiplexed library sequencing.

Similar content being viewed by others

References

  1. 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 

  2. 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 

  3. Sanyal, A., Lajoie, B.R., Jain, G. & Dekker, J. The long-range interaction landscape of gene promoters. Nature 489, 109–113 (2012).

    Article  CAS  Google Scholar 

  4. Splinter, E. & de Laat, W. The complex transcription regulatory landscape of our genome: control in three dimensions. EMBO J. 30, 4345–4355 (2011).

    Article  CAS  Google Scholar 

  5. Bulger, M. & Groudine, M. Functional and mechanistic diversity of distal transcription enhancers. Cell 144, 327–339 (2011).

    Article  CAS  Google Scholar 

  6. Ong, C.T. & Corces, V.G. Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat. Rev. Genet. 12, 283–293 (2011).

    Article  CAS  Google Scholar 

  7. Stadhouders, R. et al. Transcription regulation by distal enhancers: who's in the loop? Transcription 3, 181–186 (2012).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Gondor, A., Rougier, C. & Ohlsson, R. High-resolution circular chromosome conformation capture assay. Nat. Protoc. 3, 303–313 (2008).

    Article  Google Scholar 

  10. Sexton, T. et al. Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip. Nat. Protoc. 7, 1335–1350 (2012).

    Article  CAS  Google Scholar 

  11. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. 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 

  16. Soler, E. et al. The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation. Genes Dev. 24, 277–289 (2010).

    Article  CAS  Google Scholar 

  17. Stadhouders, R. et al. Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development. EMBO J. 31, 986–999 (2012).

    Article  CAS  Google Scholar 

  18. Ribeiro de Almeida, C. et al. The DNA-binding protein CTCF limits proximal Vκ recombination and restricts κ enhancer interactions to the immunoglobulin κ light chain locus. Immunity 35, 501–513 (2011).

    Article  CAS  Google Scholar 

  19. Hagege, H. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat. Protoc. 2, 1722–1733 (2007).

    Article  CAS  Google Scholar 

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

  21. Ecker, J.R. et al. Genomics: ENCODE explained. Nature 489, 52–55 (2012).

    Article  CAS  Google Scholar 

  22. Dostie, J. & Bickmore, W.A. Chromosome organization in the nucleus—charting new territory across the Hi-Cs. Curr. Opin. Genet Dev. 22, 125–131 (2012).

    Article  CAS  Google Scholar 

  23. Comet, I., Schuettengruber, B., Sexton, T. & Cavalli, G. A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber. Proc. Natl. Acad. Sci. USA 108, 2294–2299 (2011).

    Article  CAS  Google Scholar 

  24. Jing, H. et al. Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus. Mol. Cell 29, 232–242 (2008).

    Article  CAS  Google Scholar 

  25. Rippe, K., von Hippel, P.H. & Langowski, J. Action at a distance: DNA-looping and initiation of transcription. Trends Biochem. Sci. 20, 500–506 (1995).

    Article  CAS  Google Scholar 

  26. Dekker, J. The three 'C' s of chromosome conformation capture: controls, controls, controls. Nat. Methods 3, 17–21 (2006).

    Article  CAS  Google Scholar 

  27. Brouwer, R.W., van den Hout, M.C., Grosveld, F.G. & van Ijcken, W.F. NARWHAL, a primary analysis pipeline for NGS data. Bioinformatics 28, 284–285 (2012).

    Article  CAS  Google Scholar 

  28. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  29. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Hakim, O. et al. DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature 484, 69–74 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Visser, M., Kayser, M. & Palstra, R.J. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter. Genome Res. 22, 446–455 (2012).

    Article  CAS  Google Scholar 

  34. Lin, Y.C. et al. Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat. Immunol. 13, 1196–1204 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. van der Sloot, Z. Ozgur, E. Oole, M. van den Hout, F. Sleutels, S.Thongjuea and B. Lenhard for their help in sample processing, bioinformatics pipeline development and data analysis. R.S. received support from the Royal Netherlands Academy of Arts and Sciences (KNAW). P.K. was supported by grants from ERASysBio+/FP7 (project no. 93511024). E.S. was supported by grants from the Dutch Cancer Genomics Center, the Netherlands Genomics Initiative (project no. 40-41009-98-9082) and the French Alternative Energies and Atomic Energy Commission (CEA). This work was supported by the EU-FP7 Eutracc consortium.

Author information

Author notes

  1. Ralph Stadhouders, Petros Kolovos and Rutger Brouwer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands

    Ralph Stadhouders, Petros Kolovos, Jessica Zuin, Anita van den Heuvel, Robert-Jan Palstra, Kerstin S Wendt, Frank Grosveld & Eric Soler

  2. Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands

    Rutger Brouwer, Christel Kockx & Wilfred van Ijcken

  3. Netherlands Bioinformatics Centre (NBIC), Nijmegen, The Netherlands

    Rutger Brouwer

  4. Cancer Genomics Center, Erasmus Medical Center, Rotterdam, The Netherlands

    Frank Grosveld & Eric Soler

  5. Laboratory of Hematopoiesis and Leukemic Stem Cells (LSHL), French Alternative Energies and Atomic Energy Commission (CEA)/Institut National de la Santé et de la Recherche Médicale (INSERM) U967, Fontenay-aux-Roses, France

    Eric Soler

Authors
  1. Ralph Stadhouders
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Petros Kolovos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rutger Brouwer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jessica Zuin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anita van den Heuvel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christel Kockx
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert-Jan Palstra
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kerstin S Wendt
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Frank Grosveld
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wilfred van Ijcken
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Eric Soler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.S. and R.-J.P. adapted and optimized the protocol and library preparation for Illumina sequencing. R.S., P.K., A.v.d.H. and J.Z. used, developed and troubleshot the technique. C.K. optimized procedures for library sequencing, and R.B. developed the informatics pipeline for data processing and analysis. W.v.I., F.G., K.S.W. and E.S. supervised the projects, and participated in technology design and discussions. R.S., P.K., R.B., W.v.I., F.G., K.S.W. and E.S. drafted the manuscript.

Corresponding authors

Correspondence to Wilfred van Ijcken or Eric Soler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Scripts to analyze 3C-seq data. Within this supplementary archive, 4 python files are present that are used to analyze 3C-seq data. The findSequence.py and the regionsBetween.py files are used to generate a restriction map of the genome. The alignCounter.py script determines how many reads align to each of the restriction fragments. To run the alignCounter.py script the Pysam libraries should be installed on the system (see Materials). The libutils.py script is a shared library that should be placed in the same directory as the other py files. This library contains shared functionality between the 3 executable scripts. (ZIP 5 kb)

Supplementary Table 1

NARWHAL sample sheet. This file contains an example NARWHAL sample sheet that can be used in the primary data analysis. It serves to illustrate the specific fields that should be set in the primary data analysis procedure (Steps 65-79). (TXT 1 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stadhouders, R., Kolovos, P., Brouwer, R. et al. Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions. Nat Protoc 8, 509–524 (2013). https://doi.org/10.1038/nprot.2013.018

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.018

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