High-resolution circular chromosome conformation capture assay

Nature Protocols volume 3, pages303–313(2008)Cite this article

Abstract

The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2–3 weeks.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

Rent or Buy

All prices are NET prices.

Figure 1: The principle of the high-resolution 4C method.
Figure 2: High-resolution 4C analysis of the human chromosome 11.
Figure 3: Agarose gel electrophoresis of formaldehyde cross-linked, restriction-enzyme-digested chromatin DNA.
Figure 4: Scheme to assess the efficiency of restriction enzyme digestion by Q-PCR.

References

  1. 1

    Branco, M.R. & Pombo, A. Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations. PLoS Biol. 4, e138 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    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  Article  Google Scholar 

  6. 6

    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  Article  Google Scholar 

  7. 7

    Wurtele, H. & Chartrand, P. Genome-wide scanning of HoxB1-associated loci in mouse ES cells using an open-ended Chromosome Conformation Capture methodology. Chromosome Res. 14, 477–495 (2006).

    Article  Google Scholar 

  8. 8

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

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Ohlsson, R. & Göndör, A. The 4C technique: The 'Rosetta stone' for genome biology in 3D? Curr. Opin. Cell Biol. 19, 321–325 (2007).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Ling, J.Q. et al. CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1. Science 312, 269–272 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Kurukuti, S. et al. CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc. Natl. Acad. Sci. USA 103, 10684–10689 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    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  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Kiernan, J.A. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microscopy Today 8, 8–12 (2000).

    Article  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge help with the processing of NimbleGen data from Kuljeet Singh and discussions with many members of the RO lab. This work was supported by grants from HEROIC (EU), Swedish Research Council, Swedish Cancer Foundation, Swedish Pediatric Cancer Foundation and Lundberg's Foundation.

Author information

Affiliations

  1. Department of Development and Genetics, Uppsala University, Norbyvägen 18A, Uppsala, S-752 36, Sweden

    Anita Göndör, Carole Rougier & Rolf Ohlsson

Authors
  1. Anita Göndör
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carole Rougier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rolf Ohlsson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anita Göndör.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Göndör, A., Rougier, C. & Ohlsson, R. High-resolution circular chromosome conformation capture assay. Nat Protoc 3, 303–313 (2008). https://doi.org/10.1038/nprot.2007.540

Download citation

Further reading

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

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