Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip

Abstract

Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.

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Figure 1: Overview of the e4C procedure.
Figure 2: Flowchart of e4C timing.
Figure 3: Examples of quality controls for e4C.
Figure 4: Typical e4C and ChIP-e4C results, using Hbb-b1 sequence as bait.

References

  1. 1

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

    CAS  Article  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

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

    CAS  Article  Google Scholar 

  4. 4

    Sexton, T., Bantignies, F. & Cavalli, G. Genomic interactions: chromatin loops and gene meeting points in transcriptional regulation. Semin. Cell Dev. Biol. 20, 849–855 (2009).

    CAS  Article  PubMed  Google Scholar 

  5. 5

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

    CAS  Article  PubMed  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

    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 

  9. 9

    Ohlsson, R. & Gondor, 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

    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  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Noordermeer, D. et al. Variegated gene expression caused by cell-specific long-range DNA interactions. Nat. Cell Biol. 13, 944–951 (2011).

    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

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    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  Article  PubMed  Google Scholar 

  16. 16

    Louwers, M., Splinter, E., van Driel, R., de Laat, W. & Stam, M. Studying physical chromatin interactions in plants using chromosome conformation capture (3C). Nat. Protoc. 4, 1216–1229 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Raab, J.R. et al. Human tRNA genes function as chromatin insulators. EMBO J. (2011); 31, 330–350.

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Brazma, A. et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat. Genet. 29, 365–371 (2001).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    de Wit, E., Braunschweig, U., Greil, F., Bussemaker, H.J. & van Steensel, B. Global chromatin domain organization of the Drosophila genome. PLoS Genet. 4, e1000045 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

    Tolhuis, B. et al. Interactions among polycomb domains are guided by chromosome architecture. PLoS Genet. 7, e1001343 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M. & Altman, D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  23. 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).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Sandmann, T., Jakobsen, J.S. & Furlong, E.E. ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos. Nat. Protoc. 1, 2839–2855 (2006).

    CAS  Article  PubMed  Google Scholar 

  25. 25

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

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Carter, D., Chakalova, L., Osborne, C.S., Dai, Y.F. & Fraser, P. Long-range chromatin regulatory interactions in vivo. Nat. Genet. 32, 623–626 (2002).

    CAS  Article  Google Scholar 

  27. 27

    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  Article  Google Scholar 

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Acknowledgements

This work was supported by the UK Medical Research Council, the UK Biotechnology and Biological Sciences Research Council and by a long-term EMBO fellowship to D.U.

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Authors

Contributions

T.S., S.K. and P.F. designed the experiments, T.S. and S.K. developed the method, J.A.M. and D.U. optimized the chromatin immunoprecipitation steps, T.N. developed primers for restriction digestion efficiency tests and T.S. and P.F. wrote the manuscript.

Corresponding author

Correspondence to Peter Fraser.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Typical raw e4C and ChIP-e4C results, using Hbb-b1 sequence as bait. (PDF 360 kb)

Supplementary Table 1

List of primers used for assessing BglII restriction digestion efficiency in mouse tissues. (PDF 72 kb)

Supplementary Table 2

List of 3C primers used in Anticipated Results section. (PDF 52 kb)

Supplementary Table 3

List of ChIP primers used in Anticipated Results section. (PDF 52 kb)

Supplementary Table 4

List of primers used for assessing e4C bait enrichment in Anticipated Results section. (PDF 51 kb)

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Sexton, T., Kurukuti, S., Mitchell, J. et al. Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip. Nat Protoc 7, 1335–1350 (2012). https://doi.org/10.1038/nprot.2012.071

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