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Mapping networks of physical interactions between genomic elements using 5C technology

Nature Protocols volume 2, pages 988–1002 (2007)Cite this article

Abstract

Genomic elements separated by large genomic distances can physically interact to mediate long-range gene regulation and other chromosomal processes. Interactions between genomic elements can be detected using the chromosome conformation capture (3C) technology. We recently developed a high-throughput adaptation of 3C, 3C-carbon copy (5C), that is used to measure networks of millions of chromatin interactions in parallel. As in 3C, cells are treated with formaldehyde to cross-link chromatin interactions. The chromatin is solubilized, digested with a restriction enzyme and ligated at low DNA concentration to promote intra-molecular ligation of cross-linked DNA fragments. Ligation products are subsequently purified to generate a 3C library. The 5C technology then employs highly multiplexed ligation-mediated amplification (LMA) to detect and amplify 3C ligation junctions. The resulting 5C library of ligated primers is analyzed using either microarray detection or ultra-high-throughput DNA sequencing. The 5C protocol described here can be completed in 13 d.

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Figure 1: Schematic representation of the chromosome conformation capture (3C) methodology.
Figure 2: Diagram of the 3C-carbon copy (5C) methodology.
Figure 3: 3C-carbon copy (5C) technology primer design.
Figure 4: Diagram illustrating a 'fixed' 3C-carbon copy technology (5C) analysis between the locus control region (LCR) and the surrounding beta-globin locus.
Figure 5: Diagram illustrating the generation of dense 3C-carbon copy (5C) technology interaction maps.
Figure 6: Representative titration of a control or chromosome conformation capture (3C) library with one 3C-carbon copy (5C) technology primer pair.
Figure 7: Representative titration of a control or chromosome conformation capture (3C) library.
Figure 8: Generation of a control library from bacterial artificial chromosome (BAC) DNA.
Figure 9: Generation of a chromosome conformation capture (3C) library from mammalian cells.
Figure 10: Example of a dense interaction map analysis in a gene desert region.
Figure 11: Example of a fixed 3C-carbon copy (5C) technology analysis.

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Acknowledgements

This work was supported by a grant from the National Institutes of Health to J.D. (HG003143).

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  1. Program in Gene Function and Expression and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Lazare Research Building, 364 Plantation Street, Room 519, Worcester, 01605-4321, Massachusetts, USA

    Josée Dostie & Job Dekker

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Correspondence to Job Dekker.

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Dostie, J., Dekker, J. Mapping networks of physical interactions between genomic elements using 5C technology. Nat Protoc 2, 988–1002 (2007). https://doi.org/10.1038/nprot.2007.116

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