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.
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Branco, M.R. & Pombo, A. Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations. PLoS Biol. 4, e138 (2006).
Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).
Dekker, J. The three 'C' s of chromosome conformation capture: controls, controls, controls. Nat. Methods 3, 17–21 (2006).
Hagege, H. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat. Protoc. 2, 1722–1733 (2007).
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).
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).
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).
Lomvardas, S. et al. Interchromosomal interactions and olfactory receptor choice. Cell 126, 403–413 (2006).
Ohlsson, R. & Göndör, A. The 4C technique: The 'Rosetta stone' for genome biology in 3D? Curr. Opin. Cell Biol. 19, 321–325 (2007).
Ling, J.Q. et al. CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1. Science 312, 269–272 (2006).
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).
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).
Kiernan, J.A. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microscopy Today 8, 8–12 (2000).
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.
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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
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