Analysis of Long-range Chromatin Interactions Using Chromosome Conformation Capture

Overview

Journal Methods

Specialty Biochemistry

Date 2012 Aug 21

PMID 22903059

Citations 96

Authors

Natalia Naumova

Emily M Smith

Ye Zhan

Job Dekker

Affiliations

Soon will be listed here.

Abstract

Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.

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References

Davison L, Cooper J, Cope N, Wilson N, Smyth D, Howson J . Long-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene. Hum Mol Genet. 2011; 21(2):322-33. PMC: 3276289. DOI: 10.1093/hmg/ddr468. View

Drissen R, Palstra R, Gillemans N, Splinter E, Grosveld F, Philipsen S . The active spatial organization of the beta-globin locus requires the transcription factor EKLF. Genes Dev. 2004; 18(20):2485-90. PMC: 529536. DOI: 10.1101/gad.317004. View

Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E . Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat Genet. 2006; 38(11):1348-54. DOI: 10.1038/ng1896. View

Dekker J . GC- and AT-rich chromatin domains differ in conformation and histone modification status and are differentially modulated by Rpd3p. Genome Biol. 2007; 8(6):R116. PMC: 2394764. DOI: 10.1186/gb-2007-8-6-r116. View

Bacher C, Guggiari M, Brors B, Augui S, Clerc P, Avner P . Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nat Cell Biol. 2006; 8(3):293-9. DOI: 10.1038/ncb1365. View

Gheldof N, Smith E, Tabuchi T, Koch C, Dunham I, Stamatoyannopoulos J . Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene. Nucleic Acids Res. 2010; 38(13):4325-36. PMC: 2910055. DOI: 10.1093/nar/gkq175. View

Vu T, Nguyen A, Hoffman A . Loss of IGF2 imprinting is associated with abrogation of long-range intrachromosomal interactions in human cancer cells. Hum Mol Genet. 2009; 19(5):901-19. PMC: 2816615. DOI: 10.1093/hmg/ddp558. View

Tolhuis B, Palstra R, Splinter E, Grosveld F, de Laat W . Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol Cell. 2002; 10(6):1453-65. DOI: 10.1016/s1097-2765(02)00781-5. View

Spilianakis C, Lalioti M, Town T, Lee G, Flavell R . Interchromosomal associations between alternatively expressed loci. Nature. 2005; 435(7042):637-45. DOI: 10.1038/nature03574. View

10.

Cremer T, Cremer C . Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet. 2001; 2(4):292-301. DOI: 10.1038/35066075. View

11.

Kim S, Bultman S, Kiefer C, Dean A, Bresnick E . BRG1 requirement for long-range interaction of a locus control region with a downstream promoter. Proc Natl Acad Sci U S A. 2009; 106(7):2259-64. PMC: 2650142. DOI: 10.1073/pnas.0806420106. View

12.

Sanyal A, Bau D, Marti-Renom M, Dekker J . Chromatin globules: a common motif of higher order chromosome structure?. Curr Opin Cell Biol. 2011; 23(3):325-31. PMC: 3109114. DOI: 10.1016/j.ceb.2011.03.009. View

13.

Vakoc C, Letting D, Gheldof N, Sawado T, Bender M, Groudine M . Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1. Mol Cell. 2005; 17(3):453-62. DOI: 10.1016/j.molcel.2004.12.028. View

14.

Lieberman-Aiden E, van Berkum N, Williams L, Imakaev M, Ragoczy T, Telling A . Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009; 326(5950):289-93. PMC: 2858594. DOI: 10.1126/science.1181369. View

15.

van Steensel B, Dekker J . Genomics tools for unraveling chromosome architecture. Nat Biotechnol. 2010; 28(10):1089-95. PMC: 3023824. DOI: 10.1038/nbt.1680. View

16.

Ahmadiyeh N, Pomerantz M, Grisanzio C, Herman P, Jia L, Almendro V . 8q24 prostate, breast, and colon cancer risk loci show tissue-specific long-range interaction with MYC. Proc Natl Acad Sci U S A. 2010; 107(21):9742-6. PMC: 2906844. DOI: 10.1073/pnas.0910668107. View

17.

Dekker J . The three 'C' s of chromosome conformation capture: controls, controls, controls. Nat Methods. 2005; 3(1):17-21. DOI: 10.1038/nmeth823. View

18.

Misteli T, Soutoglou E . The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol. 2009; 10(4):243-54. PMC: 3478884. DOI: 10.1038/nrm2651. View

19.

Xu N, Donohoe M, Silva S, Lee J . Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein. Nat Genet. 2007; 39(11):1390-6. DOI: 10.1038/ng.2007.5. View

20.

Montavon T, Soshnikova N, Mascrez B, Joye E, Thevenet L, Splinter E . A regulatory archipelago controls Hox genes transcription in digits. Cell. 2011; 147(5):1132-45. DOI: 10.1016/j.cell.2011.10.023. View