Abstract
Expression of genes can be controlled by regulatory elements that are located at large genomic distances from their target genes (in cis), or even on different chromosomes (in trans). Regulatory elements can act at large genomic distances by engaging in direct physical interactions with their target genes resulting in the formation of chromatin loops. Thus, genes and their regulatory elements come in close spatial proximity irrespective of their relative genomic positions. Analysis of interactions between genes and elements will reveal which elements regulate each gene, and will provide fundamental insights into the spatial organization of chromosomes in general.
Long-range cis- and trans- interactions can be studied at high resolution using chromosome conformation capture (3C) technology. 3C employs formaldehyde crosslinking to trap physical interactions between loci located throughout the genome. Crosslinked cells are solubilized and chromatin is digested with a restriction enzyme. Chromatin is subsequently ligated under conditions that favor intramolecular ligation. After reversal of the crosslinks, the DNA is purified and interaction frequencies between specific chromosomal loci are determined by quantifying the amounts of corresponding ligation products using polymerase chain reaction (PCR). This chapter describes detailed protocols for 3C analysis of chromatin interactions in the yeast Saccharomyces cerevisiae and in mammalian cells.
Keywords
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Drissen, R., Palstra, R., Gillemano, N., Splinter, E., Grosveld, F., Philipsen, S., and de Laat, W. (2004) The active spatial organization of the β-globin locus requires the transcription factor EKLF. Genes Dev. 18, 2485-2490
Vakoc, C., Letting, D.L., Gheldof, N., Sawado, T., Bender, M.A., Groudine, M., Weiss, M.J., Dekker, J., and Blobel, G.A. (2005) Proximity among distant regulatory elements at the betaglobin locus requires GATA-1 and FOG-1. Mol. Cell. 17, 453-462
Dekker, J. (2002) Capturing chromosome conformation. Science. 295, 1306-1311
Gheldof, N., Tabuchi, T.M., and Dekker, J. (2006) The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications. Proc. Natl. Acad. Sci. USA. 103, 12463-12468
Tolhuis, B., Palstra, R.J., Splinter, E., Grosveld, F., and de Laat, W. (2002) Looping interaction between hypersensitive sites in the active beta-globin locus. Mol. Cell. 10, 1435-1465
Palstra, R.J., Tolhuis, B., Splinter, E., Nijmeijer, R., Grosveld, F., and de Laat, W. (2003) The ?-globin nuclear compartment in development and erythroid differentiation. Nat. Genet. 25, 190-194
Dostie, J., Richmond, R.A., Arnaout, R.A., Selzer, R.R., Lee, W.L., Honan, A., Rubio, E.D., Krumm, A., Lamb, J., Nusbaum, C., Green, R.D., and Dekker, J. (2006) Chromosome conformation capture carbon copy (5C): A massively parallel solution for mapping interactions between genomic elements. Genome Res. 16, 1299-1309
Liu, Z., and Garrard, W.T. (2005) Long-range interactions between three transcriptional enhancers, active Vkappa gene promoters, and a 3? boundary sequence spanning 46 kilobases. Mol. Cell. Biol. 25, 3220-3231
Murrell, A., Heeson, S., and Reik, W. (2004) Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops. Nat. Genet. 36, 889-893
Spilianakis, C.G., Lalioti, M.D., Town, T., Lee, G.R., and Flavell, R.A. (2005) Interchromosomal associations between alternatively expressed loci. Nature. 435, 637-645
Lomvardas, S., Barnea, G., Pisapia, D.J., Mendelsohn, M., Kirkland, J., and Axel, R. (2006) Interchromosomal interactions and olfactory receptor choice. Cell. 126, 403-413
Dekker, J. (2006) The three C’s of chromosome conformation capture: controls, controls, controls. nat. methods. 3, 17-21
Acknowledgments
Research in the Dekker laboratory is supported by grants from NIH (HG003143) and the Cystic Fibrosis Foundation.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Miele, A., Dekker, J. (2008). Mapping Cis- and Trans- Chromatin Interaction Networks Using Chromosome Conformation Capture (3C). In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 464. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-461-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-60327-461-6_7
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-460-9
Online ISBN: 978-1-60327-461-6
eBook Packages: Springer Protocols