High-resolution circular chromosome conformation capture assay.
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.
Göndör
Anita
A
Department of Development and Genetics, Uppsala University, Norbyvägen 18A, S-752 36 Uppsala, Sweden. anita.gondor@ebc.uu.se
Rougier
Carole
C
Ohlsson
Rolf
R
D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D002880:Chromosomes, Human, Pair 11; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D005821:Genetic Techniques; D006801:Humans; D009690:Nucleic Acid Conformation
D002843:Chromatin; D004270:DNA, Circular; D005557:Formaldehyde; D004247:DNA; D004262:DNA Restriction Enzymes