Chromosome Conformation Capture (3C) (PROT05)

Introduction

An alternative protocol (PROT31) for 3C has been adopted by the author's lab. Much of it is identical to this version. The major differences are in the amounts of DNA used at different steps. We reliably get the same results as with this version, but also a greater yield of 3C material. Yields from this version are ample for real-time PCR analysis, but greater yields are required if the 3C material is going to be processed further.

The 3C (Chromosome Conformation Capture) technique generates a population average measurement of juxtaposition frequency between any two genomic loci, thus providing information on their relative proximity in the nucleus (Dekker et al ., 2002). Cells are fixed with formaldehyde which forms DNA-protein and protein-protein cross-links between regions of the genome in proximity (see figure 1 ). Subsequent restriction enzyme digestion and intra-molecular ligation produces novel junctions between restriction fragments in proximity in the nucleus. Novel ligation products can be detected by PCR. We adapted the 3C assay (Dekker et al ., 2002) to determine the conformation of mouse chromosome 7 and in particular the co-localization of actively transcribed genes in transcription factories (Osborne et al ., 2004). The 3C assay can also be used to reveal proximity between active genes and distal genomic elements (Tolhuis et al ., 2002).

An important consideration in the interpretation of 3C data is the understanding that not all pairs of restriction fragments that provide a positive result (i.e. generate a novel PCR product) are necessarily engaged in a functional interaction in the nucleus. For example, compare results of Tolhuis et al ., 2002, with Carter et al ., 2002 in which the higher order structure of the mouse hbb locus was assayed by two different methods, 3C and RNA TRAP. Clearly, distal fragments can be cross-linked by formaldehyde simply because they are near each other in the nucleus, and presumably can "bump into" each other during the fixation process (Osborne et al ., 2004). Therefore fixation conditions are critical in the 3C assay since increased fixation leads to greater cross-linking resulting in the detection of chromatin fragments that may be in proximity in the nucleus but not necessarily engaged in a specific intermolecular interaction with implied function (see comment 1 ).

Procedure Fixation

Cells are fixed in 2% formaldehyde to generate DNA-protein and protein-protein cross-links. This results in the cross-linking of physically close chromatin segments throughout the genome.

note 1 ); Add 2.7ml of 37% formaldehyde (final conc. 2%); Fix for 5 minutes at room temp with mixing (see note 2 ); Quench with 3ml of 2M glycine (0.125M final); Spin down for 15 minutes at 3500rpm. Lysis

The cells are lysed to isolate nuclei.

Resuspend cells in 50ml of lysis buffer with freshly added protease inhibitors. Add a small magnetic stirrer and incubate on ice for 90 minutes on a stirrer plate to ensure good stirrage; Remove the magnetic flea, spin down for 15 minutes at 2500rpm.

Digestion

The DNA is completely digested with a restriction enzyme. The restriction enzyme used depends on the region of interest and the resolution required. It is important to check the ability of the chosen enzyme to digest cross-linked chromatin. I have used Bgl II.

Resuspend the nuclei in 2ml of 1X NEB buffer 3 + 0.3% SDS. Incubate at 37°C for 1 hour with shaking; Add Triton-X to a final concentration of 1.8% (180µl of 20% Triton-X) to sequester the SDS. Incubate 1 hour at 37°C with shaking; Count the nuclei using a haemocytometer; Use a 1x106 nuclei aliquot (~15µg) for digestion. Digest overnight at 37°C with shaking (see note 3 ).

Ligation

The DNA is extensively diluted to favour intra-molecular ligations i.e. only restriction fragments that are cross-linked together will be ligated.

To inactivate the restriction enzyme, add SDS to a final volume of 1.6% and heat to 65°C for 20 minutes; Take a 2µg aliquot of chromatin, make up to 800µl with ligation buffer (final conc. 2.5ng/µl); Add Triton-X to a final conc. of 1% (40µl of 20% triton). Incubate for 1 hour at 37°C; Lower temperature to 16°C and add desired amount of T4 DNA ligase. Incubate for 4 hours (see note 4 ).

DNA Purification

Cross-links are reversed and the DNA is purified prior to PCR analysis.

Add proteinase K (100µg/ml final) and incubate at 65°C overnight. Prolonged incubation at high temperature reverses formaldehyde cross-links; Treat with RNase A (0.5µg/ml final) for 30 minutes at 37°C; Phenol extract and ethanol precipitate the DNA.

PCR amplification

Ligation products are detected by PCR. Nested primers can be used with two rounds of PCR to increase sensitivity (see comment 2 ). Primers should be tested on a DNA sample that contains equimolar amounts of all expected or possible ligation products (see comment 3 ). For example BAC DNA encompassing the genomic region to be assessed should be digested and randomly ligated in vitro to generate every possible combination of ligation products. This DNA can then be used to test the efficiencies of various primer pairs. Where the genomic region(s) to be analyzed are too distal to be contained within a single large DNA clone, multiple clones can be used. Alternatively, the sequences around the individual restriction sites to be analyzed (for example genomic regions encompassing Bgl II sites) can be PCR'd up and then mixed in equimolar amounts followed by restriction digestion and ligation to provide a DNA sample with all possible combinations of ligation products.

Quantify DNA using Picogreen dsDNA Quantitation Kit (Molecular Probes). Use 300ng DNA in two rounds of PCR with nested primers. PCR primers should be designed to give rather small PCR fragments (less than 500bp) to maintain efficient amplification (see comment 4 ).

Materials & Reagents

Job Dekker , University of Massachusetts Medical School, Worcester