Authors: Vijay K. Tiwari and Stephen B. Baylin
Progress in technologies to address long-range chromosomal interactions in vivo has extensively revised concepts about different aspects of transcriptional regulation. These methods allow probing physical proximities between chromatin elements without specifically identifying the protein components that mediate such interactions. Here we describe a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to identify the proteins that bridge distant genomic regions, while simultaneously identifying such physical proximities. This method is also useful for determining if a candidate protein might mediate long-range interactions, both in cis and in trans in the nucleus. We discuss how the 6C technique can be incorporated with other techniques to discover all the chromatin regions in the nucleus that interact with a given gene or chromatin region of interest in a specific protein-dependent manner. Such information allows complete, cell-type-specific mapping of all the chromatin interactions mediated by specific proteins. The 6C assay advances our understanding of the three-dimensional aspects of the higher-order folding of chromatin and provides an important tool to examine the role of specific proteins in nuclear organization. In addition to providing a detailed protocol of the 6C technique, we discuss how this technology can be used by investigators working in the area of chromatin biology, with special interest in chromatin long-range interactions.
The 6C assay combines three different methodologies: chromosome conformation capture (3C) (Dekker et al. 2002), chromatin immunoprecipitation (ChIP), and cloning (Fig. 1). The first step involves conventional 3C methodology: The chromatin is cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin is immunoprecipitated using an antibody against the protein of interest (i.e., the suspected “bridging protein” or the protein whose mediating physical proximities the investigator wishes to map). Thereafter, the cross-links are reversed, and the DNA is purified further. The fragments obtained are then cloned into a vector harboring the same restriction enzyme site overhangs that were generated in the enzyme digestion step of the 3C portion of the protocol. The clones are further screened by digestion with the same restriction enzyme. Ideally, clones showing multiple inserts will result from the intramolecular ligation and should represent physical proximities involving the protein targeted in the immunoprecipitation steps. These clones are chosen for sequencing to reveal the identity of the partners.
Figure 1. Summary of the Combined 3C-ChIP-Cloning (6C) method.
For a review of other recent methods developed to examine long-range chromosomal interactions in vivo, see Simonis et al. (2007). For additional details on the 6C method presented here, see Tiwari et al. (2008).
Restriction Enzyme Digestion and Intramolecular Ligation
12.Resuspend the pellet in 500 μL of 1.14X restriction enzyme buffer (appropriate for the restriction enzyme to be used in Step 16).
13.Pellet the cells by centrifugation. Resuspend in 500 μL of 1.14X restriction enzyme buffer. Transfer to 1.5-mL tubes.
14.Add 7.5 μL of 20% SDS (to a final concentration of 0.3% SDS). Mix by gentle pipetting. Shake gently for 1 h at 37°C.
15.Add 50 μL of 20% Triton X-100 (to a final concentration of 1.8%). Shake gently for 1 h at 37°C.
16.Add 1200 units of restriction enzyme (i.e., 12 μL of 100 units/μL). Incubate with gentle shaking overnight at 37°C.
17.Add 40 μL of 20% SDS (to a final concentration of 1.6%). Incubate with shaking for 30 min at 65°C.
18.Transfer the sample to a 15-mL tube containing 7 mL of 1.15X ligation buffer.
19.Add 400 μL of 20% Triton X-100 (to a final concentration of 1%). Incubate with occasional gentle shaking for 1 h at 37°C.
20.Add 50 μL of T4 DNA ligase. Incubate for 4 h at 16°C.
21.Incubate for 30 min at room temperature.
Enrichment of Ligation Fragments Containing the Protein of Interest in the Complex
22.Remove 10 μL from the sample (from Step 21). Store at -20°C.
23.Remove an 800-μL aliquot from the sample (from Step 21). Add it to 7.2 mL of ChIP diluent (i.e., a 10-fold dilution).
24.Add 4-10 μg of the antibody of choice per immunoprecipitation. Rotate end-over-end overnight at 4°C.
25.Prepare the magnetic beads:
26.Add 100 μL of the blocked magnetic bead solution to each immunoprecipitate sample (from Step 24). Incubate with rotation for 3 h at 4°C.
27.Prechill one 1.5-mL tube for each immunoprecipitate.
28.Transfer ~1.5 mL of each immunoprecipitate to a separate prechilled tube. Place the tubes in the magnetic stand to collect the beads. Remove the supernatant. Add another aliquot of the remaining immunoprecipitate. Repeat until all the beads have been collected.
29.Remove the tubes from the magnetic stand. Place on ice.
30.Wash the beads four times with 500 μL of low-salt immune complex wash buffer containing protease inhibitor cocktail. Agitate gently to resuspend the beads. Place the tubes in the magnetic stand to collect the beads. Remove the supernatant.
31.Wash the beads once with 500 μL of high-salt immune complex wash buffer containing protease inhibitor cocktail. Agitate gently to resuspend the beads. Place the tubes in the magnetic stand to collect the beads. Remove the supernatant.
32.Wash the beads once with 1 mL of TE buffer containing protease inhibitor cocktail. Collect on the magnetic stand. Remove the supernatant.
33.Centrifuge at 960g for 3 min at 4°C. Remove any residual TE buffer.
34.Add 210 μL of bead eluting buffer. Incubate for 15 min at 65°C. Vortex briefly every 2 min.
35.Centrifuge at 16,000g for 1 min at room temperature.
36.Transfer 200 μL of the supernatant to a new 1.5-mL tube.
37.Thaw the inputs (from Step 22). Add 190 μL of bead eluting buffer.
38.Reverse-cross-link samples (from Step 36) and inputs (from Step 37) overnight at 65°C.
39.Dilute the SDS by adding 200 μL of TE buffer. Add 8 μL of 10 mg/mL RNase A. Incubate for 2 h at 37°C.
40.Add 8 μL of 10 mg/mL proteinase K. Incubate for 2 h at 50°C.
41.Add an equal volume of phenol:chloroform:isoamyl alcohol to the samples. Shake well. Centrifuge at 10,000 rpm for 15 min. Transfer the upper phase to a fresh tube. Repeat this step with the upper phase.
42.Precipitate the DNA by adding a one-tenth volume of 3 M sodium acetate, 1 μL glycogen, and two volumes of 100% ethanol.
43.Wash with 0.4 volume of 70% ethanol. Vortex gently after adding the ethanol.
44.Dissolve the pellet in 15 μL of nuclease-free H2O.
45.Test the success of the ChIP reaction using 1 μL of undiluted immunoprecipitate by PCR with primers specific for a chromatin region known to be occupied by the protein of interest in the cell type under investigation.
Cloning of 3C-Ligated Immunoprecipitated Fragments
46.Using standard cloning protocols, clone the 3C-ChIP products (from Step 44) into a vector that has enzyme overhangs similar to those generated in the 3C assay.
47.Use the ligated vector to transform high-efficiency competent bacterial cells.
48.For experimental samples, spread the surface of LB-agar plates containing the antibiotic of choice (i.e., LB-ampicillin or LB-kanamycin agar plates), with 45 μL of X-gal and 9 μL of IPTG per 100-mm dish before plating the transformed cells.
49.The next day, count the number of blue and white colonies in the bacterial plates from samples immunoprecipitated with the specific antibody as well as the controls (i.e., no antibody or IgG).
50.Pick several white colonies (i.e., containing the insert) from the bacterial plates.
51.Use each such colony to inoculate LB liquid medium containing the appropriate antibiotic. Incubate in a shaking incubator at 220 rpm overnight at 37°C.
Screening for Ligated Partners
53.Screen the purified plasmids (from Step 52) by digestion with the same restriction enzyme used for Step 16, using standard restriction digestion protocols.
54.Separate the resulting DNA fragments by electrophoresis in a 1% agarose gel containing SYBR Safe. Use an appropriately sized DNA ladder for reference. Visualize using UV light. See Troubleshooting.
55.Sequence the plasmids showing more than one insert.
56.Sequence the inserts using primers from two different ends of the vector (e.g., T3 and T7 promoter-specific primers for pBluescript II RI Predigested Vector).
The 6C-captured interactions should be validated by performing independent 3C assays (Tolhuis et al. 2002), in which one performs 3C-PCRs using multiple primer combinations from two different remote sequences. The primers for this purpose are designed exactly as described by Splinter et al. (2004). It also is important to establish, by using separate assays, that the interacting regions captured in the 6C assay are truly occupied by the protein of interest in cells. To this end, perform ChIP assays (Tiwari et al. 2008) using antibodies specific for the protein of interest. The immunoprecipitated DNA can then be amplified using primers spanning the restriction enzyme site found to be involved in the 3C ligation with other partner(s) in each of the 6C clones.
In early attempts of the 6C assay (Tiwari et al. 2008), screening the clones by restriction digestion identified a very low frequency of clones having multiple inserts (five out of 352). A number of reasons could account for this. First, the rest of the clones that had a single insert probably represented distinct genomic sites that were bound by the specific protein (in this case, EZH2) but were not engaged in any long-range associations. Interestingly, this might also reflect the frequency with which certain protein-dependent long-range associations take place in the nucleus. Second, the number of clones with multiple inserts might also be reduced because of difficulty in cloning (i.e., ligation and transformation) or sequencing of large fragments resulting from the ligation fragments generated by a six-cutter restriction enzyme (e.g., EcoRI). Third, intramolecular ligation after cross-linking and digestion will lead to a fraction of the DNA occurring as circular DNA that cannot be cloned; this might further reduce the number of clones in such an assay. Finally, the study could also have missed some interactions involving partners that bear DNA methylation at the EcoRI site (EcoRI is a DNA methylation-sensitive enzyme). Future work should develop strategies to tackle each of these issues to improve the overall efficiency of the method.
The 6C technique can be combined with other recently published techniques to discover all the chromatin regions in the nucleus that are brought in close physical proximity to the gene (or any other chromatin region of interest) in a specific protein-dependent manner (Fig. 2). Following immunoprecipitation with the antibody of interest, the samples can be subjected to 4C analysis (Zhao et al. 2006) or reverse-cross-linked, purified, digested with a four-cutter enzyme of choice, and processed further for either 3C-chip (Simonis et al. 2006) or the ACT assay (Ling et al. 2006). The 6C procedure can also be used to reveal whether two known chromatin regions are brought into close physical proximity by a specific protein by following the amplification criteria used in the original 3C assay, subsequent to the reversal of cross-linking and purification (Dekker et al. 2002).
Figure 2. Further applications and the future of Combined 3C-ChIP-Cloning (6C) methodology.