Chromatin Immunoprecipitation (ChIP) of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells

INTRODUCTION

This protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. The following protocol has been established for the cultured cell line Schneider 2 (S2) from Drosophila melanogaster . If other tissue is used, certain steps of the protocol may need to be optimized; the main variation is likely to be in the cross-linking step.

MATERIALS

Reagents

Antibody

ChIP cell lysis buffer

ChIP dilution buffer

ChIP hybridization buffer

ChIP wash buffer

Chloroform/isoamyl alcohol (24:1; prepare fresh before use)

Culture medium appropriate for cells or tissue being analyzed (e.g., Schneider’s Drosophila medium [GIBCO medium supplemented with 12.5% fetal bovine serum, or serum-free insect culture medium; HyQ SFX, Hyclone])

[ -³² P]dCTP (specific activity 3000 Ci/mmol; Amersham Pharmacia Biotech)

Drosophila Schneider SL2 tissue culture cells or other tissue or cell line for analysis, with appropriate growth facilities and equipment

Ethanol (100% and 70%)

Ethidium bromide (10 mg/ml)

Fixation solution, freshly prepared

Gel-loading buffer III (6X)

Glycine, powder

Glycogen (5 mg/ml; store at -20°C)

Hin dIII restriction enzyme and buffer

LiCl buffer

Ligase buffer (optional; see Step 19)

Nuclear lysis buffer

Oligonucleotide linkers (optional; see Step 18)

PBS (phosphate-buffered saline [pH 7.4])

PCR product purification kit (e.g., QiaQuick PCR purification kit, QIAGEN)

PCR reagents (e.g., Taq polymerase, NTPs, buffer, Mg⁺⁺ )

Phenol/chloroform/isoamyl alcohol

Protein A/G agarose beads (50%), pre-swollen and blocked (Santa Cruz, sc-2003)

Proteinase K (20 mg/ml; store at -20°C)

Random-primed DNA synthesis kit

RIPA buffer (05-01)

RNase, DNase-free (10 mg/ml)

SDS, 10%

Sodium acetate, 3 M (pH 5.2)

T4 DNA ligase (Roche) (optional; see Step 19)

TE (pH 8.0)

Equipment

DNA quantification software (e.g., QualityOne, Bio-Rad)

Equipment for agarose gel electrophoresis

Falcon tubes, 15-ml and 50-ml

Glass beads (150-200 µm, acid-washed)

Heating blocks, preset to 50°C, 65°C

Hybridization bottles (optional; see Step 23)

Hybridization oven, preset to 65°C (optional; see Step 23)

Nylon membrane (e.g., GeneScreen Plus, Perkin-Elmer, or Hybond-N+, Amersham Pharmacia Biotech) (optional; see Step 23)

Oven, preset to 80°C

PhosphorImager (optional; see Step 24)

Rotator, at 4°C

Shaker, at 4°C

Sonicator (e.g., Sanyo Soniprep 150, exponential microprobe, 10 amplitude microns)

Thermal cycler

UV transilluminator

METHOD

For an outline of the method, see Figure 1.

Chromatin Preparation

 

1. Grow 100 ml of Drosophila Schneider SL2 tissue culture cells in an appropriate medium, in cell culture bottles, to a density of 3 x 10⁶ to 6 x 10⁶ per milliliter.
 

2. Add the fixation solution (1/10th of volume of cells; e.g., 11 ml into 100 ml of medium; the final formaldehyde concentration should be 1%) directly to the flask and mix. Incubate fixation reaction for 10 minutes at 4°C on a shaker.
 

3. Stop the fixation by adding glycine powder to a final concentration of 125 mM. Mix well. Transfer cells to a 50-ml Falcon tube and collect by centrifuging at 800g for 5 minutes at 4°C. Wash the cells once with ice-cold PBS.
 

4. Resuspend the cell pellet in 15 ml of ice-cold cell lysis buffer, pipetting up and down until all the cells have been resuspended. Stand them on ice for 10 minutes. Collect the nuclei by centrifuging at 2000g for 5 minutes at 4°C. Carefully discard the supernatant and resuspend the pellet in 2 ml of ice-cold nuclear lysis buffer by pipetting up and down. Transfer the suspension to a 15-ml Falcon tube that has been cut down to the 10-ml mark to allow the sonicator tip to reach the suspension. Leave on ice for 10 minutes.
 

5. Add ~0.5 ml of glass beads to the cell suspension. Store on ice or sonicate immediately.
 

6. Sonicate the sample with six 30-second pulses (output near microtip limit), using a high-power sonicator. Keep the tube cool by holding it in a beaker containing an ice/water mix.
The sonicator tip should be immersed roughly one-fourth into the liquid. Avoid foaming. If foaming occurs, centrifuge the tube briefly to reduce the foam layer. Leave on ice for some minutes and sonicate again. For initial trial experiments, take an aliquot from the chromatin suspension after each sonication pulse (e.g., 10 µl). Increase the sample volume to 100 µl with TE and process as described in Step 8.
 

7. Transfer the sonicated suspension to two 15-ml Falcon tubes (leaving most of the glass beads behind), and centrifuge at 12,000-14,000g for 10 minutes at 4°C. Dilute the supernatant with dilution buffer to a final volume of 8 ml (i.e., 4X dilution). Rotate the tubes on a wheel for 10 minutes at 4°C. Take a 50-µl aliquot to check the average size of the DNA fragments (Steps 8, 9, and 10). From the remaining sample, prepare 600-µl aliquots and store at -80°C, or use the chromatin directly for immunoprecipitation.
 

8. To the 50-µl aliquot taken in Step 7, add 50 µl of TE. Incubate overnight at 65°C (if not using safelock-tubes, seal tubes with Parafilm). Add proteinase K to 500 µg/ml and SDS to 0.5% (w/v). Incubate for 3 hours at 50°C. Centrifuge briefly.
 

9. Add one volume of phenol-chloroform-isoamyl alcohol, vortex for 2 minutes, and centrifuge at 12,000-14,000g for 8 minutes. Transfer the aqueous supernatant to a new tube. Add one volume of chloroform-isoamyl alcohol, vortex for 2 minutes, and centrifuge at 12,000-14,000g for 8 minutes. To the second aqueous supernatant, add 1/10th volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol, and mix well. Leave for at least 30 minutes at -20°C. Centrifuge at 12,000-14,000g for 15 minutes at 4°C. Carefully discard the supernatant, and wash the pellet in 800 µl of 70% ethanol. Centrifuge again, and allow the pellet to air-dry for 5-10 minutes. Dissolve the pellet in 10 µl of TE.
 

10. Add to each sample 0.5 µg of DNase-free RNase, and incubate for 30 minutes at 37°C. Add 3 µl of gel loading solution. Run the sample on a 0.8% agarose gel (~15-20 cm long for best separation). When the bromophenol blue dye has migrated along two-thirds of the gel, stain the gel with 0.5 µg/ml ethidium bromide and view on a UV transilluminator. If the average length of the DNA is not short enough (there should be a smear of the molecular weight of ~300-1000 bp), the stored aliquots can be resonicated (Step 6).

^{Immunoprecipitation and Reversal of Cross-Links}

 

^{11. For each immunoprecipitation (IP), the mock control and the input control, take 300 µl of chromatin (obtained in Step 7) and add an equal volume of RIPA buffer. Add 20 µl of protein A/G agarose beads using a cut-off (wide aperture) pipette tip. Incubate for 1-2 hours at 4°C for pre-clearing, and centrifuge in a microcentrifuge at 13,000 rpm for 10 minutes at 4°C.}

^{12. Transfer the resulting supernatant to a new tube, and add the appropriate amount of antibody (usually 1 µg of an affinity-purified antibody; dilutions of 1:100 to 1:500). Use the same amount of pre-cleared chromatin in the controls, without the addition of antibody (for mock and input control), or with pre-immune serum or an appropriate nonspecific antibody. Incubate the samples from 2 to 3 hours to overnight at 4°C on a rotator.}

^{13. Centrifuge the samples in a microcentrifuge at 13,000g for 10 minutes at 4°C. Transfer the IPs to new tubes. Add 20 µl of the 50% protein A/G agarose bead solution, and incubate for a further 2-4 hours. Pellet the beads with a short centrifugation (20 seconds at maximum speed) in a benchtop centrifuge. Transfer the supernatant of the no-antibody control to a new tube and leave on ice. This material will serve as total input control. Discard the other supernatants. Wash the beads five times with 600 µl of RIPA buffer, once with 600 µl of LiCl buffer, and once with 600 µl of TE (pH 8.0), collecting the beads between washes with brief centrifugations. Finally, resuspend the beads in 100 µl of TE.}

^{14. Add 1 µg of DNase-free RNase (also to the input control), and incubate samples overnight at 65°C. The next day, adjust samples to 0.5% SDS and 0.5 mg/ml proteinase K, and incubate for a further 3 hours at 50°C. Phenol-chloroform-extract the samples as described in Step 9. Back-extract the phenol phase by adding an equal volume of TE (pH 8.0), and vortex. Combine the aqueous phases and perform one more chloroform extraction. Precipitate the DNA by adding glycogen to 100 µg/ml as carrier, 1/10th volume of 3 M sodium acetate (pH 5.2), and 2.5 volumes of 100% ethanol. Incubate at -20°C for 2 hours to overnight. Collect the DNA by centrifuging at 12,000-14,000g for 15 minutes at 4°C, and wash the pellet in 800 µl of 70% ethanol. Repeat centrifugation and discard the supernatant. Allow the pellet to air-dry for 5-10 minutes. Re-dissolve the precipitated DNA in 30 µl of TE (PCR analysis) or 9 µl of water (Southern analysis), and store at 4°C (to avoid DNA precipitation, do not freeze).}

^{PCR Analysis}

 

^{15. Perform the test, negative control, and the input-control (dilutions of 1/10, 1/100, and 1/1000 of the input) PCRs in 25-µl volumes, using the optimum magnesium concentration for each primer pair. Start by using 1 µl of the immunoprecipitated DNA as a template (in 1X reaction buffer, 0.25 mM NTPs, 1 µm primer, 0.5 units of Taq polymerase).}

Number of Cycles Denaturation Annealing Polymerization 1 2 min at 94°C     35 1 min at 94°C 1 min at 60°-65°C 1 min at 72°C 1     6 min at 72°C

^{Adjust the annealing temperature and number of cycles for each primer pair until no signal is detected for the negative control-IP DNA. Signals obtained from the test reactions under these conditions can be considered significant.}

^{16. After the amplification, add 6 µl of gel loading solution to each PCR, load half of the reaction onto a 1.5% agarose gel, and visualize amplified DNA with ethidium bromide. To increase signal intensities, the amount of template for the PCR (of all the samples, including the negative controls) can be increased.}

^{17. Quantify the resulting bands, and plot them as percentage of the input (the total of chromatin-DNA used for one IP, from Step 13).}

^{Southern Analysis}

 

^{18. Prepare the oligonucleotide linker by annealing two oligonucleotides, a 24-mer of sequence 5''-AGAAGCTTGAATTCGAGCAGTCAG-3'' and a 20-mer of sequence 5''-CTGCTCGAATTCAAGCTTCT-3'' (when ordering the synthetic oligonucleotides, take care that only the 24-mer is phosphorylated at the 5''-end). Mix equimolar amounts of these oligonucleotides in TE (e.g., 10 µl of a 100 µM stock of both oligonucleotides in 100 µl of TE), boil for 5 minutes, and allow the reaction to cool slowly to room temperature.}

^{19. Resuspend the immunopurified-chromatin DNA in 9 µl of ligase buffer containing the linker adapter at a final concentration of 0.8 µM. Add 4 units of T4 DNA ligase, and incubate for 24 hours at 4°C.}

^{20. Use the ligated mixture directly as a template in a 100-µl PCR using 1 unit of Taq polymerase, 1X corresponding buffer, and 2 mM Mg++ . The primer used is the 20-mer oligonucleotide described above, added to a final concentration of 1 µM. Amplification is performed as follows.}

Number of Cycles Denaturation Annealing Polymerization 1 2 min at 94°C     35 1 min at 94°C 1 min at 55°C 1 min at 72°C 1     6 min at 72°C

 

^{21. Extract the samples once with phenol-chloroform-isoamyl alcohol and once with chloroform-isoamyl alcohol; ethanol-precipitate as described in Step 9. Remove linkers by digesting the DNA with Hin dIII, and separate them from the amplified DNA by gel filtration (e.g., with the QiaQuick PCR purification kit; QIAGEN). An aliquot with linkers may be stored at -20°C as reservoir.}

^{22. Label the amplified DNA with [ -32 P]dCTP (specific activity 3000 Ci/mmol) using a random-primed DNA synthesis kit according to the instructions of the manufacturer.}

^{23. Separate the digested target DNA (e.g., bacterial clones, lambda clones) on an agarose gel, and transfer the DNA to nylon membranes using standard techniques (e.g., Southern Blotting: Capillary Transfer of DNA to Membranes). Bake the membrane for 2 hours at 80°C. Hybridization is best performed in glass bottles and a hybridization oven. Pre-hybridize the membrane for 3 hours at 65°C in 10 ml of hybridization buffer. Add the heat-denatured probe, from the previous step, directly to the hybridization solution, and incubate the filter overnight at 65°C.}

^{24. Wash filters once with chIP wash buffer for 10 minutes at 65°C, and at least four times, for 5 minutes each, at 65°C in the same buffer, but containing 1% SDS. After washing, seal the filters into plastic bags and expose to X-ray film or, for a more sensitive and quantitative analysis of the hybridization signals, analyze using a PhosphorImager.}

 

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Figure 1. Flowchart of chromatin immunoprecipitation protocol.