Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone

INTRODUCTION

In cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organization and gene expression in eukaryotes. Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immunofluorescence studies on cultured cells have been pivotal in unraveling the roles of histone modifications. However, to address in detail what happens at specific sites in vivo, chromatin immunoprecipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on non-fixed chromatin from animal cells or tissues (fresh or frozen) to analyze histone modifications at specific chromosomal sites. These protocols are suitable only for analyzing histones and their modifications. For other applications, chromatin immunoprecipitation should be performed on cross-linked chromatin.

RELATED INFORMATION

This ChIP protocol was derived from methodologies originally described by O’Neill and Turner (1995) . It was published earlier on the Web site of the European Network of Excellence "EPIGENOME" (www.epigenome-noe.net ), and was adapted from Umlauf et al. (2003) . An overview of the procedure is provided in Figure 1 . Following ChIP, we use different PCR-based methods that allow one to analyze a locus of interest in the precipitated chromatin (see PCR-based Analysis of Immunoprecipitated Chromatin for details).

MATERIALS

Reagents

100-bp DNA size ladder (Promega)

Agarose

Antisera (affinity-purified)

These should be raised against histone peptides with mono-, di-, or trimethylation or acetylation at a specific lysine/arginine residue of interest. In addition, include a control precipitation with an (IgG) antiserum that is not directed against chromatin proteins.

Cell culture from which nuclei are to be extracted (1 x 107 to 1 x 108 cells are required; see Steps 9-16)

Cell culture medium, appropriate for cells of interest (see Step 10)

ChIP elution buffer

ChIP incubation buffer

Dialysis-lysis buffer

DNA loading buffer (6X)

Ethanol (70%, v/v)

Ethidium bromide solution (20 mg/ml in H2 O)

Glycogen solution (20 mg/ml) (Roche)

Isopropanol

Liquid nitrogen (for frozen tissue only; see Step 1)

Micrococcal nuclease (MNase; 10 units/µl in 50% [v/v] glycerol) (Amersham Bioscience)

Store in 10- to 20-µl aliquots at �20°C. Each aliquot should be used only once to ensure equal enzyme activity in different chromatin preparations.

MNase digestion buffer

MNase stop solution

NaCl (5 M)

Nuclei preparation buffer I, prechilled on ice

Nuclei preparation buffer II, prechilled on ice

Nuclei preparation buffer III, prechilled on ice

Phenol:chloroform:isoamyl alcohol 25:24:1 (v:v:v)

For extraction of genomic DNA, the phenol should be saturated beforehand with 100 mM Tris-Cl (pH 7.5) and stored at 4°C under 10 mM Tris-Cl (pH 7.5).

Phosphate-buffered saline (PBS), cold

Protein A (e.g., CL-4B Sepharose from Amersham Bioscience) or protein G Sepharose

Protein A and protein G are bacterial cell wall proteins that bind to the Fc region of antibodies. These proteins are covalently coupled to Sepharose. The choice between protein A or protein G Sepharose depends on the nature of the antibody used for ChIP. In general, protein A works best for rabbit polyclonal antisera and for mouse monoclonal antibodies from the IgG2a, IgG2b, and IgG3 subclasses. Protein G Sepharose is preferred for mouse IgG1 monoclonal antibodies and for polyclonal antisera from mouse, rat, sheep, and goat.

Proteinase K (10 mg/ml) (optional; see Step 70)

Sodium butyrate (optional; for analyzing histone acetylation only)

To analyze histone acetylation, we recommend adding sodium butyrate (to a final concentration of 5 mM) to the solutions used for the purification of nuclei and for the preparation of input chromatin. Sodium butyrate prevents loss of histone acetylation via the nonspecific action of endogenous histone deacetylases.

Sodium dodecyl sulfate (SDS; 10 %, w/v)

TBE buffer (1X)

TE buffer (1X, pH 7.5)

Tissue samples (fresh or frozen) from which nuclei are to be extracted (see Steps 1-8)

Trypsin solution (0.05% [w/v]) (Sigma)

Tubing preparation solution I

Tubing preparation solution II

Washing buffer A

Washing buffer B

Washing buffer C

Equipment

Centrifuges:

Bench-top centrifuge with cooling system for 1.5-ml microcentrifuge tubes   Centrifuge with a swing-out bucket rotor for 15-ml polypropylene tubes   High-speed centrifuge with cooling system and a swing-out bucket rotor for 14-ml polypropylene tubes

Dialysis tubing (0.5-mm thick, 10-kDa pore width) (VWR international)

Homogenizer, prechilled on ice

We use a tissue grinder/homogenizer (from BDH) that has a glass mortar (tube) and a pestle with a hard plastic head. The clearance between pestle and mortar is 0.15-0.25 mm.

Horizontal gel electrophoresis tank for agarose gels

Ice

Magnetic stirrer (see Step 32)

Microcentrifuge tubes (1.5 ml and 2.0 ml)

Chromatin immunoprecipitations and incubations with Protein A (G) Sepharose (Steps 44-69) are performed in microcentrifuge tubes. These tubes may be siliconized beforehand (e.g., with a 2% [v/v] dichloromethylsilane solution) in order to prevent nonspecific association of chromatin and antibodies to the inner walls of the tubes. In our laboratory, we have obtained comparable results with nonsiliconized and siliconized microcentrifuge tubes.

Microscope, inverted light (optional; see Step 8)

Mortar and pestle, prechilled in liquid nitrogen (for frozen tissue only; see Step 1)

Muslin cheesecloth

Prepare the cheesecloth by rinsing with H2 O and then autoclaving.

Parafilm

Pasteur pipettes

Polypropylene tubes, 14 ml (e.g., 17 x 100-mm Falcon tubes) and 15 ml (e.g., 17 x 120-mm Falcon conical tubes)

Rotating wheels at 4°C and room temperature

Spectrophotometer

Tray for staining gels (see Step 41)

Universal tubing clamps (5 mm) (Spectrum Laboratories)

UV lamp

Vortex mixer

Water bath set at 37°C

METHOD

Nuclei Preparation from Tissues and Cells

Steps 1-8 describe the purification of nuclei from tissue, while Steps 9-16 describe the purification of nuclei from cultured cells. To prevent chromatin degradation, all steps of the nuclei purification procedure should be performed on ice, or at 4°C (e.g., precool the centrifuge rotors). In addition, one set of micropipettes should be dedicated only to the preparation of nuclei, chromatin, and ChIP analysis, to avoid DNA contamination. Wear gloves throughout all procedures, and respect the safety rules, especially when handling phenol.

Purification of Nuclei from Tissue (2 h)

1. Dissect fresh tissue (maximum 0.2 g in total) and rinse it in cold PBS. See Troubleshooting. For many tissue types, frozen tissue (snap-frozen in liquid nitrogen) can be used as well. This tissue should first be crunched into powder in a mortar filled with liquid nitrogen; this powder is used for Step 2. The mortar should be prechilled with liquid nitrogen and the tissue kept constantly under liquid nitrogen. 2. Homogenize the tissue in a prechilled glass homogenizer with 5-10 ml of ice-cold nuclei preparation buffer I, until no clumps of cells persist (~10-20 strokes). Filter the suspension through four layers of muslin cheesecloth moistened beforehand with nuclei preparation buffer I. 3. Transfer the cell suspension to a 14-ml polypropylene tube, and centrifuge the samples in a swing-out rotor (3000g , 5 min, 4°C). 4. Pour off the supernatant, and resuspend the cells in 2 ml of ice-cold nuclei preparation buffer I. Add 2 ml of ice-cold nuclei preparation buffer II, mix gently, and place the tubes on ice a maximum of 5 minutes. See Troubleshooting. 5. Prepare two new 14-ml polypropylene tubes, each containing 8 ml of ice-cold nuclei preparation buffer III. Carefully layer 2 ml of each cell suspension (from Step 4) onto each 8-ml sucrose cushion. Cover each tube with a piece of Parafilm. 6. Centrifuge the tubes in a prechilled swing-out rotor (10,000g , 20 min, 4°C). The nuclei will form a pellet at the bottom of the tube, whereas the cytoplasmic components will remain in the top layer. At this step, the nuclear pellet should be white. 7. Carefully take off the supernatant with a Pasteur pipette. This is a critical step, as the top solution (which contains the detergent IGEPAL CA-630) should not come into contact with the nuclear pellet at the bottom of the tube. One way to achieve this is to remove the supernatant in about three steps, changing the Pasteur pipette each time. See Troubleshooting. 8. Resuspend the nuclear pellet in 1 ml of MNase digestion buffer, and keep the samples on ice. If possible, MNase digestion (Step 23) should be started immediately. Nuclei can be stored for up to 1 day at 4°C. At this point, the nuclei can be counted using a microscope slide for counting cells. The number of nuclei obtained per gram of tissue varies according to tissue type. For liver, for example, this protocol yields ~2 x 109 nuclei/g tissue.

Nuclei Preparation from Cultured Cells (2 h)

9. Culture 1 x 107 to 1 x 108 cells. Ensure that the cells are not grown beyond semiconfluency. 10. Rinse the cells in PBS, add 2 ml of trypsin solution (for adhering cells only), and incubate them at 37°C. When trypsination is complete, stop the reaction by adding 5 ml of culture medium to the cells. 11. Divide the cell suspension between two 14-ml polypropylene tubes, and centrifuge the samples in a swing-out rotor (4000g , 5 min, 4°C). 12. Pour off the supernatant, and resuspend the cells in 2 ml of ice-cold nuclei preparation buffer I. Add 2 ml of ice-cold nuclei preparation buffer II, mix gently, and place the tubes on ice a maximum of 5 minutes. See Troubleshooting. 13. Prepare two new 14-ml polypropylene tubes, each containing 8 ml of ice-cold nuclei preparation buffer III. Carefully layer 2 ml of each cell suspension (from Step 4) onto each 8-ml sucrose cushion. Cover each tube with a piece of Parafilm. 14. Centrifuge the tubes in a prechilled swing-out rotor (10,000g , 20 min, 4°C). The nuclei will form a pellet at the bottom of the tube, whereas the cytoplasmic components will remain in the top layer. At this step, the nuclear pellet should be white. 15. Carefully take off the supernatant with a Pasteur pipette. This is a critical step, as the top solution (which contains the detergent IGEPAL CA-630) should not come into contact with the nuclear pellet at the bottom of the tube. One way to achieve this is to remove the supernatant in about three steps, changing the Pasteur pipette each time. See Troubleshooting. 16. Resuspend the nuclear pellet in 1 ml of MNase digestion buffer, and keep the samples on ice. If possible, MNase digestion (Step 23) should be started immediately. Nuclei can be stored for up to 1 day at 4°C. At this point, the nuclei can be counted using a microscope slide for counting cells. The number of nuclei obtained per gram of tissue varies according to cell type.

Micrococcal Nuclease (MNase) Fractionation and Purification of Chromatin

Preparation of Dialysis Tubing (2 h including cooling time)

17. Cut the tubing into pieces of convenient length (10-20 cm). 18. Boil the tubes for 10 minutes in 0.5 liter of tubing preparation solution I. 19. Rinse the tubes twice in distilled H2 O. 20. Boil the tubes for 10 minutes in 0.5 liter of tubing preparation solution II. 21. Allow the tubes to cool down, and store them in tubing preparation solution II at 4°C. Ensure that the tubes are entirely submerged. 22. Before use, wash the tubing twice, inside and out, with H2 O. Several batches of dialysis tubing can be prepared and stored at 4°C for several weeks.

MNase Fractionation (30 min)

23. Aliquot the resuspended nuclei (from Step 8 or 16) into two 1.5-ml microcentrifuge tubes (500 µl in each tube). 24. Add 1 µl of MNase enzyme (10 units/µl in 50% [v/v] glycerol) to each tube, and mix gently. 25. Incubate the two tubes in a 37° water bath. One tube should be incubated for 2 minutes; the other tube should be incubated for 5 minutes. Keep these two digestions separate in subsequent steps, until fractions are chosen for combining (Step 43). 26. Add 20 µl of MNase stop solution to each tube. 27. Chill the samples on ice.

Recovery of Soluble Chromatin Fractions (16 h)

28. Centrifuge the 1.5-ml tubes with the MNase-digested nuclei (from Step 27) to pellet the nuclei (10,000 rpm, 10 min, 4°C). 29. Transfer the supernatant into another 1.5-ml tube. Store it for up to 1 day at 4°C. This supernatant contains the first soluble fraction of chromatin, S1, which comprises small fragments only. Do not discard the pellet. 30. Carefully resuspend the pellet in 500 µl of dialysis-lysis buffer. At this stage, we normally proceed to Step 31; however, a more expedient lysis/dialysis procedure used in our laboratory has yielded chromatin fragments of comparable quality. It replaces Steps 31-34 as follows: i. After resuspending the pellet in 500 µl of dialysis-lysis buffer, place the samples for 1 hour at 4°C. ii. Centrifuge the samples (10,000 rpm, 10 min, 4°C) in a microcentrifuge. iii. Proceed with Steps 35-36. 31. Close one side of the washed dialysis tubing (from Step 22) with a universal closure clamp. Transfer the 500 µl of resuspended nuclei (from Step 30) into the dialysis tube, and close the second side with another clamp. 32. Submerge the tube in 1-2 liters of dialysis-lysis buffer. Perform dialysis for 12-16 hours at 4°C with constant mild stirring using a magnetic stirrer. 33. Transfer the dialyzed nuclei into a 1.5-ml microcentrifuge tube. 34. Centrifuge the nuclei (10,000 rpm, 10 min, 4°C) in a microcentrifuge. 35. Transfer the supernatant in a new 1.5-ml microcentrifuge tube. Store it for up to 1 day at 4°C. This is the second soluble chromatin fraction, S2, comprising the larger fragments of chromatin that were removed from the nuclei during lysis/dialysis (Step 32). 36. Resuspend the pellet in 50 µl of dialysis-lysis buffer. Store it for up to 1 day at 4°C. This is chromatin fraction P.

Quality Control of Chromatin (3 h)

37. Measure the optical density (OD) of each fraction at 260 nm using a spectrophotometer. 38. Put 0.5 µg of each fraction (S1, S2, and P) in separate 1.5-ml microcentrifuge tubes. 39. Add 2 µl of 6X DNA loading buffer and 1 µl of 10% SDS to each tube. Adjust the volume to 10 µl, and mix gently. 40. Load the samples onto a standard 1.2% (w/v) agarose gel (~10-15 cm in length) in 1X TBE, with the 100-bp DNA ladder as a size control. Let the samples migrate at 2-3 V/cm until the fastest blue marker in the DNA loading buffer has migrated about halfway down the gel. 41. Stain the gel for 30 minutes in a tray with 500 ml of H2 O to which 20 µg of ethidium bromide has been added. 42. Remove background staining from the gel by rinsing it for 15 minutes in H2 O. 43. Observe the size of the chromatin fragments in each fraction by viewing the gel under a UV lamp, and take a photograph. See Figure 2 for an example of typical S1 and S2 fractions, as well as for advice on which fractions to combine for chromatin immunoprecipitation. The pellet fraction, P, consists of chromatin fragments that are usually longer than 5 nucleosomes in length.

See Troubleshooting.

Chromatin Immunoprecipitation

Incubation of Chromatin with Antiserum (16 h)

44. Mix comparable amounts of the S1 and S2 fractions in a 1.5-ml microcentrifuge tube (4-10 µg in total). It is important to mix similar amounts of chromatin from the S1 and S2 fractions. This ensures that chromosomal regions that are both less accessible and highly accessible to MNase are present in the input chromatin that will be used for ChIP. 45. Bring the volume to 1 ml with ChIP incubation buffer. 46. Add 5-10 µg of the antibody of choice. In order to control for nonspecific background signal, it is important to include a control precipitation with an (IgG) antiserum that is not directed against chromatin proteins. 47. Close the tubes, and seal the lids with Parafilm. 48. Rotate the tubes at 20-30 rpm for 12-16 hours at 4°C. During this incubation time, the antibodies will bind to their specific epitopes. See Troubleshooting.

Preparation of Protein A (G) Sepharose (45 min)

49. Weigh 0.25 g of protein A (or G) Sepharose beads into a 14-ml polypropylene tube. 50. Add 10 ml of H2 O, and mix. 51. Centrifuge the tubes for 3 minutes at 1500g in a swing-out rotor, and discard the supernatant. 52. Repeat Steps 50 and 51 four times. 53. Add 1 ml of H2 O, and resuspend the beads. 54. Distribute 100-µl aliquots into 10 1.5-ml microcentrifuge tubes. Store these aliquots at 4°C. These aliquots are used for the extraction of antibody-bound chromatin from ChIP experiments (starting in Step 55).

Extraction of Immunoprecipitated Chromatin with Protein A (G) Sepharose (6-7 h)

55. Add 50 µl of protein A (or G) Sepharose (from Step 54) to each tube (from Step 48). 56. Rotate the tubes at 20-30 rpm for 4 hours at 4°C.