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).

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Figure 1. Flowchart of the procedures used to investigate site-specific covalent modifications (i.e., methylation and acetylation) on histones. In summary, nuclei are purified from fresh/frozen tissues or from cells, and the chromatin, after fractionation with micrococcal nuclease (MNase), is purified from the nuclei. This \"input chromatin,\" made up of fragments of up to seven nucleosomes in length, is incubated with an antiserum directed against the histone modification of interest. The antibody-bound fraction is separated from the unbound fraction and, after extraction of genomic DNA from the bound and unbound fractions, PCR technologies are applied to specifically analyze the gene or chromosomal region of interest. Precipitated DNAs can be used as probes to hybridize DNA tiling arrays (ChIP on chip) as well.

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 H₂O)

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:

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 H₂O 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)

Nuclei Preparation from Cultured Cells (2 h)

Micrococcal Nuclease (MNase) Fractionation and Purification of Chromatin

Preparation of Dialysis Tubing (2 h including cooling time)

MNase Fractionation (30 min)

Recovery of Soluble Chromatin Fractions (16 h)

Quality Control of Chromatin (3 h)

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.

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Figure 2. Photograph of native chromatin preparation with fragments of, on average, one to five nucleosomes in length. For this experiment, nuclei were purified from primary fibroblasts and incubated with MNase for 6, 9, 12, and 15 minutes (lanes 1-4, respectively). The S1 fractions were obtained directly after MNase digestion, whereas the S2 fractions were recovered by overnight dialysis. Bands corresponding to chromatin fragments of one nucleosome (mono) to five nucleosomes (penta) in length are indicated (1.2% agarose gel). Fractions 1 and 2 of S1 were combined with fractions 3 and 4 of S2 for subsequent ChIP.

See Troubleshooting.

Chromatin Immunoprecipitation

Incubation of Chromatin with Antiserum (16 h)

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

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

DNA Extraction from Precipitated Chromatin (3 h)

Assessment of Precipitated Chromatin

TROUBLESHOOTING

Problem: How much tissue (and which kind) must be used to purify enough nuclei for a ChIP experiment?

[Step 1]

Solution: This protocol works well on tissues, such as liver, brain, lung, and placenta, and also on early mammalian embryos. In our laboratory, for instance, we have performed studies on 8.5-9.5 d.p.c. mouse embryos and placentas (Umlauf et al. 2004). Some 60 dissected embryos were used for each ChIP experiment. No more than ~0.2 g of tissue should be used for the volumes and tube sizes indicated in the protocol.

Problem: How can the yield of intact nuclei be maximized?

[Steps 4 and 12]

Solution: At Step 4 (or 12) of the nuclei purification procedure, it is critical not to extend the incubation in nuclei preparation buffer II for >10 minutes. For that reason, Step 5 should be initiated after 5 minutes of incubation in order to commence centrifugation (Step 6) at exactly 10 minutes after the addition of nuclei preparation buffer II in Step 4. Longer incubations can greatly reduce the yield of intact nuclei. For many tissues (liver, kidney, placenta), a final concentration of 0.2% of the nonionic detergent IGEPAL CA-630 (in nuclei preparation buffer II) will be enough to lyse the cellular membranes during the 10-minute incubation. However, we recommend testing 0.4% IGEPAL CA-630 for other tissues. For instance, this higher concentration of detergent slightly improves the yield of nuclei from brain and muscle tissues.

Problem: Is it a problem if the top layer (containing the IGEPAL CA-630) comes into contact with the nuclei pellet?

[Steps 7 and 15]

Solution: At Step 7 (or 15), it is essential that no traces of the top layer (containing the IGEPAL CA-630) come into contact with the nuclei pellet (even small traces of IGEPAL CA-630 may aberrantly affect the subsequent digestion of chromatin by MNase). Usually, we remove the top layer and the sucrose cushion from the tube by using Pasteur pipettes. This is done by aspirating from the surface of the solution, while changing the Pasteur pipette very often. If the top layer nevertheless comes in contact with the nuclei pellet, the pellet should be gently rinsed once with 1 ml of nuclei preparation buffer III before proceeding with Step 8 (or 16).

Problem: What can be done if, after the MNase digestion, the chromatin appears to be digested too much or too little?

[Step 43]

Solution: Fractionation of chromatin depends on the batch of MNase used, the concentration of the nuclei in the tube, the time of incubation, and the tissue type from which the nuclei were purified. If one observes too much digestion of the chromatin (i.e., almost all chromatin is digested to mono- and dinucleosome fragments), a lower concentration of MNase should be used. Inversely, in case little material is obtained in the S1 fraction, the amount of enzyme should be increased.

Problem: What can be done to improve ChIP when using chicken antisera that do not bind well to either protein A or protein G?

[Step 47]

Solution: We recommend adding 5 µg of a rabbit anti-chicken antiserum directly after Step 47 for a second precipitation of 3-4 hours, before proceeding with the extraction of the antibody-bound chromatin.

Problem: When an antiserum is used for the first time, how does one verify that the histone modification it is directed against has become enriched in the antibody-bound fraction?

[Step 69]

Solution: This can be done by purifying the histone proteins from the antibody-bound fraction (from Step 69), followed by electrophoresis through acid-urea-Triton gels. After electrophoresis, proteins are Western-blotted to nylon filters, which are immunostained with the antiserum following standard procedures (Gregory et al. 2001).

DISCUSSION

There are different ways to obtain input chromatin. Several groups in the field prepare \"cross-linked chromatin,\" for example, by chemically cross-linking proteins and DNA with specific substances such as formaldehyde. However, usually only a small fraction of the chromatin is precipitated, and this method relies on random shearing after cross-linking, which does not always produce small-enough chromatin fragments at the regions of interest. For this reason, and to be able to conduct experiments on fresh and frozen tissues, we and others have preferred to make use of \"native chromatin.\" In our protocol (Fig. 1), the chromatin is fractionated by incubation of purified nuclei with micrococcal nuclease (MNase), an enzyme that cleaves preferentially at the linker DNA between the nucleosomes. By performing partial digestions with MNase, it is possible to obtain native chromatin fragments of, on average, one to five nucleosomes in length (Fig. 2). These oligo-nucleosome fragments are purified from the nuclei and are then used to perform ChIP. The choice of native chromatin as the input material for ChIP is advantageous because the epitopes, recognized by the antibody, remain intact during the chromatin preparation. As a consequence, native chromatin tends to give higher levels of precipitation for a specific histone modification than formaldehyde cross-linked chromatin. Because fractionation occurs between the nucleosomes, rather than randomly, precipitated native oligo-nucleosome fragments are also particularly suitable to be used for \"ChIP on chip.\" In a recent ChIP on chip study (Bernstein et al. 2006), both native and formaldehyde-cross-linked chromatin were precipitated with antisera against histone modifications. DNA samples extracted from the precipitated chromatin were used as probes to hybridize DNA tiling arrays covering many large chromosomal regio, ns. In this large-throughput study, results obtained with native chromatin were very similar to those obtained with cross-linked chromatin. For locus-specific, smaller-scale studies, amplification by the polymerase chain reaction (PCR) remains the method of choice. Different PCR-based approaches can be used to determine how much DNA is precipitated at a site of interest (see PCR-based Analysis of Immunoprecipitated Chromatin).

Although ChIP is presently the best methodology to analyze histone modifications at specific chromosomal loci, it has several limitations. First, unlike DNA methylation studies, ChIP does not allow analysis of histone modifications in individual cells or on individual chromosomes. ChIP studies are always performed on populations of (cultured) cells or on tissue samples comprising many cells. Moreover, although sequential precipitations with different antisera can be done (Bernstein et al. 2006), or antisera against combinations of different histone modifications can be used, it is not easy to determine whether there are specific combinations of covalent modifications on individual histones at a given locus. Again, this is because many cells are used for chromatin purification and ChIP, and chromatin is usually fractionated into fragments that comprise multiple nucleosomes. Last, it should be noted that quantification of the levels of histone modifications at specific chromosomal loci is difficult to obtain by ChIP, because levels of precipitation do not depend solely on the local abundance of the modification studied. They also depend on the quality of the prepared chromatin, on the specificity and concentration of the antiserum used, and on the global abundance of the histone modification that is being studied. Factors that can influence the outcome of the experiment are (1) the distribution of the histone modifications on the chromosomes, (2) the amount of antiserum used, and (3) the \"strength\" of the antibodies (i.e., the affinity for their epitope). On the other hand, the efficiency of precipitation of modified histones at a locus of interest greatly depends on whether the modification is common or rare in the genome. For instance, a rare modification (e.g., H3-K4 methylation) gives usually good precipitation at the site where it is present. This can be explained by the fact that, in the ChIP, the quantity of antibody added to the tube is high enough to precipitate all the chromatin that carries that specific modification. However, for a modification that is abundant in the genome, the indicated amount of antibody (5-10 µg) sometimes does not precipitate all the chromatin that has the modification. These different factors should be taken into account when comparing different chromatin immunoprecipitation experiments.

ACKNOWLEDGMENTS

We thank Richard Gregory and David Umlauf for design of methodologies, and Bryan M. Turner and Laura P. O’Neill (Birmingham, UK) for introducing us to ChIP on unfixed chromatin. The CNRS, the Association pour la Recherche sur le Cancer (ARC), and the ESF EuroCORES Programme EuroSTELLS are acknowledged for grant support.

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