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 regions. 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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