Chromatin Immunoprecipitation

KEY CONCEPTS
- Chromatin immunoprecipitation allows detection of specific protein–DNA interactions in vivo.
- “ChIP on chip” or “ChIP-seq” allows mapping of all the protein-binding sites for a given protein across the entire genome.
Most of the methods discussed thus far in this chapter are in vitro methods that allow the detection or manipulation of nucleic acids or proteins that have been isolated from cells (or produced synthetically). Many other powerful molecular techniques have been developed, however. These techniques either allow direct visualization of the in vivo behavior of macromolecules (e.g., imaging of GFP fusions in live cells) or allow researchers to take a “snapshot” of the in vivo localization or interactions of macromolecules at a particular condition or point in time.
There are numerous proteins that function by interacting directly with DNA, such as chromatin proteins, or the factors that perform replication, repair, and transcription. Although much of our
understanding of these processes is derived from in vitro reconstitution experiments, it is critical to map the dynamics of protein–DNA interactions in living cells in order to fully understand these complex functions. The powerful technique of chromatin immunoprecipitation (ChIP) was developed to capture such interactions. (Chromatin refers to the native state of eukaryotic DNA in vivo, in which it is packaged extensively with proteins; this is discussed in the Chromatin chapter.) ChIP allows researchers to detect the presence of any protein of interest at a specific DNA sequence in vivo.
FIGURE 1 shows the process of ChIP. This method depends on the use of an antibody to detect the protein of interest. As was discussed earlier for western blots , this antibody can be against the protein itself, or against an epitope-tagged target.

FIGURE 1. Chromatin immunoprecipitation detects protein–DNA interactions in the native chromatin context in vivo. Proteins and DNA are crosslinked, chromatin is broken into small fragments, and an antibody is used to immunoprecipitate the protein of interest. Associated DNA is then purified and analyzed by either identifying specific sequences by PCR (as shown), or by labeling the DNA and applying to a tiling array to detect genome-wide interactions.
The first step in ChIP is typically the crosslinking of the cell (or tissue or organism) of interest by fixing it with formaldehyde. This serves two purposes: (1) It kills the cell and arrests all ongoing processes at the time of fixation, providing the snapshot of cellular activity; and (2) it covalently links any protein and DNA that are in very close proximity, thus preserving protein–DNA interactions through the subsequent analysis. ChIP can be performed on cells or tissues under different experimental conditions (e.g., different phases of the cell cycle, or after specific treatments) to look for changes in protein–DNA interactions under different conditions.
After crosslinking, the chromatin is then isolated from the fixed material and cleaved into small chromatin fragments, usually 200 to 1,000 bp each. This can be achieved by sonication, which uses high-intensity sound waves to nonspecifically shear the chromatin.
Nucleases (either sequence-specific or sequence-nonspecific) can also be used to fragment the DNA. These small chromatin fragments are then incubated with the antibody against the protein target of interest. These antibodies can then be used to immunoprecipitate the protein by pulling the antibodies out of the solution using heavy beads coated with a protein (such as Protein A) that binds to the antibodies.
After washing away unbound material, the remaining material contains the protein of interest still crosslinked to any DNA it was associated with in vivo. This is sometimes called a “guilt by association” assay, because the DNA target is only isolated due to its interaction with the protein of interest. The final stages of ChIP entail reversal of the crosslinks so that the DNA can be purified, and specific DNA sequences can be detected using PCR.
Quantitative (real-time) PCR is usually the method of choice for detecting the DNA of a limited number of targets of interest. In addition to revealing the presence of a specific protein at a given DNA sequence (e.g., a transcription factor bound to the promoter of a gene of interest), highly specialized antibodies can provide even more detailed information. For example, antibodies can be developed that distinguish between different posttranslational modifications of the same protein. As a result, ChIP can distinguish the difference between RNA polymerase II engaged in initiation at the promoter of a gene from pol II that has entered the elongation phase of transcription, because pol II is differentially phosphorylated in these two states , and antibodies exist that recognize these phosphorylation events.
Certain variations on the ChIP procedure allow researchers to query the localization of a given protein (or modified version of a protein) across large genomic regions—or even entire genomes. In two of the most powerful variations, known as ChIP-on-chip and ChIP-seq, the only difference from a conventional ChIP is the fate of the DNA that is purified from the immunoprecipitated material. Rather than querying specific sequences in this DNA via PCR, the DNA is either labeled in bulk and hybridized to a DNA microarray (ChIP on chip; usually a genome tiling array, such as described in the previous section), or is directly subjected to deep sequencing (ChIP-seq; this is now the most popular method). Either method allows a researcher to obtain a genome-wide footprint of all of the binding sites of the protein of interest. For example, putative origins of replication (which are difficult to identify in multicellular eukaryotes) can be detected en masse by performing a ChIP against proteins in the origin recognition complex (ORC).