Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChIP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.

Introduction

DNA-protein interactions play a key role in the regulation of important cellular functions including gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. The 3-dimensional structure of chromatin is maintained by the binding of histones and other regulatory proteins to the DNA. It is vital to know how DNA-binding proteins affect the functioning of any particular gene and to identify which particular protein binds to a specific DNA sequence in vivo. Earlier methods devised to study the DNA-protein interactions involve in vitro methods, which are not within the context of the cell, and thereby have limited utility. Chromatin immunoprecipitation (ChIP) has become a very widely used technique for determining the in vivo location of binding sites of various transcription factors (1,2,3), histones (4,5), and other proteins (6). Because the proteins are captured at the sites of their binding with DNA, ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniques such as PCR and real-time PCR (7), PCR with single-stranded conformational polymorphism (8), Southern blot analysis (9), Western blot analysis (10), cloning, and microarray (1,2,3). The resulting versatility has increased the potential of this technique.

ChIP usually involves cross-linking of the chromatin-bound proteins by formaldehyde (11,12,13), followed by sonication or nuclease treatment to obtain small DNA fragments ((Figure 1)). Immunoprecipitation is then carried out using specific antibodies to the DNA-binding protein of interest. The DNA is then released from the proteins and analyzed using various methods. ChIP has also been used to study RNA-protein interactions (14).

Figure 1.

There are two main types of ChIP assays: X-ChIP and N-ChIP. The X-ChIP method utilizes fixed chromatin fragmented by sonication (15), while the N-ChIP uses native chromatin (16), which is unfixed and nuclease digested. The advantages and disadvantages of the two techniques are summarized in (Table 1). In the N-ChIP assay, immuno-precipitation is very efficient, and the precipitated DNA can be studied without further PCR amplification (16). Antibody binding to unfixed protein is generally stronger than to the fixed protein, thus the specificity of immuno-precipitation of N-ChIP is higher than that of X-ChIP. N-ChIP is most suited to study tightly bound proteins such as histones and thus is not preferred for studying nonhistone proteins, which may get rearranged during processing. Another disadvantage of the N-ChIP protocol is that not all of the nuclease-digested chromatin is solubilized, thus a subset of chromatin remains with the nuclear pellet and is therefore excluded from the ChIP analysis. X-ChIP, on the other hand, is ideal for studying all types of proteins including nonhistone proteins. Because the X-ChIP assay is more sensitive than N-ChIP, it requires fewer cells and lesser amounts of antibody. Moreover, formaldehyde fixation reduces the possibility of protein rearrangement. The disadvantage of X-ChIP is that excess cross-linking leads to difficulty in fragmentation of the DNA to the desired size (9). This causes a smaller amount of DNA retrieval, necessitating post-ChIP amplification of the recovered DNA. There is also a risk that formaldehyde might fix transient-DNA protein interactions as well, thus interfering in the detection of stable interactions (16).

Critical Parameters

Although ChIP is a highly versatile procedure, it has several limitations and requires the optimization of conditions for a successful DNA extraction. The most vital step is probably the binding of antibody, and the quality of antibody is crucial for the recovery of DNA fragments. In this section, we will discuss the critical parameters of the reaction and the alternatives available for refining the process. These parameters are summarized in (Table 2).

Fixation

The first step of the technique is the cross-linking of DNA and proteins. Formaldehyde is the most commonly used cross-linking agent (9). The most important advantage of using formaldehyde is the ease of reversibility of the cross-links and its ability to form bonds that span a distance of approximately 2 angstroms (17). This means that formaldehyde is able to bind molecules in close association with each other. Generally, formaldehyde is added to the medium in the cell culture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues and whole mouse embryos has also been done (10). In one experiment, chromatin cross-linking was carried out by the infusion of 1% formaldehyde into mouse liver in vivo, and this was followed by the dissection and mincing of the liver tissues (18). Formaldehyde causes DNA-protein, RNA-protein, as well as protein-protein cross-linking. Formaldehyde primarily targets lysine amino group and side chains of adenine, guanine, and cytosine. It is a dipolar substance and forms a Schiff's base with amino and imino groups (15). Optimal conditions for formaldehyde cross-linking need to be determined using a time-course experiment ((Figure 2)). Usually, nucleosomal proteins cross-link faster; longer incubation is needed for nonhistone proteins. Overfixation with formaldehyde can result in difficulty in fragmentation by sonication ((Figure 2)). Longer exposure to formaldehyde leads to a loss of immunoprecipitated material, although the reason for this is not understood. It is possible that prolonged cross-linking favors the binding of nucleosome-associated proteins, thereby “masking” histone epitopes. Alternatively, epitopes may be lost because of the engagement of lysines and other preferred formal-dehyde-reactive sites (15). The reaction is quenched using 0.125 M glycine (9). Reversal of cross-linking is achieved by heating at 65°C for 6 h (4).