Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

Briefly, the conventional method is as follows:

There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly cross-linked chromatin sheared by sonication called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal nuclease digestion.^{[citation needed]}

Cross-linked ChIP is mainly suited for mapping the DNA target of transcription factors or other chromatin-associated proteins, and uses reversibly cross-linked chromatin as starting material. The agent for reversible cross-linking could be formaldehyde or UV light. Then the cross-linked chromatin is usually sheared by sonication, providing fragments of 300–1000 base pairs (bp) in length. Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin. Chromatin fragments of 400–500 bp have proven to be suitable for ChIP assays as they cover two to three nucleosomes.

Cell debris in the sheared lysate is then cleared by sedimentation and protein–DNA complexes are selectively immunoprecipitated using specific antibodies to the protein(s) of interest. The antibodies are commonly coupled to agarose, sepharose, or magnetic beads. Alternatively, chromatin-antibody complexes can be selectively retained and eluted by inert polymer discs. The immunoprecipitated complexes (i.e., the bead–antibody–protein–target DNA sequence complex) are then collected and washed to remove non-specifically bound chromatin, the protein–DNA cross-link is reversed and proteins are removed by digestion with proteinase K. An epitope-tagged version of the protein of interest, or in vivo biotinylation can be used instead of antibodies to the native protein of interest.

The DNA associated with the complex is then purified and identified by polymerase chain reaction (PCR), microarrays (ChIP-on-chip), molecular cloning and sequencing, or direct high-throughput sequencing (ChIP-Seq).^{[citation needed]}

Native ChIP is mainly suited for mapping the DNA target of histone modifiers. Generally, native chromatin is used as starting chromatin. As histones wrap around DNA to form nucleosomes, they are naturally linked. Then the chromatin is sheared by micrococcal nuclease digestion, which cuts DNA at the length of the linker, leaving nucleosomes intact and providing DNA fragments of one nucleosome (200 bp) to five nucleosomes (1000 bp) in length. Thereafter, methods similar to XChIP are used for clearing the cell debris, immunoprecipitating the protein of interest, removing protein from the immunoprecipitated complex, and purifying and analyzing the complex-associated DNA.^{[citation needed]}

The major advantage of NChIP is antibody specificity. Most antibodies to modified histones are raised against unfixed, synthetic peptide antigens. The epitopes they need to recognize in the XChIP may be disrupted or destroyed by formaldehyde cross-linking, particularly as the cross-links are likely to involve lysine e-amino groups in the N-terminals, disrupting the epitopes. This is likely to explain the consistently low efficiency of XChIP protocols compared to NChIP.