Chromatin Immunoprecipitation (ChIP) is the tried and true workhorse of chromatin analysis; new and improved variations arise, but the basic idea of ChIP is at the heart of all chromatin analyses.
First, the chromatin is cross-linked using formaldehyde, preserving DNA-protein and protein-protein interactions (Jackson, 1978). The cross-linked chromatin is then sonicated or digested into small fragments. An antibody is used to immunoprecipitate chromatin fragments containing the specific protein of interest (POI). The crosslinks are then reversed and the DNA purified. The DNA sequences are identified using PCR (ChIP-qPCR), microarray (ChIP-chip), or sequencing (ChIP-seq) (Collas, 2010).
Using bioinformatics algorithms and software, “peaks” of DNA enrichment are called (Wilbanks and Facciotti, 2010). These peaks represent DNA-POI interactions in the genome; they can be hundreds of bases, depending on the resolution of the technology. DNA regions that associate with the POI are thus identified.
ChIP has more derivatives than a freshman calculus textbook. The antibody used can be against any protein or protein residues in chromatin. For example an antibody against a histone modification can be used to map its distribution under experimental or disease conditions.
The assay coupled to ChIP allows for further specialization (ie. ChiP-chip vs. ChIP-seq). Furthermore, by adding additional steps to the ChIP process, researchers have developed offshoot techniques such as ChIP-exo and ChIA-PET. These offshoots are often created in an attempt to better address a certain kind of biological questions, alter the resolution, or alter the scale of the experiment.
Advanced ChIP Technologies
ChIP-chip: The first, and most simple of the ChIP technologies (it also has the most fun to say name). ChIP-chip couples chromatin IP to microarray analysis allowing genome-wide analysis of protein or modifications of interest distribution.
ChIP-Seq: The star of chromatin analysis until ChIP-seq came along and stole the limelight. ChIP-seq uses the same chromatin IP procedures as ChIP-chip; however, it couples it with quantitative next-generation sequencing technology to detect enrichment peaks
ChIP-exo: A specialized version of ChIP used to very specifically map protein of interest (POI) binding sites in the genome via the addition of a DNA digestion step to ChIP-seq.
ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing): Combines ChIP with chromatin conformation capture (3C) technology to detect when distant DNA regions interact with each other via a protein of interest.
ChIP Additional Reading
Collas, P. (2010). The current state of chromatin immunoprecipitation. Mol. Biotechnol. 45, 87-100.
This review covers the basics of the principles of ChIP as well as many of derivate technologies and how each varies from the basic protocol.
Furey, T.S. (2012). ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat. Rev. Genet. 13, 840-852.
This review focuses on the principles of ChiP-seq, but also a good resource for some of the other related techniques. The author discusses how the data outputs vary between techniques and the advantages of each.
- Collas, P. (2010). The Current State of Chromatin Immunoprecipitation. Mol. Biotechnol. 45, 87-100.
- Jackson, V. (1978). Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent. Cell 15, 945-954.
- Wilbanks, E.G., and Facciotti, M.T. (2010). Evaluation of algorithm performance in ChIP-seq peak detection. PLoS One 5, e11471.