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 defining 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.
Briefly, the conventional method is as follows:
- DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP).
- The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion.
- Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody.
- The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo.
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 micrococcalnuclease digestion.
Cross-linked ChIP (XChIP)
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 - 500bp 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).
Native ChIP (NChIP)
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 (200bp) to five nucleosomes (1000bp) in length.
Thereafter, methods similar to XChIP are used for clearing the cell debris, immunoprecipitating the protein of interest, removing protein from the immunoprecipated complex, and purifying and analyzing the complex-associated DNA.
Comparison of XChIP and NChIP
The major advantage for NChIP is antibody specificity. It is important to note that most antibodies to modified histones are raised against unfixed, synthetic peptide antigens and that 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 compare to NChIP.
But XChIP and NChIP have different aims and advantages relative to each other. XChIP is for mapping target sites of transcription factors and other chromatin associated proteins; NChIP is for mapping target sites of histone modifiers (see Table 1).
Table 1 Advantages and disadvantages of NChIP and XChIP
|Advantages||Suitable for transcriptional factors, or any other weakly binding chromatin associated proteins.|
Applicable to any organisms where native protein is hard to prepare
|Testable antibody specificity|
Better antibody specificity as target protein naturally intact
Better chromatin and protein recovery efficiency due to better antibody specificity
|Disadvantages||Inefficient chromatin recovery due to antibody target protein epitope disruption|
May cause false positive result due to fixation of transient proteins to chromatin
Wide range of chromatin shearing size due to random cut by sonication.
|Usually not suitable for non-histone proteins|
Nucleosomes may rearrange during digestion
History and New ChIP methods
In 1984 John T. Lis and David Gilmour, at the time a graduate student in the Lis lab, used UV irradiation, a zero-length protein-nucleic acid crosslinking agent, to covalently cross-link proteins bound to DNA in living bacterial cells. Following lysis of cross-linked cells and immunoprecipitation of bacterial RNA polymerase, DNA associated with enriched RNA polymerase was hybridized to probes corresponding to different regions of known genes to determine the in vivo distribution and density of RNA polymerase at these genes. A year later they used the same methodology to study distribution of eukaryotic RNA polymerase II on fruit fly heat shock genes. These reports are considered the pioneering studies in the field of chromatin immunoprecipitation. XChIP was further modified and developed by Alexander Varshavsky and co-workers, who examined distribution of histone H4 on heat shock genes using formaldehyde cross-linking. This technique was extensively developed and refined thereafter. NChIP approach was first described by Hebbes et al., 1988, and also been developed and refined quickly. The typical ChIP assay usually take 4–5 days, and require 106~ 107 cells at least. Now new techniques on ChIP could be achieved as few as 100~1000 cells and complete within one day.
- Solid-state ChIP: This novel method ChIP uses discs of inert, porous polymer functionalized with either Protein A or G in spin columns or microplates. The chromatin-antibody complex is selectively retained by the disc and eluted to obtain enriched DNA for downstream applications such as qPCR and sequencing. The porous environment is specifically designed to maximize capture efficiency and reduce non-specific binding. Due to less manual handling and optimised protocols, ChIP can be performed in 5 hours.
- Carrier ChIP (CChIP): This approach could use as few as 100 cells by adding Drosophila cells as carrier chromatin to reduce loss and facilitate precipitation of the target chromatin. However, it demands highly specific primers for detection of the target cell chromatin from the foreign carrier chromatin background, and it takes two to three days.
- Fast ChIP (qChIP): The fast ChIP assay reduced the time by shortening two steps in a typical ChIP assay: (i) an ultrasonic bath accelerates the rate of antibody binding to target proteins—and thereby reduces immunoprecipitation time (ii) a resin-based (Chelex-100) DNA isolation procedure reduces the time of cross-link reversal and DNA isolation. However, the fast protocol is suitable only for large cell samples (in the range of 106~107). Up to 24 sheared chromatin samples can be processed to yield PCR-ready DNA in 5 hours, allowing multiple chromatin factors be probed simultaneously and/or looking at genomic events over several time points.
- Quick and quantitative ChIP (Q2ChIP): The assay uses 100,000 cells as starting material and is suitable for up to 1,000 histone ChIPs or 100 transcription factor ChIPs. Thus many chromatin samples can be prepared in parallel and stored, and Q2ChIP can be undertaken in a day.
- MicroChIP (µChIP): chromatin is usually prepared from 1,000 cells and up to 8 ChIPs can be done in parallel without carriers. The assay can also start with 100 cells, but only suit for one ChIP. It can also use small (1 mm3) tissue biopsies and microChIP can be done within one day.
- Matrix ChIP: This is a microplate-based ChIP assay with increased throughput and simplified the procedure. All steps are done in microplate wells without sample transfers, enabling a potential for automation. It enables 96 ChIP assays for histone and various DNA-bound proteins in a single day.
- Pathology-ChIP (PAT-ChIP): This technique allows ChIP from pathology formalin-fixed and paraffin-embedded tissues and thus the use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets. 
ChIP has also been applied for genome wide analysis by combining with microarray technology (ChIP-on-chip) or second generation DNA-sequencing technology (Chip-Sequencing). ChIP can also combine with paired-end tags sequencing in Chromatin Interaction Analysis using Paired End Tag sequencing (ChIA-PET), a technique developed for large-scale, de novo analysis of higher-order chromatin structures.
- Large Scale assays using ChIP is challenging using intact model organisms. This is because antibodies have to be generated for each TF, or, alternatively, transgenic model organisms expressing epitope-tagged TFs need to be produced.
- Researchers studying differential gene expression patterns in small organisms also face problems as genes expressed at low levels, in a small number of cells, in narrow time window.
- ChIP experiments cannot discriminate between different TF isoforms (Protein isoform).
- ChIP-exo, a technique that adds exonuclease treatment to the ChIP process to obtain up to single base pair resolution of binding sites
- ChIP-on-chip, combines ChIP with microarray technology
- DamID, an alternative location mapping technique that does not require specific antibodies
- RIP-Chip, a similar technique to analyze RNA-protein interactions
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ChIP-seq is a wonderful technique that allows us to interrogate the physical binding interactions between protein and DNA using next-generation sequencing. In this article, I’ll give a brief review of ChIP and introduce the chromatin immunoprecipitation sequencing technique (ChIP-seq), which combines ChIP with next-generation sequencing.
What is chromatin immunoprecipitation?
Chromatin immunoprecipitation (ChIP) allows us to determine protein-binding sites on DNA. Chromatin is the complex of DNA packaged with histone proteins into nucleosomes. ChIP makes use of reversible cross-links made between DNA and associated proteins by formaldehyde fixation of cells or tissue. The fixed chromatin is physically sheared and DNA fragments associated with a particular protein are selectively immunoprecipitated and analysed. Analysis can be on a locus-by-locus basis using PCR, but more commonly ChIP is interrogated with microarrays (ChIP-chip) or next-generation sequencing (ChIP-seq).
How does ChIP-chip work?
ChIP-on-chip, or ChIP-chip, combines chromatin immunoprecipitation with microarray (chip) analysis. In 2001, Jason Lieb in Pat Brown’s lab at Stanford published the first ChIP-chip paper (Lieb 2001). In this method, the DNA fragments that precipitate with a particular protein are applied to a microarray chip for analysis. This generates a global picture of where the protein binds, in contrast to simply interrogating single binding sites by PCR. While this was a revolutionary approach, the technique is limited by the array technologies used. Firstly, you need a microarray for the genome you want to study, and in the early to mid 2000’s this was often not the case. Secondly, those microarrays are limited by the quality of the reference genome and the ability to design probes that will work on an array. Thirdly, arrays are limited in how much of the genome can be represented, and usually tile across the genome at 100’s or 1000’s of bp resolution. There are also issues about bias in amplification of the ChIP DNA fragments, normalisation of array data, and comparability of array platforms.
How does ChIP-seq work?
Chromatin immunoprecipitation sequencing, or ChIP-seq, combines ChIP with next-generation sequencing (Barski 2007, Johnson 2007). ChIP-seq protocols have been adapted from ChIP-chip methods: proteins are cross-linked to their bound DNA by formaldehyde treatment, cells are homogenized, and chromatin is sheared and immunoprecipitated with antibody-bound magnetic beads. The immunoprecipitated DNA is then used as the input for a next-generation sequencing library prep protocol, where it is sequenced and analysed for DNA binding sites. See the figure below for a summary of the ChIP-seq workflow, and an example of ChIP-seq results (reproduced with kind permission from Dominic Schmidt (Schmidt 2009)).
Although the majority of the 400 or so papers published so far have been analysed on the Illumina platform, ChIP-seq can be performed on any next-generation sequencer (Wold 2008). ChIP-seq has been widely adopted since it was first reported in 2007. In fact, it has almost totally supplanted ChIP-Chip, since it allows genome-wide analysis and does not have the limitations discussed above.
What can you actually do with ChIP-seq
ChIP-seq is a powerful tool and versatile tool, and there are many great examples of ChIP-seq use in the literature. I have picked a couple of my favourites (from work performed in the core facility I manage) to illustrate what is possible, and have included examples where ChIP-seq has inspired the development of new methods:
- Antoni Hurtado, et al. performed knock-down of the FoxA1 “pioneer factor”, resulting in reduced binding by the estrogen receptor (ER) at over 50% of known ER binding sites. They showed that FoxA1 is an important regulator of ER-mediated transcription, suggesting it may be a new and important therapeutic target in breast cancer (Hurtado 2011).
- Dominic Shmidt, et al. used ChIP-seq to investigate the evolution of transcription factor binding. They focused on CEBPA and HNF4 binding in the liver tissue of five vertebrate species: human, mouse, dog, opossum and chicken. ChIP-chip would have been almost impossible given the different species involved and complexities in designing probes (Schmidt 2010).
Further modifications to the ChIP-seq method have led to several new methods, including ChIP-seq for RNA-protein interaction analysis, as well as DNase-seq and FAIRE-seq, both of which are used to identify regulatory regions in DNA.
In summary, ChIP-seq is a mature technique that is preferable to ChIP-chip and can be used by almost any group with an interest in DNA:Protein interaction analysis. How could you use ChIP-seq in your research?
Barski et al, High-resolution profiling of histone methylations in the human genome. Cell 129 (2007).
Hurtado et al, FOXA1 is a key determinant of estrogen receptor function and endocrine response.Nature Genetics (2011).
Johnson et al, Genome-wide mapping of in vivo protein–DNA interactions. Science (2007).
Lee et al, Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protoc (2006).
Lieb et al, Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat Genet. 2001.
Morozova & Marra, Applications of next-generation sequencing technologies in functional genomics. Genomics (2008).
Schmidt et al, ChIP-seq: Using high-throughput sequencing to discover protein–DNA interactions. Methods (2009).
Schmidt et al, Five-Vertebrate ChIP-seq Reveals the Evolutionary Dynamics of Transcription Factor Binding Science (2010).
Wold & Myers, Sequence census methods for functional genomics. Nat. Methods (2008).