Chromatin immunoprecipitation
Encyclopedia
Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation
experimental technique used to investigate the interaction between protein
s and DNA
in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factor
s on promoters or other DNA binding site
s, and possibly defining cistrome
s. 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 method is as follows: protein and associated chromatin
in a cell lysate
are temporarily bonded, the DNA-protein complexes (chromatin-protein) are then sheared and DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated, and the associated DNA fragments are purified and their sequence is determined. These DNA sequences are supposed to be associated with the protein of interest in vivo.
ed chromatin sheared by sonication
called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal
nuclease
digestion.
or UV light
. Then the cross-linked chromatin are usually sheared by sonication, providing fragments of 300–1000 base pair
s (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 nucleosome
s.
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. 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
.
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
).
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.
specificity. It is important to note that most antibodies to modified histones are raised against unfixed, synthetic peptide antigens and that the epitope
s 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 advantage against each other, XChIP is for mapping target site of transcription factors and other chromatin associated proteins, NChIP is for mapping the target site of histone modifiers (see Table 1).
Table 1 Advantages and disadvantages of NChIP and XChIP
and David Gilmour, at the time a graduate student in his lab, utilized UV irradiation to covalently cross-link proteins in contact with neighboring DNA in intact 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 could perhaps be considered the pioneering work in the field of chromatin immunoprecipitation. XChIP was further modified and developed by Alexander Varshavsky
and co-workers ,where they examined distribution of histone H4
on heat shock genes using formaldehyde cross-linking. This technique has been 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.
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.
Immunoprecipitation
Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins...
experimental technique used to investigate the interaction between protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s on promoters or other DNA binding site
DNA binding site
DNA binding sites are a type of binding site found in DNA where other molecules may bind. DNA binding sites are distinct from other binding sites in that they are part of a DNA sequence and they are bound by DNA-binding proteins...
s, and possibly defining cistrome
Cistrome
CistromeThis term http://cistrome.pbwiki.com was coined by investigators at the Dana-Farber Cancer Institute and Harvard Medical School to define the set of cis-acting targets of a trans-acting factor on a genome scale...
s. ChIP also aims to determine the specific location in the genome that various histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
modifications are associated with, indicating the target of the histone modifiers.
Briefly, the method is as follows: protein and associated chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
in a cell lysate
Cell disruption
Cell disruption is a method or process for releasing biological molecules from inside a cell.- Choice of disruption method:The production of biologically-interesting molecules using cloning and culturing methods allows the study and manufacture of relevant molecules.Except for excreted molecules,...
are temporarily bonded, the DNA-protein complexes (chromatin-protein) are then sheared and DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated, and the associated DNA fragments are purified and their sequence is determined. These DNA sequences are supposed to be associated with the protein of interest in vivo.
Typical ChIP
There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly cross-linkCross-link
Cross-links are bonds that link one polymer chain to another. They can be covalent bonds or ionic bonds. "Polymer chains" can refer to synthetic polymers or natural polymers . When the term "cross-linking" is used in the synthetic polymer science field, it usually refers to the use of...
ed chromatin sheared by sonication
Sonication
thumb|right|A sonicator at the [[Weizmann Institute of Science]] during sonicationSonication is the act of applying sound energy to agitate particles in a sample, for various purposes. In the laboratory, it is usually applied using an ultrasonic bath or an ultrasonic probe, colloquially known as...
called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal
Micrococcus
Micrococcus is a genus of bacteria in the Micrococcaceae family. Micrococcus occurs in a wide range of environments, including water, dust, and soil. Micrococci have Gram-positive spherical cells ranging from about 0.5 to 3 micrometers in diameter and are typically appear in tetrads...
nuclease
Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
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 formaldehydeFormaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...
or UV light
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
. Then the cross-linked chromatin are usually sheared by sonication, providing fragments of 300–1000 base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
s (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 nucleosome
Nucleosome
Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
s.
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
Sepharose
Sepharose is a tradename for a crosslinked, beaded-form of a polysaccharide polymer material extracted from seaweed. It is crosslinked through lysine side chains. Iodoacetyl functional groups can be added to selectively bind cysteine side chains and this method is often used to immobilize peptides....
or magnetic beads. 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
Proteinase K
Proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album . Proteinase K is able to digest native keratin , hence, the name "Proteinase K"...
.
The DNA associated with the complex is then purified and identified by polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR), microarrays (ChIP-on-chip), molecular cloning and sequencing, or direct high-throughput sequencing (ChIP-Seq
Chip-Sequencing
ChIP-Sequencing, also known as ChIP-Seq, is used to analyze protein interactions with DNA. ChIP-Seq combines chromatin immunoprecipitation with massively parallel DNA sequencing to identify the cistrome of DNA-associated proteins. It can be used to precisely map global binding sites for any...
).
Native ChIP (NChIP)
Native ChIP is mainly suited for mapping the DNA target of histoneHistone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
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 antibodyAntibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
specificity. It is important to note that most antibodies to modified histones are raised against unfixed, synthetic peptide antigens and that the epitope
Epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...
s 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
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
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 advantage against each other, XChIP is for mapping target site of transcription factors and other chromatin associated proteins, NChIP is for mapping the target site of histone modifiers (see Table 1).
Table 1 Advantages and disadvantages of NChIP and XChIP
| XChIP | NChIP | |
|---|---|---|
| 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. LisJohn T. Lis
John T. Lis is the Barbara McClintock Professor of Molecular Biology & Genetics at the Cornell University College of Agriculture and Life Sciences. Dr. Lis was a recipient of a Guggenheim Fellowship in 2000 for his research on protein templating in the propagation of gene activity.-Research:Dr...
and David Gilmour, at the time a graduate student in his lab, utilized UV irradiation to covalently cross-link proteins in contact with neighboring DNA in intact 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
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
on fruit fly heat shock genes. These reports could perhaps be considered the pioneering work in the field of chromatin immunoprecipitation. XChIP was further modified and developed by Alexander Varshavsky
Alexander Varshavsky
Alexander Varshavsky is a Russian-American biochemist and recipient of the Albert Lasker Award for Basic Medical Research, the Wolf Prize in Medicine and the Louisa Gross Horwitz Prize from Columbia University in 2001 for his research on ubiquitination...
and co-workers ,where they examined distribution of histone H4
Histone H4
Histone H4 is one of the 5 main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N terminal tail, H4 is a structural component of the nucleosome, and is subject to covalent modification, including acetylation and methylation,...
on heat shock genes using formaldehyde cross-linking. This technique has been 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.
- Carrier ChIP (CChIP): This approach could use as few as 100 cells by adding DrosophilaDrosophilaDrosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
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 biopsiesBiopsyA biopsy is a medical test involving sampling of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically...
and microChIP can be done within one day.
- Matrix ChIP: This is a microplateMicrotiter plateA Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
-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.
ChIP has also been applied for genome wide analysis by combining with microarray technology (ChIP-on-chip
ChIP-on-chip
ChIP-on-chip is a technique that combines chromatin immunoprecipitation with microarray technology . Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo...
) or second generation DNA-sequencing technology (Chip-Sequencing
Chip-Sequencing
ChIP-Sequencing, also known as ChIP-Seq, is used to analyze protein interactions with DNA. ChIP-Seq combines chromatin immunoprecipitation with massively parallel DNA sequencing to identify the cistrome of DNA-associated proteins. It can be used to precisely map global binding sites for any...
). ChIP can also combine with paired-end tags
Paired-end Tags
Paired-end tags, also known as PET, refer to the short sequences at the 5’ and 3’ ends of the DNA fragment of interest, which can be a piece of genomic DNA or cDNA. These short sequences are called tags or signatures because, in theory, they should contain enough sequence information to be uniquely...
sequencing in Chromatin Interaction Analysis using Paired End Tag sequencing
ChIA-PET
Chromatin Interaction Analysis by Paired-End Tag Sequencing is a technique that incorporates chromatin immunoprecipitation -based enrichment, chromatin proximity ligation, Paired-End Tags, and ultra-high-throughput sequencing to determine de novo long-range chromatin interactions genome-wide...
(ChIA-PET), a technique developed for large-scale, de novo analysis of higher-order chromatin structures.
See also
- RIP-ChipRIP-ChipRIP-Chip is immunoprecipitation of an RNA-binding protein coupled to reverse transcription and a microarray. It has been used to find interactions between RNA and protein ....
, a similar technique to analyze RNA-protein interactions - DamIDDamIDDamID is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase...
, an alternative location mapping technique that does not require specific antibodies