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Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
s on promoters or other
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 (1) they are part of a DNA sequence (e.g. a genome) and (2) they are bound by DNA-binding ...
s, and possibly define
cistrome In simple words, the cistrome refers a collection of regulatory elements of a set of genes, including transcription factor binding-sites and histone modifications. More specifically, "the set of cis-acting targets of a trans-acting factor on a genom ...
s. ChIP also aims to determine the specific location in the genome that various
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn a ...
modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of
epigenomics Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigen ...
and learning more about epigenetic phenomena. Briefly, the conventional method is as follows: # DNA and associated proteins on
chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
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 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 such as the extraction of multiple compounds from plants, microalgae and seawe ...
or nuclease digestion. #
Cross-linked In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
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''.


Typical ChIP

There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly
cross-link In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
ed chromatin sheared by
sonication 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 such as the extraction of multiple compounds from plants, microalgae and seawe ...
called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal nuclease 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-link In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
ed chromatin as starting material. The agent for reversible cross-linking could be
formaldehyde Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
or
UV light Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiation i ...
. Then the cross-linked chromatin is usually sheared by sonication, providing fragments of 300 - 1000
base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
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 nucleosomes. Cell debris in the sheared lysate is then cleared by sedimentation and protein–DNA complexes are selectively immunoprecipitated using specific
antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
to the protein(s) of interest. The antibodies are commonly coupled to
agarose Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is ...
,
sepharose Sepharose is a tradename for a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed. Its brand name is a portmanteau derived from Separation-Pharmacia-Agarose. A common application for the material is in chro ...
, or magnetic beads. Alternatively, chromatin-antibody complexes can be selectively retained and eluted by inert polymer discs.Revolutionary solid state platform for chromatin immunopreciptation. 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 In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
is reversed and proteins are removed by digestion with
proteinase K In molecular biology Proteinase K (, ''protease K'', ''endopeptidase K'', ''Tritirachium alkaline proteinase'', ''Tritirachium album serine proteinase'', ''Tritirachium album proteinase K'') is a broad-spectrum serine protease. The enzyme was dis ...
. An
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 epitope is the specific piece of the antigen to which an antibody binds. The p ...
-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 The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR),
microarrays A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of genes from a sample (e.g. from a tissue). It is a two-dimensional array on a solid substrate—usually a glass slide or silicon ...
(
ChIP-on-chip ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA ''in vivo' ...
), molecular cloning and sequencing, or direct high-throughput sequencing (
ChIP-Seq ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated prote ...
).


Native ChIP (NChIP)

Native ChIP is mainly suited for mapping the DNA target of
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn a ...
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 immunoprecipitated complex, and purifying and analyzing the complex-associated DNA.


Comparison of XChIP and NChIP

The major advantage of NChIP is
antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
specificity. It is important to note that most antibodies to modified histones are raised against unfixed, synthetic peptide antigens and that the
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 epitope is the specific piece of the antigen to which an antibody binds. The p ...
s they need to recognize in the XChIP may be disrupted or destroyed by formaldehyde
cross-link In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
ing, particularly as the
cross-link In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
s are likely to involve
lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −C ...
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. 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).


Comparison of ChIP-seq and ChIP-chip

Chromatin Immunoprecipitation sequencing, also known as
ChIP-seq ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated prote ...
, is an experimental technique used to identify transcription factor binding events throughout an entire
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
. Knowing how the proteins in the human body interact with DNA to regulate gene expression is a key component of our knowledge of human diseases and biological processes.
ChIP-seq ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated prote ...
is the primary technique to complete this task, as it has proven to be extremely effective in resolving how proteins and transcription factors influence phenotypical mechanisms. Overall ChIP-seq has risen to be a very efficient method for determining these factors, but there is a rivaling method known as ChIP-on-chip.
ChIP-on-chip ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA ''in vivo' ...
, also known as ChIP-chip, is an experimental technique used to isolate and identify genomic sites occupied by specific DNA-binding proteins in living cells. ChIP-on-chip is a relatively newer technique, as it was introduced in 2001 by Peggy Farnham and Michael Zhang. ChIP-on-chip gets its name by combining the methods of Chromatin Immunoprecipitation and
DNA microarray A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to ...
, thus creating ChIP-on-chip. The two methods seek similar results, as they both strive to find protein binding sites that can help identify elements in the human genome. Those elements in the human genome are important for the advancement of knowledge in human diseases and biological processes. The difference between ChIP-seq and ChIP-chip is established by the specific site of the protein binding identification. The main difference comes from the efficacy of the two techniques, ChIP-seq produces results with higher sensitivity and spatial resolution because of the wide range of genomic coverage. Even though ChIP-seq has proven to be more efficient than ChIP-chip, ChIP-seq is not always the first choice for scientists. The cost and accessibility of ChIP-seq is a major disadvantage, which has led to the more predominant use of ChIP-chip in laboratories across the world. Table 1 Advantages and disadvantages of NChIP and XChIP


History and New ChIP methods

In 1984
John T. Lis John T. Lis (born in Willimantic, Connecticut) 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 ...
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 In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
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 the distribution of eukaryotic
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukaryo ...
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 Alexander Jacob Varshavsky (russian: link=no, Александр Яковлевич Варшавский; born 8 November 1946) is a Russian-American biochemist, noted for his discovery of the N-end rule of ubiquitination. A native of Moscow, he ...
and co-workers, who examined the distribution of
histone H4 Histone H4 is one of the five main histone proteins involved in the structure of chromatin in eukaryote, eukaryotic cells. Featuring a main globular domain and a long N-terminus, N-terminal tail, H4 is involved with the structure of the nucleos ...
on
heat shock genes In thermodynamics, heat is defined as the form of energy crossing the boundary of a thermodynamic system by virtue of a temperature difference across the boundary. A thermodynamic system does not ''contain'' heat. Nevertheless, the term is ...
using formaldehyde cross-linking. This technique was extensively developed and refined thereafter. NChIP approach was first described by Hebbes ''et al''., 1988, and has also been developed and refined quickly. The typical ChIP assay usually takes 4–5 days and requires 106~ 107 cells at least. Now new techniques on ChIP could be achieved as few as 100~1000 cells and completed within one day. * Bead-free 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 optimized protocols, ChIP can be performed in 5 hours. * Carrier ChIP (CChIP): This approach could use as few as 100 cells by adding ''
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
'' 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 In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
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 A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a dise ...
and microChIP can be done within one day. * Matrix ChIP: This is a
microplate A microplate, also known as a microtiter plate (''Microtiter'' is a registered trademark in the United States, therefore it should not be used generically without attribution), microwell plate or multiwell, is a flat plate with multiple "wells" ...
-based ChIP assay with increased throughput and a simplified procedure. All steps are done in microplate wells without sample transfers, enabling 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 ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). 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, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated prot ...
). ChIP can also combine with
paired-end tags Paired-end tags (PET) (sometimes "Paired-End diTags", or simply "ditags") are the short sequences at the 5’ and 3' ends of a DNA fragment which are unique enough that they (theoretically) exist together only once in a genome, therefore making th ...
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.


Limitations

* 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 A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isof ...
).


See also

*
ChIP-exo ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest (transcription factor) binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites ...
, a technique that adds exonuclease treatment to the ChIP process to obtain up to single base pair resolution of binding sites *
ChIP-on-chip ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA ''in vivo' ...
, combines ChIP with microarray technology *
DamID DNA adenine methyltransferase identification, often abbreviated DamID, 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 D ...
, an alternative location mapping technique that does not require specific antibodies *
RIP-Chip RIP-chip (RNA immunoprecipitation chip) is a molecular biology technique which combines RNA immunoprecipitation with a microarray. The purpose of this technique is to identify which RNA sequences interact with a particular RNA binding protein of in ...
, a similar technique to analyze RNA-protein interactions


References


External links

*
EpigenomeNOE.com

Chromatin Immunopreciptation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications

Chromatin Immunoprecipitation (ChIP) of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells
{{DEFAULTSORT:Chromatin Immunoprecipitation Biochemical separation processes Protein methods Genomics techniques Molecular biology techniques Protein–protein interaction assays Immunologic tests