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ChIP-exo is a
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 genom ...
based method for mapping the locations at which a protein of interest (
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 ...
) binds to the genome. It is a modification of the
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 ...
protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. It employs the use of
exonucleases Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is th ...
to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing,
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 silico ...
, and
PCR PCR or pcr may refer to: Science * Phosphocreatine, a phosphorylated creatine molecule * Principal component regression, a statistical technique Medicine * Polymerase chain reaction ** COVID-19 testing, often performed using the polymerase chain r ...
. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds. __TOC__


Theory

Chromatin immunoprecipitation (
ChIP 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 genom ...
) techniques have been in use since 1984 to detect protein-DNA interactions. There have been many variations on ChIP to improve the quality of results. One such improvement,
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 ...
(ChIP-chip), combines ChIP with microarray technology. This technique has limited sensitivity and specificity, especially ''in vivo'' where microarrays are constrained by thousands of proteins present in the nuclear compartment, resulting in a high rate of false positives. Next came
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 pro ...
(ChIP-seq), which combines ChIP with high-throughput sequencing. However, the heterogeneous nature of sheared DNA fragments maps binding sites to within ±300 base pairs, limiting specificity. Secondly, contaminating DNA presents a grave problem since so few genetic loci are cross-linked to the protein of interest, making any non-specific genomic DNA a significant source of background noise. To address these problems, Rhee and Pugh revised the classic
nuclease protection assay Nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence e ...
to develop ChIP-exo. This new ChIP technique relies on a lambda
exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is t ...
that degrades only, and all, unbound double-stranded DNA in the 5′-3′ direction. Briefly, a protein of interest (engineering one with an epitope tag can be useful for immunoprecipitation) is crosslinked in vivo to its natural binding locations across a genome using formaldehyde. Cells are then collected, broken open, and the chromatin sheared and solubilized 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 ...
. An antibody is then used to immunoprecipitate the protein of interest, along with the crosslinked DNA. DNA PCR adaptors are then ligated to the ends, which serve as a priming point for second strand DNA synthesis after the exonuclease digestion. Lambda exonuclease then digests double DNA strands from the 5′ end until digestion is blocked at the border of the protein-DNA covalent interaction. Most contaminating DNA is degraded by the addition of a second single-strand specific exonuclease. After 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 ...
ing is reversed, the primers to the PCR adaptors are extended to form double stranded DNA, and a second adaptor is ligated to 5′ ends to demarcate the precise location of exonuclease digestion cessation. The library is then amplified by PCR, and the products are identified by
high throughput sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine ...
. This method allows for resolution of up to a single base pair for any protein binding site within any genome, which is a much higher resolution than either ChIP-chip or ChIP-seq.


Advantages

ChIP-exo has been shown to give up to single base pair resolution in identifying protein binding locations. This is in contrast to ChIP-seq which can locate a protein's binding site only to with ±300 base pairs. Contamination of non-protein-bound DNA fragments can result in a high rate of false positives and negatives in ChIP experiments. The addition of exonucleases to the process not only improves resolution of binding-site calling, but removes contaminating DNA from the solution before sequencing. Proteins that are inefficiently bound to a nucleotide fragment are more likely to be detected by ChIP-exo. This has allowed, for example, the recognition of more CTCF transcription factor binding sites than previously discovered. Due to the higher resolution and reduced background, less depth of sequencing coverage is needed when using ChIP-exo.


Limitations

If a protein-DNA complex has multiple locations of cross-linking within a single binding event, then it can appear as though there are multiple distinct binding events. This likely results from these proteins being denatured and cross-linking at one of the available binding sites within the same event. The exonuclease would then stop at one of the bound sites, depending on which site the protein is cross-linked to. As with any ChIP-based method, a suitable antibody for the protein of interest needs to be available in order to use this technique.


Applications

Rhee and Pugh introduce ChIP-exo by performing analyses on a small collection of transcription factors: Reb1, Gal4, Phd1, Rap1 in yeast and CTCF in human. Reb1 sites were often found in clusters and these clusters had ~10-fold higher occupancy than expected. Secondary sites in clusters were found ~40 bp from a primary binding site. Binding motifs of Gal4 showed a strong preference for three of the four nucleotides, suggesting a negative interaction between Gal4 and the excluded nucleotide. Phd1 recognizes three different motifs which explains previous reports of the ambiguity of Phd1's binding motif. Rap1 was found to recognize four motifs. Ribosomal protein genes bound by this protein had a tendency to use a particular motif with a stronger consensus sequence. Other genes often used clusters of weaker consensus motifs, possibly to achieve a similar occupancy. Binding motifs of CTCF employed four "modules". Half of the bound CTCF sites used modules 1 and 2, while the rest used some combination of the four. It is believed that CTCF uses its zinc fingers to recognize different combinations of these modules. Rhee and Pugh analyzed pre-initiation complex (PIC) structure and organization in ''Saccharomyces'' genomes. Using ChIP-exo, they were able to, among other discoveries, precisely identify TATA-like features in promoters reported to be TATA-less.


See also

*
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 genom ...
*
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 pro ...
*
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 ...
*
Protein-DNA interaction DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, becaus ...


References


External links


DNA-protein interactions in high definition





Important Gene-Regulation Proteins Pinpointed by New Method


* ttp://www.peconicgenomics.com Peconic Genomics {{DEFAULTSORT:ChIP-exo Protein methods Molecular biology Molecular biology techniques Genomics DNA sequencing Bioinformatics