This article describes chromatin fragmentation, cross-linking, ChIP antibodies, and draws a comparison between X-ChIP and N-ChIP techniques.
What is ChIP?
The ChIP follows a simple principle - the selective enrichment of a chromatin percentage comprising of a specific protein. A target protein along with its relevant DNA is immunoprecipitated with an antibody. This is subsequently recovered and examined, for instance by sequencing, microarrays, or PCR to determine the type of genomic loci the protein binds to.
Euchromatin is the most common type of chromatin analyzed by ChIP. It contains active genes and features an open and extended structure so as to play a key role in DNA repair, transcription, and gene replication. Many inactive genes are present in heterochromatin, making it difficult to study by ChIP, but not due to its repetitive DNA sequence and condensed state.
ChIP: A useful technique
The temporal and spatial dynamics of the interactions between chromatin and its related factors are dissected by ChIP. This method enables mapping very small changes at a single promoter or tracking a single transcription factor over the whole human genome. It is to be noted that the output is an average one created from the analysis of cell population. Being a versatile technique, ChIP can provide a deep understanding of the way genes are controlled in their natural environment.
How does ChIP work?
ChIP is applied to find out if a specified protein attaches to a specific DNA sequence in vivo.
The following steps are included in the ChIP protocol:
- Total chromatin is isolated
- Chromatin is fragmented (to attain resolution)
- The resulting chromatin fragments are immunoprecipitated
- Analysis of the immunoprecipitated chromatin fraction to establish the amount of a target DNA sequence (or sequences) in relation to its abundance in the input chromatin
The ChIP technique may sound complicated, but can be mastered using the right tools. A kit could be used if ChIP is not a recognized method in a specific laboratory.
Useful tips for a successful ChIP experiment
To stabilize the interactions between DNA and proteins, these two are often cross-linked prior to analysis. There are two different ways to perform the ChIP procedure based on whether users choose to cross-link their chromatin sample. The technique is called ChIP (N-ChIP), but is known as crosslinking ChiP (X-ChIP) is the sample is cross-linked.
To cross-link or not to cross-link
The goal of cross-linking is to bind the target antigen to its chromatin binding site. As a rule, cross-linking is not required for histones because they are already tightly related to the DNA. However, other DNA binding proteins with a weaker affinity for histones or DNA would need to be cross-linked. This cross-linking holds the DNA binding proteins in place and prevents protein dissociation from the chromatin binding site.
If the target interaction lies further away from the DNA, the ChIP won’t be as effective without cross-linking.
Cross-linking may be required if ChIP is used for histone modifications, but it may not be needed for non-histone proteins such as proteins and transcription factors contained in DNA binding complexes.
How to cross-link
Formaldehyde should be used since it forms reversible links. As UV cross-linking is irreversible, it is not appropriate. It must be noted that there are alternative cross-linkers to formaldehyde—these may prove valuable if the researcher had to cross-link over numerous intermolecular distances.
While formaldehyde is an excellent DNA-protein cross-linker, it is not an efficient protein-protein cross-linker because of its small size (2 Å). As a result, it is often hard to ChIP proteins that do not directly bind to DNA.
Can I cross-link too much?
The answer is yes. Being a time-critical process, cross-linking should only be performed for a few minutes. If cross-linking is done excessively, it can lead to a number of problems including reduction in sonication efficiency and antigen availability. For instance, epitopes may be changed or concealed, which could affect the potential of the antibody to bind the antigen. This in turn promotes a reduction in the material of the sample.
A time-course experiment should always be performed in order to optimize cross-linking conditions. It is recommended to cross-link the samples for 2–30 minutes. By adding glycine, the formaldehyde is quenched and the cross-linking reaction is terminated.
To further help the purification of DNA, cross-links between DNA and proteins are disrupted by treatment with proteinase K. This enzyme cleaves the peptide bonds next to the carboxylic group of aromatic and aliphatic amino acids.
It is important to fragment the chromatin so that interactions are made accessible to antibody reagents. Chromatin can be fragmented by either digesting it or sonicating with micrococcal nuclease. The selected method will mostly rely on the type of ChIP experiment being conducted.
Regardless of the type of method being used, a fragmentation time course should be run whenever an experiment is set up.
N-ChIP with enzymatic digestion
Enzymatic digestion using micrococcal nuclease should be more than enough to fragment the sample for conducting N-ChIP. Since N-ChIP does not require cross-linking, no potential effects on the enzyme accessing its target will occur.
Single monosomes (~175 base pairs) can be generated by leveraging the enzymatic method, affording the highest resolution in a traditional ChIP. However, there are certain chromatin binders such as transcription factors that usually bind inter-nucleosomal DNA, which means purified mono-ucleosomes are not appropriate.
Nucleosomes are also dynamic and if there is no cross-linking they are more likely to reorganize during the enzymatic digestion. However, this could present a potential problem when the genomic areas need to be mapped, and appropriate controls will also be required to track any changes (refer detection controls for quantitative PCR).
Random chromatin sections will not be produced by enzymatic cleavage. Micrococcal nuclease prefers specific areas of genome sequence over others and will not be able to digest DNA equally or evenly. In addition, over representation of certain loci as well as missed data will not give entirely accurate results.
How to get consistency in digestions
After procuring the stock enzyme, it should be aliquoted and a new time course must be run with a fresh aliquot whenever an experiment is set up. While the quality of the enzyme may differ in due course in storage, the risk of difference within the chromatin preparations (degree of compaction etc.) is much higher. A single chromatin sample should never be treated as being the same as all other samples before it.
X-ChIP should be performed as a control experiment when N-ChIP is being done to assess any unnecessary or dynamic changes that result from lack of cross-linking.
X-ChIP and sonication
Generally, sonication is required for X-ChIP because cross-linking of formaldehyde tends to limit the access of enzymes like micrococcal nuclease to their targets which means enzymatic digestion will not be efficient on cross-linked samples. It is often assumed that randomly-sized DNA fragments are produced by sonication – with no genomic section being preferentially cleaved, albeit this is seldom seen in practice.
The DNA fragments produced by sonicating, averaging 500–700 base pairs (2–3 nucleosomes), are larger than those produced through the enzymatic cleavage. The resolution of the ChIP protocol is directly affected by the size of the DNA fragments produced. Up to 1.5 kb fragments can resolve well for most applications in ChIP.
Despite the fact that sonication is highly suitable for X-ChIP, and that enzymatic digestion is not effective in the case of fully cross-linked samples, micrococcal nuclease digestion can prove useful when incomplete or gentle cross-linking is needed; it can also enhance resolution in tandem with sonication.
Foaming should be avoided at all costs since it decreases the energy transfer within the solution, lowering the sonication efficiency.
It is possible to snap freeze the sonicated chromatin in liquid nitrogen and preserve it at -80 °C for up to a couple of months. However, multiple cycles of freeze thaw should be avoided.
Antibodies for ChIP
ChIP makes use of antibodies to capture both the proteins and the interacting DNA. Ideally, these antibodies have to be fully characterized and should be able to function in ChIP. If available, an antibody that is labeled as ChIP-grade and fully characterized should be used.
For N-ChIP, it is advised to characterize antibody specificity through peptide competition in western blot. Preferably, specific antibodies meant for ChIP have to be affinity-purified, but majority of laboratories utilize sera as their antibody source and then use stringent buffers to resolve the background issues that may otherwise occur.
Even full characterization will not provide a complete picture of whether or not an antibody will work in X-ChIP, because the cross-linking effects can be significant to such an extent that specific epitopes may be lost and different epitopes may be produced. One way to test whether or not an uncharacterized antibody will function in ChIP would be to conduct a ChIP with the antibody and then perform a western blot using the same antibody.
Don’t have a ChIP-grade antibody?
If antibodies are not available, those that work in IHC and IP also serve as good candidates. For N-ChIP, it is recommended to characterize antibody specificity through peptide competition in western blot. Preferably, specific antibodies intended for ChIP should be affinity-purified, but most laboratories utilize sera as their antibody source and then use stringent buffers to resolve the background issues that may occur otherwise (refer other frequently asked questions). For histone modifications, antibodies have to be completely tested for specificity, for instance by peptide array.
Polyclonal versus monoclonal antibodies
Monoclonal antibodies are capable of recognizing just one epitope within a population of polyclonal antibodies, but there are also several antibodies that can detect different epitopes. A polyclonal antibody population will be able to reduce the probability that all specific epitopes will be concealed by the cross-linking process, and therefore there is a greater chance of obtaining a positive result in X-ChIP. On the other hand, higher batch to batch consistency is usually observed in monoclonal antibodies.
What antibody controls can be used?
As a positive antibody control for the method, popular positive controls histone H3 tri-methyl K9 (H3K9me3) and tri-methyl K4 (H3K4me3) can be used when exploring inactive and active genes, respectively.
It must be noted that these antibodies are not negative and positive controls as such, because this will rely on the locus being studied – if there is no H3K4me3 at the specific target locus, even the world’s best anti-H3K4me3 ChIP-grade antibody will not be able to immunoprecipitate anything from this area, and thus will be an inappropriate positive control.
As a negative antibody control, an antibody should be used that detects a non-chromatin epitope ,like an anti-GFP antibody, for example.
Remodeling of chromatin may either move or remove the histones at a specific locus of interest, for example, an active promoter. Therefore, a control antibody should be used against histone H3 – an example of a non-modified histone – to validate the preservation of nucleosomes at specific genomic loci.
Whenever histone modifications are being studied, the histone content has to be normalized. The anti-H3 antibody (ab1791) can be used to accomplish this.
The antibody is working for ChIP but the signal is weak—how to remedy this?
The first step is to try a different ChIP type. For instance, X-ChIP can be tried if N-ChIP is being performed. Another option is to look in a different site. The antigen may be present but not on the genome loci that are being observed. Trying out different antibodies, when available, is a good practice to spot the one that works best in ChIP. Lastly, there is a possibility that the target epitope is being concealed in X-ChIP: the cross-linkage time course may need to be further optimized.
What concentration of antibody should be used in ChIP experiment?
First, 3–5 µg of antibody should be used for each 25–35 mg of pure monosomes used. If a quantitative ChIP is being performed, it has to be ensured that the amount of chromatin matches with the same amount of antibody. Just like many methods, the amount of antibody should be optimized at the beginning itself, if possible.
Even if the antibody immunoprecipitates the target protein in formaldehyde fixed chromatin, this does not necessarily signify that the ChIP experiment has actually worked. This is because the target protein may not have cross-linked to the DNA.
If there is high background, further washes may be required. On the other hand, sonicated chromatin could also be pre-cleared by incubating it with Protein A/G beads for a period of 1 hour before immunoprecipitation. During this additional step, non-specific binding to the Protein A/G beads will be eliminated.
Controls for quantitative PCR
There are certain genomic areas that purify better than others, while some nucleosomes may reorganize during enzymatic fragmentation. Therefore, PCR primers should be generated to a number of regions both in the starting material and the purified/ChIPped material, as controls for erroneous results. The starting cells are lysed to produce the starting material and collect a sample for easy PCR of control areas in parallel with ChIP.
Considering the potential differences in the starting material, it is important to normalize the data for the amount of starting material so that any errors introduced by uneven sample volumes can be eliminated. Data can be normalized by considering the final amplicon value and dividing it by the amplicon value of the input material. In the case of histone modifications, the immunoprecipitated material is often normalized to the amount of the input material and the amount of the related immunoprecipitated histone. For instance, ChIP with an H3K4me3 antibody is expressed in relation to the amount of the input material and the amount of H3 immunoprecipitated.
Quantifying the amounts (and quality) of starting material is important for effective interpretation of the results.
Other frequently asked questions
What histone control sample should be used for ChIP?
As a positive control histone sample, calf thymus histone preparation is recommended for assessing antibody specificity in western blot. During immunoprecipitation of histone modifications, purified histone H1 and H3 can be utilized as positive controls to ensure the quality of the experimental preparation of histone (histone H1 is often used for X-ChIP).
What buffer is recommended?
Buffers should be as stringent as possible to obtain better results, i.e., buffers containing higher concentrations of detergent and salt will lead to cleaner results. The buffer should be optimized for each new ChIP experiment because a compromise has to be reached between detrimental effects and low backgrounds on the target. NP-40 can serve as a detergent, while RIPA is often used for X-ChIP.
What other treatments might affect the ChIP results?
ChIP is not generally affected by butyrate, TSA, or colcemid addition.
Sepharose beads should never be centrifuged at high rpm (do not surpass 6,000 rpm) as this will not only compact the beads but also damage them. Relatively low concentrations of SDS can affect certain antibodies.
Advantages and disadvantages
Testable and predictable and antibody specificity
Not useful for non-histone proteins
Efficient precipitation of protein and DNA
Selective nuclease digestion may bias input chromatin
High resolution (175 bp/monosomes)
Nucleosomes may rearrange during digestion
Good for non-histone proteins binding indirectly or weakly to DNA
May be inefficient antibody binding due to epitope disruption
Cross-linking reduces nucleosome rearrangements
Fixes transient (artifactual) interactions to give a false picture of steady state levels
Good for organisms where native chromatin is hard to prepare (e.g., yeasts)
Lower resolution chromatin preparation by sonication
Enzymatic digestion of cross-linked DNA is difficult
Adapted from O'Neill and Turner Methods, 2003, pp76–82
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