All tags Epigenetics ChIP tips and tricks written by Dr Andy Bannister

ChIP tips and tricks written by Dr Andy Bannister

Cross-linking, chromatin fragmentation, ChIP antibodies and detection and a comparison between N-ChIP and X-ChIP. Compiled by Dr. Andy Bannister from the Gurdon Institute.

What is ChIP?

The principle of ChIP is simple: the selective enrichment of a chromatin fraction containing a specific protein. An antibody is used to immunoprecipitate a protein of interest together with its associated DNA. It is then recovered and analyzed for example by PCR, microarrays or sequencing to find out at what genomic loci the protein was bound to.

The most commonly ‘ChIPped’ chromatin is euchromatin. This contains active genes and maintains an open and extended structure in order to play an important role in transcription, DNA repair and gene replication.

Heterochromatin contains many inactive genes and is difficult to analyze by ChIP, not least because of its condensed state and repetitive DNA sequence.

Read further for insights into the ChiP technique that complement our C-ChIP and N-ChIP protocols. Written in collaboration with Andy Bannister.


Why is ChIP such a useful technique?

ChIP dissects the spatial and temporal dynamics of the interactions between chromatin and its associated factors.

The technique allows us to map minute-by-minute changes at a single promoter or follow a single transcription factor over the entire human genome. It must be remembered that the output is an average one generated from the analysis of a population of cells.

ChIP is versatile and can give us significant insight into how genes are regulated in their natural context.

How does ChIP work?

ChIP is used to determine whether a given protein binds to a specific DNA sequence in vivo.

The ChIP procedure consists of the following steps:

  1. Isolation of total chromatin
  2. Fragmentation of the chromatin (to achieve resolution)
  3. Immunoprecipitation of the resulting chromatin fragments
  4. Analysis of the immunoprecipitated fraction to determine the amount of a target DNA sequence (or sequences) relative to its abundance in the input chromatin.

ChIP is a technique that may sound intimidating, but with the right tools it can be mastered. If ChIP is not an established technique in your lab, you might consider using a kit.


Useful tips for a successful ChIP experiment

Cross-linking

Cross-linking of DNA and proteins is often required to stabilize their interactions before analysis. ChIP can be performed in two different ways depending on whether you opt to cross-link your chromatin sample.

If you cross-link your sample then the technique is termed “X-ChIP,” otherwise it is referred to as “N-ChIP.”

To cross-link or not to cross-link?
The aim of cross-linking is to fix the antigen of interest to its chromatin binding site. Histones themselves generally do not need to be cross-linked, as they are already tightly associated with the DNA.

Other DNA binding proteins that have a weaker affinity for DNA or histones may need to be cross-linked. This holds them in place and avoids protein dissociating from the chromatin binding site.

The further away from the DNA your interaction of interest lies then the less effective ChIP will be without cross-linking.

E.g. ChIP for histone modifications is unlikely to require cross-linking whereas non-histone proteins such as transcription factors and proteins contained in DNA binding complexes will probably need cross-linking.

How do I cross-link?
Use formaldehyde, as the links it forms are reversible. See our N-ChIP and X-ChIP protocols for details. UV cross-linking is not appropriate as it is irreversible. It should be noted that alternative cross-linkers to formaldehyde do exist - these may be useful if the researcher need to cross-link over various intermolecular distances.

Can I cross-link too much?
Yes. Cross-linking is a time-critical procedure. Cross-linking should generally only be carried out for a few minutes. Excessive cross-linking can lead to several issues including reduction in antigen availability and sonication efficiency. For example, epitopes may be masked or changed, affecting the ability of the antibody to bind the antigen, which, in turn, causes a reduction in the material in your sample.

Always perform a time-course experiment to optimize cross-linking conditions. We would suggest cross-linking the samples for 2-30 min.

Glycine is added to quench the formaldehyde and terminate the cross-linking reaction.

Formaldehyde is a very good DNA – protein cross-linker but due to its small size (2 Å) it is not a very efficient protein – protein cross-linker. It is therefore often difficult to ChIP proteins that do not bind directly to DNA.

To further aid DNA purification, cross-links between proteins and DNA are disrupted by treatment with proteinase K, which cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids.


Chromatin fragmentation

Fragmentation of the chromatin is required to make interactions accessible to antibody reagents. To fragment chromatin, you can either sonicate it or digest it with using micrococcal nuclease. Which method you choose will largely depend upon the type of ChIP experiment being performed.

Whatever method you are using be sure to run a fragmentation time course every time you set up an experiment.

N-ChIP with enzymatic digestion

Enzymatic digestion, with micrococcal nuclease, should be sufficient to fragment your sample for performing N-ChIP. N-ChIP does not call for cross-linking and so there will be no potential effects on the enzyme accessing its target.

Using the enzymatic technique it is possible to generate single monosomes (~175 base pairs), providing the highest resolution in a standard ChIP. However, certain chromatin binders such as transcription factors often bind inter-nucleosomal DNA so purified mono-nucleosomes are not suitable.

Also, nucleosomes are dynamic and without cross-linking they may rearrange during the enzymatic digestion. This is a potential problem if you wish to map areas of the genome, and suitable controls must be used to monitor any changes (see Detection — controls for quantitative PCR).

Enzymatic cleavage will not produce “random” sections of chromatin. Micrococcal nuclease favors certain areas of genome sequence over others and will not digest DNA evenly or equally.

Results may not be entirely accurate as certain loci could be over represented and some data may be missed.


How do I get consistency in my digestions?

Be sure to aliquot your stock enzyme after purchase and run a new time course with a fresh aliquot every time you set up an experiment. Although enzyme quality may vary over time in storage, the risk of variation within chromatin preparations (degree of compaction etc.) is far higher — one chromatin sample should not be treated as being the same as all others before it.

X-ChIP should be carried out as a control experiment when doing N-ChIP to assess any dynamic and unwanted changes resulting from the absence of cross-linking.

X-ChIP and sonication

Typically, sonication is necessary for X-ChIP as formaldehyde cross-linking restricts the access of enzymes such as micrococcal nuclease to their targets, meaning that enzymatic digestion will normally be inefficient on cross-linked samples.

Sonication is generally believed to create randomly sized DNA fragments, with no section of the genome being preferentially cleaved, although in practice this is rarely observed. The fragments created by sonicating, which average 500–700 base pairs (2–3 nucleosomes), are typically larger than those created via enzymatic cleavage. The size of the fragments that are created directly affects the resolution of the ChIP procedure; fragments up to 1.5 kb resolve well for most purposes in ChIP.

Although sonication is most appropriate for X-ChIP and enzymatic digestion is ineffective on fully cross-linked samples, micrococcal nuclease digestion can be useful when gentle/incomplete cross-linking is carried out and it can improve resolution in combination with sonication.

Avoid foaming as it results in a decrease of energy transfer within the solution and will decrease the sonication efficiency.

Sonicated chromatin can be snap frozen in liquid nitrogen and stored at -80°C for up to 2 months. Avoid multiple cycles of freeze thaw.

Antibodies for ChIP


Antibodies are used in ChIP to capture proteins and the interacting DNA and should ideally be fully characterized. Make sure the antibody works in ChIP. If available, use an antibody that has been fully characterized and labeled as ChIP-grade.

Characterizing antibody specificity using peptide competition in western blot is recommended for N-ChIP. Ideally, specific antibodies for ChIP should be affinity-purified; however many laboratories use sera as their antibody source and then overcome background problems that may arise with stringent buffers.

Even full characterization will not tell you whether or not an antibody will function in X-ChIP, as the effects of cross-linking can be dramatic to the extent that different epitopes may be generated and specific epitopes may be lost. To test whether an uncharacterized antibody can ChIP, you can perform a ChIP with the antibody followed by a western blot with the same antibody.


Don’t have a ChIP-grade antibody?
If there is none available, then antibodies that work in IP and in IHC are good candidates.  Characterizing antibody specificity using peptide competition in western blot is recommended for N-ChIP. Ideally, specific antibodies for ChIP should be affinity-purified; however many laboratories use sera as their antibody source and then overcome background problems that may arise with stringent buffers (see other frequently asked questions). Antibodies for histone modifications need to be thoroughly tested for specificity e.g. by peptide array.

Polyclonal vs. monoclonal antibodies
Whereas monoclonal antibodies recognize only a single epitope, within a polyclonal antibody population there will be a number of antibodies that recognize different epitopes. A polyclonal population will reduce the probability that all specific epitopes will be masked by the process of cross-linking, so there is a better chance of a positive result in X-ChIP. However, monoclonal antibodies usually have higher batch to batch consistency.

What antibody controls could I use?
As a positive antibody control for the technique, Histone H3 tri-methyl K4 (H3K4me3) and tri-methyl K9 (H3K9me3) are popular positive controls to use when ChIPping active and inactive genes respectively.

Remember that these antibodies are not positive and negative controls per se, as this will depend on the locus you are studying: if there is no H3K4me3 at the particular locus of interest, the best anti-H3K4me3 ChIP-grade antibody in the world will not immunoprecipitate anything from this region and therefore will not be an appropriate positive control.

As a negative control, use an antibody that recognizes a non-chromatin epitope such as an anti-GFP antibody.

Chromatin remodeling may move or remove histones at a particular locus e.g. an active promoter, so use a control antibody against a non modified histone such as Histone H3 to check for the preservation of nucleosomes at particular genomic loci.

When analyzing histone modifications, you need to normalize to histone content. This can be done with the anti-H3 antibody (ab1791).


The antibody is working for ChIP but the signal is weak — how can I remedy this?

As a first step you can try a different type of ChIP. For example, if you are performing N-ChIP try X-ChIP. Alternatively, look in a different location. It may be that the antigen is present but not on the genome loci that you are looking at. It is good practice to try different antibodies, when available, to find the one that works best in ChIP. Finally it might be that the epitope of interest is being masked in X-ChIP: it may be necessary to further optimize the cross-linkage time course.

What concentration of antibody should I use in my ChIP experiment?
To start with, use 3–5 µg of antibody for every 25–35 mg of pure monosomes used. If you are doing a quantitative ChIP then ultimately you may need to match the amount of chromatin with the same amount of antibody. As with many techniques, it is essential to optimize the amount of antibody at the start if possible.

Even if the antibody is able to immunoprecipitate the protein of interest in formaldehyde fixed chromatin this does not mean that the ChIP experiment has worked, as it is possible that your protein of interest is not cross-linked to the DNA.

If high background is observed, additional washes may be needed. Alternatively, sonicated chromatin may also be pre-cleared by incubating with Protein A/G beads for 1 hr prior to immunoprecipitation. Any non-specific binding to the beads will be removed during this additional step.


Detection

Controls for quantitative PCR
Certain areas of the genome will purify better than others, and some nucleosomes may re-arrange during enzymatic fragmentation. As a result, it is important to generate PCR primers to several regions in the starting material, as well as the purified/ChIPped material, as controls for spurious results. Generate starting material by lysing the starting cells and take a sample for simple PCR of control regions in parallel with ChIP.

Data
 With variations in starting material possible, data should always be normalized for the amount of starting material, to remove errors introduced due to uneven sample quantities. To normalize your data, take the final amplicon value and divide it by the amplicon value of input material.  For histone modifications, the ChIPped material is usually normalized to the input amount and the amount of the relevant ChIPped histone. For example, a ChIP with an H3K4me3 antibody will be expressed relative to the input amount and the amount of H3 ChIPped.

Measuring the amounts (and quality) of starting material is the key to interpreting your results effectively.


Other frequently asked questions


What histone control sample should be used for ChIP?
Calf thymus histone preparation should be used as a positive control histone sample for checking antibody specificity in western blot. When ChIPping for histone modifications, purified histone H3 and H1 can be used as positive controls for the quality of the experimental histone preparation (histone H1 is usually used for X-ChIP).

What buffer is recommended?
The more stringent the buffer used the better (i.e. higher concentrations of salt and detergent in the buffer will lead to cleaner results). It is essential that the buffer is optimized for every new ChIPping experiment as a compromise must be found between low backgrounds and detrimental effects on the target. NP40 can be used as a detergent and RIPA is also commonly used for X-ChIP.

What other treatments might affect my ChIP results?
TSA, butyrate or colcemid addition do not generally affect ChIP.

Do not centrifuge sepharose beads at high rpm (do not exceed 6,000 rpm) as this will compact the beads and damage them.

Some antibodies are affected by relatively low concentrations of SDS.


Advantages and disadvantages


AdvantagesDisadvantages

N-ChIP

Predictable and testable antibody specificityNot useful for non-histone proteins

Efficient precipitation of DNA and proteinSelective nuclease digestion may bias input chromatin

High resolution (175 bp/monosomes)Nucleosomes may rearrange during digestion

X-ChIP

Good for non-histone proteins binding weakly or indirectly to DNAMay be inefficient antibody binding due to epitope disruption

Cross-linking minimizes nucleosome rearrangementsFixes transient (artifactual) interactions to give a false picture of steady state levels

Good for organisms where native chromatin is difficult to prepare (e.g., yeasts)

Lower resolution chromatin preparation by sonication



Difficult to enzymatically digest cross-linked DNA

Adapted from O'Neill and Turner, Methods, 2003, pp76–82



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