Studying epigenetics using ChIP

Find out about the different epigenetic factors which require analysis by ChIP and help determine which ChIP method is right for you in our guide to ChIP.

Find out about the different epigenetic factors, which require ChIP analysis, and determine which ChIP method is right for you.

Epigenetics application guide

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What is ChIP?

​​​Chromatin immunoprecipitation (ChIP) is a powerful technique that allows researchers to examine the interactions between epigenetic regulators and DNA in their natural context. With ChIP, researchers can identify specific genes and sequences where a protein of interest binds, across the entire genome, providing critical clues to their regulatory functions and mechanisms. By dissecting the temporal and spatial dynamics of protein-DNA interactions, ChIP provides insights into core biological processes and disease pathology.

​ChIP is exceptionally versatile, with use in a broad scope of applications. From looking at sequence-specific protein binding to global regulatory processes, ChIP gives researchers the tools to integrate discoveries and paint a comprehensive picture of complex epigenetic regulatory systems.

Applications of ChIP

ChIP has played a central role in elucidating gene regulation, transcriptional machinery, and chromatin structure. Here are some of the key proteins you can detect using ChIP:

Combined analysis

Combining ChIP analysis of multiple proteins is a powerful way to build a complete picture of genomic regulation. This approach enables researchers to study how different types of proteins, and protein complexes, interact spatially and temporally at specific sites along a particular gene, or across the entire genome, to regulate gene transcription (Barski et al., 2007).

ChIP expands the scope and precision of epigenetic research

ChIP facilitates the analysis of epigenetic mechanisms on a variety of scales, with unrivalled precision. These are some of the things you can achieve using ChIP:

Protocol overview: how ChIP works

The ChIP procedure utilizes an antibody to immunoprecipitate a protein of interest, such as a transcription factor, along with its associated DNA. The associated DNA is then recovered and analyzed by PCR, microarray or sequencing to determine the genomic sequence and location where the protein was bound. The procedure can be broken down into five parts (Figure 4).

1. Cross-linking

In some cases, cross-linking of DNA and proteins may be required to stabilize their interactions, particularly for proteins that interact as part of large protein complexes and do not directly contact DNA. Crosslinking fixes these molecular interactions and freezes them at a particular point in time and is termed cross-linking ChIP (X-ChIP). In contrast, native ChIP (N-ChIP) which is performed without prior crosslinking.

Crosslinking is generally performed with formaldehyde, which reversibly crosslinks protein to DNA, RNA, and other proteins. Other chemicals like cisplatin can be used to selectively crosslink only between DNA and protein. Dual crosslinking with additional reagents may be required to study interactions between DNA and particularly large protein/RNA complexes. UV crosslinking is irreversible and therefore not compatible with ChIP.

2. Chromatin Fragmentation

Chromatin must be fragmented into small pieces for efficient immunoprecipitation and precise mapping of the target antigen to the genome. The size of the DNA fragments will ultimately determine the resolution of genomic mapping, so it is important to optimize the fragmentation protocol.

Steps 1 and 2 are incredibly important to optimize for each experiment to get the highest quality DNA possible for subsequent ChIP analysis.

3. Immunoprecipitation

The protein of interest and DNA fragment to which it is bound are then immunoprecipitated. The chromatin mixture is incubated with an antibody to the protein of interest, and either agarose or magnetic beads overnight at 4oC to form bead/antibody/protein/DNA complexes. The beads are then collected by either centrifugation, for Protein A, Protein G, or Protein A/G agarose beads, or by magnetic tube rack, for magnetic beads, to immunoprecipitate the antibody/protein/DNA complex. Non-specific binding is removed with subsequent washes.

4. DNA Recovery and Purification

The antibody/protein/DNA complex is eluted from the beads with SDS and heat. Crosslinking of protein and DNA must then be reversed with NaCl and heat for X-ChIP experiments. Any protein and RNA present are then degraded with proteinase K and RNase A, and the remaining DNA is purified with either phenolchloroform extraction or PCR purification kit.

5. Analyze the DNA

The purified DNA is then analyzed by qPCR, hybrid array (ChIP-on-ChIP) or next-generation sequencing (ChIP-seq) to identify and quantify the sequences that have been immunoprecipitated. These sequences are mapped to the genome to identify the genes and regions where the protein of interest was bound.

Sample Preparation: X-ChIP vs N-ChIP

The aim of cross-linking is to fix the antigen of interest to its chromatin binding site. Histones themselves generally do not require cross-linking, as they are already tightly associated with the DNA. Other DNA binding proteins that with weaker affinities for DNA or histones may require cross-linking to hold them in place.

Chromatin fragmentation differences

While N-ChIP and X-ChIP both require chromatin fragmentation to make interactions accessible to antibodies, they require different fragmentation procedures utilizing micrococcal nuclease or sonication, respectively (Neill et al., 2003).

N-ChIP

For N-ChIP experiments, enzymatic digestion with micrococcal nuclease should sufficiently fragment your sample into single nucleosomes (monosomes containing ~175 base pairs of DNA).

X-ChIP

Formaldehyde cross-linking restricts access of enzymes such as micrococcal nuclease to their targets, making enzymatic digestion ineffective in X-ChIP experiments. Instead, sonication is used to generate random DNA fragments of 500–700 base pairs (2–3 nucleosomes).

Regardless of which fragmentation method is chosen, it is important to always run a fragmentation time course to optimize fragment size when setting up an experiment.

Table 5: Advantages and disadvantages of N-ChIP vs X-ChIP.

N-ChIP
X-ChIP
Advantages
Efficient precipitation of DNA and histone proteins
For non-histone proteins. Can be performed on all cell types, tissues, and organisms.
Specificity of the binding is more predictable
Enables DNA-protein, RNA-protein, and protein-protein cross-linking
High resolution (~175bp/mononucleosome)
Reduced chances of chromatin rearrangements
Disadvantages
Only for histones
Over fixation can prevent effective sonication
Selective nuclease digestion may bias input chromatin
Formaldehyde can alter the binding properties of the antigen
High concentrations of nuclease may over-digest chromatin
Lower resolution compared to N-ChIP

Antibody selection

Not all antibodies are appropriate for ChIP experiments, and many antibodies are not of ChIP-quality or validated for ChIP applications. Choosing the right ChIP-grade antibody is essential for the success of your ChIP experiment. If not commercially available, or if you would like to try an antibody that is not yet ChIP-tested:

​​Controls

ChIP protocols and data analysis can be complex, so it is critical to include the right controls to ensure that the experiment worked as intended.

Sample controls

It is crucial to include an input sample control that has not been immunoprecipitated in all DNA recovery steps for comparison to pulldown sample results. It is ultimately this comparison that normalizes the data to provide interpretable results.

When immunoprecipitating for histone modifications, purified histone H3, and H1 can be used as positive controls for the quality of the histone preparation (histone H1 is commonly used for X-ChIP). Meanwhile, calf thymus histone preparation should be used as a positive control histone sample for checking antibody specificity in western blot.

Antibody controls

Various antibody controls are important to ensure that immunoprecipitation was successful and to rule out the possibility of contamination. Here are some examples of key controls:

Also, chromatin remodeling may move or remove histones at a particular locus (eg an active promoter). To confirm the preservation of nucleosomes at particular genomic loci, use a control antibody against a non-modified histone such as histone H3. When analyzing histone modifications, normalize to histone content with an anti-H3 antibody.

Quantitative PCR controls

If analyzing data by qPCR, additional controls are necessary to ensure the quality of data analysis. Certain areas of the genome will purify better than others, and some nucleosomes may rearrange 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/ChIP 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.

Also, during the qPCR stages, it is essential to perform positive and negative control qPCR for genomic loci where you know the protein of interest should or should not be bound. It is also necessary to perform a non-template control qPCR as a negative control to ensure there is no contamination in the PCR.

Protocol optimization

ChIP protocols must be optimized at multiple stages to achieve the best results. Here are a few things that may need a little extra optimization to give you the best ChIP results.

Cross-linking (X-ChIP only)

Formaldehyde is recommended for reversible cross-linking. Formaldehyde is an efficient DNA-protein crosslinker but not an effective protein-protein crosslinker making it difficult to ChIP proteins that do not bind directly to DNA. Alternative cross-linkers may be useful for cross-linking over various intermolecular distances.

Cross-linking is a time-critical procedure and should generally only last a few minutes. Excessive cross-linking can create several issues, including a reduction in antigen availability and sonication efficiency. For example, epitopes may be masked or altered, reducing antibody binding to the antigen and ensuing extraction of material from your sample.

Chromatin fragmentation

It is critical to optimize your chromatin input by fragmenting the chromatin to the appropriate size. Fragment sizes should be less than 1 kb, but ideally, 200-1000bp. The best resolution can be achieved with MNase digestion to single nucleosome level of 175 bp. Perform a time course of chromatin digestion over 2–30 minutes, purify DNA and run a gel alongside a DNA ladder to determine which conditions and timing achieve the optimal DNA fragment size (Figure 5). Different factors require optimization between N-ChIP and X-ChIP protocols.

Figure 5: Example of sonication time-course experiment. U2OS cells were sonicated for 5, 10, 15 and 20 min. The cross-links were reversed and the purified DNA was resolved on a 1.5% agarose gel. The fragment size decreases during the time course. The optimal fragment size is observed at 15 min.

​​Figure 5: Example of sonication time-course experiment. U2OS cells were sonicated for 5, 10, 15 and 20 min. The cross-links were reversed and the purified DNA was resolved on a 1.5% agarose gel. The fragment size decreases during the time course. The optimal fragment size is observed at 15 min.

Antibody concentration

It is important to titer the antibody to optimize the signal to noise ratio. Start with 3–5 µg of antibody for every 25–35 mg of pure monosomes. For quantitative ChIP, you may need to match the amount of chromatin with the same amount of antibody. ChIP typically requires a large amount of primary antibody (1-10 µg per ug of beads). As with many techniques, it is essential to optimize the amount of antibody at the beginning before you run your experiment.

Wash buffers.

Determine the correct composition for appropriate stringency of wash steps, typically between 250–500 mM NaCl or LiCl. Higher concentrations of salt and detergent will give cleaner results. However, balance must be achieved between low background and detrimental effects on the target. If the buffer is too stringent, it will destroy specific antibody interactions, resulting in low signal. If the buffer is not stringent enough, non-specific interactions will remain, resulting in high background. NP-40 can be used as a detergent, while RIPA is commonly used for X-ChIP.

ChIP with low cell numbers

Standard ChIP workflows require a large number of cells. Approximately 106 to 107cells as starting material, below which the assay is hindered by high background binding, poor enrichment efficiencies, and loss of enriched library complexity. However, these large sample sizes can be difficult to obtain, specifically when examining precious sample types like transgenic mouse tissues or clinical samples. To adjust for lower sample inputs, a number of strategies can be applied.

1. Improving enrichment efficiency and minimizing sample loss

Several adjustments to the ChIP workflow can increase enrichment efficiency and minimize sample loss for low input samples (Mao et al., 2013 and Dirks et al., 2016).

2. Readout and downstream data processing platforms

In addition to the assay itself, the choice and optimization of downstream processing (ie sequencing, array, or PCR) and bioinformatic analysis are also important.

ChIP from tissue

Examining epigenetic mechanisms in specific tissues can reveal essential elements of tissue-specific genetic programming, development, and biological processes. ChIP can be a valuable tool for examining roles and mechanisms of tissue-specific transcription factors, gene activation and other aspects of epigenetic regulation. To perform ChIP from tissue samples requires specialized chromatin preparation protocols to ensure quality input material and reliable results.

The amount of tissue required will depend upon protein abundance, antibody affinity and the efficiency of cross-linking. The following protocol was optimized using 5–15 µg of chromatin for each ChIP assay, with 30 mg of liver tissue for each ChIP/antibody. Exact chromatin concentration should be determined for each tissue type before starting the X-ChIP assay.

Click here for our ChIP from tissue samples protocol.

ChIP readout

Once pulled down DNA fragments have been immunoprecipitated and purified, they can be analyzed by several different methods.

qPCR

Utilizes gene or target-specific primers to amplify known target loci among pulldown DNA

Limitations:

ChIP-on-Chip

Employs microarrays to examine the presence of many loci of interest, specific domains, etc. across the genome. Pulldown and control samples are amplified and labeled with complementary fluorescent probes. Samples are combined and hybridized to a microarray of interest. The ratio of fluorescent signals indicates enriched regions where proteins of interest have bound.

Limitations:

ChIP-seq

Most commonly used method for genome-wide analysis with improved base-pair resolution and none of the limitations of ChIP-on-Chip. Pulldown DNA and control samples are amplified, followed by high throughput sequencing of the fragments, which are then aligned to the genome. Overlapping fragments form a peak, indicating where the protein of interest was bound to the genome.

Figure 6: ChIP seq overview. After ChIP is carried out, the precipitated DNA can be used for library preparation, sequencing and then these sequences are mapped to a reference genome to then undergo peak calling to determine binding sites of your protein of interest.

Figure 6: ChIP seq overview. After ChIP is carried out, the precipitated DNA can be used for library preparation, sequencing and then these sequences are mapped to a reference genome to then undergo peak calling to determine binding sites of your protein of interest.

Data analysis

Data should always be normalized for the amount of starting material to eliminate errors introduced by uneven sample quantities. To normalize data, take the final amplicon value and divide it by the amplicon value of input material. For histone modifications, the immunoprecipitated material is usually normalized to the input amount and the amount of the relevant immunoprecipitated histone. For example, ChIP with an H3K4me3 antibody will be expressed relative to the input amount and the amount of H3 immunoprecipitated. Measuring the amounts (and quality) of starting material is the key to interpreting your results effectively.

Tutorial for ChIP-seq data analysis using online software

While ChIP-seq data analysis can be complex, it is arguably the most important part of the experiment. Robust data analysis is key to accurate and reliable results. A combination of online tools makes data interpretation accessible to bioinformatics specialists and wet lab biologists alike. There are many resources that demonstrate how to extract reliable results from ChIP-seq data, and how to interpret data sets for successful ChIP-seq analysis (Hurtado et al., 2010 and Yan et al., 2013).

For a step-by-step tutorial on ChIP-seq data analysis, watch our webinar here.

This webinar covers the following ChIP-seq data analysis workflow:

  1. Quality control of sequencing reads using FastQC, with a side-by-side comparison of a successful and failed experiment.

  2. How to align or map the reads for a reference genome using Galaxy/Bowtie.

  3. How to perform peak calling to identify enriched regions for the protein of interest using Galaxy/macs.

  4. Binding signal visualization with UCSC genome browser.

  5. De novo motif discovery: how to find the A motif enriched within the binding regions with MEME-ChIP.

  6. How to assign binding sites to genes and get enriched genome ontology terms to discover potential biological function with GREAT.

  7. Heatmap generation for binding signal representation with seqMINER.

References

  1. Barski A,, Cuddapah S,, Cui K,, et al. High-resolution profiling of histone methylations in the human genome Cell  18 (129),823-37 (2007)
  2. Bolduc, N. Preparation of low-input and ligation-free librarIes using template-switching technology In Current protocols in molecular biology. Wiley & Sons  7 (26), (2016)
  3. Cejas, P Chromatin Immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles Nature Medicine  22 (685), (2016)
  4. Dirks, R. Genome-wide epigenomic profiling for biomarker discovery Clinical Epigenetics 8 (122), (2016)
  5. Gilfillian, G. Limitations and possibilities of low cell number CHIP-SEQ BMC Genomcis  13 (645), (2012)
  6. Hurtado A,, Holmes KA,, Ross-Innes CS,, et al. FOXA1 is a key determinant of estrogen receptor function and endocrine response Nat Genet  43 (1),27-33 (2010)
  7. Kidder, B ChIP-Seq: Technical considerations for obtaining high quality data Nature Immunology  12 (918), (2011)
  8. Mao Accounting for immunoprecipitation inefficiences in the statistical analysis of ChIP-Seq data BMC Bionformatics  14 (169), (2013)
  9. Neill O. P. L,, Turner M. B. Immunoprecipitation of native chromatin: NChIP Methods  31 (1),76-82 (2003)
  10. Reverberi, R. Factors affecting the antigen-antibody reaction Blood transfusion  5 (227), (2007)
  11. Schmidl, C ChIPmentation: fast, robust, low-input ChIP-Seq for histones and transcription factors Nature Methods  12 (963), (2015)
  12. Stelloo, S. Androgen receptor profiling predicts prostate cancer outcome EMBO Mol Med.  7 (1450), (2015)
  13. Xiong, X. A scalable epitope tagging approach for high throughput ChIP-Seqanalysis ACS Synth biol , (2017)
  14. Yan J,, Enge M,, Whitington T,, et al. Transcription factor binding in human cells occurs in dense clusters formed around cohesion anchor sites Cell  154 (4),801-13 (2013)