The epigenome regulates the expression of genes without altering their underlying DNA sequence. Comprising a dynamic landscape of chemical modifications and structural changes to DNA and its associated histone proteins, the epigenome wields profound influence over cellular identity and function. Epigenetic mechanisms, such as DNA methylation and histone modifications, form an elaborate regulatory network that fine-tunes gene expression. These modifications can be influenced by environmental factors, developmental stages and even the experiences of previous generations.
Studying the epigenome allows us to decipher how genes are switched on or off in various cells and tissues. It offers insights into diseases like cancer, in which epigenetic changes can drive abnormal cell behaviour. By delving into this regulatory network, researchers have the power to develop innovative therapies and a deeper understanding of the intricate molecular networks that dictate our lives.
Figure 1. The four major epigenetic layers and how to profile them. Credit: Mehrmohamadi, et al.1
There are a multitude of different ways a researcher can study the epigenome. Most methods involve sequencing the genome under specific conditions to isolate the regions that are ‘under the influence’ of a specific epigenetic regulator. We provide a brief overview of some of the most common methods below.
DNA methylation studies
DNA methylation refers to the addition of a methyl group to a cytosine molecule in the DNA. Methylation can repress gene expression through, for example, the blocking of promoter regions. This repression can extend from partial limitations on RNA expression through to complete silencing of a gene. Perhaps the most common method for assessing the level of DNA methylation at any point in the genome is bisulphite conversion and sequencing, which we dive into below.
The first step of bisulphite conversion and sequencing is to denature DNA to create single strands that are more accessible for analysis. Then, the DNA is treated with bisulphite – a chemical agent that selectively converts unmethylated cytosine residues to uracil, which is normally only found in RNA. However, methylated cytosines are protected from this conversion.
The DNA then undergoes a series of chemical reactions to convert uracil to thymine, effectively converting all unmethylated cytosines to thymines. This process is called desulphonation. The treated DNA is then subjected to PCR amplification to generate enough material for subsequent sequencing.
The sequence data obtained from bisulphite-treated DNA reveals the locations of the converted cytosines (originally unmethylated) as thymines, while the methylated cytosines remain as cytosines. By comparing the bisulphite-treated sequence to the reference genome sequence, researchers can determine the methylation status of individual cytosines within the DNA sample. By determining the location of these methylation markers, researchers can make deductions about the impact of the modification. Levels of methylation can then be intuited from this data.
Chromatin accessibility assays
DNA is tightly coiled and packed into chromosomes to fit within the nucleus. Part of this compaction process involves coiling DNA into chromatin fibres. A tightly packed chromatin fibre is inaccessible for gene expression. However, the chromatin fibres are dynamic and can uncoil to ‘open up’ areas of the genome for easy transcription. Chromatin accessibility assays are a popular method for identifying these regions of open chromatin. This can have serious implications in the regulation of gene expression and subsequent disease. The most widely used chromatin accessibility assay is ATAC-Seq21.
The process begins with the collection of cells or tissues of interest. These cells are typically fixed and then permeabilised to make the chromatin accessible for subsequent steps.
Tn5 transposase is a special enzyme used in ATAC-seq that is typically loaded with adapter sequences and inserts itself into the open regions of the genome – in other words, where chromatin is not tightly packed – and then fragments the DNA. This step is called “tagmentation” (a combination of “tagging” and “fragmentation”).
After tagmentation, the DNA fragments are PCR-amplified using primers that anneal to the added adapter sequences. This step creates a library of DNA fragments that represent the accessible regions of chromatin in the sample.
The library is then subjected to high-throughput DNA sequencing, which yields millions of short DNA sequences that are representative of the open chromatin regions in the sample.
The sequencing data is analysed to identify the regions of the genome that were accessible during the experiment. This information can be used to map regulatory elements, such as promoters, enhancers and transcription factor binding sites, and to gain insights into the regulation of gene expression, since ‘open’ regions of the genome are now identified.
Other chromatin accessibility assays include DNAse-Seq22 and MNase-Seq23.
Histone modification assays
The first stage of packaging DNA into the nuclei is for the DNA to be bound to, and wrapped around, histone proteins. How accessible the DNA is for transcription is also subject to these histone proteins, and modifications to the histones change their interactions with DNA. Common modifications include H3K9me3, H3K27me3 and H3K4me3, which can change the transcriptional state of the chromatin-associated DNA.
Chromatin immunoprecipitation sequencing (ChIP-seq24) is a commonly used method to detect these histone modifications. ChIP-seq also allows us to detect where certain proteins, such as transcription factors, bind to the DNA.
The first step of ChIP-seq is cross-linking the proteins (chromatin) to the DNA in your sample by adding formaldehyde to the cells or tissue. This will preserve the protein-DNA interactions in their state at the time of sampling.
The next step is the fragmentation of the cross-linked protein-DNA sample into small pieces (typically 100-500 base pairs) using a sonicator or enzymatic shearing. Then, using antibodies specific to the protein or marker of interest, you can immunoprecipitate the protein-DNA complexes. This will result in the selective enrichment of DNA fragments associated with the protein or histone marker.
Wash away any nonspecific or unbound DNA fragments, leaving only the protein-bound DNA fragments attached to the antibody-coated beads before reversing the formaldehyde cross-links to separate the DNA from the proteins. This is typically done by heating the samples.
Then, purify the immunoprecipitated DNA fragments from the solution and prepare a sequencing library from the purified DNA fragments. This often involves DNA end repair, adapter ligation and PCR amplification.
The final step is to sequence the ChIP-enriched DNA library. This allows you to identify specific areas of the DNA that were bound by chromatin or other proteins, allowing you to map out where the protein was bound, or where histone modification markers were present.
Again, there are alternatives to ChIP-seq such as CUT&Run25 and CUT&Tag26. CUT&Tag – which stands for Cleavage Under Targets and Tagmentation – is now one of the most popular methods to perform this kind of analysis. In CUT&Tag, cells are permeabilises and the chromatin is bound by an antibody in situ, which is then conjugated to a protein A-Tn5 transposase fusion enzyme. The Tn5-transposase then cleaves and tags the DNA. The DNA is then released from the complex and the tagged sequences can be PCR amplified and sequenced in order to determine where the chromatin was bound.
The following two sections were adapted from our Spatial and Single-Cell Analysis Playbook, which is due to be released in late September 2023.
Single-cell and spatial epigenomic profiling tools have been released at increasing rates over the last decade. The underlying biology of these techniques is similar to those discussed above, but these methods allow one to truly capture the epigenetic environment within a single-cell. Below we will summarise some of these methods.
Figure 2. Publication dates and cell numbers of single-cell epigenomic methods. Colour indicates epigenetic layer being profiled by each technology and symbol for single-cell isolation technology. Image credit: Bond, et al. 2
For single-cell DNA methylation detection, early approaches, such as single-cell whole genome bisulfite sequencing (scWGBS-seq – Farlik, et al. 3) and the reduced range version (scRRBS-seq – Guo, et al. 4), used library prep methods to overcome the DNA loss caused by bisulphate sequencing to enable methylation reads from single cells. These early methods typically had high coverage of major CpG islands, but at low throughput and low coverage of sparse CpGs. Modern methods for DNA methylation, such as sci-MET5 and scCGI-seq6, use combinatorial sequencing to increase the throughput but at a lower coverage per cell.
For detection of histone modifications, scChIP-seq7 is the single-cell alternative, which involves passing cells through micrococcal nuclease to reduce background noise and make the technique viable for individual cells. It is a high-throughput technique, but at the cost of lower coverage per cell. The alternative CUT&Tag method also has a complementary single-cell implementation (scCUT&Tag 8). It has recently been expanded to scMulti-CUT&Tag9 and scCUT&Tag2for110 to allow for sequencing of multiple chromatin factors and for the active and repressive genomic elements, respectively. A similar assay, scGET-seq11, also profiles whether chromatin is in an open or closed state to compute a new metric, chromatin velocity, which measures epigenetic plasticity of cells.
For chromatin accessibility, a single-cell implementation of ATAC-seq was released in 2015, sc-ATAC-seq12, and is performed on isolated cells, which allows high read coverage but low throughput. However, a combinatorial form was later introduced, sci-ATAC-seq, which allows high throughput at the cost of coverage13. sci-Atac-seq3 is the most recent iteration of the technology, using a three-level combinatorial indexing assay to profile cells at extraordinary throughput and low-cost to allow chromatin profiling of whole embryos14.
A significant advance in the epigenomic space in the last 12-18 months has been the blossoming of spatial epigenomic methods to profile chromatin accessibility and histone modifications.
Two significant early methods in this space were Spatial-ATAC-seq18 and Spatial-CUT&Tag19. These methods rely on the original CUT&Tag and ATAC-seq chemistry but capture that information with a spatial resolution of 20µm.
The Spatial-ATAC assay allows unbiased genome-wide mapping of chromatin accessibility and has been used to profile chromatin in the whole mouse embryo to identify spatial organization of cell types as well as their states and fates19. SpatialCUT&Tag provides a targeted approach, visualizing spatial histone modifications using specific antibodies. This provides insights into protein-DNA interactions, transcription factor binding, and the epigenetic modifications linked to the targeted proteins. This can provide a more granular understanding of epigenetic biomarkers of disease and histone modifications controlling chromatin structure.
Alternative methods exist for histone modifications, such as epigenomic MERFISH 20, an imaging based method that combines the CUT&Tag methodology with MERFISH. This method achieves remarkable subcellular resolution (Figure 5) and has been used to identify new promoter-enhancer hubs in the mouse brain. However, this method lacks the unbiased nature of the above methods, requiring prior knowledge to select epigenomic loci.
Figure 3. Overview of Epigenomic MERFISH. This technique allows the visualisation of epigenetically modified DNA at subcellular and tissue level resolutions .Image Credit: Lu, et al. 20
Epigenome-wide association studies
After identifying epigenetic signatures using the above methods, you can carry out an epigenome-wide association study to discern whether these epigenetic alternations are in fact linked to any traits or disease. Much like a genome-wide association study, EWAS involves the identification of variants that differ in prominence between cases and controls. Check out our How-to: Perform a GWAS feature for more information on how to conduct one of these studies.
Additionally, the use of epigenomic profiling is invaluable in adding new depth to cell atlases, particularly in adding single-cell and spatial resolution. The epigenomic revolution will likely contribute to the development of personalised therapies, particularly for cancer.
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