ATAC-Seq for epigenome characterization

Assay for Transposase-accessible Chromatin using Sequencing

Epigenetics is a study that looks at the modification along the DNA backbone without involving the change of its constituents (such as mutation, insertion or deletion). In order to understand the effects of epigenetic modification on biological phenotypes, many methods are applied. For example, chromatin immunoprecipitation (Figure 1) and reduced representation bisulfite sequencing (Figure 2) are two methods for us to investigate epigenetic modifications on a genome-wide scale. However, we have to have an idea about what epigenetic mechanisms behind our experimental systems.

Figure 1 (https://blog.benchsci.com/chromatin-immunoprecipitation-chip-principles-and-how-to-obtain-quality-results)

Figure 2 (https://nxtgnt.ugent.be/oxidative-reduced-representation-bisulfite-sequencing/)

ATAC-Seq is useful

The ATAC-Seq gives a whole picture of the information about chromatin accessibility across the genome. Thus, we can still commence our study without knowing or guessing the mechanism in advance. One way to utilize ATAC-Seq is to apply it as a first-pass screening approach to find changes in chromatin accessibility between samples, followed by subsequent experiments to prove the findings.

1. First published in 2013 (Nature Methods)

2. Solve the issue of high amount of starting material, complex and time-consuming protocols

3. Relies on the hyperactive Tn5 transposase already used for tagmentation-based NGS library preparation methods (This process can be used to sequence with adapters added)

4. The addition of the adapters — in open chromatin regions — no steric hindrance of the transposase would occur — enzyme to preferentially access these regions.

5. High-resolution map of nucleosome positions and transcription factor binding profiles.

6. From 1–50 million cells to 500–50,000

How ATAC-Seq is performed?

ATAC-Seq consists of five essential steps:

1. Sample preparation
2. Transposition-Let Taq5 find and cut active chromatin areas
3. Library preparation-Amplification of the regions
4. Sequencing-Identify genomic regions with active transcription
5. Data analysis-Downstream bioinformatic analyses

Why ATAC-Seq is important?

Phenotypes of cells are defined in part by their cell states. But what is cell state? Cell state is how we define a cellular condition by looking at the level of development, differentiation or disease. The states are basically governed by the gene expression dynamics, which is regulated by complicated but orchestrated built-in programs. It’s the thermodynamics, biochemistry and molecular biology at play. We don’t need to worry about them. The question is: How do we comprehend their roles intertwined within the tiny structure?

Transcription factors

It has been made clear in recent years that the regulatory programs are controlled by the activity of transcription factors (TFs). TFs are specialized set of proteins that function in interpreting and altering the state of chromatin. The epigenetic state of the chromatin is regulated by chemical modification of both DNA and histone proteins. The logic behind this control is that every individual cell is highly specialized and unique; and is highly context-dependent. That means, with the same genetic code for each cell, the manners with which a cell chooses to become a certain cell with identifiable identity is critical to an entity as a whole, such as a mouse. It is possible that both active gene regulatory elements such as enhancers, promoters and insulators, and inactive regions of silenced or poised chromatin are both present.

How to assay the epigenome?

The most specific way to assay the epigenome is through chromatin immunoprecipitation with sequencing (ChIP-Seq) (Figure 3). This method uses an antibody-based pull-down manner to identify the location and abundance of a particular histone/DNA modification or TFs. The fragments pulled via antibody are marked and prepared for a library construction, which was then sequenced and aligned to the reference genome.

When there are overlaps for genomic regions, peaks of signal can be identified. This signal indicates the target of interest at a particular locus for bulk tissues or cells.

Newer method to refine the conventional ChIP-Seq process is chromatin immunocleavage techniques (ChIC), which includes CUT&RUN and CUT&TAG. This approach attaches a nuclease or Tn5 tansposase to a target antibody through a Protein-A. Different from ChIP-Seq, ChIC does not involve the step of immunoprecipitation, decreasing the amount of materials required while at the same time increasing the resolution of the protein binding site.

Figure 3 (https://www.nature.com/articles/nrg2641)

Agnostic profiling techniques have been developed to map the gene regulatory landscape by identifying all TF binding sites without specifying the precise TF identities. DNase-Seq and MNase-Seq are two of these techniques that use high-throughput sequencing to measure different chromatin states. DNase-Seq measures hypersensitive sites by mapping the DNA fragments protected by TFs or nucleosomes and is considered the gold-standard technique for TF footprinting. MNase-Seq, on the other hand, maps nucleosome-occupied regions by cleaving DNA not protected by nucleosomes or DNA-binding proteins. These agnostic profiling techniques provide a broad picture of the gene regulatory landscape and have been used extensively to identify putative gene regulatory elements.

Application of ATAC-Seq

ATAC-Seq is a technique used to assay the regions of the genome that are bound by TFs and to compare changes in these landscapes under different conditions. It uses in vitro transposition of sequencing adapters into native chromatin to create sequenceable DNA fragments. The Tn5 transposase is used to cut and paste the DNA, which creates sequence handles for amplification during library preparation. It is believed that the binding of TFs to DNA creates nucleosome-free regions that are associated with increased Tn5 transposition. ATAC-seq provides a simple and scalable way to study changes in the gene regulatory landscape.

Figure 4 (https://www.nature.com/articles/s41596-022-00692-9)

Detailed steps of ATAC-Seq

1. Isolation of nuclei from cells and maintain the chromatin structure
2. Exposing the isolated nuclei to the Tn5 transposase which fragments the chromatin and inserts sequences containing PCR handles.
3. Amplifying and sequencing only fragments with i5/P5 and i7/P7 ATAC-Seq adapters
4. Designating genomic regions enriched for many Tn5 transposition events as peaks of chromatin accessibility
5. Analyzing the library fragments for peaks of chromatin accessibility (ATAC-Seq peaks)
6. Determining differential peaks of chromatin accessibility between cell types or conditions.

In conclusion, ATAC-Seq can give us a general view of epigenetic modulation within the genome of interest. It is simple and fast. Further knowledge of mechanisms behind is required to further our study based on this technique.