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Profiling chromatin accessibility and DNA methylation—simultaneously

Headshots of (L-R): Emily Hodges wearing earrings and a beige top with a leopard skin print; Lindsey Guerin wearing a white sleeveless top; Kelly Barnett wearing a light blue denim shirt. He's standing in front of a lab bench with yellow, green, and red boxes stacked on the shelves.
L-R: Emily Hodges, Lindsey Guerin, Kelly Barnett

By Aaron Conley

Research led by Emily Hodges, assistant professor of biochemistry, first-author Lindsey Guerin, a graduate student in the Hodges lab, and Kelly Barnett, a recent graduate of the Hodges lab, developed and tested a new method called ATAC-Me, which profiles multiple epigenetic features, including DNA methylation, simultaneously from a single DNA source. Hodges’s recent work using this new method challenges the classical, text-book role of DNA methylation in gene regulation and cellular differentiation and may fundamentally change how we view its function.

Their article was published last year in Nature Protocols. We sat down with Hodges and Guerin to learn more about this exciting research.

What issue/problem does your research address?

Dysregulated DNA methylation, a type of epigenetic mark, has been implicated in diseases ranging from cancers to developmental disorders, such as Rett Syndrome, yet its regulatory function remains poorly understood. The protocol we’ve outlined in this paper, ATAC-Me, provides a method to profile DNA methylation and other epigenetic features, which will ultimately help us understand the function of DNA methylation and its link to these diseases.

The epigenome is multi-dimensional, with individual molecular components operating on different levels to control transcription. By combining specialized techniques, you can measure these epigenetic features to better understand how they jointly regulate genes. ATAC-Me, however, is an integrated method to probe DNA methylation and chromatin accessibility from a single DNA fragment library; chromatin accessibility looks at the parts of the genome that are accessible for the transcription of genes, many of which are inaccessible or “turned off.” CpG sites—that is, locations in the genome where the sequence is a cytosine nucleotide followed by a guanine nucleotide—in accessible regions, profiled by ATAC-Me, display a range of methylation values depending on the cellular and genomic context. Most of the genome is highly methylated, making regions with low methylation of particular interest because they typically represent DNA that is bound by proteins involved in transcriptional control. The epigenetic regulation of these regions varies widely between cell types . Methylation values thus offer an additional layer of epigenetic information about accessible regions’ regulatory states.

The standard method for profiling DNA methylation requires high sequencing coverage, which means that, for humans, we need about 500 million sequence reads (short, overlapping pieces of DNA that correspond to the entire genome). This can quickly become expensive, limiting the number of samples many labs are able to sequence.

In the first stage of the protocol, ATAC-Me selects for open regions, providing data on chromatin accessibility. Then, these open regions are subjected to a methylation quantification step. This allows both data types to be profiled simultaneously, and, since only open region fragments (around 2% of the whole genome) are forwarded on to stage two, fewer sequencing reads are required.

The approach is well-suited for studies that aim to capture chromatin and DNA methylation dynamics in tandem during cellular differentiation. An ATAC-Me run can be completed in two days with standard molecular biology equipment and expertise, and the resulting data can be interpreted with basic bioinformatics skills and publicly available software. We have made our pipeline available on GitHub for others to reference.

What were your findings and why are they unique?  

This is a protocol paper building on a previous publication that demonstrates the utility of the approach in different systems. We developed a detailed method for profiling chromatin and DNA methylation simultaneously, expanding the cell types to which it can be applied.

A 3-part diagram showing a segment of DNA in each part. Top: a “closed enhancer,” represented by DNA tightly wrapped around nucleosomes, is to the left of a downstream, target gene that is turned off (represented by a red arrow, pointing to the right, with a small x on top). Middle: a “poised enhancer,” represented by “open” DNA flanked by nucleosomes and with yellow “popsicle” markers on the open segment, is on the left of a target gene that is turned off. Bottom: an “active enhancer,” represented by “open” DNA flanked by nucleosomes and with no popsicles (but with a shape on top that says “TF,” transcription factor, on top of the open DNA), is on the left of a target gene that is turned on (represented by a green arrow pointing to the right).
Canonically, DNA methylation (yellow popsicles) is understood to silence gene expression. In this model, a region must first become accessible and must then lose methylation to activate transcription of the target gene. Figure created using

What do you hope will be achieved with the research results in the short and long terms?

Our goal is to better understand the role of DNA methylation in gene regulation and cellular differentiation. Although DNA methylation has been studied for decades, the field still struggles to understand its ultimate function. Initial work in the field led to a model in which DNA methylation silences transcription by inhibiting the binding of transcription factors to the DNA, which are essential for beginning the process of gene transcription. However, these studies focused primarily on CpG-dense regions, termed “CpG islands,” which are fairly stable between different cell types. There is more variability in methylation outside of CpG islands, often in “distal” regulatory elements, located far from the genes they regulate.

In this paper we use a human embryonic stem cell model, which can be differentiated into many different cell types. To undergo differentiation, cells change their methylation profiles, primarily at these “distal” elements, along with other epigenetic features. This results in a differential methylation pattern in which a region is methylated in stem cells but has low methylation in neurons (for example). Understanding how patterns of methylation and other epigenetic features relate to gene expression could help us understand how cells with one genome give rise to all other specialized cells in the body.


This work was supported by National Institutes of Health.

Go Deeper

The article “Dual detection of chromatin accessibility and DNA methylation using ATAC-Me” was published in Nature Protocols on October 18, 2021.