Tools and Techniques for Studying DNA Methylation Status

DNA methylation is among the epigenetic mechanisms impacted by cancer (as well as vice versa). Identification of DNA methylation aberrations has the potential to serve as prognostic and diagnostic biomarkers. Meanwhile, the ability to affect methylation as a therapeutic target has already entered the clinic.

This article will look at how researchers study methylation status, as well touch on some advances in the development of DNA methyltransferase (DNMT) inhibitors.

Epigenetics, DNA methylation, and cancer

Epigenetics generally refers to reversible mechanisms—that is, that don’t alter the genetic sequence—that impact gene expression. Examples include the effects of non-coding RNA, post-translational modifications of chromatin proteins, as well as covalent attachments of small chemical groups to specific nucleotides in the DNA.

Perhaps the best known and most studied of the latter is the addition of a methyl group to the 5th position of the cytosine (C) carbon ring to form 5-methyl cytosine (5mC). This usually (but not always) occurs in the context of a cytosine followed by a guanine (G)—termed a CpG site. Highly methylated promoters tend to be associated with transcriptional repression, while gene body methylation is linked to transcriptional activity.

DNA methylation is involved in a host of normal physiological functions, from embryonic development to cellular differentiation. But inappropriate methylation is associated with multiple disease process including inflammatory diseases and cancer. In fact, aberrant DNA methylation is considered a hallmark of cancer development and progression, leading to, for example, inactivation or repression of tumor suppressor genes, and affecting chromatin stability. It is also believed that aberrant methylation is a contributing factor in the development of cancer.

Signs of aberrant DNA methylation can be detected in urine and blood, for example, before other signs of cancer are apparent. Thus it’s not surprising that methylation patterns are being investigated as potential biomarkers of disease, and as a way to understand the disease and its progression. How is this accomplished?

Looking for 5mC

To examine DNA methylation, generally the DNA is first prepared in a way that allows 5mC to be distinguished from unmethylated Cs, and then the results read out. Before deciding on preparation and read-out, certain questions must be asked such as: How much of the genome is to be examined? How many samples are being examined? What is the resolution required? Are you looking for a comparative or an absolute quantification? Is hands-on time or time-to-result an important consideration?

Louise Williams, principal development scientist at New England Biolabs, broadly groups what I have called preparation into three distinct categories, with each category itself composed of multiple methods:

• Enzymatic or chemical detection of cytosine and its modified bases
• Methylation specific restriction enzyme (MSRE)-based approaches
• Affinity enrichment-based methods

Conversion

5mC and C cannot be distinguished by current standard next-generation sequencing (NGS) technologies (although methods are being developed to do just that using so-called third-generation single-molecule long-read sequencing). Thus different chemical and enzymatic techniques have been developed to convert the DNA to a form that can be distinguished from the reference genome.

“The gold standard is measuring DNA methylation by whole genome bisulfite sequencing [WGBS],” says Peter A. Jones, professor of epigenetics and CSO of the Van Andel Institute. Here, sodium bisulfite is used to deaminate Cs to uracils (Us); 5mC is protected from deamination, with the end result being that 5mCs are sequenced as Cs while the originally unmethylated Cs are read as thymines (Ts). Bisulfite treatment is harsh, requiring extreme pH and temperatures, which can degrade the DNA. WGBS assumes that the whole genome is to be queried, at the single base level, and that the DNA is to be sequenced—all of which will be explored further.

An alternative is enzymatic methyl-sequencing [EM-seq] in which the TET2 enzyme and an oxidation enhancer covert 5mC to a product that cannot be deaminated by the APOBEC enzyme. APOBEC converts unmodified Cs to Us, leaving 5mCs detectable as Cs, explains Williams. There are variations on the theme using different sets of enzymes, as well as hybrid methods that combine different enzymatic and chemical steps. Among the advantages of these conversions is that they are relatively gentle.

Recently the roles of 5-hydroxymethylcytosine (5hmC) and other cytosine modifications have come under active investigation. Several protocols have been developed to distinguish 5hmC from 5mC, for example, or 5hmC and 5mC from C.

MSRE

Some restriction enzymes are sensitive to methylation status, cleaving only at a methylated restriction site or cleaving only a site without methylation. Differences in restriction patterns can be observed in several ways.

“For MSRE-PCR studies, DNA is digested and then gene-specific PCR primers are used to amplify DNA of interest. If the DNA is digested by a methylation sensitive enzyme, then PCR cannot amplify the expected DNA fragment, which therefore indicates that the site being interrogated is not methylated,” Williams explains, noting that PCR and qPCR are low throughput compared with NGS.

MSRE can also be combined with sequencing (MRE-seq) to identify non-methylated DNA regions: while non-methylated target regions will be digested, the methylated DNA regions remain intact, she adds.

While these approaches offer high sensitivity and are of relatively low cost, they are limited to regions with a MSRE—about 2 million CpGs in humans.

Enrichment

If your research doesn’t require single nucleotide resolution, consider an affinity-based enrichment method such as methyl-DNA immunoprecipitation (Me-DIP). Fragmented DNA is immunoprecipitated using 5mC-specific antibodies, and once released can be either queried by an array-based assay or converted into a sequencing library (for Me-DIP-seq), explains Williams.

A similar approach, termed MDB-seq, is to use a methyl-CpG binding domain protein to capture methylated cytosines.

Affinity enrichment methods are cost-effective, but display a bias toward hypermethylated regions and are unable to predict absolute methylation level.

Many these approaches and sub-approaches can be mixed and matched, combined, or adapted according to the user’s needs. For example, an enzymatic conversion can used for a genome-wide sequencing study, and many methods can be used to prepare input for a variety of different array types. Digital PCR can be used in place of standard or qPCR, offering greater sensitivity and better ability to overcome inhibitors found in complex matrices such as urine and blood, points out Kimberley Gutierrez, applications manager at Stilla.

Methylation panels

In oncology, diseased tissue is often limiting, and so it behooves the researcher to make the most of precious samples. They want to “distill down from large discovery efforts to a manageable number of methylation markers, to develop a signature” that can help define a disease and ultimately formulate a therapeutic course of action, suggest a prognosis, and track the progress, says Jeff Smith, global lead of NGS Precision Medicine initiatives at Thermo Fisher.

Yet WGBS comes with substantial financial and analytical burdens, and it is notoriously difficult to multiplex PCR beyond a few markers.

Thermo Fisher has developed targeted, customizable amplicon-based methylation panels that use bisulfite converted DNA as input, and NGS as readout. They include a plugin to simplify the analytic workflow, performing alignment and methylation calling for amplicons. The panels can query up to 200 markers simultaneously, although Smith notes that ultimately the number needed can be drastically reduced: “Once you get down to that discrete signature of biomarkers, there's really no need to look at 450,000 different positions when you can get the same answer with 10 or 15.”

DNMTs and DNMTis

5mC is created by members of the DNA methyltransferase (DNMT) family transferring a methyl group from S-adenosyl-L-methionine (SAM) to cytosine. “All cancers—I don’t think there are any exceptions—have methylation defects,” says Jones.

DNMT inhibitors (DNMTis) have been around for a long time: “I’ve been working with them for 40-odd years,” recalls Jones. They didn’t get much attention until they were shown to be effective against myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CML), for which they are now approved clinical treatments. The mode of action of these nucleotide analogs is to form a covalent complex with DNA that inhibits methylation and promotes degradation of the DNMT. Because DNA synthesis is necessary, they only work on actively dividing cells—a plus for cancer treatments.

Another class of DNMTi—under active development but not yet approved—are non-nucleotide analogs. These are generally small molecules that bind directly to the catalytic site of the DNMT. Unlike nucleotide analogs, many of these promise selectivity of the DNMTs they inhibit. There are more than 250 compounds with measured activity against DNMTs in annotated public chemical databases, and given the interest of pharmaceutical companies, likely many more are not reported.

There are many ways to study both regional and specific DNA methylation status, leading to a better understanding of normal physiology as well as diseases like cancer. As our ability to control that methylation progresses, so should our ability to ameliorate such diseases.