Epigenetics is the study of heritable changes in the phenotype of a cell or an organism that are not encoded by the genome (hence epi which means ‘above’ in Greek, and genetikos which means ‘origin’).
In this article, we’ll discuss DNA methylation, a common epigenetic modification: both what it is, and how it can be detected and quantified.
What are epigenetic modifications?
Our genome stores information in the form of DNA molecules. Most of us are familiar with the genomic architecture, which consists of DNA wrapped around histone proteins to form nucleosomes, which are then packaged into highly condensed chromatin. Our genome sequence remains generally constant, with the exception of genetic mutations that can lead to deleterious results such as cancer. Epigenetic changes, however, which are not encoded in the DNA sequence, are reversible and dynamic, and are also heritable. These epigenetic modifications reflect adaptations to our environment, life-style, diet, and other external factors. Thus, the mammalian genome can be modified at either the chromatin or the DNA level.
What is DNA methylation?
DNA consists of four nucleotide building blocks: cytosine, guanine, adenine, and thymine. Cytosine residues can undergo an epigenetic modification called methylation, catalyzed by the enzyme DNA methyltransferase, that results in the addition of a methyl group to the carbon-5 position, yielding 5-methylcytosine (5-mC). Additionally, 5-mC can be enzymatically oxidized to 5-hydroxymethylcytosine (5-hmC) by the TET1/2/3 enzymes. These DNA methylation events are thought to regulate gene expression, and there is increasing evidence that these modifications are linked to embryonic stem cell function, development, normal tissue function, and disease progression. However, 5-hmC and 5-mC are believed to have different functional roles in the mammalian genome, and it is possible that these changes may represent early epigenetic biomarkers for different pathogeneses.
How can you detect and quantify DNA methylation?
In our recent webinar on Bitesize Bio, Dr. Sriharsa Pradham of New England Biolabs discussed various techniques for detecting 5-hmC and 5-mC modifications, both globally and within specific sequences. Dr. Pradham presented a very robust and easy-to-use protocol that employs the EpiMark 5-hmC and 5-mC analysis kit.
How it works
- Glucosylation. Genomic DNA contains a mixture of unmethylated, 5-methylated, and 5-hydroxymethylated cytosine residues. In order to distinguish between these individual states, the DNA sample is first treated with UDP-glucose and T4 phage beta glucosyltransferase enzyme (T4 BGT), which adds glucose to the hydroxymethyl site. This converts all of the 5-hmC residues to 5-glucosyl-mC (5-hmC), while the 5-mC sites remain intact. Your negative control is set up in the absence of T4 BGT, thus leaving all 5-mC sites unmodified.
- Methylation-sensitive restriction digestion. During the first step, we glucosylated all 5-hmC sites to 5-ghmC sites using T4 BGT and UDP-glucose, converting all cleavable 5-hmC locations to non-cleavable 5-ghmC sites. Next up, the DNA is divided into fractions, and each fraction is digested with a methylation-sensitive restriction enzyme. The way this works is by using two restriction enzymes, MspI and HsaII, that recognize the same restriction site (CCGG) but have differential methylation sensitivity. These enzymes are used because their specificity and ability to cleave DNA can be altered when cytosine in the recognition site is modified. For example, MspI cleaves 5-hmC, 5-mC, and unmethylated cytosine but is blocked by 5-ghmC. HpaII can only cleave unmethylated C residues. Thus, in the samples treated with BGT, MspI will cleave all methylated sites but not glycosylated-hydroxymethylated sites. In contrast, HpaII will only cut unmethylated sites because cleavage is blocked by 5-hmC, 5-mC, and 5-ghmC modifications. By comparing the amount of DNA cleaved by either one of these enzymes, then, you can identify the total methylated or hydroxymethylated content of your DNA sample (uncut control DNA is used to identify total amount of DNA present).
- Detection by PCR. Finally, you can use end-point or real-time PCR to determine and quantitate the amount of 5-mC and 5-hmC across different tissues, states, or genomes. To do this, you use PCR primers flanking a CCGG site of interest (and amplifying a fragment of about 100–200 bp). If the site is unmethylated, then your template DNA will be digested by HpaII and you will not amplify any product. However, if the CpG site contains 5-hydroxymethylcytosine, your target template will not be cleaved, allowing amplification of your gene of interest. End-point PCR can be used to determine which sample is enriched for the 5-hydroxymethylcytosine modification. Following restriction digestion with MspI and HpaII and end-point PCR, you should see a band representing the amplified gene of interest in the samples enriched for 5-hmC, where restriction cleavage was blocked by glucosylation of 5-hmC (MspI). To get the exact percentage of 5-hmC and 5-mC present across different samples for the gene of interest, quantitative Real-Time PCR is used.
The power of this approach is that it can determine enrichment for specific methylation modifications locally and on a genome-wide level. For instance, you could determine global 5-hmC levels in normal vs. cancer samples, possibly identifying epigenetic biomarkers for a particular cancer.
How could you use this technology in your reseaarch?
ps: Don't forget to check out our webinar on detecting 5-hmC and 5-mC modifications!