DNA methylation is in the crosshairs of cancer researchers. The addition of methyl and hydroxymethyl groups at cytosine bases in the genome is an epigenetic modification that changes the expression of the methylated genes. Indeed, most DNA methylation occurs in CpG islands of promoter regions of genes. Methylation by DNA methyltransferase enzymes typically silences gene expression, possibly by rendering a gene less physically accessible to translation. Conversely, expression of a methylated gene can be turned back on by demethylation through a multi-step process mediated by the TET, TDG, and BER enzymes.
Aberrant DNA methylation patterns are known to be associated with the development of cancer, but it is not as simple as switching one gene on or off. For example, hypermethylation inhibits tumor suppressor genes, yet overall global hypomethylation of the genome can stimulate expression of certain oncogenes. Here’s a look at some tools and technologies researchers are relying on to study the role of DNA methylation in gene regulation and cancer today.
Measuring methylation levels
Cell Signaling Technology (CST) offers a range of tools to measure levels of DNA methylation, such as monoclonal antibodies to DNA modifications. These include antibodies to the 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), and N6-methyladenosine (m6A) modifications. “Our antibodies against methylated DNA and RNA can be used to analyze differences in bulk DNA and RNA methylation in normal versus cancerous cells or tissues, using DNA or RNA dot blot, IF, or MeDIP,” says Chris Fry, director of epigenetics product development at Cell Signaling Technology. “The majority of our rabbit monoclonal antibodies are recombinantly expressed, enabling us to provide a ready supply of antibody and ensure lot-to-lot consistency.” For example, the CST 5-mC antibody is an important tool for methyl-DNA immunoprecipitation (MeDIP) assays, used to analyze changes in methylation changes at specific DNA sites.
ELISA assays are another type of tool for measuring DNA methylation levels. EpiGentek offers a wide range of DNA methylation tools, including the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit. An advantage of the kit is that it uses intact DNA as input. “This preserves samples in their native state, [rather than] requiring additional processing such as DNA denaturation or nuclease digestion, that compromises sample integrity,” says Michael Spelios, a scientist at EpiGentek. “Customers looking to rapidly and accurately quantify methylated DNA from virtually any species often utilize this kit as a cost-effective initial assessment before proceeding downstream with in-depth and expensive sequencing applications.”
Enriching for methylation sites
Enriching for sites of DNA methylation as an epigenetic marker can streamline research. Tools such as bisulfite conversion sequencing can differentiate between methylated and non-methylated cytosines in the genetic code. Zymo Research offers many tools for studying DNA methylation, including bisulfite conversion based library preparation kits for next-generation sequencing. The new Zymo-Seq RRBS™ Library Kit is particularly useful for researchers doing quick screens or pilot studies. RRBS uses restriction enzyme digestion to return CpG-enriched regions where DNA methylation occurs predominantly in mammals. “The Zymo-Seq RRBS™ Library Kit enriches for CpG-rich regions across the genome, and reduces the sequencing cost dramatically compared to whole-genome bisulfite sequencing, while still covering a significant portion of those important gene regulatory regions,” says Xiaojing Yang, scientist at Zymo Research. Toward the end of 2020, Zymo also plans to release a whole-genome bisulfite sequencing library prep kit, the Zymo-Seq WGBS™ Library Kit.
EpiGentek’s BisulPlus™ Loci 5mc & 5hmC Detection PCR Kit is designed to detect both DNA methylation at 5-mC sites, as well as hydroxymethylation at 5-hmC sites. “[It] uses an innovative approach by sequentially combining bisulfite and APOBEC treatments that differentially target unmodified cytosines and 5-mC, respectively, leaving 5-hmC intact and allowing for the discrimination of each modification,” says Spelios.
DNA methylation and gene regulation
Proteins that interact with DNA at methylated sites play an important role in gene regulation and the development or suppression of cancer. Three commonly studied types include writer proteins (DNMTs) that add methyl groups; eraser proteins (TETs) involved in removing methyl groups; and reader proteins (methyl-CpG-binding domain proteins) involved in de novo methylation patterns during development.
CST offers antibodies to readers, writers, and erasers of DNA methylation to study their localization, abundance, and potential roles in cancer when comparing normal and cancer cells. “Our antibodies against the writer, reader, and eraser proteins can be used to determine changes in cellular localization and abundance that occur in cancer versus normal cells,” says Fry, such as the CST monoclonal antibody to the writer protein DNMT3A. Recently a research group in Taiwan used this antibody to study the epigenetic perturbations of the DNMT3A-R882 mutation in myeloid leukemia cells.
CST antibodies are used in assays designed to analyze the interactions of such proteins at sites of DNA methylation. Their chromatin immunoprecipitation sequencing (ChIP-seq) assay kits, along with antibodies to reader, writer, or eraser proteins allow researchers to analyze protein-DNA interactions across the genome. A recent study from China used the CST monoclonal antibody to eraser protein TET2 to study how TET2 and AID proteins cooperatively regulate the expression of the FANCA gene in diffuse large B cell lymphoma.
CST antibodies can also be used with the new Cleavage Under Targets & Release Using Nuclease (CUT&RUN) Assay Kit, which provides fast detection of DNA-protein interactions using a low number of cells. The CUT&RUN assay is faster than ChIP because it doesn’t require formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation steps. “Instead of fragmenting the chromatin as done in the ChIP assay, CUT&RUN utilizes an antibody-targeted digestion of chromatin, resulting in much lower background signal than seen in the ChIP-assay,” says Fry. “As a result, CUT&RUN requires only 1/10th of the sequencing depth that is required for ChIP-seq assays.” The ongoing development of new assays, and better tools to accomplish them faster and more efficiently, are helping cancer researchers to home in on the role of DNA methylation in cancer.