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Home > Organization > Divisions and Independent Research Units > Group for Development of Molecular diagnostics and Individualized Therapy > Division of Epigenomics > Research Projects > Development of method for analysis of aberrant DNA methylation

Development of method for analysis of aberrant DNA methylation

An accurate analysis of DNA methylation enables us to unravel fundamental mechanisms of cancers and to make accurate diagnoses. There are two major categories of DNA methylation analysis. One is an analysis of individual genomic regions and the other is genome-wide DNA methylation analysis.

We played a pioneering role in the development of genome-wide analysis of DNA methylation. In 1997, we developed methylation-sensitive representational difference analysis (MS-RDA) (Ushijima et al., 1997 , Ushijima and Yamashita, 2009). Subsequently, we applied the method to various cancers, and identified many genes with aberrant DNA methylation (Takai et al., 2001 , Kaneda et al., 2002 , Miyamoto et al., 2003 , Hagihara et al., 2004 , Abe et al., 2005 , Miyamoto et al., 2005 , Furuta et al., 2006 ). Recently, using our accumulated knowledge, we made a significant improvement on DNA methylation microarray analysis (Iida et al., 2018).

In addition to analysis of DNA methylation itself, analysis of the factor of alteration of DNA methylation is also important. We are developing such a method.


Methods of analysis of each region of genes

Regarding the methods of analysis of individual genomic regions, several methods are available, such as bisulfite sequencing, methylation-specific PCR (MSP), quantitative MSP, COBRA, and Pyrosequencing. Individual methods have different versatility of regions analyzed, sensitivity, precision, and necessary instruments for analysis. All of these methods are based on the bisulfite reaction that converts unmethylated cytosine to uracil.

Bisulfite sequencing can identify the methylation status of individual CpG sites in a specific region, but requires labor and time. MSP can be performed by a simple procedure, and has a high sensitivity that can detect one methylated molecule out of 1,000 unmethylated molecules. However, it is susceptible to false positive results, and quantification cannot be done. Quantitative MSP can quantify DNA methylation levels within a wide range, from 1% to 100%, with high precision. Therefore, we frequently use this method in our laboratory. COBRA method has a limitation in the sequences it can analyze, but it can easily quantify DNA methylation levels. Pyrosequencing needs special reagents and an instrument, but can quantify DNA methylation levels of specific CpG sites with good sensitivity and high precision.

Reference: Hattori and Ushijima, 2018

Methods of analysis of each region of genes

Fig. Methods of analysis of each region of genes

Methods for analysis of individual genomic regions can be categorized by their flexibility and quantitativity. We mainly use the methods in red line boxes.


Methods of the genome-wide DNA methylation analysis

Methods of genome-wide screening for differences in DNA methylation consist of two steps, detection of DNA methylation and genome-wide screening. DNA methylation statuses can be detected based on several principles, affinity to methylated DNA, such as an anti-5-methylcytocine antibody, methylation-sensitive restriction enzymes, bisulfite conversion reaction, and induction of gene expression by treatment with a demethylating agent. Genome-wide screening can be conducted by several methods, such as microarray, next-generation deep sequencer, two-dimensional electrophoresis, and DNA subtraction. These methods have quite different versatility of resolution, quantitativity, cost-effectiveness, and necessary instruments.

For example, methylated DNA immunoprecipitation-microarray analysis (MeDIP microarray)  is a method using immunoprecipitation of methylated DNA by an antibody of 5-methylcytocine and oligonucleotide microarray. At present, the MeDIP microarray has reasonable resolution, quantitativity and cost-effectiveness. Methylation-sensitive representational difference analysis (MS-RDA) is a method using methylation-sensitive restriction enzymes and DNA subtraction. This method is inferior to methods involving microarray in resolution and simpleness of procedure, but even now, has an advantage in an application to the species whose genome sequences are not available. If one wants to identify methylation-silenced genes and a cell line is available, expression microarray screening of genes re-expressed after treatment of the cell line with a demethylating agent is effective.


Development of detection systems for chemicals that induce abnormality in DNA methylation

Chemicals that induce genetic abnormality are designated as mutagens, and chemicals that induce epigenetic abnormality are designated as "epimutagens" (Holliday, 1991 ). Only a limited number of epimutagens are currently known, including cytidine analogues, metal compounds such as arsenic or nickel, and drugs such as valproate (an antiepileptic agent) and procainamide (an antiarrhythmic agent), and so on. The number is very small compared with that of mutagens, and this is possibly because there are no efficient assay systems for epimutagens. Therefore, it is critically important to develop an efficient detection system for epimutagens. We have already developed a prototype detection system for demethylating agents (Fig. ) (Okochi-Takada et al., 2004 ). Currently, we are developing a highly sensitive system, which can be used in a high-throughput platform.

Development of method for analysis of aberrant DNA methylation

Fig. Establishment of an assay system for CpG demethylating agents

Under a methylated endogenous promoter CpG islands (CGI), a fluorescence-expressing marker gene was introduced. The addition of 5-aza-dC to introduced cells led to demethylation of the CGI, and expression of the marker gene. As expected, fluorescence was confirmed by fluorescence microscopy.