DNA Methylation: Mechanisms and Applications in Disease and Research

What is DNA Methylation?

DNA Methylation is the process by which specific bases in a DNA sequence acquire a methyl group by covalent bonding under the action of DNA methylation transferase (DNMT). DNA methylation is a form of chemical modification of DNA, which is a very conservative epigenetic modification capable of altering the activity of DNA fragments without changing the DNA sequence, thereby altering genetic expression. It plays a crucial role in regulating biological processes such as gene expression, embryonic development, cell proliferation, cell differentiation, maintenance of genome stability, and defense against invasion by exogenous DNA viruses. DNA methylation can occur at the C-5 position of cytosine, the N-6 position of adenine, the N-7 position of guanine, etc., which are catalyzed by different DNA methylation enzymes to produce 5-methylcytosine (5-mC), N6-methyladenine (N6-mA), and 7-methylguanine (7-mG), respectively. The DNA methylation involved in general research mainly refers to the methylation process of the 5th carbon atom of cytosine in the CpG site (cytosine-phosphate-guanine site, i.e., the site in the DNA sequence where cytosine is immediately followed by guanine), and the product is called 5-methylcytosine (5-mC). 5-mC is widely found in the genomes of plants, animals and other eukaryotic organisms, and is one of the most studied forms of DNA methylation modification.

DNA methylation pathways. (Moore, L.D.; et al, 2013)

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Mechanisms of DNA Methylation

DNA methylation occurs when cytosine protrudes from the DNA double helix into a cleft that can bind to the enzyme, and catalyzed by cytosine methyltransferases (Dnmts), which transfer the active methyl group from the S-adenosylmethanethionine to the cytosine 5-position to form 5-methylcytosine (5-mC).The mode of methylation of DNA is achieved by DNA methyltransferases.

(1) DNA methylation enzymes are divided into 2 classes, including maintenance DNA methyltransferases (Dnmt1) and slave methylation enzymes (Dnmt3a, Dnmt3b, and Dnmt3L, among others).

(2) DNA methylation reactions are of 2 types. One type is where DNA that is unmethylated on both strands is methylated and is called de novo methylation. Slave methylation is the methylation modification of unmethylated DNA, which occurs mainly in the early stages of embryonic development and is used to set the methylation status of cells in the early embryo. The other type is double-stranded DNA whose one strand is already methylated and the other unmethylated strand is methylated, this type is called retention methylation.

What is DNA Demethylation?

DNA methylation modification mainly refers to 5-methylcytosine (5mC), which is a reversible epigenetic modification. This process is known as DNA demethylation, and the exact mechanism is still being studied, and the factors that cause demethylation differ from organism to organism.DNA demethylation is caused by the lack of and reduction in the amount of intracellular DNA methyltransferase in cells, which is important for turning on the expression of specific genes and the initiation of reprogramming. Currently there are two main ways used for demethylation, the active pathway as well as the passive pathway.

(1) Active pathway: it is the process of removing methyl groups by the action of demethylases.

(2) Passive pathway: the action of DNMT1 is blocked due to the adhesion of methylated DNA by the nuclear factor NF (a protein that acts as a multidirectional transcriptional regulator), which prevents the DNA near the adhesion site from being completely methylated.

The mechanism of active DNA demethylation has long been one of the most fundamental questions in the field of epigenetics. In contrast to passive demethylation, active DNA demethylation is a rapid and cell division-independent process.

Biological Role of DNA Methylation

DNA methylation plays a crucial role in the maintenance of normal cell function, X-chromosome inactivation in female individuals, repression of parasitic DNA sequences, stability of genome structure, genetic imprinting, embryonic development, and the onset and progression of tumors and diseases that are closely related.

DNA Methylation and Tumorigenesis

Cancer begins as a single cell that undergoes many changes that make its phenotype different from its normal precursor. Although this process can be driven by key genes that control cell growth, many of the changes in expression may be due to epigenetic alterations (primarily DNA methylation). Altered DNA methylation status is prevalent in tumors and is characterized by a decrease in overall methylation levels versus an increase in local methylation levels.

• Inactivation of certain genes in cancer cells due to hypermethylation of CpG islands in regulatory regions that are unmethylated in normal tissues. Example: retinoblastoma tumor gene 1 (RB1).
• Aberrant CpG island methylation promotes cancer development and progression by affecting genes involved in key cellular processes. Example: death-associated protein kinase 1 (DAPK1). In addition, in tumor cells, oncogenes are activated by being in a hypomethylated state and oncogenes are repressed by being in a hypermethylated state.

Although DNA methylation may not play a dominant role in all cancers, there is no doubt that changes in these modification patterns ultimately affect cellular susceptibility and tumor phenotype.

DNA Methylation and Embryonic Development

During embryonic development, genome-wide DNA methylation levels are drastically altered, with the most dramatic alterations occurring during the gamete formation and early embryonic development stages.

The establishment of erroneous methylation patterns may cause human diseases such as fragile X chromosome syndrome.

DNA Methylation and Stability of Genetic Material

Research has proved that bacterial DNA replication initiation is related to DNA methylation and the interaction between DNA and bacterial plasma membrane, DNA methylation as a label determines the replication initiation point and controls the replication initiation, which makes the DNA replication consistent with the cell division; DNA mismatch repair is an important means of correcting the DNA replication errors in the process of cell proliferation.

After replication, double-stranded DNA remains hemi-methylated for a short period of time (a few minutes), and the mismatch repair system is thus able to distinguish the old strand from the new strand, providing a molecular marker for the erroneous bases incorporated in the new strand.

DNA Methylation and Regulation of Gene Expression

DNA methylation provides an effective inhibitory mechanism for the long-term silencing of non-coding regions (e.g. introns, etc.).

Applications of DNA Methylation

DNA methylation has a wide range of applications in disease development, exposure and response to environmental factors, development and differentiation, and disease marker research. Clinically, it is mainly applied to the diagnosis of tumors.

Early Diagnosis and Screening

Normal cells undergo cancerous transformation and become tumor cells. Tumor cells also experience "old age, sickness and death", and substances in tumor cells may be released into the bloodstream, such as tumor cell DNA, which flows with the blood circulation, and this is what we call circulating tumor DNA (ctDNA). The methylation patterns of different types of cancers are highly specific, and the ctDNA levels of cancers at different stages vary. Therefore, early diagnosis as well as staging of tumors can be achieved by liquid biopsy to detect the levels and methylation characteristics of ctDNA.

Prognostic Risk Prediction

The methylation profile of ctDNA can predict the risk of postoperative recurrence and death of cancer patients, which can help to adjust the treatment plan, assess the need for postoperative chemotherapy and determine the chemotherapy regimen.

Evaluation of Treatment Efficacy

Liquid biopsy collection of ctDNA can be repeated several times, which is very helpful for evaluating treatment efficacy during the course of the disease and monitoring the patient's physical condition in real time.

DNA Methylation Test

There are various methods for DNA methylation analysis techniques, which are broadly categorized as follows:

Analysis Techniques Based on Base Separation

High performance liquid chromatography (HPLC) hydrolyzes DNA into individual deoxyribonucleosides, separates and detects deoxycytidine and 5-methyldeoxycytidine thereby calculating the 5-mC content of the genome. in 1980, Kuo et al. first used high performance liquid chromatography (HPLC) with ultraviolet (UV) detection to analyze the 5-mC content of the genome. With the development of mass spectrometry (MS) technology, the application of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has led to a significant enhancement of the selectivity and sensitivity of the 5mC detection. Due to its high accuracy and sensitivity, HPLC-MS is the gold standard method for the detection of genome-wide 5-mC content.

Analytical Techniques Based on Methylation-Sensitive Restriction Endonuclease Processing

Methylation-sensitive restriction endonuclease (MSRE) is a class of restriction endonucleases that are sensitive to the presence of methylated bases in their recognition sites, and can specifically recognize methylated sequences by using digestive treatment, which can generally recognize only one methylated base site. Methylation-sensitive endonuclease digestion and amplification method is easy to operate and has a wide range of applications, but it can only qualitatively or semi-quantitatively detect the methylation status of a certain site of the target fragment, but not quantitatively, and if the enzyme digestion is incomplete, the results will be affected.

Sequencing Technology Based on Bisulfite Treatment

Classical bisulfite sequencing is currently the most important method for studying DNA methylation. The principle of bisulfite sequencing (BS-seq) is to utilize the efficient deamination of non-methylcytosine by bisulfite to generate uracil, which is read as thymine in subsequent sequencing, while both 5mC and 5hmC are insensitive to bisulfite, and DNA methylation can be judged by direct sequencing of PCR products. This method is considered as the "gold standard" for DNA methylation detection because of its high accuracy and its ability to know the methylation status of each CG, CHG and CHH site of the target fragment.

Sequencing Technology Based on Affinity Enrichment Processing

This technology includes DNA methylation immunoprecipitation microarray detection or sequencing and methylated DNA-specific binding protein enrichment sequencing. The method is cost-effective and can be used for methylation detection of large-volume samples. The disadvantages are that it is not quantitative, the resolution is low (usually around 150 bp, which cannot reach single-base resolution), and it is difficult to obtain information on hypomethylated regions.

Third Generation Sequencing Technology

Second-generation sequencing (NGS) has become a standard tool for detecting DNA methylation levels due to its inexpensive price, mature and reliable experimental process, but the short-read technology has its own inherent limitations, such as assembly from scratch, haploid phasing, and difficulty in detecting structural differences. Third-generation sequencing technologies mainly include single-molecule real-time sequencing (SMRT) and single-molecule nanopore sequencing. Compared with second-generation sequencing technologies, the high error rate and high cost of third-generation sequencing technologies limit their applications.

Reference

1. Moore, L.D.; et al. DNA Methylation and Its Basic Function. Neuropsychopharmacology. 2013, 38: 23–38.

DNA Methylation: Mechanisms and Applications in Disease and Research

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