为了使您在Abcam官网的浏览体验更顺畅，请使用最新版本的浏览器比如 Google Chrome
DNA methylation is a process that plays an important role in several biological phenomena. For example, in prokaryotes, it is involved in processes such as virulence, cell cycle regulation, gene expression and protection from foreign DNA introduction (DNA-host specificity). In higher eukaryotes, DNA methylation is involved in the regulation of several cellular processes such as chromatin stability, imprinting, X chromosome inactivation and carcinogenesis.
In mammals, DNA methylation occurs mainly on the fifth carbon of the cytosine base, forming 5-methylcytosine or 5-methylcytidine (5-mC). It is found almost exclusively at CpG dinucleotides and is a key epigenetic marker and regulator of gene expression. Methylated CpG clusters at gene promoters, or CpG islands, have been associated with gene inactivation.
DNA methylation is catalyzed by a family of enzymes called DNA methyltransferases and include DNMT1, DNMT3a and DNMT3b. DNMT3a and DNMT3b are de novo methyltransferases that are able to methylate previously unmethylated CpG dinucleotides. In contrast DNMT1 is a maintenance methyltransferase, and it modifies hemi-methylated DNA during replication.
Figure 1. DNA methylation and demethylation overview.
DNA demethylation is believed to involve the successive oxidation of 5-mC to 5-hydroxymethyl- (5-hmC), 5-formyl- (5-fC) and 5-carboxy- (5-caC) cytosine in a process that involves the Tet family of enzymes including Tet1, Tet2 and Tet3 (Figure 1). A glycosylase of the base excision repair (BER) pathway, TDG, was also recently found to be involved in an alternative demethylation pathway, where it can repair the 5-hydroxymethyluracil which can result from deamination of 5-hmC
Main approaches to study DNA methylation are:
Sodium bisulfite conversion is one of the most useful tools for analyzing cytosine methylation. This method is based on treating DNA with sodium bisulfite in order to determine its methylation pattern.
Treatment of DNA with sodium bisulfite leads to the deamination of cytosine residues and converts them to uracil, while 5-mC residues remain the same (Figure 2). The treatment generates specific changes in the DNA sequence, potentially providing single-nucleotide resolution information about the methylation status of a DNA region.
Figure 2. Schematic of bisulphite conversion for the analysis of DNA methylation.
Bisulfite-modified DNA can be analyzed by PCR methods that can discriminate the methylation state of cytosines in specific genomic regions. Alternatively, bisulfite treatment can be coupled to next-generation sequencing to achieve single nucleotide resolution mapping of cytosine methylation across the whole genome.
This method involves the use of methylation-sensitive restriction endonucleases to produce DNA fragments based on the methylation status of the sequence. In general, a methylation-sensitive enzyme and a methylation-insensitive isoschizomer are used to digest the same DNA sample.
This technique can be used in combination with DNA sequencing, micro arrays, PCR or Southern blotting to provide information on the methylation status for the whole genome or for specific genomic loci.
The use of methylated DNA-binding proteins or antibodies that specifically recognize methylated DNA is another common method for studying methylated DNA. Antibodies can be used in an ELISA-type assay to specifically detect the percentage of methylated or hydroxymethylated cytosine residues in the sample: the higher the signal, the higher percentage of methylated DNA.
Antibodies can be also used to immunoprecipitate methylated DNA thereby enriching a DNA sample in methylated DNA. This technique is also known as MeDIP, or methylated DNA immunoprecipitation, and it can be coupled to locus specific PCR or to whole genome approaches such as microarrays or next-generation sequencing. Similar techniques have been developed using methylated DNA-binding proteins instead of antibodies.