Methylation refers to the process of catalytic transfer of methyl from active methyl compounds to other compounds. It can form various methyl compounds, or chemically modify some proteins or nucleic acids to form methylation products. In biological systems, methylation is catalyzed by enzymes. This methylation involves heavy metal modification, gene expression regulation, protein function regulation and RNA processing. [1]
Methylation includes DNA methylation And protein methylation. (1) DNA methylation: In vertebrates, DNA methylation generally occurs at the CpG site (cytosine phosphate guanine site, that is, the site where cytosine is followed by guanine in the DNA sequence). Transformation of cytosine to 5-Methylcytosine 。 About 80% - 90% of the CpG sites in human genes have been methylated, but in some specific regions, such as those rich in cytosine and guanine CpG Island Is not methylated. This is related to promoters in 56% of mammalian genes including all widely expressed genes. 1% - 2% of the human genome is a CpG group, and CpG methylation is inversely proportional to transcriptional activity. [2] (2) Protein methylation: Protein methylation generally refers to the methylation of arginine or lysine in the protein sequence. Arginine can be methylated once (called monomethyl arginine) or twice (arginine methyltransferases (PRMTs) simultaneously transfer two methyl groups to the same nitrogen atom at the end of arginine polypeptide to become asymmetric methyl arginine, Or add a methyl at each nitrogen end to form symmetrical dimethylarginine) lysine can be methylated once, twice or three times by the catalysis of lysine transferase. Among histones, protein methylation has been studied most. Under the catalysis of histone transferase, S-adenosylmethionine Methyl group transfer to histone. Some histone residues can be inhibited or activated by methylation gene expression Thus, it becomes epigenetic. Protein methylation is a form of post-translational modification. [3] Methylation is an important modification of protein and nucleic acid, which regulates the expression and shutdown of genes and is closely related to many diseases such as cancer, aging and Alzheimer's disease. It is one of the important research contents of epigenetics. The most common methylation modifications are DNA methylation and histone methylation.
DNA methylation can turn off the activity of some genes, while demethylation can induce gene reactivation and expression. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability and the interaction mode between DNA and protein, thus controlling gene expression. Studies have confirmed that the methylation of cytosine in CpG dinucleotide leads to more than 1/3 of human genetic diseases caused by base conversion. DNA methylation mainly forms 5-methylcytosine (5-mC) and a small amount of N6-methyladenine (N6-mA) and 7-methylguanine (7-mG). In eukaryotes, 5-methylcytosine mainly appears in CpG sequence, CpXpG, CCA/TGG and GATC.
DNA methylation refers to that the organism takes s-adenosylmethionine (SAM) as the Methyl donor The process of transferring methyl groups to specific bases. DNA methylation can occur at N-6 position of adenine, N-7 position of guanine, C-5 position of cytosine, etc. However, in mammals, DNA methylation mainly occurs on the C of 5 '- CpG-3' to generate 5-methylcytosine (5mC). In mammals, CpG exists in two forms: one is dispersed in DNA sequence; The other is highly concentrated, which is called CpG island. In normal tissues, 70%~90% of scattered CpG is methylated, while CpG islands are often unmethylated (except for some special regions and genes). Under normal circumstances, the CpG dinucleotides of the "junk" sequence of the human genome are relatively rare and always in the methylation state. On the contrary, the size of the human genome is about 100-1000 bp, and the CpG island rich in CpG dinucleotides is always in the unmethylated state, and the CpG island is often located near the transcriptional regulation area, which is related to 56% of the coding genes of the human genome, Therefore, it is very important to study the methylation status of CpG island in gene transcription region. The analysis results of human genome sequence sketch show that there are about 28890 CpG islands in the human genome, and most chromosomes have 5-15 CpG islands per 1Mb, with an average of 10.5 CpG islands per Mb. The number of CpG islands has a good correlation with gene density.
DNA methylation is mainly catalyzed by the DNA methyltransferase family. Researchers found three types of DNA methyltransferases (Dnmt1, Dnmt2, Dnmt3a, Dnmt3b) in eukaryotes; Dnmt2 can bind to specific sites on DNA, but the specific role is still unclear; Dnmt3a and Dnmt3b are re methylation enzymes, which re methylation the demethylated CpG site, that is, participate in de novo methylation of DNA. During mammalian germ cell development and preimplantation embryo stage, the methylation pattern within the genome is reprogrammed through large-scale demethylation and subsequent remethylation process to generate cells with developmental potential; In the process of cell differentiation, the methylation status of genes will be passed on to offspring cells. Due to the close relationship between DNA methylation and human development and tumor diseases, especially the inactivation of tumor suppressor gene transcription caused by CpG island methylation, DNA methylation has become an epigenetic and Epigenomics The important research content of this paper. Histone methylation refers to the methylation of Arg or Lys residues at the N-terminal of H3 and H4 histones, which is mediated and catalyzed by histone methyltransferase. The functions of histone methylation are mainly embodied in heterochromatin formation, gene imprinting, X chromosome inactivation and transcriptional regulation. In addition to histone methyltransferase, demethylase was also found. It was previously thought that histone Methylation It is stable and irreversible. It is the discovery of this demethylase that makes the histone methylation process more dynamic. (1) Methylation specific PCR (MSP)
When genomic DNA is treated with bisulfite, all unmethylated cytosines are converted to uracil, while methylated cytosines remain unchanged; Then we designed primers for methylation and unmethylation sequences for PCR. The MSP amplification product is detected by electrophoresis. If the amplification segment can be obtained by using the primer for the methylated DNA chain after treatment, it indicates that the site has methylation; On the contrary, there is no methylation at the detected site.
(2) Bisulfite sequencing PCR (BSP)
When genomic DNA is treated with bisulfite, the unmethylated cytosine is converted to uracil, while the methylated cytosine remains unchanged. Then, BSP primers were designed for PCR. During the amplification process, all uracil was converted into thymine. Finally, sequencing the PCR product can determine whether the CpG site was methylated, which is called BSP direct sequencing method. The success rate of sequencing can be improved by cloning PCR products into vectors and sequencing them. This method is called BSP cloning sequencing.
(3) High Resolution Melting (HRM)
A pair of primers for bisulfite modified DNA double strand were designed at the non CpG island location. The fragment in the middle of the pair of primers contains the CpG island of interest. If these CpG islands are methylated, the unmethylated cytosine will be transformed into thymine after PCR amplification after being treated with bisulfite, while the methylated cytosine will not change, and the GC content in the sample will change, leading to the change of melting temperature.
(4) Direct genome sequencing
Direct genome sequencing is the research method of DNA methylation that has been used in the past. Maxam Gilbert chemical lysis method is used to process genomic DNA, and link mediated PCR is used to amplify the signal strength, and then sequence analysis is carried out. This method is based on the fact that 5mC is not cracked in the standard Maxam Gilbert cytosine chemical cracking reaction, so 5mC can be identified by the lack of a band on the sequencing gel corresponding to the cytosine degradation reaction product. If MnO is used four - Piperidine method, on the contrary, so when detecting 5mC, the two methods can provide complete complementary detection information. After combining this method with LM-PCR, the requirement of genomic DNA (1-2 ng) was greatly reduced. When 5mC and C are at the same site on different DNA molecules at the same time, the site must have at least 25% of the 5mC to be N two H four Method; MnO four - Fabi N two H four The method is more sensitive. Because these two chemical modification methods of genomic DNA have the characteristics of inhibiting the extension of DNA polymerase, methylation analysis can be carried out by direct genome sequencing technology without DNA piperidine cleavage. [4]
