DNA methylation is an epigenetic modification that involves DNA methyltransferase (DNMT) catalyzing S-adenosylmethionine (SAM) as a methyl donor, selectively adding methyl groups to the cytosine of two nucleotides in the CG region of DNA, The gene mainly forms 5-methylcytosine (5-mC) (commonly found in the 5 '- CG-3' sequence of genes) and a small amount of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG). The gene contains many CpG structures, and the 5-carbon atoms of the two cytosines in 2CpG and 2GPC are usually methylated, and the two methyl groups form specific three-dimensional structures in the double stranded groove of DNA. 60% to 90% of CpGs in the genome are methylated, while unmethylated CpGs cluster together to form CpG islands, located in the core sequence of structural gene promoters and transcription initiation points. DNA methylation can cause changes in the chromatin structure of corresponding regions in the genome, causing DNA to lose the cleavage sites of ribozyme ö restriction endonucleases and sensitive sites of DNA enzymes, resulting in highly helical chromatin condensation into clusters and loss of transcriptional activity. 5-methylated cytosine deamines to produce thymine, which may lead to gene displacement mutations and base mismatches: T2G, if not corrected during cell division, can trigger genetic diseases or cancer, and the way organisms methylate is stable and heritable.

5-methylcytosine is the only chemically modified base present in eukaryotic DNA. CG dinucleotide is the most important methylation site, which is unevenly distributed in the genome, with regions of high methylation, low methylation, and non methylation. In mammals, mC accounts for about 2-7% of the total amount of C. DNA methylation is the main mode of epigenetic modification, which can alter genetic expression without altering the DNA sequence. Epigenetic coding is a part of the exogenetic mechanism. The DNA methylation process adds methyl groups to DNA molecules, such as on the 5 'carbon of the cytosine ring: this 5' direction of DNA methylation is visible in all vertebrates. Approximately 1% of DNA bases in human cells are methylated. In mature cellular tissues, DNA methylation generally occurs at the CpG dinucleotide site; Non CpG methylation is more common in embryonic stem cells. The methylation of cytosine in plants can be divided into symmetrical CpG (or CpNpG) or asymmetric CpNpNp forms (C and G are bases; p is phosphate; N refers to any nucleotide). The methylation of specific cytoskeleton can be determined using bisulfite sequencing. DNA methylation may silence genes, leading to their loss of function. In addition, there are also some organisms that do not have DNA methylation.

There are two types of DNA methyltransferases:

(1) DNM T1, persistent DNA methyltransferase - acts on DNA double strands with only one strand methylated, causing complete methylation and participating in the methylation of newly synthesized strands in DNA replication double strands. DNM T1 may directly act in conjunction with HDAC (histone deacetyltransferase) to block transcription;

(2) DNM T3a and DNM T3b are de novo methyltransferases that can methylate CpG, causing it to semi methylate and then fully methylate. De novo methyltransferase may be involved in the regulation of cell growth and differentiation, among which DNM T3b plays an important role in tumor gene methylation.

There are two ways for DNA demethylation:

(1) Passive pathway: Due to the adhesion of nuclear factor NF to methylated DNA, the DNA near the adhesion point cannot be completely methylated, thereby blocking the action of DNM T1;

(2) Active pathway: It is the process of removing methyl groups through the action of demethylases. Methylated CpG adhesion proteins play an important role in the process of DNA methylation inhibiting gene expression. Although methylated DNA can directly act on methylation sensitive transcription factors E2F, CREB, A P2, CM yc ö M yn, NF2 κ B. Cmyb and Ets lose their ability to bind to DNA, thereby blocking transcription. However, methylated CpG adhesion molecules can act on methylated non sensitive transcription factors (SP1, CTF, YY1), inactivating them and thus blocking transcription. Five methylated CpG adhesion proteins with a constant methylated DNA binding domain (MBD) have been discovered. Among them, M ECP2, MBD1, MBD2, and MBD3 are involved in transcriptional repression related to methylation; MBD1 has glycosyltransferase activity, which can remove T from the mismatched base pair T ö G. Mutations in the MBD4 gene are also associated with mitochondrial instability in tumor development. In MBD2 deficient mouse cells, the absence of the M ECP1 complex cannot effectively prevent the expression of methylation genes. This indicates that methylated CpG adhesion proteins play an important role in the selection of DNA methylation modes, as well as in the interaction between DNA methylation, histone deacetylation, and chromatin recombination.

DNA methylation detection method: BSP

Principles of methylation detection methods

The bisulfite genomic sequencing PCR (BSP) method utilizes the principle that unmethylated cytosine can be deaminated by sodium bisulfite to form uracil, and amplifies with two specific primers before sequencing. The sequencing method overcomes the disadvantage of only being able to detect individual loci, which must be restriction endonuclease recognition loci, and can detect the methylation status of any gene sequence.