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DNA Methyltransferase Pathway


Figure1. DNA Methyltransferase pathway.

DNA Methyltransferase overview

DNA methylation is a post-replication modification common in prokaryotic and eukaryotic genomes and is involved in many important physiological processes in the body, including regulation of gene expression, gene imprinting, maintenance of chromosomal integrity, and X-chromosome inactivation. According to the structure and function, DNA methyltransferases (Dnmts) in mammals fall into two main categories, DNA methylation maintenance enzyme Dnmt1 and DNA de novo methylases Dnmt3a, Dnmt3b and Dnmt3L. In addition, Dnmt2 also has weak DNA methyltransferase activity, which has been found to methylate the tRNAAsp anti-codon loop in recent years. These Dnmts are important for mammalian growth and development, and their dysfunction will lead to many diseases such as embryonic developmental disorders and cancers. Therefore, Dnmts may become an important molecular target and play an important role in the treatment and prevention of diseases.

DNA Methyltransferase family

In mammals, four Dnmts have been found to be classified into two broad categories based on differences in structure and function, represented by Dnmt1 and Dnmt3, respectively. The former is mainly involved in the maintenance of methylation status and is also necessary for CPG site de novo methylation, and is related to the extension of methylation status; the latter includes Dnmt3a, Dnmt3b, and Dnmt3L, etc., which is the main de novo methylation. Enzymes and the attribution and function of Dnmt2 are not yet clear. Dnmt1 consists of two parts structurally, the N-terminal large domain contains several regulatory sites, and the C-terminus is a catalytic domain. There are three different splice isoforms in Dnmt1, namely Dnmt1s, Dnmt1o, and Dnmt1p. Dnmt1s is expressed in somatic cells, Dnmt1p is only found in the crude line spermatocytes, and Dnmt1o is specifically expressed in oocytes and preimplantation embryos. Dnmt1p does not have Dnmt1 activity because its first exon ORF is too short and may interfere with normal translation. Dnmt1s is a key enzyme that maintains methylation after replication. It undergoes high-speed methylation along DNA. The CGCTC site tends to terminate this process. It may be the terminator of Dnmt1 action, but it is also the repressor protein CTCF binding site. CTCF or an independent pathway prevents DNA methylation from spreading to the unmethylated region of DNA. Dnmt3 family is highly homologous to bacterial 5mc methyltransferase. Dnmt3a and Dnmt3b have a similar structure, a highly variable N-terminus, including a PWWP site, a Zn binding site (containing multiple Cys, six CXXC structures), and a C-terminus with catalytic function. The N-terminus of Dnmt3L has only a motif PHD domain like the plant homology domain, and the C-terminus extends only to the conserved motif VIII, lacking an efficient catalytic domain. Thus, Dnmt3L itself has no catalytic activity. Dnmt3a is mainly located in heterochromatin, and Dnmt3a2 is the main enzyme in embryonic development. It is in euchromatin, mainly in embryonic stem cells, and is required for genomic imprinting. The results of RT-PCR showed that Dnmt3a was highly abundantly expressed in the egg cells and then gradually decreased in the 8-cell stage. Dnmt3L is highly expressed in the chorion of the testis and placenta and is de novo methylated with Dnmt3. Dnmt3L can increase the methylation activity of Dnmt3a and Dnmt3b by 1.5~3 times in a dose-dependent manner, and its C-terminus is associated with the enhanced Dnmt3 activity. Structurally, Dnmt3a forms a tetramer with Dnmt3L, the Dnmt3a dimer is in the middle, and the Dnmt3L dimer is on the outside, and the two are in contact through the C-end. Through the C-terminal G718-L719-Y720 structure, Dnmt3L can stabilize the conformation of the Dnmt3a activity site, including the nucleophilic group Cys706. The combination of Dnmt3a-Dnmt3L provides a stable and broad polarity effect that enhances the binding of Dnmt3a to SAM. This facilitates Dnmt3 involvement in chromatin remodeling. In addition, studies have shown that the deletion of Dnmt3a and Dnmt3b leads to down-regulation of the methylation level of Dnmt3L promoter region, which leads to incomplete inhibition of Dnmt3L transcription in ES cells, and Dnmt3L itself is involved in this methylation regulation, which is fully demonstrated as an important participant in DNA methylation and will be subject to various apparent regulation.

DNA Methyltransferase pathway

  1. DNA Methyltransferase pathway cascade
    Gene closure and activation is an extremely complex process. Dnmts affects the expression of this gene by modulating the level of DNA methylation in the CpG island of the promoter region of the gene. Within the genome, there are many CpG sites, which are often clustered. It is generally believed that the GC content of at least 200 bp DNA sequence exceeds 50%, and the CG observation/CG expectation value exceeds 0.6, which is called CpG island, which is more common in the promoter of the gene and has a length of 500~1000 bp. 56% of human gene promoter region has a CPG island. Regardless of gene expression, most CpG islands remain hypomethylated during development. However, there are also some gene promoter regions that are differentially methylated, mainly those that have important functions in development, such as homeobox (HOX), paired box (PAX), and their related genes. The data shows that the non-methylation and hypermethylation states are relatively stable, the low-density methylation is very unstable, and the probability of demethylation and de novo methylation is similar. Typically, promoter methylation causes the gene to be in a transcriptionally repressed state. For example, hypermethylation of the reelin and GAD67 promoter regions often causes a decrease in their mRNA expression. In the human hepatocyte cell line HepG2, many liver function metabolism genes are expressed from transcriptional repression to transcriptional activation after treatment with Dnmts inhibitor (5aza-dc). This suggests that Dnmts-mediated DNA methylation may limit the expression of fetal liver genes and shut down some of these genes. Gene imprinting refers to the modification of the alleles from the father and mother when it is passed to the offspring through sperm and eggs. This effect allows the offspring to express only one of the parent or maternal alleles. Up to now, there are more than 70 imprinted genes found in mammals, and their expression controls the occurrence and development of embryos. The CCCTC binding factor (CTCF) binds to the hypomethylated DMR1 region, establishing a heterochromatin boundary that limits the transcriptional activation of IGF2 by the H19 downstream enhancer; on the paternal chromosome, the hypermethylated DMR1 region cannot bind to CTCF. Therefore, IGF2 can activate transcription using an enhancer downstream of H19. Genomic imprinting is an important mechanism of gene regulation. Due to the role of imprinting, only one of the parent’s genes will be expressed. The mechanism of gene imprinting is not fully understood, but DNA methylation is an important factor even if it is not the main mechanism of action. During development, the embryo undergoes a process of demethylation and remethylation. In mature germ cells, it is often a high methyl state. Once conceived, the entire genome of the embryo is demethylated, and after embryo implantation, methylation occurs again. Dnmts related to embryonic development mainly include three kinds, Dnmt1o, Dnmt3a2, and Dnmt3L, and their deletion often leads to embryonic death. In the early stages of development, Dnmt1o is one of the main enzymes that maintain the methylated form of the imprinted gene. Studies have shown that Dnmt3a can methylate H19 and Dlk1/Gtl2 DMR and short tandem repeat series SineB1. Dnmt3b is required for microsatellite DNA repeat series methylation, and it is considered that Dnmt3a in germ cells is mainly Dnmt3a2. At physiological salt concentrations, Dnmt3a2 activity is inhibited.
  2. Pathway regulation
    Recent studies have also revealed the interaction of Dnmt with gene silencing related factors. EZH2 (Enhancer of zeste homolog 2) in the polycomb repressive complexes of the PcG protein 2 and 3 can interact with Dnmt to inhibit gene expression. PRC1 activation-related NSPc1 (nervous system polycomb 1) protein binds to DNMT1 and EZH2 in the HOXA7 promoter in HeLa cells. The binding of DNMT1 to the HOXA7 promoter decreases when NSPc1 is deleted, and the binding level of NSPC1 when DNMT1 is absent also declines. These findings suggest that there is also an interaction between the gene silencing mechanism of Dnmt involved in DNA methylation and the gene silencing mechanism involved in PcG. Some factors can act on Dnmt, resulting in changes in the amount or activity of the enzyme, which affects the level of DNA methylation and affects gene expression. The HDAC inhibitor apicidin down-regulates DNMT1 levels in HeLa cell lines, and the absence of DNMT1 leads to tumor cell death. SNF2 (sucrose nonfermenting protein 2) family-associated ATP-dependent chromatin remodeling protein hHNF2H (Human sucrose nonfermenting protein 2 homolog) can bind to the N-terminus of DNMT1. When hSNF2H is present, the affinity of DNMT1 for single nucleotides is increased. The study found that another SNF2 family-associated ATP-dependent chromatin remodeling protein LSH (lymphoid-specific helicase) does not directly lead to DNA methylation, but indirectly leads to methylation by increasing the number of Dnmt. In a recent study, the coding gene aof 2 of mouse LSD1 (Lysine-specific demethylase 1, also known as KDM1 and AOF2) was knocked out and DNA methylation levels decreased, and Dnmt1 levels decreased. Histone methylase SET7 also regulates the stability of DNMT1. SET7 inhibits the amount and activity of Dnmt1 by directly interacting with DNMT1 and specifically methylating lysine 142 of DNMT1. These factors can be involved in gene silencing indirectly by regulating the quantity and activity of Dnmt, and Dnmt is also involved in the regulation of gene expression at other levels through these factors.
  3. Relationship with disease
    Colorectal cancer
    Hypermethylation of the hMLH1 promoter occurs in 15% to 20% of rectal tumors. DNA methylation inhibitors have yet to be therapeutic targets for colorectal cancer.
    Immunodeficiency syndrome
    The immunodeficiency syndrome is caused by a mutation in the catalytic domain of Dnmt3b, and its phenotype includes unstable chromosomes 1, 9, and 16. In addition, the overall methylation level of the genome in the senescent cells is reduced, while some genes are hypermethylated. Correspondingly, Dnmt1 activity decreases, while Dnmt3a and Dnmt3b activities increase significantly.

References:

  1. Yunlei Z, Zhenning X, Mo W, et al. DNA methyltransferase activity assay based on visible light-activated photoelectrochemical biosensor. Biosensors & Bioelectronics. 2014, 53(9):263-267.
  2. Singer B D, Mock J R, Aggarwal N R, et al. Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation. Am J Respir Cell Mol Biol. 2015, 52(5):641-652.
  3. Deng H, Yang X, Gao Z. MoS2 nanosheets as an effective fluorescence quencher for DNA methyltransferase activity detection. Analyst. 2015, 140(9):3210-3215.
  4. Murakami J, Asaumi J, Maki Y, et al. Influence of CpG island methylation status in O6-methylguanine-DNA methyltransferase expression of oral cancer cell lines. Oncology Reports. 2004, 12(2):339-345.

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