Characteristic DNA methylation patterns confirmed in placental tissue of patients with recurrent miscarriage of unknown cause
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In addition, when we extracted genes with large differences in DNA methylation between the recurrent abortion group and the normal pregnancy group, we succeeded in identifying SPATS2L as a representative gene.
A research group at Nagoya University and Nagoya City University conducted a DNA measurement of villi (derived from the fetus) of patients with unexplained recurrent miscarriage. → Continue reading
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DNA methylation(DNA methyl?) means in DNAbaseto the carbon atoms ofMethyl groupA chemical reaction in which modifications are added.EukaryoteからProkaryotes, are found widely in living things, down to viruses.Especially for eukaryotes,CpG islandCommonly found in genomic regions such as segments,EpigeneticsIt is considered to be an essential mechanism for accurately shaping the body of complex organisms.canceris thought to be involved in the formation and progression of
DNA methylation isCytosine OfPyrimidinefifth position in the ringcarbonatom orAdenine OfPuddingfifth position in the ringnitrogento the atomMethyl group(Cytosine and adenine areDNA4 types ofbase).This modifier isCell divisionIt is inherited even after passing through.DNA methylation is usuallyJoined bodyremoved during formation,Outbreakrebuilt through cell division that continues duringHowever, recent studies have shown that in the zygote, rather than complete removal of the methyl group,Hydroxylationis shown to be happening. DNA methylation isHigher organismsnormal occurrence in細胞 OfDifferentiationplays an extremely important role in DNA methylation is細胞``Where am I?''Memoryas stable as possiblegeneAlter expression patterns or decrease gene expression.for example,Embryoduring the outbreakpancreasLangerhans IslandA cell programmed to become a pancreatic islet of Langerhans remains a pancreatic islet for the life of the organism, even if it continues to receive no signal to be an islet of Langerhans.Furthermore, DNA methylation increases the host'sgenometaken inウ イ ル スand suppress the expression of genes of other harmful elements. DNA methylation alsoChromatinform the foundation of the structure.This allows the cell toMulticellular organismcan form a myriad of features necessary for DNA methylation also affects almost all types ofcancerplay a pivotal role in the development of.
DNA methylation involves the addition of methyl groups to DNA—for example, the 5th carbon atom of the pyrimidine ring of cytosine—which in this case has the specific effect of decreasing gene expression.Methylation at the 5-position of cytosine was observed in allvertebrateis found inIn adult somatic tissues, DNA methylation is normallydinucleotide site (cytosine-Phosphodiester bond-Guanine).Non-CpG methylation isEmbryonic stem cellswidespread in.
60-90% of all CpG sites are methylated in mammals.Methylated cytosine residues spontaneouslyAmino groupis removedThymineIt becomes a residue.Therefore, CpG dinucleotides in turn mutate to TpG dinucleotides.This is evident from the low frequency of CpG dinucleotides in the human genome (CpG dinucleotides are present at only 21% of the expected frequency)..On the other hand, for the spontaneous deamination of unmethylated cytosinesUracilThis mutation is quickly recognized by cells,repairIs done.
Unmethylated CpG is often the 5' of many genes Exists inCpG islandIt is collected as a cluster calledcancerIn many disease processes such as the genepromoterCpG islands that areCell divisioninherited by daughter cells ofGene silencinghappens. Alterations in DNA methylation are recognized as an important component of cancer development.Hypermethylation is associated with promoters and genes (Tumor suppressor gene), whereas hypomethylation generally occurs earlyChromosomeassociated with instability and loss of imprinting.But hypomethylation could also be a target for epigenetic therapy.
DNA methylation can be detected in genes in two ways.Transcriptionaffect. First, DNA methylation itself is physicallytranscription proteinThe second, and perhaps more important, factor that prevents DNA from binding to genes is the binding of methylated DNA to methyl-CpG-binding domain protein (MBD).Next, the MBD protein is located at the locusHistonemodify theHistone deacetylaseAnd otherChromatin reconstructionRecruit additional proteins such as protein, packed and inactivatedChromatin(silent chromatin) is formed.The connection between this DNA methylation and chromatin structure is very important.In particular, deletion of methyl-CpG-binding protein 2 (MeCP2)Rett syndromeis related tomethyl-CpG-binding domain protein 2 (MBD2)mediates transcriptional silencing of hypermethylated genes in cancer.
DNA methylation in cancer
DNA methylation is a key regulator of gene transcription, aberrant DNA methylation is associated with unplanned gene silencing, high levels of5-MethylcytosineA large body of evidence reveals that genes containing DNA methylation isEmbryodevelopment, and in somatic cells, patterns of DNA methylation are generally passed on to daughter cells with high fidelity.Aberrant DNA methylation patterns are associated with manyHuman capitalIt is associated with malignancies and has two distinct forms, hypermethylation and hypomethylation compared to normal tissue.Hypermethylation is one of the major epigenetic modifications that act on the promoter regions of tumor suppressor genes to repress transcription.Hypermethylation usually occurs at CpG islands in promoter regions and is associated with gene inactivation.Extensive hypomethylation is also associated with cancer development and malignant transformation through different mechanisms.As an example of gene inactivation by methylation of the gene promoter region, humanBreast cancer, in uterine cancerEstrogenReceptor-deficient, in non-hereditary breast cancerBRCA1inactivation can be given.
DNA methyltransferase in mammals
Maintained methylation activity of all cellsDNA replicationRequired for preservation of DNA methylation after cycling.DNA methyltransferase In the absence of (DNMT), the replication machinery produces unmethylated daughter strands, of which passive demethylation occurs.DNMT1is a putative maintenance methyltransferase required to replicate DNA methylation patterns to daughter strands during DNA replication. Model mice lacking two copies of DNMT1 are embryonic lethal at approximately day 2, because DNMT1 activity is required for mammalian cell development.
DNMT3aandDNMT3bis the DNA methylation that shapes the pattern of DNA methylation during early development. de novo It is believed to be a methyltransferase.DNMT3Lis homologous to other DNMT3 proteins but lacks catalytic activity.Instead, DNMT3L isde novo It assists methyltransferases by increasing their ability to bind to DNA and stimulating their activity.Finally, tRNA(cytosine-5-)-methyltransferase (DNMT2 (TRDMT1)) is a protein that has been identified as a DNA methyltransferase homolog and contains all 10 sequence motifs common to all DNA methyltransferases.However, DNMT2 (TRDMT1) does not methylate DNA, instead of aspartate-tRNAAnticodonMethylates cytosine-38 in the loop.
Many tumor suppressor genes areAttempts have been made to re-express these tumor suppressor genes by inhibiting the DNMT protein. 5-aza-2'-deoxycytidine () are nucleoside derivatives and are catalyticβ-eliminationBy interfering with the step and trapping the DNMT and DNA in a covalent complex, the enzyme is degraded, resulting in inhibition of DNMT activity.However, although decitabine is active, it must be incorporated into the cell's genome, causing genetic mutations in daughter cells if the cell does not die.In addition, decitabine is dose-limiting due to its myelotoxicity.From these pitfallsMessenger RNA (mRNA) degraded翻 訳targeted DNMTs that interfere withantisense RNATreatment development has progressed.However, it is currently unclear whether treatments targeting DNMT1 alone are sufficient to reactivate tumor suppressor genes silenced by DNA methylation.
モデルplantIsArabidopsis (Arabidopsis thaliana), our understanding of DNA methylation has advanced significantly.DNA methylation in plants differs from that in mammals.DNA methylation in mammals occurs primarily at cytosine nucleotides at CpG sites, whereas in plants cytosines are methylated at CpG, CpHpG, and CpHpH sites (H represents a nucleotide other than guanine).
A major molecule that transfers a methyl group to DNA and covalently binds it. Arabidopsis DNA methyltransferase enzymes are DRM2, MET1, CMT3. The DRM2 and MET1 proteins share high homology with the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 proteinBotanical kingdomis a protein unique toAs mentioned in the previous section, there are currently two classes of DNA methyltransferases. 2) create new methyl tags in DNA de novo and 2) a maintenance enzyme that recognizes methylation sites on the parent strand of DNA and transfers the new methylation to the daughter strand after DNA replication. Only DRM2 de novo Considered to be a DNA methyltransferase. DRM2 has also been shown to be involved in maintaining methylation markers after DNA replication along with MET1 and CMT3.Other DNA methyltransferases are expressed in plants but have no known function (Chromatin DatbaseSee).
how the cellsde novo Although it is not clear whether it determines the position of DNA methylation, evidence to date suggests that at many (but not all) positions, (RNA-directed DNA methylation: RdDM) is suggested to be involved. In RdDM, a specificRNAA transcript is produced from a genomic DNA template, and this RNA forms a secondary structure called a double-stranded RNA molecule.Double-stranded RNA is a small interfering RNA (siRNA) or microRNA (miRNAs) pathway of the original genomic location that produced this RNA de novo Directs DNA methylation.A mechanism of this kind isRNA virusとTransposonare thought to be important in cellular defense against either or both Both RNA viruses and transposons often form double-stranded RNA against the host genome.By a poorly understood mechanism, their RNA transcription is terminated, rendering them inactive in the cell and protecting the genome from mutational effects by methylation of the genomic loci where they are docked.
manyFungushave low levels of cytosine methylation (0.1-0.5%), whereas other fungi have ~5% genome methylation.This methylation value varies between species and between isolates of the same species.DNA methylation has also been shown to be involved in developmental stage-dependent regulation of gene expression in fungi..
Beer yeast (saccharomyces) andFission yeast (Schizosaccharomyces) have very low DNA methylation levels, but the modelFilamentous fungusNeurospora crassa (Neurospora crassa) has a well-characterized methylation system.Several genes control methylation in Neurospora, and the DNA methyltransferase dim-2 mutation abolishes all DNA methylation but does not affect growth or sexual reproduction.Although repetitive DNA is very rare in the Neurospora genome, half of the methylation occurs in repetitive DNA containing transposon remnants and DNA centromeres. Neurospora has become an important model system for the study of DNA methylation, as it allows us to assess other important phenomena in genetic backgrounds in which DNA methylases are deficient.
manyEubacteria(bacteria) andArchaea(archaea) also has methylation of adenine and cytosine.Here, specific DNA sequences are cyclically methylated throughout the genome.Some of these methylations areDNA methyltransferase(methylase) andRestriction enzymesIt is composed of two enzymes calledRestriction modification systemby a system calledDNA methyltransferaserecognizes a specific sequence and methylates one of the bases in or near this sequence.When unmethylated foreign DNA is introduced into cells in this manner, sequence-specificRestriction enzymesdisassembled and cut byBacterial genomic DNA is not recognized by these restriction enzymes.Methylation of native DNA acts as a primitive immune system in this species, protecting the bacteria themselves.Bacteriophageprotects against infection byagain,giant virusdouble-stranded containingDNA virusDNA methylation, including that caused by restriction-modification systems, is known to occur widely in.
E. coli E. E. coli coli DNA adenine methyltransferase (Dam) does not belong to the restriction/qualification system ~32 kDa kDais the enzyme ofE. E. coli coli The target recognition sequence of Dam is GATC and methylation occurs at the N6 position of the adenine of this sequence (G meATC).The 3 base pairs flanking this sequence also affect DNA-Dam binding. Dam isDNA mismatch repairand plays several important roles in bacterial processes such as DNA replication timing and gene expression. by DNA replicationE. E. coli coliGATC sites in the genome go from fully methylated to half methylated (hemimethylated).This is because the adenine introduced into the new DNA strand is unmethylated.Remethylation occurs within 2-4 seconds while replication errors in the new DNA strand are repaired.Methylation or lack of methylation allows the cell's repair machinery to distinguish between template and nascent strands.Alterations in bacterial Dam activity have been shown to increase spontaneous mutation rates.Bacterial viability is compromised in the dam mutant. The lack of other specific DNA repair enzyme activities in dam mutants provides further evidence for the role of Dam in DNA repair.
A region of DNA that remains hemimethylated for a long time is enriched with GATC sitesOrigin of replicationis.It lies at the heart of the DNA replication timing machinery in bacteria. SeqA prevents methylation by binding to and sequestering replication origins.This mechanism limits DNA replication to once per cell cycle because the hemimethylated origin of replication is inactive.specific genes, e.g.E. E. coli coli Ofsexual ciliaThe expression of the gene encoding the geneOperonis regulated by methylation of GATC sites present in the promoter region of The environmental conditions of cells immediately after DNA replication determine whether methylation of regions proximal or distal to the promoter region by Dam is prevented.Once the methylation pattern is created, transcription of the sex cilia gene is locked into an on or off state until the DNA is replicated again.E. E. coli coliIn , these sex cilia operons have important roles in the pathogenesis of urinary tract infections. Dam inhibitors areAntibioticshas been proposed to function as[By whom?].
DNA cytosine methylase, on the other hand, targets the CCAGG and CCTGG sites and methylates the C5 position of cytosine (CmeC(A/T)GG).Another methyltransferase, EcoKI, methylates adenines in AAC(N6A)GTGC and GCAC(N6A)GTT sequences.
Most strains used in molecular biology are derivatives of the K-12 strain and possess both Dam and Dcm, but there are also commercially available strains with dam-/dcm- activity.Indeed, DNA extracted from dam+/dcm+ strains can be demethylated by transforming into dam−/dcm− strains..
DNA methylation can be detected by the following tests currently used in scientific research.
- The MSP method converts unmethylated cytosines of CpG dinucleotides to uracil (UpG)sodium bisulfiteand DNA chemical reaction followed by normalPCRbased on law.Since methylated cytosines are not converted by this treatment, primers can be designed to overlap the CpG site of interest to determine whether they are methylated or not.
- (Whole genome bisulfite sequencing, WGBS)
- The WGBS method is the same as the MSP methodsodium bisulfite (sodium bisulfite) is used.By sequencing a DNA fragment in which only unmethylated cytosines have been converted to uracil by treatment with sodium bisulfite, the cytosine methylation rate can be determined genome-wide with single base resolution.Since unmethylated cytosine converted to uracil is read out as thymine, mapping to the reference genome requires mapping software (Bismark, BSMAP etc.) should be used.
- The HELP assay is based on the ability of the restriction enzyme HpaII to recognize and cut differentially between methylated and unmethylated CpG DNA sites.
- ChIP (Chromatin immunoprecipitation)-on-chip assays are commercially availableantibodyTo use.
- (Restriction landmark genomic scanning, RLGS)
- The RLGS method is complicated and rarely used at present.It is similar in concept to the HELP assay in that it uses restriction enzymes to distinguish between methylated and unmethylated CpG sites.
- (Methylated DNA immunoprecipitation, MeDIP)
- The MeDIP method is similar to chromatin immunoprecipitation,DNA microarray (MeDIP-chip) andDNA sequencing Immunoprecipitation is used to isolate methylated DNA fragments that are input to DNA detection methods such as (MeDIP-seq).
- Molecular break light assay
- The molecular break light assay uses the restriction enzyme DpnI, which has specificity for fully methylated (adenine methylation) GATC sites.The oligonucleotides are labeled with fluorescent and quenching molecules.Adenine methyltransferase methylates molygonucleotides, which become substrates for DpnI. Cleavage of oligonucleotides by DpnI increases fluorescence intensity.
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