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DNA methylation

August 18, 2023

DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. In mammals, DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis.

Representation of a DNA molecule that is methylated. The two white spheres represent methyl groups. They are bound to two cytosine nucleotide molecules that make up the DNA sequence.

As of 2016, two nucleobases have been found on which natural, enzymatic DNA methylation takes place: adenine and cytosine. The modified bases are N6-methyladenine[1], 5-methylcytosine[2] and N4-methylcytosine.[3]

Unmodified base  
  Adenine, A   Cytosine, C
Modified forms
  N6-Methyladenine, 6mA   5-Methylcytosine, 5mC   N4-Methylcytosine, 4mC

Two of DNA's four bases, cytosine and adenine, can be methylated. Cytosine methylation is widespread in both eukaryotes and prokaryotes, even though the rate of cytosine DNA methylation can differ greatly between species: 14% of cytosines are methylated in Arabidopsis thaliana, 4% to 8% in Physarum,[4] 7.6% in Mus musculus, 2.3% in Escherichia coli, 0.03% in Drosophila, 0.006% in Dictyostelium[5] and virtually none (0.0002 to 0.0003%) in Caenorhabditis[6] or fungi such as Saccharomyces cerevisiae and S. pombe (but not N. crassa).[7][8]: 3699  Adenine methylation has been observed in bacterial, plant, and recently in mammalian DNA,[9][10] but has received considerably less attention.

Methylation of cytosine to form 5-methylcytosine occurs at the same 5 position on the pyrimidine ring where the DNA base thymine's methyl group is located; the same position distinguishes thymine from the analogous RNA base uracil, which has no methyl group. Spontaneous deamination of 5-methylcytosine converts it to thymine. This results in a T:G mismatch. Repair mechanisms then correct it back to the original C:G pair; alternatively, they may substitute A for G, turning the original C:G pair into a T:A pair, effectively changing a base and introducing a mutation. This misincorporated base will not be corrected during DNA replication as thymine is a DNA base. If the mismatch is not repaired and the cell enters the cell cycle the strand carrying the T will be complemented by an A in one of the daughter cells, such that the mutation becomes permanent. The near-universal use of thymine exclusively in DNA and uracil exclusively in RNA may have evolved as an error-control mechanism, to facilitate the removal of uracils generated by the spontaneous deamination of cytosine.[11] DNA methylation as well as many of its contemporary DNA methyltransferases have been thought to evolve from early world primitive RNA methylation activity and is supported by several lines of evidence.[12]

In plants and other organisms, DNA methylation is found in three different sequence contexts: CG (or CpG), CHG or CHH (where H correspond to A, T or C). In mammals however, DNA methylation is almost exclusively found in CpG dinucleotides, with the cytosines on both strands being usually methylated. Non-CpG methylation can however be observed in embryonic stem cells,[13][14][15] and has also been indicated in neural development.[16] Furthermore, non-CpG methylation has also been observed in hematopoietic progenitor cells, and it occurred mainly in a CpApC sequence context.[17]

Conserved function of DNA methylation

 
Typical DNA methylation landscape in mammals

The DNA methylation landscape of vertebrates is very particular compared to other organisms. In mammals, around 75% of CpG dinucleotides are methylated in somatic cells,[18] and DNA methylation appears as a default state that has to be specifically excluded from defined locations.[15][19] By contrast, the genome of most plants, invertebrates, fungi, or protists show “mosaic” methylation patterns, where only specific genomic elements are targeted, and they are characterized by the alternation of methylated and unmethylated domains.[20][21]

 
Cytosine methylation then deamination to Thymine

High CpG methylation in mammalian genomes has an evolutionary cost because it increases the frequency of spontaneous mutations. Loss of amino-groups occurs with a high frequency for cytosines, with different consequences depending on their methylation. Methylated C residues spontaneously deaminate to form T residues over time; hence CpG dinucleotides steadily deaminate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency).[22] (On the other hand, spontaneous deamination of unmethylated C residues gives rise to U residues, a change that is quickly recognized and repaired by the cell.)

CpG islands

Main article: CpG islands

In mammals, the only exception for this global CpG depletion resides in a specific category of GC- and CpG-rich sequences termed CpG islands that are generally unmethylated and therefore retained the expected CpG content.[23] CpG islands are usually defined as regions with: 1) a length greater than 200bp, 2) a G+C content greater than 50%, 3) a ratio of observed to expected CpG greater than 0.6, although other definitions are sometimes used.[24] Excluding repeated sequences, there are around 25,000 CpG islands in the human genome, 75% of which being less than 850bp long.[22] They are major regulatory units and around 50% of CpG islands are located in gene promoter regions, while another 25% lie in gene bodies, often serving as alternative promoters. Reciprocally, around 60-70% of human genes have a CpG island in their promoter region.[25][26] The majority of CpG islands are constitutively unmethylated and enriched for permissive chromatin modification such as H3K4 methylation. In somatic tissues, only 10% of CpG islands are methylated, the majority of them being located in intergenic and intragenic regions.

Repression of CpG-dense promoters

DNA methylation was probably present at some extent in very early eukaryote ancestors. In virtually every organism analyzed, methylation in promoter regions correlates negatively with gene expression.[20][27] CpG-dense promoters of actively transcribed genes are never methylated, but, reciprocally, transcriptionally silent genes do not necessarily carry a methylated promoter. In mouse and human, around 60–70% of genes have a CpG island in their promoter region and most of these CpG islands remain unmethylated independently of the transcriptional activity of the gene, in both differentiated and undifferentiated cell types.[28][29] Of note, whereas DNA methylation of CpG islands is unambiguously linked with transcriptional repression, the function of DNA methylation in CG-poor promoters remains unclear; albeit there is little evidence that it could be functionally relevant.[30]

DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene,[31] and second, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that can modify histones, thereby forming compact, inactive chromatin, termed heterochromatin. This link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has been implicated in Rett syndrome; and methyl-CpG-binding domain protein 2 (MBD2) mediates the transcriptional silencing of hypermethylated genes in "cancer."

Repression of transposable elements

DNA methylation is a powerful transcriptional repressor, at least in CpG dense contexts. Transcriptional repression of protein-coding genes appears essentially limited to very specific classes of genes that need to be silent permanently and in almost all tissues. While DNA methylation does not have the flexibility required for the fine-tuning of gene regulation, its stability is perfect to ensure the permanent silencing of transposable elements.[32] Transposon control is one of the most ancient functions of DNA methylation that is shared by animals, plants and multiple protists.[33] It is even suggested that DNA methylation evolved precisely for this purpose.[34]

Genome expansion

DNA methylation of transposable elements has been known to be related to genome expansion. However, the evolutionary driver for genome expansion remains unknown. There is a clear correlation between the size of the genome and CpG, suggesting that the DNA methylation of transposable elements led to a noticeable increase in the mass of DNA.[35]

Methylation of the gene body of highly transcribed genes

A function that appears even more conserved than transposon silencing is positively correlated with gene expression. In almost all species where DNA methylation is present, DNA methylation is especially enriched in the body of highly transcribed genes.[20][27] The function of gene body methylation is not well understood. A body of evidence suggests that it could regulate splicing[36] and suppress the activity of intragenic transcriptional units (cryptic promoters or transposable elements).[37] Gene-body methylation appears closely tied to H3K36 methylation. In yeast and mammals, H3K36 methylation is highly enriched in the body of highly transcribed genes. In yeast at least, H3K36me3 recruits enzymes such as histone deacetylases to condense chromatin and prevent the activation of cryptic start sites.[38] In mammals, DNMT3a and DNMT3b PWWP domain binds to H3K36me3 and the two enzymes are recruited to the body of actively transcribed genes.

In mammals

 
Dynamic of DNA methylation during mouse embryonic development. E3.5-E6, etc., refer to days after fertilization. PGC: primordial germ cells

During embryonic development

Main article: DNA methylation reprogramming

DNA methylation patterns are largely erased and then re-established between generations in mammals. Almost all of the methylations from the parents are erased, first during gametogenesis, and again in early embryogenesis, with demethylation and remethylation occurring each time. Demethylation in early embryogenesis occurs in the preimplantation period in two stages – initially in the zygote, then during the first few embryonic replication cycles of morula and blastula. A wave of methylation then takes place during the implantation stage of the embryo, with CpG islands protected from methylation. This results in global repression and allows housekeeping genes to be expressed in all cells. In the post-implantation stage, methylation patterns are stage- and tissue-specific, with changes that would define each individual cell type lasting stably over a long period.[39] Studies on rat limb buds during embryogenesis have further illustrated the dynamic nature of DNA methylation in development. In this context, variations in global DNA methylation were observed across different developmental stages and culture conditions, highlighting the intricate regulation of methylation during organogenesis and its potential implications for regenerative medicine strategies.[40]

Whereas DNA methylation is not necessary per se for transcriptional silencing, it is thought nonetheless to represent a “locked” state that definitely inactivates transcription. In particular, DNA methylation appears critical for the maintenance of mono-allelic silencing in the context of genomic imprinting and X chromosome inactivation.[41][42] In these cases, expressed and silent alleles differ by their methylation status, and loss of DNA methylation results in loss of imprinting and re-expression of Xist in somatic cells. During embryonic development, few genes change their methylation status, at the important exception of many genes specifically expressed in the germline.[43] DNA methylation appears absolutely required in differentiated cells, as knockout of any of the three competent DNA methyltransferase results in embryonic or post-partum lethality. By contrast, DNA methylation is dispensable in undifferentiated cell types, such as the inner cell mass of the blastocyst, primordial germ cells or embryonic stem cells. Since DNA methylation appears to directly regulate only a limited number of genes, how precisely DNA methylation absence causes the death of differentiated cells remain an open question.

Due to the phenomenon of genomic imprinting, maternal and paternal genomes are differentially marked and must be properly reprogrammed every time they pass through the germline. Therefore, during gametogenesis, primordial germ cells must have their original biparental DNA methylation patterns erased and re-established based on the sex of the transmitting parent. After fertilization, the paternal and maternal genomes are once again demethylated and remethylated (except for differentially methylated regions associated with imprinted genes). This reprogramming is likely required for totipotency of the newly formed embryo and erasure of acquired epigenetic changes.[44]

In cancer

Main articles: DNA methylation in cancer and Regulation of transcription in cancer

In many disease processes, such as cancer, gene promoter CpG islands acquire abnormal hypermethylation, which results in transcriptional silencing that can be inherited by daughter cells following cell division.[45] Alterations of DNA methylation have been recognized as an important component of cancer development. Hypomethylation, in general, arises earlier and is linked to chromosomal instability and loss of imprinting, whereas hypermethylation is associated with promoters and can arise secondary to gene (oncogene suppressor) silencing, but might be a target for epigenetic therapy.[46] In developmental contexts, dynamic changes in DNA methylation patterns also have significant implications. For instance, in rat limb buds, shifts in methylation status were associated with different stages of chondrogenesis, suggesting a potential link between DNA methylation and the progression of certain developmental processes.[47]

Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms.[48] Typically, there is hypermethylation of tumor suppressor genes and hypomethylation of oncogenes.[49]

Generally, in progression to cancer, hundreds of genes are silenced or activated. Although silencing of some genes in cancers occurs by mutation, a large proportion of carcinogenic gene silencing is a result of altered DNA methylation (see DNA methylation in cancer). DNA methylation causing silencing in cancer typically occurs at multiple CpG sites in the CpG islands that are present in the promoters of protein coding genes.

Altered expressions of microRNAs also silence or activate many genes in progression to cancer (see microRNAs in cancer). Altered microRNA expression occurs through hyper/hypo-methylation of CpG sites in CpG islands in promoters controlling transcription of the microRNAs.

Silencing of DNA repair genes through methylation of CpG islands in their promoters appears to be especially important in progression to cancer (see methylation of DNA repair genes in cancer).

In atherosclerosis

Epigenetic modifications such as DNA methylation have been implicated in cardiovascular disease, including atherosclerosis. In animal models of atherosclerosis, vascular tissue, as well as blood cells such as mononuclear blood cells, exhibit global hypomethylation with gene-specific areas of hypermethylation. DNA methylation polymorphisms may be used as an early biomarker of atherosclerosis since they are present before lesions are observed, which may provide an early tool for detection and risk prevention.[50]

Two of the cell types targeted for DNA methylation polymorphisms are monocytes and lymphocytes, which experience an overall hypomethylation. One proposed mechanism behind this global hypomethylation is elevated homocysteine levels causing hyperhomocysteinemia, a known risk factor for cardiovascular disease. High plasma levels of homocysteine inhibit DNA methyltransferases, which causes hypomethylation. Hypomethylation of DNA affects genes that alter smooth muscle cell proliferation, cause endothelial cell dysfunction, and increase inflammatory mediators, all of which are critical in forming atherosclerotic lesions.[51] High levels of homocysteine also result in hypermethylation of CpG islands in the promoter region of the estrogen receptor alpha (ERα) gene, causing its down regulation.[52] ERα protects against atherosclerosis due to its action as a growth suppressor, causing the smooth muscle cells to remain in a quiescent state.[53] Hypermethylation of the ERα promoter thus allows intimal smooth muscle cells to proliferate excessively and contribute to the development of the atherosclerotic lesion.[54]

Another gene that experiences a change in methylation status in atherosclerosis is the monocarboxylate transporter (MCT3), which produces a protein responsible for the transport of lactate and other ketone bodies out of many cell types, including vascular smooth muscle cells. In atherosclerosis patients, there is an increase in methylation of the CpG islands in exon 2, which decreases MCT3 protein expression. The downregulation of MCT3 impairs lactate transport and significantly increases smooth muscle cell proliferation, which further contributes to the atherosclerotic lesion. An ex vivo experiment using the demethylating agent Decitabine (5-aza-2 -deoxycytidine) was shown to induce MCT3 expression in a dose dependent manner, as all hypermethylated sites in the exon 2 CpG island became demethylated after treatment. This may serve as a novel therapeutic agent to treat atherosclerosis, although no human studies have been conducted thus far.[55]

In heart failure

In addition to atherosclerosis described above, specific epigenetic changes have been identified in the failing human heart. This may vary by disease etiology. For example, in ischemic heart failure DNA methylation changes have been linked to changes in gene expression that may direct gene expression associated with the changes in heart metabolism known to occur.[56] Additional forms of heart failure (e.g. diabetic cardiomyopathy) and co-morbidities (e.g. obesity) must be explored to see how common these mechanisms are. Most strikingly, in failing human heart these changes in DNA methylation are associated with racial and socioeconomic status which further impact how gene expression is altered,[57] and may influence how the individual's heart failure should be treated.

In aging

In humans and other mammals, DNA methylation levels can be used to accurately estimate the age of tissues and cell types, forming an accurate epigenetic clock.[58]

A longitudinal study of twin children showed that, between the ages of 5 and 10, there was divergence of methylation patterns due to environmental rather than genetic influences.[59] There is a global loss of DNA methylation during aging.[49]

In a study that analyzed the complete DNA methylomes of CD4+ T cells in a newborn, a 26 years old individual and a 103 years old individual were observed that the loss of methylation is proportional to age.[60] Hypomethylated CpGs observed in the centenarian DNAs compared with the neonates covered all genomic compartments (promoters, intergenic, intronic and exonic regions).[61] However, some genes become hypermethylated with age, including genes for the estrogen receptor, p16, and insulin-like growth factor 2.[49]

In exercise

High intensity exercise has been shown to result in reduced DNA methylation in skeletal muscle.[62] Promoter methylation of PGC-1α and PDK4 were immediately reduced after high intensity exercise, whereas PPAR-γ methylation was not reduced until three hours after exercise.[62] At the same time, six months of exercise in previously sedentary middle-age men resulted in increased methylation in adipose tissue.[63] One study showed a possible increase in global genomic DNA methylation of white blood cells with more physical activity in non-Hispanics.[64]

In B-cell differentiation

A study that investigated the methylome of B cells along their differentiation cycle, using whole-genome bisulfite sequencing (WGBS), showed that there is a hypomethylation from the earliest stages to the most differentiated stages. The largest methylation difference is between the stages of germinal center B cells and memory B cells. Furthermore, this study showed that there is a similarity between B cell tumors and long-lived B cells in their DNA methylation signatures.[17]

In the brain

Two reviews summarize evidence that DNA methylation alterations in brain neurons are important in learning and memory.[65][66] Contextual fear conditioning (a form of associative learning) in animals, such as mice and rats, is rapid and is extremely robust in creating memories.[67] In mice[68] and in rats[69] contextual fear conditioning, within 1–24 hours, it is associated with altered methylations of several thousand DNA cytosines in genes of hippocampus neurons. Twenty four hours after contextual fear conditioning, 9.2% of the genes in rat hippocampus neurons are differentially methylated.[69] In mice,[68] when examined at four weeks after conditioning, the hippocampus methylations and demethylations had been reset to the original naive conditions. The hippocampus is needed to form memories, but memories are not stored there. For such mice, at four weeks after contextual fear conditioning, substantial differential CpG methylations and demethylations occurred in cortical neurons during memory maintenance, and there were 1,223 differentially methylated genes in their anterior cingulate cortex.[68] Mechanisms guiding new DNA methylations and new DNA demethylations in the hippocampus during memory establishment were summarized in 2022.[70] That review also indicated the mechanisms by which the new patterns of methylation gave rise to new patterns of messenger RNA expression. These new messenger RNAs were then transported by messenger RNP particles (neuronal granules) to synapses of the neurons, where they could be translated into proteins.[70] Active changes in neuronal DNA methylation and demethylation appear to act as controllers of synaptic scaling and glutamate receptor trafficking in learning and memory formation.[65]

DNA methyltransferases (in mammals)

 
Possible pathways of cytosine methylation and demethylation. Abbreviations: S-Adenosyl-L-homocysteine (SAH), S-adenosyl-L-methionine (SAM), DNA methyltransferase (DNA MTase), Uracil-DNA glycosylase (UNG)

In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities – maintenance methylation and de novo methylation.[71]

Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the DNA methyltransferase (DNMT), the replication machinery itself would produce daughter strands that are unmethylated and, over time, would lead to passive demethylation. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.

It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Mice and rats have a third functional de novo methyltransferase enzyme named DNMT3C, which evolved as a paralog of Dnmt3b by tandem duplication in the common ancestral of Muroidea rodents. DNMT3C catalyzes the methylation of promoters of transposable elements during early spermatogenesis, an activity shown to be essential for their epigenetic repression and male fertility.[72][73] It is yet unclear if in other mammals that do not have DNMT3C (like humans) rely on DNMT3B or DNMT3A for de novo methylation of transposable elements in the germline. Finally, DNMT2 (TRDMT1) has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.[74]

Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-Aza-2'-deoxycytidine (decitabine) is a nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation. However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.

In plants

Significant progress has been made in understanding DNA methylation in the model plant Arabidopsis thaliana. DNA methylation in plants differs from that of mammals: while DNA methylation in mammals mainly occurs on the cytosine nucleotide in a CpG site, in plants the cytosine can be methylated at CpG, CpHpG, and CpHpH sites, where H represents any nucleotide but not guanine. Overall, Arabidopsis DNA is highly methylated, mass spectrometry analysis estimated 14% of cytosines to be modified.[8]: abstract 

The principal Arabidopsis DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 protein is unique to the plant kingdom. There are currently two classes of DNA methyltransferases: 1) the de novo class or enzymes that create new methylation marks on the DNA; 2) a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughter strands after DNA replication. DRM2 is the only enzyme that has been implicated as a de novo DNA methyltransferase. DRM2 has also been shown, along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication.[75] Other DNA methyltransferases are expressed in plants but have no known function (see the ).

It is not clear how the cell determines the locations of de novo DNA methylation, but evidence suggests that for many (though not all) locations, RNA-directed DNA methylation (RdDM) is involved. In RdDM, specific RNA transcripts are produced from a genomic DNA template, and this RNA forms secondary structures called double-stranded RNA molecules.[76] The double-stranded RNAs, through either the small interfering RNA (siRNA) or microRNA (miRNA) pathways direct de-novo DNA methylation of the original genomic location that produced the RNA.[76] This sort of mechanism is thought to be important in cellular defense against RNA viruses and/or transposons, both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, through an as yet poorly understood mechanism, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect. Recently, it was described that methylation of the DNA is the main determinant of embryogenic cultures formation from explants in woody plants and is regarded the main mechanism that explains the poor response of mature explants to somatic embryogenesis in the plants (Isah 2016).

In insects

Further information: Epigenetics in insects

Diverse orders of insects show varied patterns of DNA methylation, from almost undetectable levels in flies to low levels in butterflies and higher in true bugs and some cockroaches (up to 14% of all CG sites in Blattella asahinai).[77]

Functional DNA methylation has been discovered in Honey Bees.[78][79] DNA methylation marks are mainly on the gene body, and current opinions on the function of DNA methylation is gene regulation via alternative splicing[80]

DNA methylation levels in Drosophila melanogaster are nearly undetectable.[81] Sensitive methods applied to Drosophila DNA Suggest levels in the range of 0.1–0.3% of total cytosine.[82] This low level of methylation[83] appears to reside in genomic sequence patterns that are very different from patterns seen in humans, or in other animal or plant species to date. Genomic methylation in D. melanogaster was found at specific short motifs (concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine) and is independent of DNMT2 activity. Further, highly sensitive mass spectrometry approaches,[84] have now demonstrated the presence of low (0.07%) but significant levels of adenine methylation during the earliest stages of Drosophila embryogenesis.

In fungi

Many fungi have low levels (0.1 to 0.5%) of cytosine methylation, whereas other fungi have as much as 5% of the genome methylated.[85] This value seems to vary both among species and among isolates of the same species.[86] There is also evidence that DNA methylation may be involved in state-specific control of gene expression in fungi.[citation needed] However, at a detection limit of 250 attomoles by using ultra-high sensitive mass spectrometry DNA methylation was not confirmed in single cellular yeast species such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, indicating that yeasts do not possess this DNA modification.[8]: abstract 

Although brewers' yeast (Saccharomyces), fission yeast (Schizosaccharomyces), and Aspergillus flavus[87] have no detectable DNA methylation, the model filamentous fungus Neurospora crassa has a well-characterized methylation system.[88] Several genes control methylation in Neurospora and mutation of the DNA methyl transferase, dim-2, eliminates all DNA methylation but does not affect growth or sexual reproduction. While the Neurospora genome has very little repeated DNA, half of the methylation occurs in repeated DNA including transposon relics and centromeric DNA. The ability to evaluate other important phenomena in a DNA methylase-deficient genetic background makes Neurospora an important system in which to study DNA methylation.

In other eukaryotes

DNA methylation is largely absent from Dictyostelium discoidium[89] where it appears to occur at about 0.006% of cytosines.[5] In contrast, DNA methylation is widely distributed in Physarum polycephalum[90] where 5-methylcytosine makes up as much as 8% of total cytosine[4]

In bacteria

 
All methylations in a prokaryote. In some prokaryotic organisms, all three previously known DNA methylation types are represented (N4-methylcytosine: m4C, 5-methylcytosine: m5C and N6-methyladenine: m6A). Six examples are shown here, two of which belong to the Archaea domain and four of which belong to the Bacteria domain. The information comes from Blow et al. (2016).[91] In the left column are the species names of the organisms, to the right there are examples of methylated DNA motifs. The full names of the archaea and bacterial strains are according to the NCBI taxonomy: "Methanocaldococcus jannaschii DSM 2661", "Methanocorpusculum labreanum Z", "Clostridium perfringens ATCC 13127", "Geopsychrobacter electrodiphilus DSM 16401", "Rhodopseudomonas palustris CGA009" and "Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150"

Adenine or cytosine methylation are mediated by restriction modification systems of many bacteria, in which specific DNA sequences are methylated periodically throughout the genome.[92] A methylase is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzymes and cleaved. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage.

E. coli DNA adenine methyltransferase (Dam) is an enzyme of ~32 kDa that does not belong to a restriction/modification system. The target recognition sequence for E. coli Dam is GATC, as the methylation occurs at the N6 position of the adenine in this sequence (G meATC). The three base pairs flanking each side of this site also influence DNA–Dam binding. Dam plays several key roles in bacterial processes, including mismatch repair, the timing of DNA replication, and gene expression. As a result of DNA replication, the status of GATC sites in the E. coli genome changes from fully methylated to hemimethylated. This is because adenine introduced into the new DNA strand is unmethylated. Re-methylation occurs within two to four seconds, during which time replication errors in the new strand are repaired. Methylation, or its absence, is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands. It has been shown that altering Dam activity in bacteria results in an increased spontaneous mutation rate. Bacterial viability is compromised in dam mutants that also lack certain other DNA repair enzymes, providing further evidence for the role of Dam in DNA repair.

One region of the DNA that keeps its hemimethylated status for longer is the origin of replication, which has an abundance of GATC sites. This is central to the bacterial mechanism for timing DNA replication. SeqA binds to the origin of replication, sequestering it and thus preventing methylation. Because hemimethylated origins of replication are inactive, this mechanism limits DNA replication to once per cell cycle.

Expression of certain genes, for example, those coding for pilus expression in E. coli, is regulated by the methylation of GATC sites in the promoter region of the gene operon. The cells' environmental conditions just after DNA replication determine whether Dam is blocked from methylating a region proximal to or distal from the promoter region. Once the pattern of methylation has been created, the pilus gene transcription is locked in the on or off position until the DNA is again replicated. In E. coli, these pili operons have important roles in virulence in urinary tract infections. It has been proposed[by whom?] that inhibitors of Dam may function as antibiotics.

On the other hand, DNA cytosine methylase targets CCAGG and CCTGG sites to methylate cytosine at the C5 position (C meC(A/T) GG). The other methylase enzyme, EcoKI, causes methylation of adenines in the sequences AAC(N6)GTGC and GCAC(N6)GTT.

In Clostridioides difficile, DNA methylation at the target motif CAAAAA was shown to impact sporulation, a key step in disease transmission, as well as cell length, biofilm formation and host colonization.[93]

Molecular cloning

Most strains used by molecular biologists are derivatives of E. coli K-12, and possess both Dam and Dcm, but there are commercially available strains that are dam-/dcm- (lack of activity of either methylase). In fact, it is possible to unmethylate the DNA extracted from dam+/dcm+ strains by transforming it into dam-/dcm- strains. This would help digest sequences that are not being recognized by methylation-sensitive restriction enzymes.[94][95]

The restriction enzyme DpnI can recognize 5'-GmeATC-3' sites and digest the methylated DNA. Being such a short motif, it occurs frequently in sequences by chance, and as such its primary use for researchers is to degrade template DNA following PCRs (PCR products lack methylation, as no methylases are present in the reaction). Similarly, some commercially available restriction enzymes are sensitive to methylation at their cognate restriction sites and must as mentioned previously be used on DNA passed through a dam-/dcm- strain to allow cutting.

Detection

DNA methylation can be detected by the following assays currently used in scientific research:[96]

  • Mass spectrometry is a very sensitive and reliable analytical method to detect DNA methylation. MS, in general, is however not informative about the sequence context of the methylation, thus limited in studying the function of this DNA modification.
  • Methylation-Specific PCR (MSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR.[97] However, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated.
  • Whole genome bisulfite sequencing, also known as BS-Seq, which is a high-throughput genome-wide analysis of DNA methylation. It is based on the aforementioned sodium bisulfite conversion of genomic DNA, which is then sequenced on a Next-generation sequencing platform. The sequences obtained are then re-aligned to the reference genome to determine the methylation status of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil.
  • Reduced representation bisulfite sequencing, also known as RRBS knows several working protocols. The first RRBS protocol was called RRBS and aims for around 10% of the methylome, a reference genome is needed. Later came more protocols that were able to sequence a smaller portion of the genome and higher sample multiplexing. EpiGBS was the first protocol where you could multiplex 96 samples in one lane of Illumina sequencing and were a reference genome was no longer needed. A de novo reference construction from the Watson and Crick reads made population screening of SNP's and SMP's simultaneously a fact.
  • The HELP assay, which is based on restriction enzymes' differential ability to recognize and cleave methylated and unmethylated CpG DNA sites.
  • GLAD-PCR assay, which is based on a new type of enzymes – site-specific methyl-directed DNA endonucleases, which hydrolyze only methylated DNA.
  • ChIP-on-chip assays, which is based on the ability of commercially prepared antibodies to bind to DNA methylation-associated proteins like MeCP2.
  • Restriction landmark genomic scanning, a complicated and now rarely used assay based upon restriction enzymes' differential recognition of methylated and unmethylated CpG sites; the assay is similar in concept to the HELP assay.
  • Methylated DNA immunoprecipitation (MeDIP), analogous to chromatin immunoprecipitation, immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP-chip) or DNA sequencing (MeDIP-seq).
  • Pyrosequencing of bisulfite treated DNA. This is the sequencing of an amplicon made by a normal forward primer but a biotinylated reverse primer to PCR the gene of choice. The Pyrosequencer then analyses the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user. If there is a mismatch, it is recorded and the percentage of DNA for which the mismatch is present is noted. This gives the user a percentage of methylation per CpG island.
  • Molecular break light assay for DNA adenine methyltransferase activity – an assay that relies on the specificity of the restriction enzyme DpnI for fully methylated (adenine methylation) GATC sites in an oligonucleotide labeled with a fluorophore and quencher. The adenine methyltransferase methylates the oligonucleotide making it a substrate for DpnI. Cutting of the oligonucleotide by DpnI gives rise to a fluorescence increase.[98][99]
  • Methyl Sensitive Southern Blotting is similar to the HELP assay, although uses Southern blotting techniques to probe gene-specific differences in methylation using restriction digests. This technique is used to evaluate local methylation near the binding site for the probe.
  • MethylCpG Binding Proteins (MBPs) and fusion proteins containing just the Methyl Binding Domain (MBD) are used to separate native DNA into methylated and unmethylated fractions. The percentage methylation of individual CpG islands can be determined by quantifying the amount of the target in each fraction.[citation needed] Extremely sensitive detection can be achieved in FFPE tissues with abscription-based detection.
  • High Resolution Melt Analysis (HRM or HRMA), is a post-PCR analytical technique. The target DNA is treated with sodium bisulfite, which chemically converts unmethylated cytosines into uracils, while methylated cytosines are preserved. PCR amplification is then carried out with primers designed to amplify both methylated and unmethylated templates. After this amplification, highly methylated DNA sequences contain a higher number of CpG sites compared to unmethylated templates, which results in a different melting temperature that can be used in quantitative methylation detection.[100][101]
  • Ancient DNA methylation reconstruction, a method to reconstruct high-resolution DNA methylation from ancient DNA samples. The method is based on the natural degradation processes that occur in ancient DNA: with time, methylated cytosines are degraded into thymines, whereas unmethylated cytosines are degraded into uracils. This asymmetry in degradation signals was used to reconstruct the full methylation maps of the Neanderthal and the Denisovan.[102] In September 2019, researchers published a novel method to infer morphological traits from DNA methylation data. The authors were able to show that linking down-regulated genes to phenotypes of monogenic diseases, where one or two copies of a gene are perturbed, allows for ~85% accuracy in reconstructing anatomical traits directly from DNA methylation maps.[103]
  • Methylation Sensitive Single Nucleotide Primer Extension Assay (msSNuPE), which uses internal primers annealing straight 5' of the nucleotide to be detected.[104]
  • Illumina Methylation Assay measures locus-specific DNA methylation using array hybridization. Bisulfite-treated DNA is hybridized to probes on "BeadChips." Single-base base extension with labeled probes is used to determine methylation status of target sites.[105] In 2016, the Infinium MethylationEPIC BeadChip was released, which interrogates over 850,000 methylation sites across the human genome.[106]
  • Using nanopore sequencing, researchers have directly identified DNA and RNA base modifications at nucleotide resolution, including 5mC, 5hmC, 6mA, and BrdU in DNA, and m6A in RNA, with detection of other natural or synthetic epigenetic modifications possible through training basecalling algorithms.[107]

Differentially methylated regions (DMRs)

Differentially methylated regions, which are genomic regions with different methylation statuses among multiple samples (tissues, cells, individuals or others), are regarded as possible functional regions involved in gene transcriptional regulation. The identification of DMRs among multiple tissues (T-DMRs) provides a comprehensive survey of epigenetic differences among human tissues.[108] For example, these methylated regions that are unique to a particular tissue allow individuals to differentiate between tissue type, such as semen and vaginal fluid. Current research conducted by Lee et al., showed DACT1 and USP49 positively identified semen by examining T-DMRs.[109] The use of T-DMRs has proven useful in the identification of various body fluids found at crime scenes. Researchers in the forensic field are currently seeking novel T-DMRs in genes to use as markers in forensic DNA analysis. DMRs between cancer and normal samples (C-DMRs) demonstrate the aberrant methylation in cancers.[110] It is well known that DNA methylation is associated with cell differentiation and proliferation.[111] Many DMRs have been found in the development stages (D-DMRs)[112] and in the reprogrammed progress (R-DMRs).[113] In addition, there are intra-individual DMRs (Intra-DMRs) with longitudinal changes in global DNA methylation along with the increase of age in a given individual.[114] There are also inter-individual DMRs (Inter-DMRs) with different methylation patterns among multiple individuals.[115]

QDMR (Quantitative Differentially Methylated Regions) is a quantitative approach to quantify methylation difference and identify DMRs from genome-wide methylation profiles by adapting Shannon entropy.[116] The platform-free and species-free nature of QDMR makes it potentially applicable to various methylation data. This approach provides an effective tool for the high-throughput identification of the functional regions involved in epigenetic regulation. QDMR can be used as an effective tool for the quantification of methylation difference and identification of DMRs across multiple samples.[117]

Gene-set analysis (a.k.a. pathway analysis; usually performed tools such as DAVID, GoSeq or GSEA) has been shown to be severely biased when applied to high-throughput methylation data (e.g. MeDIP-seq, MeDIP-ChIP, HELP-seq etc.), and a wide range of studies have thus mistakenly reported hyper-methylation of genes related to development and differentiation; it has been suggested that this can be corrected using sample label permutations or using a statistical model to control for differences in the numbers of CpG probes / CpG sites that target each gene.[118]

DNA methylation marks

DNA methylation marks – genomic regions with specific methylation patterns in a specific biological state such as tissue, cell type, individual – are regarded as possible functional regions involved in gene transcriptional regulation. Although various human cell types may have the same genome, these cells have different methylomes. The systematic identification and characterization of methylation marks across cell types are crucial to understanding the complex regulatory network for cell fate determination. Hongbo Liu et al. proposed an entropy-based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues/cells and identified 757,887 genome segments.[119] Nearly 75% of the segments showed uniform methylation across all cell types. From the remaining 25% of the segments, they identified cell type-specific hypo/hypermethylation marks that were specifically hypo/hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks. Further analysis revealed that the cell type-specific hypomethylation marks were enriched through H3K27ac and transcription factor binding sites in a cell type-specific manner. In particular, they observed that the cell type-specific hypomethylation marks are associated with the cell type-specific super-enhancers that drive the expression of cell identity genes. This framework provides a complementary, functional annotation of the human genome and helps to elucidate the critical features and functions of cell type-specific hypomethylation.

The entropy-based Specific Methylation Analysis and Report Tool, termed "SMART", which focuses on integrating a large number of DNA methylomes for the de novo identification of cell type-specific methylation marks. The latest version of SMART is focused on three main functions including de novo identification of differentially methylated regions (DMRs) by genome segmentation, identification of DMRs from predefined regions of interest, and identification of differentially methylated CpG sites.[120]

In identification and detection of body fluids

DNA methylation allows for several tissues to be analyzed in one assay as well as for small amounts of body fluid to be identified with the use of extracted DNA. Usually, the two approaches of DNA methylation are either methylated-sensitive restriction enzymes or treatment with sodium bisulphite.[121] Methylated sensitive restriction enzymes work by cleaving specific CpG, cytosine and guanine separated by only one phosphate group, recognition sites when the CpG is methylated. In contrast, unmethylated cytosines are transformed to uracil and in the process, methylated cytosines remain methylated. In particular, methylation profiles can provide insight on when or how body fluids were left at crime scenes, identify the kind of body fluid, and approximate age, gender, and phenotypic characteristics of perpetrators.[122] Research indicates various markers that can be used for DNA methylation. Deciding which marker to use for an assay is one of the first steps of the identification of body fluids. In general, markers are selected by examining prior research conducted. Identification markers that are chosen should give a positive result for one type of cell. One portion of the chromosome that is an area of focus when conducting DNA methylation are tissue-specific differentially methylated regions, T-DMRs. The degree of methylation for the T-DMRs ranges depending on the body fluid.[122] A research team developed a marker system that is two-fold. The first marker is methylated only in the target fluid while the second is methylated in the rest of the fluids.[104] For instance, if venous blood marker A is un-methylated and venous blood marker B is methylated in a fluid, it indicates the presence of only venous blood. In contrast, if venous blood marker A is methylated and venous blood marker B is un-methylated in some fluid, then that indicates venous blood is in a mixture of fluids. Some examples for DNA methylation markers are Mens1(menstrual blood), Spei1(saliva), and Sperm2(seminal fluid).

DNA methylation provides a relatively good means of sensitivity when identifying and detecting body fluids. In one study, only ten nanograms of a sample was necessary to ascertain successful results.[123] DNA methylation provides a good discernment of mixed samples since it involves markers that give “on or off” signals. DNA methylation is not impervious to external conditions. Even under degraded conditions using the DNA methylation techniques, the markers are stable enough that there are still noticeable differences between degraded samples and control samples. Specifically, in one study, it was found that there were not any noticeable changes in methylation patterns over an extensive period of time.[122]

The detection of DNA methylation in cell-free DNA and other body fluids has recently become one of the main approaches to Liquid biopsy.[124] In particular, the identification of tissue-specific and disease-specific patterns allows for non-invasive detection and monitoring of diseases such as cancer.[125] If compared to strictly genomic approaches to liquid biopsy, DNA methylation profiling offers a larger number of differentially methylated CpG sites and differentially methylated regions (DMRSs), potentially enhancing its sensitivity. Signal deconvolution algorithms based on DNA methylation have been successfully applied to cell-free DNA and can nominate the tissue of origin of cancers of unknown primary, allograft rejection, and resistance to hormone therapy.[126]

Computational prediction

DNA methylation can also be detected by computational models through sophisticated algorithms and methods. Computational models can facilitate the global profiling of DNA methylation across chromosomes, and often such models are faster and cheaper to perform than biological assays. Such up-to-date computational models include Bhasin, et al.,[127] Bock, et al.,[128] and Zheng, et al.[129][130] Together with biological assay, these methods greatly facilitate the DNA methylation analysis.

See also

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Further reading

  • Law JA, Jacobsen SE (March 2010). "Establishing, maintaining and modifying DNA methylation patterns in plants and animals". Nature Reviews Genetics. 11 (3): 204–20. doi:10.1038/nrg2719. PMC 3034103. PMID 20142834.
  • Straussman R, Nejman D, Roberts D, Steinfeld I, Blum B, Benvenisty N, Simon I, Yakhini Z, Cedar H (May 2009). "Developmental programming of CpG island methylation profiles in the human genome". Nature Structural & Molecular Biology. 16 (5): 564–71. doi:10.1038/nsmb.1594. PMID 19377480. S2CID 8804930.
  • Patra SK (April 2008). "Ras regulation of DNA-methylation and cancer". Experimental Cell Research. 314 (6): 1193–201. doi:10.1016/j.yexcr.2008.01.012. PMID 18282569.
  • Patra SK, Patra A, Rizzi F, Ghosh TC, Bettuzzi S (June 2008). "Demethylation of (Cytosine-5-C-methyl) DNA and regulation of transcription in the epigenetic pathways of cancer development". Cancer and Metastasis Reviews. 27 (2): 315–34. doi:10.1007/s10555-008-9118-y. hdl:11381/1797001. PMID 18246412. S2CID 22435914.

External links

 
Wikimedia Commons has media related to DNA methylation.
  • DNA+Methylation at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
  • ENCODE threads explorer Non-coding RNA characterization. Nature (journal)
  • PCMdb Pancreatic Cancer Methylation Database.
  • Nagpal G, Sharma M, Kumar S, Chaudhary K, Gupta S, Gautam A, Raghava GP (February 2014). "PCMdb: pancreatic cancer methylation database". Scientific Reports. 4: 4197. Bibcode:2014NatSR...4E4197N. doi:10.1038/srep04197. PMC 3935225. PMID 24569397.
  • SMART Specific Methylation Analysis and Report Tool
  • Human Methylation Mark Atlas
  • DiseaseMeth Human disease methylation database
  • EWAS Atlas A knowledgebase of epigenome-wide association studies

August 18, 2023
methylation, biological, process, which, methyl, groups, added, molecule, methylation, change, activity, segment, without, changing, sequence, when, located, gene, promoter, typically, acts, repress, gene, transcription, mammals, essential, normal, development. DNA methylation is a biological process by which methyl groups are added to the DNA molecule Methylation can change the activity of a DNA segment without changing the sequence When located in a gene promoter DNA methylation typically acts to repress gene transcription In mammals DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting X chromosome inactivation repression of transposable elements aging and carcinogenesis Representation of a DNA molecule that is methylated The two white spheres represent methyl groups They are bound to two cytosine nucleotide molecules that make up the DNA sequence As of 2016 two nucleobases have been found on which natural enzymatic DNA methylation takes place adenine and cytosine The modified bases are N6 methyladenine 1 5 methylcytosine 2 and N4 methylcytosine 3 Unmodified base Adenine A Cytosine CModified forms N6 Methyladenine 6mA 5 Methylcytosine 5mC N4 Methylcytosine 4mCTwo of DNA s four bases cytosine and adenine can be methylated Cytosine methylation is widespread in both eukaryotes and prokaryotes even though the rate of cytosine DNA methylation can differ greatly between species 14 of cytosines are methylated in Arabidopsis thaliana 4 to 8 in Physarum 4 7 6 in Mus musculus 2 3 in Escherichia coli 0 03 in Drosophila 0 006 in Dictyostelium 5 and virtually none 0 0002 to 0 0003 in Caenorhabditis 6 or fungi such as Saccharomyces cerevisiae and S pombe but not N crassa 7 8 3699 Adenine methylation has been observed in bacterial plant and recently in mammalian DNA 9 10 but has received considerably less attention Methylation of cytosine to form 5 methylcytosine occurs at the same 5 position on the pyrimidine ring where the DNA base thymine s methyl group is located the same position distinguishes thymine from the analogous RNA base uracil which has no methyl group Spontaneous deamination of 5 methylcytosine converts it to thymine This results in a T G mismatch Repair mechanisms then correct it back to the original C G pair alternatively they may substitute A for G turning the original C G pair into a T A pair effectively changing a base and introducing a mutation This misincorporated base will not be corrected during DNA replication as thymine is a DNA base If the mismatch is not repaired and the cell enters the cell cycle the strand carrying the T will be complemented by an A in one of the daughter cells such that the mutation becomes permanent The near universal use of thymine exclusively in DNA and uracil exclusively in RNA may have evolved as an error control mechanism to facilitate the removal of uracils generated by the spontaneous deamination of cytosine 11 DNA methylation as well as many of its contemporary DNA methyltransferases have been thought to evolve from early world primitive RNA methylation activity and is supported by several lines of evidence 12 In plants and other organisms DNA methylation is found in three different sequence contexts CG or CpG CHG or CHH where H correspond to A T or C In mammals however DNA methylation is almost exclusively found in CpG dinucleotides with the cytosines on both strands being usually methylated Non CpG methylation can however be observed in embryonic stem cells 13 14 15 and has also been indicated in neural development 16 Furthermore non CpG methylation has also been observed in hematopoietic progenitor cells and it occurred mainly in a CpApC sequence context 17 Contents 1 Conserved function of DNA methylation 1 1 CpG islands 1 2 Repression of CpG dense promoters 1 3 Repression of transposable elements 1 4 Genome expansion 1 5 Methylation of the gene body of highly transcribed genes 2 In mammals 2 1 During embryonic development 2 2 In cancer 2 3 In atherosclerosis 2 4 In heart failure 2 5 In aging 2 6 In exercise 2 7 In B cell differentiation 2 8 In the brain 3 DNA methyltransferases in mammals 4 In plants 5 In insects 6 In fungi 7 In other eukaryotes 8 In bacteria 8 1 Molecular cloning 9 Detection 10 Differentially methylated regions DMRs 11 DNA methylation marks 11 1 In identification and detection of body fluids 12 Computational prediction 13 See also 14 References 15 Further reading 16 External linksConserved function of DNA methylation Edit Typical DNA methylation landscape in mammalsThe DNA methylation landscape of vertebrates is very particular compared to other organisms In mammals around 75 of CpG dinucleotides are methylated in somatic cells 18 and DNA methylation appears as a default state that has to be specifically excluded from defined locations 15 19 By contrast the genome of most plants invertebrates fungi or protists show mosaic methylation patterns where only specific genomic elements are targeted and they are characterized by the alternation of methylated and unmethylated domains 20 21 Cytosine methylation then deamination to ThymineHigh CpG methylation in mammalian genomes has an evolutionary cost because it increases the frequency of spontaneous mutations Loss of amino groups occurs with a high frequency for cytosines with different consequences depending on their methylation Methylated C residues spontaneously deaminate to form T residues over time hence CpG dinucleotides steadily deaminate to TpG dinucleotides which is evidenced by the under representation of CpG dinucleotides in the human genome they occur at only 21 of the expected frequency 22 On the other hand spontaneous deamination of unmethylated C residues gives rise to U residues a change that is quickly recognized and repaired by the cell CpG islands Edit Main article CpG islands In mammals the only exception for this global CpG depletion resides in a specific category of GC and CpG rich sequences termed CpG islands that are generally unmethylated and therefore retained the expected CpG content 23 CpG islands are usually defined as regions with 1 a length greater than 200bp 2 a G C content greater than 50 3 a ratio of observed to expected CpG greater than 0 6 although other definitions are sometimes used 24 Excluding repeated sequences there are around 25 000 CpG islands in the human genome 75 of which being less than 850bp long 22 They are major regulatory units and around 50 of CpG islands are located in gene promoter regions while another 25 lie in gene bodies often serving as alternative promoters Reciprocally around 60 70 of human genes have a CpG island in their promoter region 25 26 The majority of CpG islands are constitutively unmethylated and enriched for permissive chromatin modification such as H3K4 methylation In somatic tissues only 10 of CpG islands are methylated the majority of them being located in intergenic and intragenic regions Repression of CpG dense promoters Edit DNA methylation was probably present at some extent in very early eukaryote ancestors In virtually every organism analyzed methylation in promoter regions correlates negatively with gene expression 20 27 CpG dense promoters of actively transcribed genes are never methylated but reciprocally transcriptionally silent genes do not necessarily carry a methylated promoter In mouse and human around 60 70 of genes have a CpG island in their promoter region and most of these CpG islands remain unmethylated independently of the transcriptional activity of the gene in both differentiated and undifferentiated cell types 28 29 Of note whereas DNA methylation of CpG islands is unambiguously linked with transcriptional repression the function of DNA methylation in CG poor promoters remains unclear albeit there is little evidence that it could be functionally relevant 30 DNA methylation may affect the transcription of genes in two ways First the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene 31 and second and likely more important methylated DNA may be bound by proteins known as methyl CpG binding domain proteins MBDs MBD proteins then recruit additional proteins to the locus such as histone deacetylases and other chromatin remodeling proteins that can modify histones thereby forming compact inactive chromatin termed heterochromatin This link between DNA methylation and chromatin structure is very important In particular loss of methyl CpG binding protein 2 MeCP2 has been implicated in Rett syndrome and methyl CpG binding domain protein 2 MBD2 mediates the transcriptional silencing of hypermethylated genes in cancer Repression of transposable elements Edit DNA methylation is a powerful transcriptional repressor at least in CpG dense contexts Transcriptional repression of protein coding genes appears essentially limited to very specific classes of genes that need to be silent permanently and in almost all tissues While DNA methylation does not have the flexibility required for the fine tuning of gene regulation its stability is perfect to ensure the permanent silencing of transposable elements 32 Transposon control is one of the most ancient functions of DNA methylation that is shared by animals plants and multiple protists 33 It is even suggested that DNA methylation evolved precisely for this purpose 34 Genome expansion Edit DNA methylation of transposable elements has been known to be related to genome expansion However the evolutionary driver for genome expansion remains unknown There is a clear correlation between the size of the genome and CpG suggesting that the DNA methylation of transposable elements led to a noticeable increase in the mass of DNA 35 Methylation of the gene body of highly transcribed genes Edit A function that appears even more conserved than transposon silencing is positively correlated with gene expression In almost all species where DNA methylation is present DNA methylation is especially enriched in the body of highly transcribed genes 20 27 The function of gene body methylation is not well understood A body of evidence suggests that it could regulate splicing 36 and suppress the activity of intragenic transcriptional units cryptic promoters or transposable elements 37 Gene body methylation appears closely tied to H3K36 methylation In yeast and mammals H3K36 methylation is highly enriched in the body of highly transcribed genes In yeast at least H3K36me3 recruits enzymes such as histone deacetylases to condense chromatin and prevent the activation of cryptic start sites 38 In mammals DNMT3a and DNMT3b PWWP domain binds to H3K36me3 and the two enzymes are recruited to the body of actively transcribed genes In mammals Edit Dynamic of DNA methylation during mouse embryonic development E3 5 E6 etc refer to days after fertilization PGC primordial germ cellsDuring embryonic development Edit Main article DNA methylation reprogramming DNA methylation patterns are largely erased and then re established between generations in mammals Almost all of the methylations from the parents are erased first during gametogenesis and again in early embryogenesis with demethylation and remethylation occurring each time Demethylation in early embryogenesis occurs in the preimplantation period in two stages initially in the zygote then during the first few embryonic replication cycles of morula and blastula A wave of methylation then takes place during the implantation stage of the embryo with CpG islands protected from methylation This results in global repression and allows housekeeping genes to be expressed in all cells In the post implantation stage methylation patterns are stage and tissue specific with changes that would define each individual cell type lasting stably over a long period 39 Studies on rat limb buds during embryogenesis have further illustrated the dynamic nature of DNA methylation in development In this context variations in global DNA methylation were observed across different developmental stages and culture conditions highlighting the intricate regulation of methylation during organogenesis and its potential implications for regenerative medicine strategies 40 Whereas DNA methylation is not necessary per se for transcriptional silencing it is thought nonetheless to represent a locked state that definitely inactivates transcription In particular DNA methylation appears critical for the maintenance of mono allelic silencing in the context of genomic imprinting and X chromosome inactivation 41 42 In these cases expressed and silent alleles differ by their methylation status and loss of DNA methylation results in loss of imprinting and re expression of Xist in somatic cells During embryonic development few genes change their methylation status at the important exception of many genes specifically expressed in the germline 43 DNA methylation appears absolutely required in differentiated cells as knockout of any of the three competent DNA methyltransferase results in embryonic or post partum lethality By contrast DNA methylation is dispensable in undifferentiated cell types such as the inner cell mass of the blastocyst primordial germ cells or embryonic stem cells Since DNA methylation appears to directly regulate only a limited number of genes how precisely DNA methylation absence causes the death of differentiated cells remain an open question Due to the phenomenon of genomic imprinting maternal and paternal genomes are differentially marked and must be properly reprogrammed every time they pass through the germline Therefore during gametogenesis primordial germ cells must have their original biparental DNA methylation patterns erased and re established based on the sex of the transmitting parent After fertilization the paternal and maternal genomes are once again demethylated and remethylated except for differentially methylated regions associated with imprinted genes This reprogramming is likely required for totipotency of the newly formed embryo and erasure of acquired epigenetic changes 44 In cancer Edit Main articles DNA methylation in cancer and Regulation of transcription in cancer In many disease processes such as cancer gene promoter CpG islands acquire abnormal hypermethylation which results in transcriptional silencing that can be inherited by daughter cells following cell division 45 Alterations of DNA methylation have been recognized as an important component of cancer development Hypomethylation in general arises earlier and is linked to chromosomal instability and loss of imprinting whereas hypermethylation is associated with promoters and can arise secondary to gene oncogene suppressor silencing but might be a target for epigenetic therapy 46 In developmental contexts dynamic changes in DNA methylation patterns also have significant implications For instance in rat limb buds shifts in methylation status were associated with different stages of chondrogenesis suggesting a potential link between DNA methylation and the progression of certain developmental processes 47 Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms 48 Typically there is hypermethylation of tumor suppressor genes and hypomethylation of oncogenes 49 Generally in progression to cancer hundreds of genes are silenced or activated Although silencing of some genes in cancers occurs by mutation a large proportion of carcinogenic gene silencing is a result of altered DNA methylation see DNA methylation in cancer DNA methylation causing silencing in cancer typically occurs at multiple CpG sites in the CpG islands that are present in the promoters of protein coding genes Altered expressions of microRNAs also silence or activate many genes in progression to cancer see microRNAs in cancer Altered microRNA expression occurs through hyper hypo methylation of CpG sites in CpG islands in promoters controlling transcription of the microRNAs Silencing of DNA repair genes through methylation of CpG islands in their promoters appears to be especially important in progression to cancer see methylation of DNA repair genes in cancer In atherosclerosis Edit Epigenetic modifications such as DNA methylation have been implicated in cardiovascular disease including atherosclerosis In animal models of atherosclerosis vascular tissue as well as blood cells such as mononuclear blood cells exhibit global hypomethylation with gene specific areas of hypermethylation DNA methylation polymorphisms may be used as an early biomarker of atherosclerosis since they are present before lesions are observed which may provide an early tool for detection and risk prevention 50 Two of the cell types targeted for DNA methylation polymorphisms are monocytes and lymphocytes which experience an overall hypomethylation One proposed mechanism behind this global hypomethylation is elevated homocysteine levels causing hyperhomocysteinemia a known risk factor for cardiovascular disease High plasma levels of homocysteine inhibit DNA methyltransferases which causes hypomethylation Hypomethylation of DNA affects genes that alter smooth muscle cell proliferation cause endothelial cell dysfunction and increase inflammatory mediators all of which are critical in forming atherosclerotic lesions 51 High levels of homocysteine also result in hypermethylation of CpG islands in the promoter region of the estrogen receptor alpha ERa gene causing its down regulation 52 ERa protects against atherosclerosis due to its action as a growth suppressor causing the smooth muscle cells to remain in a quiescent state 53 Hypermethylation of the ERa promoter thus allows intimal smooth muscle cells to proliferate excessively and contribute to the development of the atherosclerotic lesion 54 Another gene that experiences a change in methylation status in atherosclerosis is the monocarboxylate transporter MCT3 which produces a protein responsible for the transport of lactate and other ketone bodies out of many cell types including vascular smooth muscle cells In atherosclerosis patients there is an increase in methylation of the CpG islands in exon 2 which decreases MCT3 protein expression The downregulation of MCT3 impairs lactate transport and significantly increases smooth muscle cell proliferation which further contributes to the atherosclerotic lesion An ex vivo experiment using the demethylating agent Decitabine 5 aza 2 deoxycytidine was shown to induce MCT3 expression in a dose dependent manner as all hypermethylated sites in the exon 2 CpG island became demethylated after treatment This may serve as a novel therapeutic agent to treat atherosclerosis although no human studies have been conducted thus far 55 In heart failure Edit In addition to atherosclerosis described above specific epigenetic changes have been identified in the failing human heart This may vary by disease etiology For example in ischemic heart failure DNA methylation changes have been linked to changes in gene expression that may direct gene expression associated with the changes in heart metabolism known to occur 56 Additional forms of heart failure e g diabetic cardiomyopathy and co morbidities e g obesity must be explored to see how common these mechanisms are Most strikingly in failing human heart these changes in DNA methylation are associated with racial and socioeconomic status which further impact how gene expression is altered 57 and may influence how the individual s heart failure should be treated In aging Edit In humans and other mammals DNA methylation levels can be used to accurately estimate the age of tissues and cell types forming an accurate epigenetic clock 58 A longitudinal study of twin children showed that between the ages of 5 and 10 there was divergence of methylation patterns due to environmental rather than genetic influences 59 There is a global loss of DNA methylation during aging 49 In a study that analyzed the complete DNA methylomes of CD4 T cells in a newborn a 26 years old individual and a 103 years old individual were observed that the loss of methylation is proportional to age 60 Hypomethylated CpGs observed in the centenarian DNAs compared with the neonates covered all genomic compartments promoters intergenic intronic and exonic regions 61 However some genes become hypermethylated with age including genes for the estrogen receptor p16 and insulin like growth factor 2 49 In exercise Edit High intensity exercise has been shown to result in reduced DNA methylation in skeletal muscle 62 Promoter methylation of PGC 1a and PDK4 were immediately reduced after high intensity exercise whereas PPAR g methylation was not reduced until three hours after exercise 62 At the same time six months of exercise in previously sedentary middle age men resulted in increased methylation in adipose tissue 63 One study showed a possible increase in global genomic DNA methylation of white blood cells with more physical activity in non Hispanics 64 In B cell differentiation Edit A study that investigated the methylome of B cells along their differentiation cycle using whole genome bisulfite sequencing WGBS showed that there is a hypomethylation from the earliest stages to the most differentiated stages The largest methylation difference is between the stages of germinal center B cells and memory B cells Furthermore this study showed that there is a similarity between B cell tumors and long lived B cells in their DNA methylation signatures 17 In the brain Edit Two reviews summarize evidence that DNA methylation alterations in brain neurons are important in learning and memory 65 66 Contextual fear conditioning a form of associative learning in animals such as mice and rats is rapid and is extremely robust in creating memories 67 In mice 68 and in rats 69 contextual fear conditioning within 1 24 hours it is associated with altered methylations of several thousand DNA cytosines in genes of hippocampus neurons Twenty four hours after contextual fear conditioning 9 2 of the genes in rat hippocampus neurons are differentially methylated 69 In mice 68 when examined at four weeks after conditioning the hippocampus methylations and demethylations had been reset to the original naive conditions The hippocampus is needed to form memories but memories are not stored there For such mice at four weeks after contextual fear conditioning substantial differential CpG methylations and demethylations occurred in cortical neurons during memory maintenance and there were 1 223 differentially methylated genes in their anterior cingulate cortex 68 Mechanisms guiding new DNA methylations and new DNA demethylations in the hippocampus during memory establishment were summarized in 2022 70 That review also indicated the mechanisms by which the new patterns of methylation gave rise to new patterns of messenger RNA expression These new messenger RNAs were then transported by messenger RNP particles neuronal granules to synapses of the neurons where they could be translated into proteins 70 Active changes in neuronal DNA methylation and demethylation appear to act as controllers of synaptic scaling and glutamate receptor trafficking in learning and memory formation 65 DNA methyltransferases in mammals Edit Possible pathways of cytosine methylation and demethylation Abbreviations S Adenosyl L homocysteine SAH S adenosyl L methionine SAM DNA methyltransferase DNA MTase Uracil DNA glycosylase UNG In mammalian cells DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities maintenance methylation and de novo methylation 71 Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle Without the DNA methyltransferase DNMT the replication machinery itself would produce daughter strands that are unmethylated and over time would lead to passive demethylation DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9 due to the requirement of DNMT1 activity for development in mammalian cells It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity Instead DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity Mice and rats have a third functional de novo methyltransferase enzyme named DNMT3C which evolved as a paralog of Dnmt3b by tandem duplication in the common ancestral of Muroidea rodents DNMT3C catalyzes the methylation of promoters of transposable elements during early spermatogenesis an activity shown to be essential for their epigenetic repression and male fertility 72 73 It is yet unclear if in other mammals that do not have DNMT3C like humans rely on DNMT3B or DNMT3A for de novo methylation of transposable elements in the germline Finally DNMT2 TRDMT1 has been identified as a DNA methyltransferase homolog containing all 10 sequence motifs common to all DNA methyltransferases however DNMT2 TRDMT1 does not methylate DNA but instead methylates cytosine 38 in the anticodon loop of aspartic acid transfer RNA 74 Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis there have been attempts to re express these genes by inhibiting the DNMTs 5 Aza 2 deoxycytidine decitabine is a nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the b elimination step of catalysis thus resulting in the enzymes degradation However for decitabine to be active it must be incorporated into the genome of the cell which can cause mutations in the daughter cells if the cell does not die In addition decitabine is toxic to the bone marrow which limits the size of its therapeutic window These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation However it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation In plants EditSignificant progress has been made in understanding DNA methylation in the model plant Arabidopsis thaliana DNA methylation in plants differs from that of mammals while DNA methylation in mammals mainly occurs on the cytosine nucleotide in a CpG site in plants the cytosine can be methylated at CpG CpHpG and CpHpH sites where H represents any nucleotide but not guanine Overall Arabidopsis DNA is highly methylated mass spectrometry analysis estimated 14 of cytosines to be modified 8 abstract The principal Arabidopsis DNA methyltransferase enzymes which transfer and covalently attach methyl groups onto DNA are DRM2 MET1 and CMT3 Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1 respectively whereas the CMT3 protein is unique to the plant kingdom There are currently two classes of DNA methyltransferases 1 the de novo class or enzymes that create new methylation marks on the DNA 2 a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughter strands after DNA replication DRM2 is the only enzyme that has been implicated as a de novo DNA methyltransferase DRM2 has also been shown along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication 75 Other DNA methyltransferases are expressed in plants but have no known function see the Chromatin Database It is not clear how the cell determines the locations of de novo DNA methylation but evidence suggests that for many though not all locations RNA directed DNA methylation RdDM is involved In RdDM specific RNA transcripts are produced from a genomic DNA template and this RNA forms secondary structures called double stranded RNA molecules 76 The double stranded RNAs through either the small interfering RNA siRNA or microRNA miRNA pathways direct de novo DNA methylation of the original genomic location that produced the RNA 76 This sort of mechanism is thought to be important in cellular defense against RNA viruses and or transposons both of which often form a double stranded RNA that can be mutagenic to the host genome By methylating their genomic locations through an as yet poorly understood mechanism they are shut off and are no longer active in the cell protecting the genome from their mutagenic effect Recently it was described that methylation of the DNA is the main determinant of embryogenic cultures formation from explants in woody plants and is regarded the main mechanism that explains the poor response of mature explants to somatic embryogenesis in the plants Isah 2016 In insects EditFurther information Epigenetics in insects Diverse orders of insects show varied patterns of DNA methylation from almost undetectable levels in flies to low levels in butterflies and higher in true bugs and some cockroaches up to 14 of all CG sites in Blattella asahinai 77 Functional DNA methylation has been discovered in Honey Bees 78 79 DNA methylation marks are mainly on the gene body and current opinions on the function of DNA methylation is gene regulation via alternative splicing 80 DNA methylation levels in Drosophila melanogaster are nearly undetectable 81 Sensitive methods applied to Drosophila DNA Suggest levels in the range of 0 1 0 3 of total cytosine 82 This low level of methylation 83 appears to reside in genomic sequence patterns that are very different from patterns seen in humans or in other animal or plant species to date Genomic methylation in D melanogaster was found at specific short motifs concentrated in specific 5 base sequence motifs that are CA and CT rich but depleted of guanine and is independent of DNMT2 activity Further highly sensitive mass spectrometry approaches 84 have now demonstrated the presence of low 0 07 but significant levels of adenine methylation during the earliest stages of Drosophila embryogenesis In fungi EditMany fungi have low levels 0 1 to 0 5 of cytosine methylation whereas other fungi have as much as 5 of the genome methylated 85 This value seems to vary both among species and among isolates of the same species 86 There is also evidence that DNA methylation may be involved in state specific control of gene expression in fungi citation needed However at a detection limit of 250 attomoles by using ultra high sensitive mass spectrometry DNA methylation was not confirmed in single cellular yeast species such as Saccharomyces cerevisiae or Schizosaccharomyces pombe indicating that yeasts do not possess this DNA modification 8 abstract Although brewers yeast Saccharomyces fission yeast Schizosaccharomyces and Aspergillus flavus 87 have no detectable DNA methylation the model filamentous fungus Neurospora crassa has a well characterized methylation system 88 Several genes control methylation in Neurospora and mutation of the DNA methyl transferase dim 2 eliminates all DNA methylation but does not affect growth or sexual reproduction While the Neurospora genome has very little repeated DNA half of the methylation occurs in repeated DNA including transposon relics and centromeric DNA The ability to evaluate other important phenomena in a DNA methylase deficient genetic background makes Neurospora an important system in which to study DNA methylation In other eukaryotes EditDNA methylation is largely absent from Dictyostelium discoidium 89 where it appears to occur at about 0 006 of cytosines 5 In contrast DNA methylation is widely distributed in Physarum polycephalum 90 where 5 methylcytosine makes up as much as 8 of total cytosine 4 In bacteria Edit All methylations in a prokaryote In some prokaryotic organisms all three previously known DNA methylation types are represented N4 methylcytosine m4C 5 methylcytosine m5C and N6 methyladenine m6A Six examples are shown here two of which belong to the Archaea domain and four of which belong to the Bacteria domain The information comes from Blow et al 2016 91 In the left column are the species names of the organisms to the right there are examples of methylated DNA motifs The full names of the archaea and bacterial strains are according to the NCBI taxonomy Methanocaldococcus jannaschii DSM 2661 Methanocorpusculum labreanum Z Clostridium perfringens ATCC 13127 Geopsychrobacter electrodiphilus DSM 16401 Rhodopseudomonas palustris CGA009 and Salmonella enterica subsp enterica serovar Paratyphi A str ATCC 9150 Adenine or cytosine methylation are mediated by restriction modification systems of many bacteria in which specific DNA sequences are methylated periodically throughout the genome 92 A methylase is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence Foreign DNAs which are not methylated in this manner that are introduced into the cell are degraded by sequence specific restriction enzymes and cleaved Bacterial genomic DNA is not recognized by these restriction enzymes The methylation of native DNA acts as a sort of primitive immune system allowing the bacteria to protect themselves from infection by bacteriophage E coli DNA adenine methyltransferase Dam is an enzyme of 32 kDa that does not belong to a restriction modification system The target recognition sequence for E coli Dam is GATC as the methylation occurs at the N6 position of the adenine in this sequence G meATC The three base pairs flanking each side of this site also influence DNA Dam binding Dam plays several key roles in bacterial processes including mismatch repair the timing of DNA replication and gene expression As a result of DNA replication the status of GATC sites in the E coli genome changes from fully methylated to hemimethylated This is because adenine introduced into the new DNA strand is unmethylated Re methylation occurs within two to four seconds during which time replication errors in the new strand are repaired Methylation or its absence is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands It has been shown that altering Dam activity in bacteria results in an increased spontaneous mutation rate Bacterial viability is compromised in dam mutants that also lack certain other DNA repair enzymes providing further evidence for the role of Dam in DNA repair One region of the DNA that keeps its hemimethylated status for longer is the origin of replication which has an abundance of GATC sites This is central to the bacterial mechanism for timing DNA replication SeqA binds to the origin of replication sequestering it and thus preventing methylation Because hemimethylated origins of replication are inactive this mechanism limits DNA replication to once per cell cycle Expression of certain genes for example those coding for pilus expression in E coli is regulated by the methylation of GATC sites in the promoter region of the gene operon The cells environmental conditions just after DNA replication determine whether Dam is blocked from methylating a region proximal to or distal from the promoter region Once the pattern of methylation has been created the pilus gene transcription is locked in the on or off position until the DNA is again replicated In E coli these pili operons have important roles in virulence in urinary tract infections It has been proposed by whom that inhibitors of Dam may function as antibiotics On the other hand DNA cytosine methylase targets CCAGG and CCTGG sites to methylate cytosine at the C5 position C meC A T GG The other methylase enzyme EcoKI causes methylation of adenines in the sequences AAC N6 GTGC and GCAC N6 GTT In Clostridioides difficile DNA methylation at the target motif CAAAAA was shown to impact sporulation a key step in disease transmission as well as cell length biofilm formation and host colonization 93 Molecular cloning Edit Most strains used by molecular biologists are derivatives of E coli K 12 and possess both Dam and Dcm but there are commercially available strains that are dam dcm lack of activity of either methylase In fact it is possible to unmethylate the DNA extracted from dam dcm strains by transforming it into dam dcm strains This would help digest sequences that are not being recognized by methylation sensitive restriction enzymes 94 95 The restriction enzyme DpnI can recognize 5 GmeATC 3 sites and digest the methylated DNA Being such a short motif it occurs frequently in sequences by chance and as such its primary use for researchers is to degrade template DNA following PCRs PCR products lack methylation as no methylases are present in the reaction Similarly some commercially available restriction enzymes are sensitive to methylation at their cognate restriction sites and must as mentioned previously be used on DNA passed through a dam dcm strain to allow cutting Detection EditDNA methylation can be detected by the following assays currently used in scientific research 96 Mass spectrometry is a very sensitive and reliable analytical method to detect DNA methylation MS in general is however not informative about the sequence context of the methylation thus limited in studying the function of this DNA modification Methylation Specific PCR MSP which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG followed by traditional PCR 97 However methylated cytosines will not be converted in this process and primers are designed to overlap the CpG site of interest which allows one to determine methylation status as methylated or unmethylated Whole genome bisulfite sequencing also known as BS Seq which is a high throughput genome wide analysis of DNA methylation It is based on the aforementioned sodium bisulfite conversion of genomic DNA which is then sequenced on a Next generation sequencing platform The sequences obtained are then re aligned to the reference genome to determine the methylation status of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil Reduced representation bisulfite sequencing also known as RRBS knows several working protocols The first RRBS protocol was called RRBS and aims for around 10 of the methylome a reference genome is needed Later came more protocols that were able to sequence a smaller portion of the genome and higher sample multiplexing EpiGBS was the first protocol where you could multiplex 96 samples in one lane of Illumina sequencing and were a reference genome was no longer needed A de novo reference construction from the Watson and Crick reads made population screening of SNP s and SMP s simultaneously a fact The HELP assay which is based on restriction enzymes differential ability to recognize and cleave methylated and unmethylated CpG DNA sites GLAD PCR assay which is based on a new type of enzymes site specific methyl directed DNA endonucleases which hydrolyze only methylated DNA ChIP on chip assays which is based on the ability of commercially prepared antibodies to bind to DNA methylation associated proteins like MeCP2 Restriction landmark genomic scanning a complicated and now rarely used assay based upon restriction enzymes differential recognition of methylated and unmethylated CpG sites the assay is similar in concept to the HELP assay Methylated DNA immunoprecipitation MeDIP analogous to chromatin immunoprecipitation immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays MeDIP chip or DNA sequencing MeDIP seq Pyrosequencing of bisulfite treated DNA This is the sequencing of an amplicon made by a normal forward primer but a biotinylated reverse primer to PCR the gene of choice The Pyrosequencer then analyses the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user If there is a mismatch it is recorded and the percentage of DNA for which the mismatch is present is noted This gives the user a percentage of methylation per CpG island Molecular break light assay for DNA adenine methyltransferase activity an assay that relies on the specificity of the restriction enzyme DpnI for fully methylated adenine methylation GATC sites in an oligonucleotide labeled with a fluorophore and quencher The adenine methyltransferase methylates the oligonucleotide making it a substrate for DpnI Cutting of the oligonucleotide by DpnI gives rise to a fluorescence increase 98 99 Methyl Sensitive Southern Blotting is similar to the HELP assay although uses Southern blotting techniques to probe gene specific differences in methylation using restriction digests This technique is used to evaluate local methylation near the binding site for the probe MethylCpG Binding Proteins MBPs and fusion proteins containing just the Methyl Binding Domain MBD are used to separate native DNA into methylated and unmethylated fractions The percentage methylation of individual CpG islands can be determined by quantifying the amount of the target in each fraction citation needed Extremely sensitive detection can be achieved in FFPE tissues with abscription based detection High Resolution Melt Analysis HRM or HRMA is a post PCR analytical technique The target DNA is treated with sodium bisulfite which chemically converts unmethylated cytosines into uracils while methylated cytosines are preserved PCR amplification is then carried out with primers designed to amplify both methylated and unmethylated templates After this amplification highly methylated DNA sequences contain a higher number of CpG sites compared to unmethylated templates which results in a different melting temperature that can be used in quantitative methylation detection 100 101 Ancient DNA methylation reconstruction a method to reconstruct high resolution DNA methylation from ancient DNA samples The method is based on the natural degradation processes that occur in ancient DNA with time methylated cytosines are degraded into thymines whereas unmethylated cytosines are degraded into uracils This asymmetry in degradation signals was used to reconstruct the full methylation maps of the Neanderthal and the Denisovan 102 In September 2019 researchers published a novel method to infer morphological traits from DNA methylation data The authors were able to show that linking down regulated genes to phenotypes of monogenic diseases where one or two copies of a gene are perturbed allows for 85 accuracy in reconstructing anatomical traits directly from DNA methylation maps 103 Methylation Sensitive Single Nucleotide Primer Extension Assay msSNuPE which uses internal primers annealing straight 5 of the nucleotide to be detected 104 Illumina Methylation Assay measures locus specific DNA methylation using array hybridization Bisulfite treated DNA is hybridized to probes on BeadChips Single base base extension with labeled probes is used to determine methylation status of target sites 105 In 2016 the Infinium MethylationEPIC BeadChip was released which interrogates over 850 000 methylation sites across the human genome 106 Using nanopore sequencing researchers have directly identified DNA and RNA base modifications at nucleotide resolution including 5mC 5hmC 6mA and BrdU in DNA and m6A in RNA with detection of other natural or synthetic epigenetic modifications possible through training basecalling algorithms 107 Differentially methylated regions DMRs EditDifferentially methylated regions which are genomic regions with different methylation statuses among multiple samples tissues cells individuals or others are regarded as possible functional regions involved in gene transcriptional regulation The identification of DMRs among multiple tissues T DMRs provides a comprehensive survey of epigenetic differences among human tissues 108 For example these methylated regions that are unique to a particular tissue allow individuals to differentiate between tissue type such as semen and vaginal fluid Current research conducted by Lee et al showed DACT1 and USP49 positively identified semen by examining T DMRs 109 The use of T DMRs has proven useful in the identification of various body fluids found at crime scenes Researchers in the forensic field are currently seeking novel T DMRs in genes to use as markers in forensic DNA analysis DMRs between cancer and normal samples C DMRs demonstrate the aberrant methylation in cancers 110 It is well known that DNA methylation is associated with cell differentiation and proliferation 111 Many DMRs have been found in the development stages D DMRs 112 and in the reprogrammed progress R DMRs 113 In addition there are intra individual DMRs Intra DMRs with longitudinal changes in global DNA methylation along with the increase of age in a given individual 114 There are also inter individual DMRs Inter DMRs with different methylation patterns among multiple individuals 115 QDMR Quantitative Differentially Methylated Regions is a quantitative approach to quantify methylation difference and identify DMRs from genome wide methylation profiles by adapting Shannon entropy 116 The platform free and species free nature of QDMR makes it potentially applicable to various methylation data This approach provides an effective tool for the high throughput identification of the functional regions involved in epigenetic regulation QDMR can be used as an effective tool for the quantification of methylation difference and identification of DMRs across multiple samples 117 Gene set analysis a k a pathway analysis usually performed tools such as DAVID GoSeq or GSEA has been shown to be severely biased when applied to high throughput methylation data e g MeDIP seq MeDIP ChIP HELP seq etc and a wide range of studies have thus mistakenly reported hyper methylation of genes related to development and differentiation it has been suggested that this can be corrected using sample label permutations or using a statistical model to control for differences in the numbers of CpG probes CpG sites that target each gene 118 DNA methylation marks EditDNA methylation marks genomic regions with specific methylation patterns in a specific biological state such as tissue cell type individual are regarded as possible functional regions involved in gene transcriptional regulation Although various human cell types may have the same genome these cells have different methylomes The systematic identification and characterization of methylation marks across cell types are crucial to understanding the complex regulatory network for cell fate determination Hongbo Liu et al proposed an entropy based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues cells and identified 757 887 genome segments 119 Nearly 75 of the segments showed uniform methylation across all cell types From the remaining 25 of the segments they identified cell type specific hypo hypermethylation marks that were specifically hypo hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks Further analysis revealed that the cell type specific hypomethylation marks were enriched through H3K27ac and transcription factor binding sites in a cell type specific manner In particular they observed that the cell type specific hypomethylation marks are associated with the cell type specific super enhancers that drive the expression of cell identity genes This framework provides a complementary functional annotation of the human genome and helps to elucidate the critical features and functions of cell type specific hypomethylation The entropy based Specific Methylation Analysis and Report Tool termed SMART which focuses on integrating a large number of DNA methylomes for the de novo identification of cell type specific methylation marks The latest version of SMART is focused on three main functions including de novo identification of differentially methylated regions DMRs by genome segmentation identification of DMRs from predefined regions of interest and identification of differentially methylated CpG sites 120 In identification and detection of body fluids Edit DNA methylation allows for several tissues to be analyzed in one assay as well as for small amounts of body fluid to be identified with the use of extracted DNA Usually the two approaches of DNA methylation are either methylated sensitive restriction enzymes or treatment with sodium bisulphite 121 Methylated sensitive restriction enzymes work by cleaving specific CpG cytosine and guanine separated by only one phosphate group recognition sites when the CpG is methylated In contrast unmethylated cytosines are transformed to uracil and in the process methylated cytosines remain methylated In particular methylation profiles can provide insight on when or how body fluids were left at crime scenes identify the kind of body fluid and approximate age gender and phenotypic characteristics of perpetrators 122 Research indicates various markers that can be used for DNA methylation Deciding which marker to use for an assay is one of the first steps of the identification of body fluids In general markers are selected by examining prior research conducted Identification markers that are chosen should give a positive result for one type of cell One portion of the chromosome that is an area of focus when conducting DNA methylation are tissue specific differentially methylated regions T DMRs The degree of methylation for the T DMRs ranges depending on the body fluid 122 A research team developed a marker system that is two fold The first marker is methylated only in the target fluid while the second is methylated in the rest of the fluids 104 For instance if venous blood marker A is un methylated and venous blood marker B is methylated in a fluid it indicates the presence of only venous blood In contrast if venous blood marker A is methylated and venous blood marker B is un methylated in some fluid then that indicates venous blood is in a mixture of fluids Some examples for DNA methylation markers are Mens1 menstrual blood Spei1 saliva and Sperm2 seminal fluid DNA methylation provides a relatively good means of sensitivity when identifying and detecting body fluids In one study only ten nanograms of a sample was necessary to ascertain successful results 123 DNA methylation provides a good discernment of mixed samples since it involves markers that give on or off signals DNA methylation is not impervious to external conditions Even under degraded conditions using the DNA methylation techniques the markers are stable enough that there are still noticeable differences between degraded samples and control samples Specifically in one study it was found that there were not any noticeable changes in methylation patterns over an extensive period of time 122 The detection of DNA methylation in cell free DNA and other body fluids has recently become one of the main approaches to Liquid biopsy 124 In particular the identification of tissue specific and disease specific patterns allows for non invasive detection and monitoring of diseases such as cancer 125 If compared to strictly genomic approaches to liquid biopsy DNA methylation profiling offers a larger number of differentially methylated CpG sites and differentially methylated regions DMRSs potentially enhancing its sensitivity Signal deconvolution algorithms based on DNA methylation have been successfully applied to cell free DNA and can nominate the tissue of origin of cancers of unknown primary allograft rejection and resistance to hormone therapy 126 Computational prediction EditDNA methylation can also be detected by computational models through sophisticated algorithms and methods Computational models can facilitate the global profiling of DNA methylation across chromosomes and often such models are faster and cheaper to perform than biological assays Such up to date computational models include Bhasin et al 127 Bock et al 128 and Zheng et al 129 130 Together with biological assay these methods greatly facilitate the DNA methylation analysis See also Edit5 Hydroxymethylcytosine 5 Methylcytosine 7 Methylguanosine Decrease in DNA Methylation I DDM1 a plant methylation gene Demethylating agent Differentially methylated regions DNA demethylation DNA methylation reprogramming Epigenetics of which DNA methylation is a significant contributor Epigenetic clock a method to calculate age based on DNA methylation Epigenome Genome Genomic imprinting an inherited repression of an allele relying on DNA methylation MethBase DNA Methylation database hosted on the UCSC Genome Browser MethDB DNA Methylation database N6 MethyladenosineReferences Edit D B 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2005 Prediction of methylated CpGs in DNA sequences using a support vector machine PDF FEBS Letters 579 20 4302 8 doi 10 1016 j febslet 2005 07 002 PMID 16051225 S2CID 14487630 Bock C Paulsen M Tierling S Mikeska T Lengauer T Walter J March 2006 CpG island methylation in human lymphocytes is highly correlated with DNA sequence repeats and predicted DNA structure PLOS Genetics 2 3 e26 doi 10 1371 journal pgen 0020026 PMC 1386721 PMID 16520826 Zheng H Jiang SW Wu H 2011 Enhancement on the predictive power of the prediction model for human genomic DNA methylation International Conference on Bioinformatics and Computational Biology BIOCOMP 11 Zheng H Jiang SW Li J Wu H 2013 CpGIMethPred computational model for predicting methylation status of CpG islands in human genome BMC Medical Genomics 6 Suppl 1 Suppl 1 S13 doi 10 1186 1755 8794 6 S1 S13 PMC 3552668 PMID 23369266 Further reading EditLaw JA Jacobsen SE March 2010 Establishing maintaining and modifying DNA methylation patterns in plants and animals Nature Reviews Genetics 11 3 204 20 doi 10 1038 nrg2719 PMC 3034103 PMID 20142834 Straussman R Nejman D Roberts D Steinfeld I Blum B Benvenisty N Simon I Yakhini Z Cedar H May 2009 Developmental programming of CpG island methylation profiles in the human genome Nature Structural amp Molecular Biology 16 5 564 71 doi 10 1038 nsmb 1594 PMID 19377480 S2CID 8804930 Patra SK April 2008 Ras regulation of DNA methylation and cancer Experimental Cell Research 314 6 1193 201 doi 10 1016 j yexcr 2008 01 012 PMID 18282569 Patra SK Patra A Rizzi F Ghosh TC Bettuzzi S June 2008 Demethylation of Cytosine 5 C methyl DNA and regulation of transcription in the epigenetic pathways of cancer development Cancer and Metastasis Reviews 27 2 315 34 doi 10 1007 s10555 008 9118 y hdl 11381 1797001 PMID 18246412 S2CID 22435914 External links Edit Wikimedia Commons has media related to DNA methylation DNA Methylation at the U S National Library of Medicine Medical Subject Headings MeSH ENCODE threads explorer Non coding RNA characterization Nature journal PCMdb Pancreatic Cancer Methylation Database Nagpal G Sharma M Kumar S Chaudhary K Gupta S Gautam A Raghava GP February 2014 PCMdb pancreatic cancer methylation database Scientific Reports 4 4197 Bibcode 2014NatSR 4E4197N doi 10 1038 srep04197 PMC 3935225 PMID 24569397 SMART Specific Methylation Analysis and Report Tool Human Methylation Mark Atlas DiseaseMeth Human disease methylation database EWAS Atlas A knowledgebase of epigenome wide association studies Retrieved from https en wikipedia org w index php title DNA methylation amp oldid 1169976018, wikipedia, wiki, book, books, library,

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