DNA demethylation
This article may be too technical for most readers to understand.(May 2017) |
For
Methylated
Almost 100% DNA demethylation occurs by a combination of passive dilution and active enzymatic removal during the
Embryonic development
Early embryonic development
The mouse
Demethylation of the maternal genome occurs by a different process. In the mature
DNMT3b begins to be expressed in the blastocyst.[9] Methylation begins to increase at 3.5 days after fertilization in the blastocyst, and a large wave of methylation then occurs on days 4.5 to 5.5 in the epiblast, going from 12% to 62% methylation, and reaching maximum level after implantation in the uterus.[10] By day seven after fertilization, the newly formed primordial germ cells (PGC) in the implanted embryo segregate from the remaining somatic cells. At this point the PGCs have about the same level of methylation as the somatic cells.
Gametogenesis
The newly formed primordial germ cells (PGC) in the implanted embryo devolve from the somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast toward the
In addition, from embryo day 9.5 to 13.5 there is an active form of demethylation. As indicated below in "Molecular stages of active reprogramming," two enzymes are central to active demethylation. These are a ten-eleven translocation methylcytosine dioxygenase (TET) and thymine-DNA glycosylase (TDG). One particular TET enzyme, TET1, and TDG are present at high levels from embryo day 9.5 to 13.5,[12] and are employed in active demethylation during gametogenesis.[11] PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle of the mouse at embryonic day 13.5. [13]
Learning and Memory
Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either accumulated slowly (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time. Rats subjected to one instance of contextual fear conditioning create an especially strong long-term memory. At 24 hours after training, 9.17% of the genes in the genomes of rat hippocampus neurons were found to be differentially methylated. This included more than 2,000 differentially methylated genes at 24 hours after training, with over 500 genes being demethylated.[4] Similar results to that in the rat hippocampus were also obtained in mice with contextual fear conditioning.[14]
The hippocampus region of the brain is where contextual fear memories are first stored (see figure of the brain, this section), but this storage is transient and does not remain in the hippocampus. In rats contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just one day after conditioning, but rats retain a considerable amount of contextual fear when hippocampectomy is delayed by four weeks.[15] In mice, examined at 4 weeks after conditioning, the hippocampus methylations and demethylations were reversed (the hippocampus is needed to form memories but memories are not stored there) while substantial differential CpG methylation and demethylation occurred in cortical neurons during memory maintenance. There were 1,223 differentially methylated genes in the anterior cingulate cortex of mice four weeks after contextual fear conditioning. Thus, while there were many methylations in the hippocampus shortly after memory was formed, all these hippocampus methylations were demethylated as soon as four weeks later.
Demethylation in Cancer
The human genome contains about 28 million CpG sites, and roughly 60% of the CpG sites are methylated at the 5 position of the cytosine. [16] During formation of a cancer there is an average reduction of the number of methylated cytosines of about 5% to 20%,[17] or about 840,00 to 3.4 million demethylations of CpG sites.
DNMT1 methylates CpGs on hemi-methylated DNA during DNA replication. Thus, when a DNA strand has a methylated CpG, and the newly replicated strand during semi-conservative replication lacks a methyl group on the complementary CpG, DNMT1 is normally recruited to the hemimethylated site and adds a methyl group to cytosine in the newly synthesized CpG. However, recruitment of DNMT1 to hemimethylated CpG sites during DNA replication depends on the UHRF1 protein. If UHRF1 does not bind to a hemimethylated CpG site, then DNMT1 is not recruited and cannot methylate the newly synthesized CpG site. The arginine methyltransferase PRMT6 regulates DNA methylation by methylating the arginine at position 2 of histone 3 (H3R2me2a).[18] (See Protein methylation#Arginine.) In the presence of H3R2me2a UHRF1 can not bind to a hemimethylated CpG site, and then DNMT1 is not recruited to the site, and the site remains hemimethylated. Upon further rounds of replication the methylated CpG is passively diluted out. PRMT6 is frequently overexpressed in many types of cancer cells.[19] The overexpression of PRMT6 may be a source of DNA demethylation in cancer.
Molecular stages of active reprogramming
Three molecular stages are required for actively, enzymatically reprogramming the DNA methylome. Stage 1: Recruitment. The enzymes needed for reprogramming are recruited to genome sites that require demethylation or methylation. Stage 2: Implementation. The initial enzymatic reactions take place. In the case of methylation, this is a short step that results in the methylation of cytosine to 5-methylcytosine. Stage 3: Base excision DNA repair. The intermediate products of demethylation are catalysed by specific enzymes of the base excision DNA repair pathway that finally restore cystosine in the DNA sequence.
Stage 2 of active demethylation
Demethylation of 5-methylcytosine to generate
TET family
TET dioxygenase isoforms include at least two isoforms of TET1, one of TET2 and three isoforms of TET3.[22][23] The full-length canonical TET1 isoform appears virtually restricted to early embryos, embryonic stem cells and primordial germ cells (PGCs). The dominant TET1 isoform in most somatic tissues, at least in the mouse, arises from alternative promoter usage which gives rise to a short transcript and a truncated protein designated TET1s. The isoforms of TET3 are the full length form TET3FL, a short form splice variant TET3s, and a form that occurs in oocytes and neurons designated TET3o. TET3o is created by alternative promoter use and contains an additional first N-terminal exon coding for 11 amino acids. TET3o only occurs in oocytes and neurons and is not expressed in embryonic stem cells or in any other cell type or adult mouse tissue tested. Whereas TET1 expression can barely be detected in oocytes and zygotes, and TET2 is only moderately expressed, the TET3 variant TET3o shows extremely high levels of expression in oocytes and zygotes, but is nearly absent at the 2-cell stage. It is possible that TET3o, high in neurons, oocytes and zygotes at the one cell stage, is the major TET enzyme utilized when very large scale rapid demethylations occur in these cells.
Stage 1 of demethylation - recruitment of TET to DNA
The TET enzymes do not specifically bind to 5-methylcytosine except when recruited. Without recruitment or targeting, TET1 predominantly binds to high CG promoters and CpG islands (CGIs) genome-wide by its CXXC domain that can recognize un-methylated CGIs.[24] TET2 does not have an affinity for 5-methylcytosine in DNA.[25] The CXXC domain of the full-length TET3, which is the predominant form expressed in neurons, binds most strongly to CpGs where the C was converted to 5-carboxycytosine (5caC). However, it also binds to un-methylated CpGs.[23]
For a TET enzyme to initiate demethylation it must first be recruited to a methylated CpG site in DNA. Two of the proteins shown to recruit a TET enzyme to a methylated cytosine in DNA are OGG1 (see figure Initiation of DNA demethylation at a CpG site)[26] and EGR1.[27]
OGG1
Oxoguanine glycosylase (OGG1) catalyses the first step in base excision repair of the oxidatively damaged base 8-OHdG. OGG1 finds 8-OHdG by sliding along the linear DNA at 1,000 base pairs of DNA in 0.1 seconds.[28] OGG1 very rapidly finds 8-OHdG. OGG1 proteins bind to oxidatively damaged DNA with a half maximum time of about 6 seconds.[29] When OGG1 finds 8-OHdG it changes conformation and complexes with 8-OHdG in its binding pocket.[30] OGG1 does not immediately act to remove the 8-OHdG. Half maximum removal of 8-OHdG takes about 30 minutes in HeLa cells in vitro,[31] or about 11 minutes in the livers of irradiated mice.[32] DNA oxidation by reactive oxygen species preferentially occurs at a guanine in a methylated CpG site, because of a lowered ionization potential of guanine bases adjacent to 5-methylcytosine.[33] TET1 binds (is recruited to) the OGG1 bound to 8-OHdG (see figure).[26] This likely allows TET1 to demethylate an adjacent methylated cytosine. When human mammary epithelial cells (MCF-10A) were treated with H2O2, 8-OHdG increased in DNA by 3.5-fold and this caused about 80% demethylation of the 5-methylcytosines in the MCF-10A genome.[26]
EGR1
The gene early growth response protein 1 (EGR1) is an immediate early gene (IEG). EGR1 can rapidly be induced by neuronal activity.[34] The defining characteristic of IEGs is the rapid and transient up-regulation—within minutes—of their mRNA levels independent of protein synthesis.[35] In adulthood, EGR1 is expressed widely throughout the brain, maintaining baseline expression levels in several key areas of the brain including the medial prefrontal cortex, striatum, hippocampus and amygdala.[35] This expression is linked to control of cognition, emotional response, social behavior and sensitivity to reward.[35] EGR1 binds to DNA at sites with the motifs 5′-GCGTGGGCG-3′ and 5'-GCGGGGGCGG-3′ and these motifs occur primarily in promoter regions of genes.[34] The short isoform TET1s is expressed in the brain. EGR1 and TET1s form a complex mediated by the C-terminal regions of both proteins, independently of association with DNA.[34] EGR1 recruits TET1s to genomic regions flanking EGR1 binding sites.[34] In the presence of EGR1, TET1s is capable of locus-specific demethylation and activation of the expression of downstream genes regulated by EGR1.[34]
DNA demethylation intermediate 5hmC
As indicated in the Figure above, captioned "Demethylation of 5-methylcytosine," the first step in active demethylation is a TET oxidation of 5-methylcytosine (5mC) to
Stage 3 base excision repair
The third stage of DNA demethylation is removal of the intermediate products of demethylation generated by a TET enzyme by
Demethylation after exercise
Physical exercise has well established beneficial effects on learning and memory (see Neurobiological effects of physical exercise). BDNF is a particularly important regulator of learning and memory.[40] As reviewed by Fernandes et al.,[41] in rats, exercise enhances the hippocampus expression of the gene Bdnf, which has an essential role in memory formation. Enhanced expression of Bdnf occurs through demethylation of its CpG island promoter at exon IV[41] and this demethylation depends on steps illustrated in the two figures.[20]
In a panel of healthy adults, negative associations were found between total DNA methylation and exposure to traffic related air pollution. DNA methylation levels were associated both with recent and chronic exposure to Black Carbon as well as benzene. [42]
Peripheral sensory neuron regeneration
After injury,
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