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DNA demethylation is the process of removal of a methyl group from nucleotides in DNA. Both DNA demethylation and methylation play important roles in mammalian development and differentiation, as well as in cognition and neuroregeneration (after injury to peripheral nerves in mammals). DNA methylation on cytosine at CpG sites on a gene promoter leads to the silencing of gene expression, while DNA demethylation of a gene promoter is linked to transcriptional activation and gene expression. These are called epigenetic changes. The DNA methylation status that controls gene expression is copied during DNA replication and is transmitted to daughter cells along with the DNA sequence. This area is currently being investigated for its role in disease progression and for potential treatments, such as cancer therapy.
- 1 Passive and active demethylation
- 2 Examples DNA Demethylation
- 3 Possible mechanisms of active DNA demethylation
- 4 DNA hydroxymethylation
- 5 Cognition
- 6 Peripheral sensory neuron regeneration
- 7 References
Passive and active demethylationEdit
DNA demethylation can occur through passive or active mechanisms. The passive process takes place in the absence of methylation of newly synthesised DNA strands by DNMT1 during several replication rounds (for example, upon 5-Azacytidine treatment), leading to dilution of the methylation signal. Active DNA demethylation is mediated by multiple enzymes and can occur independent of DNA replication.
Examples DNA DemethylationEdit
All the cases of DNA demethylation can be classified as global (genome wide) or locus-specific (when just specific sequences are demethylated). The genome-wide DNA demethylation occurs:
- In mammals:
- Possibly in amphibia - during midblastula transition.
Examples of specific DNA demethylation:
Possible mechanisms of active DNA demethylationEdit
There are several proposed hypothetical mechanisms of active DNA demethylation:
A Direct removal of 5-methylcytosine
- Direct removal of methyl group. This process has quite low thermodynamic probability.
- Removal of methylated bases (either by direct removal of methylcytosine, or through cytosine deamination followed by removal of thymine from thymine/guanosine mismatch), followed by insertion of unmethylated one using base excision repair machinery (BER).
- Removal of entire DNA patch and following filling it with new nucleotides by nucleotide excision repair (NER) or mismatch repair (MMR).
B Removal of 5-methylcytosine via further modified cytosine bases
Oxidation of the methyl group generates 5-Hydroxymethylcytosine. Several mechanisms have been proposed to mediate demethylation of 5-hydroxymethylcytosines. This base can be either deaminated by AID/Apobec enzymes to give 5-Hydroxymethyluracil. Alternatively, TET enzymes can further oxidize 5-hydroxymethylcytosine to 5-Formylcytosine and 5-Carboxylcytosine.
- Both the deamination and the oxidation products have been shown to be repaired by TDG, a glycosylase which is involved in base excision repair. A base excision mediated demethylation mechanism would yield double strand breaks if it occurs on large scale in CpG islands.
- The carboxyl and formyl groups of 5-Formylcytosine and 5-Carboxylcytosine could be enzymatically removed without excision of the base. Precedent for similar reactions is found in biosynthetic pathways.
DNA hydroxymethylation has been proposed to act as a specific epigenetic mark opposing DNA methylation, rather than a passive intermediate in the de-methylation pathway. DNA hydroxymethylation in vivo is sometimes associated with labile nucleosomes, which are more easy to disassemble and to be out-competed by transcription factors during cell development. Hydroxymethylation has been associated with pluripotency of stem cells. Furthermore, changes in hydroxymethylation have been associated with cancer.
In mammals, DNA methyltransferases (which add methyl groups to DNA bases) exhibit a strong sequence preference for cytosines within the particular DNA sequence cytosine-phosphate-guanine (CpG sites). Methylation of cytosines occurs at 60–90% of CpG sites depending on the tissue type. In the mammalian brain, ~62% of CpGs are methylated. Methylation of CpG sites tends to stably silence genes.
Active DNA methylation and demethylation is required for the cognition process of memory formation and maintenance. In rats, contextual fear conditioning can trigger lifelong memory for the event with a single trial, and methylation changes appear to be correlated with triggering particularly long-lived memories. With contextual fear conditioning, after 24 hours, DNA isolated from the rat brain hippocampus region had 2097 differentially methylated genes, with a portion being demethylated. Similar results in the hippocampus were obtained with contextual fear conditioning in mice. As shown with the rats, 9.2% of the genes in the rat hippocampus neurons are differentially methylated 24 hours after contextual fear conditioning. 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, where there were many methylations in the hippocampus shortly after memory was formed, all these hippocampus methylations were demethylated as soon as 4 weeks later.
The process of demethylation is illustrated in the two figures shown in this section. First, the guanine in the CpG site is oxidized to form 8-oxo-dG (or its tautomer 8-OHdG) (see first figure). TET1 is a key enzyme involved in demethylating 5mCpG. However, TET1 is only able to act on 5mCpG if the guanine has first been oxidized (presumably by an ROS) to form 8-OHdG, resulting in a 5mCp-8-OHdG dinucleotide (see first figure). After formation of 5mCp-8-OHdG, the base excision repair enzyme OGG1 binds to the 8-OHdG lesion without immediate excision. Adherence of OGG1 to the 5mCp-8-OHdG site recruits TET1, allowing TET1 to oxidize the 5mC adjacent to 8-OHdG, as shown in the second figure. As reviewed by Bayraktar and Kreutz, in the brain, further reactions in DNA demethylation are primarily dependent upon TET enzymes in the steps indicated in the second figure. The formation of the final product, unmethylated cytosine, depends on base excision repair (BER) as the terminal step.
Altered protein expression in neurons, (likely initiated by 8-oxo-dG-dependent demethylation of CpG sites in gene promoters within neuron DNA), is central to memory formation.
Demethylation after exerciseEdit
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. As reviewed by Fernandes et al., 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 and this demethylation depends on steps illustrated in the two figures.
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. 
Peripheral sensory neuron regenerationEdit
After injury, neurons in the adult peripheral nervous system can switch from a dormant state with little axonal growth to robust axon regeneration. DNA demethylation in mature mammalian neurons removes barriers to axonal regeneration. This demethylation, in regenerating mouse peripheral neurons, depends upon TET3 to generate 5-hydroxymethylcytosine (5hmC) in DNA. 5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs such as Atf3, Bdnf, and Smad1, that regulate the axon growth potential of neurons.
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