*2.1. DNA Methylation*

DNA methylation involves the addition of methyl groups to the cytosine residues of DNA [24]. Initial studies of X-chromosome inactivation in mice provided the evidence that DNA could be silenced without changes in the DNA sequence itself [25,26]. Later studies showed that DNA methylation can act as a mechanism through which this silencing may occur [27,28]. Importantly, DNA methylation marks can be copied from the parent strand to the daughter strand. Another notable finding is the identification of the main methylation target: the sequence of CpG, shorthand for 5 -C-phosphate-G-3 , that is, cytosine and guanine separated by only one phosphate group [27,28]. These findings eventually led to the development of methylation-sensitive restriction enzymes, which became important tools in epigenetic analyses [29,30].

DNA methlytransferases are responsible for moving methyl groups from S-adenosyl methionine (SAM) to CpG islands on DNA. For example, DNA methlytransferases 1, 3A, and 3B (DNMT1/3A/3B) add methyl groups to the carbon at position 5 of cytosine resides which are adjacent to guanine residues to produce 5-methylcytosine (5mC) [31,32]. While CpG is the main target, methylation may occur on CpHpG, where H may be A, T, or C [31]. The methylation mark 5mC is generally associated with gene repression within the gene promoter [31,33]. Alternatively, actively transcribed genes may be methylated within the transcriptional region which includes parts actually translated into protein (exons) and parts that will be cut out of the mRNA and are not translated into protein

(introns) [31]. The methylation pattern of a cell may vary in response to stress, as different genes are turned on and off. For example, it was shown that in the nuclei of mouse cardiomyocytes, 127 genes gained methylation and 313 lost methylation of their transcriptional region during the postnatal period [34]. While methylation at transcription start sites represses transcription, it is yet unclear whether methylation within exons serve clear functions, as high methylation seems to be positively correlated with transcription. One suggestion is that DNA methylation within exons may affect alternative splicing [35].

Methylation patterns can also change under stress and disease, as shown in the methylation of promoters of tumor suppressor genes in cancer [36]. Importantly, DNA methylation is reversible, with the removal reaction catalyzed by histone lysine demethylases (KDM) [37], providing therapeutic potential to prevent or change the pathological DNA methylation that may be related to the diseases.

Initial methylation detection assays utilized restriction endonucleases with known methylation sensitive CpG sites and their methylation insensitive isoschizomers [30]. Genomic DNA was then subjected to cleavage by both sets of restriction enzymes, and the differences between the fragments would outline where the methylated cytosines were, as revealed by Southern blot analysis, 2-dimensional gel electrophoresis, polymerase chain reaction (PCR), and, more recently, next-generation sequencing [31]. Alternatively, methylated cytosines may be targeted using specific antibodies by a process known as methylated DNA immunoprecipitation (MeDIP) or affinity enrichment [38]. One of the most common current methods of detecting epigenetic modifications is bisulfite sequencing. Unmethylated cytosine residues are converted to uracil using sodium bisulfite and alkaline treatment, while methylated cytosines are left intact. The bisulfite-treated DNA is then sequenced to reveal the methylated cytosines [31].

In addition to DNA methylation (5mC), other DNA modifications include hydroxymethylation (5hmC) and formylcytosine (5fC) [39]. Ten eleven translocation (TET) enzymes oxidize 5mCs to 5hmCs, and further catalyze the conversion to 5fC and 5-carboxylcytosine (5-caC) which can then be replaced by unmethylated cytosine after undergoing thymine DNA glycosylase (TDG)-mediated base excision and DNA base excision repair [33,40]. The TET enzymes may be significant to future therapies as they allow exploitation of the oxidation of 5mCs as a means of reversing gene repression by DNA methylation, should the repression of signature genes be identified.
