Not all genes are active at all times. DNA methylation is one of several epigenetic mechanisms that cells use to control gene expression.
There are many ways that gene expression is controlled in eukaryotes, but methylation of DNA (not to be confused with histone methylation) is a common epigenetic signaling tool that cells use to lock genes in the "off" position. In recent decades, researchers have learned a great deal about DNA methylation, including how it occurs and where it occurs, and they have also discovered that methylation is an important component in numerous cellular processes, including embryonic development , genomic imprinting , X-chromosome inactivation, and preservation of chromosome stability . Given the many processes in which methylation plays a part, it is perhaps not surprising that researchers have also linked errors in methylation to a variety of devastating consequences, including several human diseases.
Prior to 1980, there were a number of clues that suggested that methylation might play a role in the regulation of gene expression . For example, J. D. McGhee and G. D. Ginder compared the methylation status of the beta-globin locus in cells that did and did not express this gene. Using restriction enzymes that distinguished between methylated and unmethylated DNA, the duo showed that the beta-globin locus was essentially unmethylated in cells that expressed beta-globin but methylated in other cell types (McGhee & Ginder, 1979). This and other evidence of the time were indirect suggestions that methylation was somehow involved in gene expression.
Shortly after McGhee and Ginder published their results, a more direct experiment that examined the effects of inhibiting methylation on gene expression was performed using 5-azacytidine in mouse cells. 5-azacytidine is one of many chemical analogs for the nucleoside cytidine. When these analogs are integrated into growing DNA strands, some, including 5-azacytidine, severely inhibit the action of the DNA methyltransferase enzymes that normally methylate DNA. (Interestingly, other analogs, like Ara-C, do not negatively impact methylation.) Because most DNA methylation was known to occur on cytosine residues, scientists hypothesized that if they inhibited methylation by flooding cellular DNA with 5-azacytidine, then they could compare cells before and after treatment to see what impact the loss of methylation had on gene expression. Knowing that gene expression changes are responsible for cellular differentiation , these researchers used changes in cellular phenotypes as a proxy for gene expression changes (Table 1; Jones & Taylor, 1980).
Table 1: Effect of Cytidine Analogs on Cell Differentiation and DNA Methylation
| Chemical Added | Number of Differentiated Cells | Amount of Methylation Measured |
| 3 μM cytidine (control) | 0 | 100% |
| 0.3 μM Ara-C | 0 | 127% |
| 3 μM 5-azacytidine | 22,141 | 33% |
This straightforward experiment demonstrated that it was not the removal of cytidine residues alone that resulted in changes in cell differentiation (because Ara-C did not have an impact on differentiation); rather, only those analogs that impacted methylation resulted in such changes. These experiments opened the door for investigators to better understand exactly how methylation impacts gene expression and cellular differentiation.
Today, researchers know that DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5-methylcytosine by DNA methyltransferase (DNMT) enzymes. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide , resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands. Different members of the DNMT family of enzymes act either as de novo DNMTs, putting the initial pattern of methyl groups in place on a DNA sequence, or as maintenance DNMTs, copying the methylation from an existing DNA strand to its new partner after replication . Methylation can be observed by staining cells with an immunofluorescently labeled antibody for 5-methylcytosine. In mammals, methylation is found sparsely but globally, distributed in definite CpG sequences throughout the entire genome , with the exception of CpG islands, or certain stretches (approximately 1 kilobase in length) where high CpG contents are found. The methylation of these sequences can lead to inappropriate gene silencing , such as the silencing of tumor suppressor genes in cancer cells.
Currently, the mechanism by which de novo DNMT enzymes are directed to the sites that they are meant to silence is not well understood. However, researchers have determined that some of these DNMTs are part of chromatin-remodeling complexes and serve to complete the remodeling process by performing on-the-spot DNA methylation to lock the closed shape of the chromatin in place.
The roles and targets of DNA methylation vary among the kingdoms of organisms. As previously noted, among Animalia, mammals tend to have fairly globally distributed CpG methylation patterns. On the other hand, invertebrate animals generally have a "mosaic" pattern of methylation, where regions of heavily methylated DNA are interspersed with nonmethylated regions. The global pattern of methylation in mammals makes it difficult to determine whether methylation is targeted to certain gene sequences or is a default state, but the CpG islands tend to be near transcription start sites, indicating that there is a recognition system in place.
Plantae are the most highly methylated eukaryotes, with up to 50% of their cytosine residues exhibiting methylation. Interestingly, in Fungi, only repetitive DNA sequences are methylated, and in some species , methylation is absent altogether, or it occurs on the DNA of transposable elements in the genome. The mechanism by which the transposons are recognized and methylated appears to involve small interfering RNA ( siRNA ). The whole silencing mechanism invoking DNMTs could be a way for these organisms to defend themselves against viral infections, which could generate transposon-like sequences. Such sequences can do less harm to the organism if they are prevented from being expressed, although replicating them can still be a burden (Suzuki & Bird, 2008). In other fungi, such as fission yeast , siRNA is involved in gene silencing, but the targets include structural sequences of the chromosomes, such as the centromeric DNA and the telomeric repeats at the chromosome ends.
