DNA methylation refers to the process of adding a methyl group to specific regions of DNA. This process can bring about changes in the activity of DNA without changing the sequence of nucleotides. Although DNA methylation has been known to occur since the 1950s, its mechanism and roles are still under investigation.
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DNMT3 is a DNA methlytransferase enzyme found in eukaryotes. (Credit: petarg | Shutterstock).
DNA methylation in prokaryotes
In bacteria, DNA methylation is used as a signal for the regulation of a specific DNA-protein interaction. Methylation systems typically comprise of a DNA methylase and one or more DNA binding proteins that can overlap the target methylation site on DNA, subsequently blocking methylation of that site.
Methylation of the target site inhibits protein binding, which can result in two alternative methylation states of the target site – methylated and nonmethylated. This leads to a DNA methylation pattern that dictates which genes are expressed, and therefore how a microorganism interacts with the environment.
Origins of DNA methylation in bacteria
The methylation of DNA provides an epigenetic reversible control of the genetic program. This modification in bacteria can modulate various processes, such as the regulation of gene expression and DNA replication. In 1953, the first evidence of DNA methylation was found in bacteria during phage infection. In this case, DNA methylation originated as a method to protect the bacterial DNA from the extraneous DNA of the phage.
The aforementioned defense mechanism is termed the restriction-modification system (R-M system), and it consists of two components: DNA methyltransferase and restriction endonuclease. The methyltransferase methylates a specific section of DNA, while the restriction endonuclease cleaves the DNA based on the sequence.
As the bacteria consists of both the restriction and modification systems, they can effectively differentiate between the ‘self’ and ‘non-self’ DNA to restrict the foreign or bacteriophage infection. This mechanism in principle work similar to the innate immune response.
Types of restriction-modification systems in bacteria
Restriction-modification systems in bacteria have manifold biological roles - from the defense against various genetic elements that are considered parasitic to halting gene transfer and stopping lineage homogenization.
These systems can be divided into four types:
Type I systems are complex and consist of separate restriction endonuclease and methyltransferase subunits along with a common DNA recognition specificity (S) subunit. This system recognizes motifs that are bipartite and can cleave at distances that are far away from the binding region.
The type II restriction-modification system has restriction endonuclease and methyltransferase enzymes. These show identical binding specificity towards DNA and bind to short, non-palindromic DNA sequences. Cleavage also happens outside of the DNA binding region in this system.
The Type III system consists of a methyltransferase that contains the DNA binding specificity domain and complexes with the restriction endonuclease for cleaving. This system also binds to short, palindromic motifs and cut outside the region of DNA binding. The type IV systems cleave modified DNA and they do not have a methyltransferase part.
Prevalence of DNA methylation in bacteria
In one study, researchers looked at more than 200 diverse bacteria and other prokaryotes, such as archaea. They found that DNA methylation was present in more than 90% of the organisms and that there were DNA binding sites for more than 600 restriction systems, indicating the enormous amount of diversity.
The scientists also observed numerous cases of DNA methylation (even in the absence of a restriction system); these patterns indicate the role of this system to regulate the genome for other purposes in prokaryotes.
How DNA methylation works
DNA methylation in eukaryotes
The methylation of DNA in eukaryotes is often used to switch-off the signaling activity of the genes. DNA methylation has been shown to have an important role in several eukaryotic processes, such as embryonic development, genome imprinting, inactivation of X-chromosome, and generally preserving the stability of chromosomes. The process is very specific in mammals, where the methylation process for 75% of all CpG dinucleotides is in somatic cells.
Considering the scale of impact of DNA methylation, it is not surprising that these effects are also linked to several diseases, including those found in humans. In mammals, the global methylation pattern makes it challenging to determine whether methylation is a default state or actually targeted towards specific sequences of genes. However, the CpG islands usually occur close to transcription start sites, suggesting that there is a system for recognition.
Discovery of DNA methylation in eukaryotes
To understand the exact roles of DNA methylation, scientists J. D. McGhee and G. D. Ginder looked at the methylation status of cells that expressed and did not express the beta-globin gene. They found that in cells that beta-globin locus was unmethylated in the cells that expressed beta-globin, while the methylated cells did not express beta-globin. These results suggested that methylation could be involved in the expression of genes.
Subsequently, the effect of methylation was studied using more direct methods, such as using chemical analogs. 5-azacytidine is a chemical analog for nucleoside cytidine. When incorporated into the DNA strand, it can inhibit the action of DNA methyltransferase enzymes. Thus, this can be used to infer the importance of DNA methyltransferases in the gene expression profile. Using this method, researchers found that DNA methylation could affect the cell differentiation process.
Mechanism of DNA methylation in eukaryotes
DNA methylation usually occurs at the cytosine bases in the eukaryotic genome, which leads to the formation of 5-methylcytosine. As altered residues are often lying adjacent to guanine nucleotides, this results in methylated cytosine residues that lie diagonally to each other.
There are two modes of DNA methylation in eukaryotic cells. The first method is called de novo methylation, which involves rearranging the methylation pattern during the formation of embryo or differentiation in adult cells. Two classes of methyltransferases, DNMT3a and DNMT3b have been shown to be involved in de novo methylation.
Conversely, maintenance methylation falls in the second category. This class of DNA methyltransferases maintains the methylation pattern once it is established. DNM1 was the first mouse methyltransferase that was described by Bestor and colleagues. This enzyme was found to have high maintenance methylation activity and is critically important for mouse development.
There are also two mechanisms to remove the existing methylation pattern. The first method involves passive demethylation where DNM1 does not maintain the methylation pattern. In the second method, certain demethylases actively perform demethylation to remove the patterns.
Visualizing the methylation pattern
Methylation can be observed directly by staining the cells for immunofluorescently labeled 5-methylcytosine. Mammals often have a global distribution of CpG or cytosine nucleoside followed by guanine nucleotide in a linear sequence. However, invertebrates harbor a mosaic pattern of methylation where there are regions of heavy methylation followed by non-methylated regions. Plants are also highly methylated, with up to 50% of cytosine residues in plants being methylated. Only repetitive sequences are methylated in fungi, and some species completely lack methylation.
Role of DNA methylation in human disease
As methylation has critical roles during gene expression and during cell differentiation, any errors in this process can lead to a disease state. Some of the diseases that are known to be associated with defects in gene imprinting include cancer, lupus, muscular dystrophy, several birth defects, etc. DNA methylation has even been implicated in many cardiovascular diseases, including atherosclerosis.
Understanding the process of epigenetic changes can be invaluable for understanding and preventing these complications. A vast amount of research currently performed to understand the link between DNA methylation and diseases is focused on cancer and tumor suppressor genes.
The tumor suppressor genes are the genes that are usually silenced in the cells with cancer due to hypermethylation; on the other hand, the genomes of cancer cells have overall hypomethylated genes compared to the normal cells.
Genes involved in cell cycle regulation, tumor cell invasion, DNA repair, and other events form an exception as they are hypermethylated in cancer cells. In these cases, silencing due to hypermethylation propagates metastasis. For example, hypermethylation is visible during the early stages of colon cancer and even has the potential to serve as a biomarker of the disease.
How Methylation Silences Genes
Eukaryotes versus Prokaryotes
In eukaryotes, the DNA methylation only occurs on the cytosine residues and specifically for the CpG sequences. Whereas in prokaryotes, methylation of the adenine residues is the primary epigenetic signal. Eukaryotes only use a few DNA methyltransferases; the numbers are much higher in bacteria where most of them have high sequence specificity. For example, important human gastric pathogen Helicobacter pylori has a large repertoire of DNA methyltransferase genes, with different strains containing different and rather unique sequences.
However, the protective function of DNA methylation is similar across prokaryotes and eukaryotes. For example, inserted viral sequences in humans and rodents can be methylated to silence the introduced genes. The same mechanisms to silence the transgenes have been found in mice as well. Therefore, the recognition and elimination functions of DNA methylation machinery seem to be conserved in evolution.
As the eukaryotic genome is much more complex compared to the prokaryotic genome, several studies have proposed the role of methylated cytosine as a “fifth base” in the eukaryotic genome. Furthermore, many experiments show that the methylation of cytosine is involved in the functional reorganization of the eukaryotic genome.