Structural Biochemistry/Enzyme Regulation/Methylation

Methylation is a regulatory process that can prevent certain processes such as degradation or catalysis from occurring. In the restriction-modification system of bacteria, the bacterial DNA is methylated at adenine bases by methylase, thereby preventing the DNA from being degraded by restriction endonucleases. These restriction enzymes have active sites that recognize the specific amino acid-based conformations in DNA. The enzymes can bind and cleave the phosphodiester bonds of the backbone at recognized (cognate) DNA. Methylated groups lose one hydrogen bond linkage with the enzyme and thus decrease the binding energy, resulting in a lower enzyme affinity and no cleavage.

Methylation also occurs in amino acid synthesis and in gene expression. In the latter, cytosine is methylated at C5. This 5-methylcytosine interferes with the proteins that bind to start transcription. In amino acid synthesis, methylases and other methyl group carriers attach methyl groups.

DNA Methylation Overview

Eukaryotic cellular DNA is usually stored in the nucleus, is wrapped around by a chromatin, which is a histone octamer. The chromatin is a crucial part in genetic modification in cells with the help of enzymes in order to make suitable changes to the cell, DNA, RNA, and proteins, such as the modification of transcription, or even the complete inactivation of a gene. One of these chromatin histone mechanism modifications can be a direct change on the mechanism of DNA methylation, an epigenetic mechanism. Proper DNA methylation is extremely important in functional cellular health, as no methylation (or even too much (hypermethylation) or insufficient methylation) could lead to serious diseases such as the creation of carcinogenic cancer cells.

DNA Methyltransferase

Ribbon representation of a DNA Methyltransferase

Enzymes that add methyl groups to DNA are called DNA methyltransferases. The mechanisms of DNA methyltransferases vary for each organism, but the enzyme generally binds unspecifically before traveling along the DNA strand to find the specific sequence to be methylated. As stated above, methylation of DNA is carried out so that the cell can differentiate between foreign and infectious DNA, and for gene expression purposes. But methylation of the wrong sequence may lead to chromosomal problems, including unfavorable interactions with Histone proteins that are essential for chromosomes to fold properly. Hence these enzymes are highly specific. DNA methyltransferases are present in most organisms including mammals.

Methyltransferases attach methyl groups to specific sequence in conjunction with endonucleases that must scan for DNA sequences that are intended for cutting. Methyltransferases belong in several different types that have different functions. Although their domain sites may have structural differences, DNMT3 which is similar to DNMT1 contains a regulatory region attached to a catalytic domain. Among types, there are also subcategories that can mediate methylation-independent gene repression. Others may be used for genomic imprinting. Some enzymes may methylate strictly RNA and not DNA. DNMT1 identifies methylate groups and perform nucleophilic attacks among marked nucleotides.

The Role of Methylation in Gene Expression

Meythlation is extremely important when it comes to gene expression. not all genes are active at all times which is why DNA methylation is one of the several mechanics that allow cells to control gene expression. Although there are many ways that a gene can be expressed in particularly eukaryotes, the methylation of DNA is a common epigenetic signaling tool that can allow cells to lock genes in the off position. Key experiments were needed in order to provide the early clues for what the role of methylation had on gene expression. One such experiment was conducted by McGhee and Ginder in 1979 where they compared the methylation status of beta-globin loci in cells that did and that did not express the gene. By utilizing restriction enzymes that distinguished between methylated and unmethylated DNA, the two scientists were capable of dictating that the beta globin locus gene was not being expressed in the cells that were unmethylated. In addition to this experiment, more supporting evidence suggested the same conclusion. The same mechanism was performed on 5-azacytidine, a chemical analog of the nucleoside cytidine, in mouse cells. Similar observations were found with this experiment in the sense that the cytosine residues that were not methylated on the nucleoside prevented the gene to be expressed.

DNA methylation is the addition of a methyl group to the 5th carbon in cytosines in DNA into a covalent bond. This mechanism is catalyzed by DNMTs (DNA methyltransferases) by utilizing the CpG dinucleotide sequence. CpGs that aren’t methylated usually contain ahigher GC base concentration and are clustered together, forming CpG islands, which are usually found at the starting sites for transcription and promoter binding. DNA methylation at CGI promoters have been known to cause gene expression silencing, such as the silencing of mechanisms such as X-chromosomes and genomic imprinting. DNA methylation can silence DNA through various mechanisms, such as utilizing 5meC to inhibit transcription factor binding or using Methyl Binding Domain Proteins (MDB) to repress the DNA using repressive chromatin-modifying complexes. Overall, DNA methylation is responsible for important cellular functions involving the genetic sequence of eukaryotes, such as genetic modification and even silencing.

How and Where are Genes Methylated

Now that it is understood that DNa methylation is key for gene expression, the questions comes about where exactly must methylation occur on the gene to induce expression. Today researchers know that DNA methylation occurs primarily at the cytosine bases of eukaryotic DNA, which are converted to 5-methycytosine by DNA methyltransferase (DNMT) enzymes. These altered cytosine residues are normally adjacent to guanine nucleotides (due to base pair mechanics) which essentially results in two methylated cytosine residues that sit diagonally from each on opposing DNA strands.

The roles and targets of DNA methylation varies among the kingdoms of organisms. For examples, among Animalia, mammals tend to have distributed CpG methylation patterns as mentioned above, however in invertebrate animals, a mosiac pattern of methylation is found where regions of heavily methylated DNA are interspersed with nonmethylated regions. In other words, no distinct pattern is seen in this kingdom of species. It should also be noted that surprisingly the kingdom plantae are organisms that are found to have the highest amount of methylation, up to 50% of cystosine residues undergoing methylation.

DNA methylation and Disease

When one considers the magnitude of importance DNA methylation has upon gene expression, one must also ask of the consequences of errors found in methylation and what can arise from such error. Errors in methylation give rise to a number of devastating consequences, including various disease associated with tumor suppressor genes. It has been found that tumor suppressor genes are often turned off in cancer cells due to hypermethylation of cytosine residues. hypermethylation is also found at other very critical gene expressions such as in the cell cycle regulatory genes, the tumor cell invasion genes, DNA repair genes and other genes that involve the propogation of metastasis. In addition, the genomes of cancel cells have also shown a decrease in methylation, or hypomethylation, compared to normal healthy cells (with the exception of hypermethylation found on the for mentioned genes). The combination of hypomethylation and hypermythelation describes the potent nature of a cancer cell.

Gene-Body DNA Methylation

DNA methylation in active genes can be analyzed with the use of bisulfites to sequence the DNA and methyls attached to the DNA. With bisulfite sequencing, it was discovered that CpG methylation occurred in active genes and much downstream the sites of transcription (as long as the methylation didn’t silence the gene). These discoveries showed that DNA methylation can occur and various promoters (like human X chromosomes, which has many transcribable regions). Due to the fact that bisulfite sequencing is unbiased, it also showed methylated as well as non-methylated CpG sites, showing that; with MeDIP-Seq and MRE-Seq, non-methylated CpG is prevalent at most promoters.

References

McGhee, J. D., & Ginder, G. D. Specific DNA methylation sites in the vicinity of the chicken beta-globin genes. Nature 280, 419–420 (1979)

Biochemistry 6th Edition, Berg et al.