Methylation
Addition of a methyl group (-CH3) to the DNA base, cytosine, creates a modified base called 5-methylcytosine, found in both prokaryotes and eukaryotes. This process is catalyzed by enzymes called methylases. In bacteria, methylation is part of a protective mechanism against foreign DNA. In mammalian cells, methylation of cytosine takes place specifically when it is located immediately 5' to a guanine, a structure called a CpG site; dense clusters of these sites are called CpG islands.
DNA, once methylated, tends to stay methylated through subsequent cell divisions, due to work of the maintenance methylase, DNA methyltransferase 1 (Dnmt 1). As DNA is replicated, it becomes transiently hemi-methylated, with methyl groups on the template strands but not the daughter strands. Dnmt 1 recognizes these structures and methylates CpG sites on daughter strands, thereby maintaining the methylation patterns.
Methylation is generally a silencing mechanism in mammalian cells, inhibiting transcriptional activity from the modified genes, including transposons and retroviruses that have accumulated in the mammalian genome. It is important for specific tissue and developmental patterns of gene expression and plays essential roles in X chromosome inactivation, genomic imprinting, genome stability and chromosome segregation. Normal methylation depends upon the functioning of a wide metabolic network in the cell that involves enzymes, vitamins, and nutrients such as folates.
One way that methylated DNA can affect transcription is if binding proteins recognize methylated sites, bind to the DNA and also bind to enzymes (deacetylases) that then alter nearby histones. This changes the local chromatin structure and renders methylated DNA inaccessible to the transcriptional machinery. Sometimes, however, methylation is associated with active alleles, in which case it likely interferes with recruitment of transcriptional repressors.
Structural genes are generally hypomethylated in tissues where they are actively expressed and hypermethylated in tissues where they are inactive. As part of the normal regulatory mechanism, methylation frequently acts to suppress the tissue-specific genes whose activity is not required in the particular cell type.
In mammals, X-chromosome inactivation in females is a dosage compensation mechanism, since females have two X chromosomes whereas males have one. Methylation of the inactive X, particularly at gene promoters, seems to function as a maintenance mechanism for X inactivation.
Genetic imprinting is another normal process, whereby the two parental alleles are differentiated for selective expression. The importance of methylation in at least the maintenance of imprints is clear, and the nature of the de novo imprint is a subject of active research. Imprinted genes are rich in CpG islands near to clustered direct repeats and most show differences in DNA methylation status between maternal and paternal alleles. To re-set the imprint during transmission between generations, the previous imprint is erased with genome-wide demethylation in germ cells. A de novo imprint is then reestablished using specific methylases, though how the sites for methylation are recognized is not yet clear. Once methylated in the parental germ cells, the patterns are maintained after fertilization by Dnmt 1.
Failure of the normal methylation mechanisms of the cell can lead to a variety of consequences. In most tumor cells, methylation patterns are disrupted, typically showing both genome-wide hypomethylation and region-specific hypermethylation. Each may contribute to tumor progression. Hypomethylation is likely involved with the widespread chromosomal alterations, whereas hypermethylation of specific promoters, notably in tumor suppressor genes can functionally inactivate the genes, providing growth advantage to the cell. Methylator phenotype describes a pathway that results in instability through the simultaneous silencing of multiple genes, and is involved with tumors with sporadic mismatch repair deficiency. Because of the involvement of hypermethylation in some cancers, demethylating drugs such as 5-azacytidine are being tested in clinical trials.
The loss of normal methylation control is responsible for a number of non-cancer genetic syndromes. Fragile X syndrome is caused by an expanded trinucleotide (CGG) repeat region in the X-linked FMR1 gene. The expanded region is hypermethylated, thereby repressing the gene's expression. Patients with ICF Syndrome (immunodeficiency, centromeric instability, and facial anomalies) show selective undermethylation of pericentromeric satellite DNA, with related chromosome abnormalities, due to mutations in a de novo methyltransferase gene (Dnmt3b). Rett syndrome involves mutations in the gene for a protein that binds to methylated DNA, and which is involved in the chain of regulatory events. Genes that are normally imprinted express from only one parental allele, and are susceptible to the effects of genetic alterations of the complimentary allele. Example of such abnormalities include Prader-Willi, Angelman and Beckwith-Wiedemann syndromes.
he importance of DNA methylation as a regulatory mechanism in many aspects of cell function has become increasingly apparent with research in the past few years. The major question still puzzling scientists regards the exact nature of the basic signal that alerts the cell to first methylate or demethylate a particular site.
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