Introduction of Methylation: In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with specifically a methyl group, rather than a larger carbon chain, replacing a hydrogen atom. In biological systems, methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA metabolism. Methylation of heavy metals can also occur outside of biological systems. Types of methylation: 1. Biological methylation: The methylation of DNA sequences is an important component of epigenetic control that can be inherited or occur over time. Methylation contributing to epigenetic inheritance: Methylation contributing to epigenetic inheritance can occur by following ways: 1. DNA methylation 2.Protein methylation.3.Histone methylation 1. DNA methylation: Methylation of DNA is one of the parameters that control transcription. Methylation in the vicinity of the promoter is associated with the absence of transcription. This is one of several regulatory events that influence the activity of a promoter; like the other regulatory events, typically this will apply to both (allelic) copies of the gene. However, methylation also occurs as an epigenetic event that can distinguish alleles whose sequences are identical. This can result in differences in the expression of the paternal and maternal alleles DNA methylation in vertebrates typically occurs at CpG sites (cytosine-phosphate-guanine sites; that is, where a cytosine is directly followed by a guanine in the DNA sequence); this methylation results in the conversion of the cytosine to 5-methylcytosine. The formation of Me-CpG is catalyzed by the enzyme DNA methyltransferase. The bulk of mammalian DNA has about 40% of CpG sites methylated but there are certain areas, know as CpG islands which are GC rich (made up of about 65% CG residues) where none are methylated. These are associated with the promoters of 56% of mammalian genes, including all ubiquitously expressed genes. 1-2% of the human genome are CpG clusters and there is an inverse relationship between CpG methylation and transcriptional activity.
2. Protein methylation: It typically takes place on arginine or lysine amino acid residues in the protein sequence. Arginine can be methylated once (monomethylated arginine) or twice, with either both methyl groups on one terminal nitrogen (asymmetric dimethylated arginine) or one on both nitrogens (symmetric dimethylated arginine) by peptidylarginine methyltransferases (PRMTs). Lysine can be methylated once, twice or three times by lysine methyltransferases. Protein methylation has been most well studied in the histones. The transfer of methyl groups from S-adenosyl methionine to histones is catalyzed by enzymes known as histon methyltransferases. Histones which are methylated on certain residues can act epigenetically to repress or activate gene expression. Protein methylation is one type of post-translational modification.
3. Histone methylation: Methylation of both histones and DNA is associated with inactivity. Sites that are methylated in histones include two lysines in the tail of H3 and an arginine in the tail of H4. Methylation of H3 9Lys is a feature of condensed regions of chromatin, including heterochromatin as seen in bulk and also smaller regions that are known not to be expressed. The histone methyltransferases enzyme that targets this lysine is called SUV39H1. Its catalytic site has a region called the SET domain. Other histone methyltransferases act on arginine. In addition, methylation may occur on 79Lys in the globular core region of H3; this may be necessary for the formation of heterochromatin at telomeres. Acetylation of histones is associated with gene activation.Methylation of DNA and of histones is associated with heterochromatin. Active chromatin is acetylated on the tails of histones H3 and H4. Inactive chromatin is methylated on 9Lys of histone H3.
2. Chemical methylation: Chemical methylation of tissue samples is a method used for reducing certain histological staining artifacts The methylation machinery: How are the developmental changes of methylation levels affected? Faithful propagation of the methylation state of a CpG dinucleotide occurs directly after DNA replication by an enzymatic methyl transfer reaction at cytosine residues in the unmethylated nascent DNA strand across from methylated CpG dinucleotides.This activity, which uses hemi-methylated CpG dinucleotides as a substrate, is referred to as maintenance methylation activity. Loss of methylation can occur by the failure of maintenance methylation, but acquisition of DNA methylation at a previously non-methylated CpG can not be accomplished by maintenance methylation activity. This requires a methylation activity that can recognize unmethylated CpG dinucleotides and is referred to as de novo methylation activity. Currently, only one DNA (cytosine-5) methyltransferase (MTase) has been identified in mammalian cells. This MTase shows both maintenance and de novo methylation activity in vitro. However, it seems likely that its main function in vivo is maintenance methylation. Maintenance methylation is apparently essential for normal embryonic development in mammals because mice deficient for the enzyme die soon after gastrulation, when the burst of de novo methylation activity has ceased. There is no evidence for de novo DNA methylation activity of the known mammalian MTase in vivo and genetic evidence obtained in our laboratory with cells and mice homozygous for a mutated MTase gene suggest that de novo MTase activity is not impaired. This suggests that a separate de novo MTase activity may be encoded by an as yet unidentified gene. This is an important issue, since regional hypermethylation seen in cancer cells can be attributed to the known MTase enzyme only if it is capable of such de novo DNA methylation in vivo. The role of DNA methylation in gene expression and gene silencing: DNA methylation in both plant and mammalian systems has long been associated with changes in gene expression, chromatin structure alterations, activation of transposable elements, genomic imprinting, and carcinogenesis. In eukaryotes and in particular in higher plants, 5-methylcytosine is the predominant modified base. DNA methylation can inhibit transcription directly by blocking the binding of transcription factors by modifying their target sites. However, DNA methylation alone is often not sufficient to block transcription, but it is likely the nature of chromatin formed on a methylated template that renders it transcriptionally inactive. DNA methylation and gene expression: Housekeeping genes have a non-methylated CpG island tightly associated with their promoter. Since such genes tend to be expressed ubiquitously and since autosomal CpG islands are normally never found to be methylated, housekeeping genes are thought not to be regulated by DNA methylation. The past decade has seen a plethora of reports documenting the mostly inverse correlation between DNA methylation and gene expression levels of tissue-specific genes. In a recent survey by Yeivin and Razin, a large majority of tissue specific genes showed a correlation between gene activity and hypomethylation of the promoter region. A weaker correlation was seen with CpG dinucleotides throughout the gene body. Importantly, widespread de novo methylation of CpG islands has been observed in many cells growing in tissue culture. These CpG islands are not methylated in normal tissues in vivo and have been found to be associated with genes that are not essential for growth in culture suggesting that gene silencing by methylation can be of selective advantage for cell growth. The Role of DNA Methylation in Gene Expression:
Not all genes are active at all times. DNA methylation is one of several epigenetic mechanisms that cells use to control gene expression.1Introduction2 5-azacytidine Experiments Provide Early Clues to the Role of Methylation in Gene Expression 3 How and Where Are Genes Methylated? 4 The Role of Methylation in Gene Expression
5 DNA Methylation and Histones 6 DNA Methylation and Disease 7 Summary 8References and Recommended Reading 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. For many years, methylation was believed to play a crucial role in repressing gene expression, perhaps by blocking the promoters at which activating transcription factors should bind. Presently, the exact role of methylation in gene expression is unknown, but it appears that proper DNA methylation is essential for cell differentiation and embryonic development. Moreover, in some cases, methylation has observed to play a role in mediating gene expression. Evidence of this has been found in studies that show that methylation near gene promoters varies considerably depending on cell type, with more methylation of promoters correlating with low or no transcription (Suzuki & Bird, 2008). Also, while overall methylation levels and completeness of methylation of particular promoters are similar in individual humans, there are significant differences in overall and specific methylation levels between different tissue types and between normal cells and cancer cells from the same tissue. Researchers have also determined that mice that lack a particular DNMT have reduced methylation levels and die early in development (Suzuki & Bird, 2008). This is not the case for all eukaryotes, however; some organisms, such as the yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans, are thought to have no methylated DNA at all (although, at least in yeast, there are sequences in their genomes that are homologous to those that code for the DNMT enzymes). Early Clues to the Role of DNA Methylation in Gene Expression: Early clues to the role of methylation in gene expression were provided by 5-azacytidine experiments. 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 (Jones & Taylor, 1980). How and Where Are Genes Methylated? 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. Epigenetic gene silencing results from the inhibition of transcription or from posttranscriptional RNA degradation. DNA methylation is one of the most central and frequently discussed elements of gene silencing in both plants and mammals. Because DNA methylation has not been detected in yeast, Drosophila or Caenorhabditis elegies, the standard genetic workhorses, plants are important models for revealing the role of DNA methylation in the epigenetic regulation of genes in vivo. The outcome of transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS) is a reduction in the accumulation of gene transcripts. This epigenetic silencing may persist over many cell divisions or plant generations.TGS is defined as an inhibition of transcription, whereas PTGS involves the posttranscriptional degradation of RNA species but does not effect transcription rate .Recent studies indicate that TGS and PTGS are mechanistically and probably functionally related because they are correlated with some of the same events, including changes in DNA methylation. TGS is associated with the hypermethylation of promoter sequences, and PTGS is hemimethylated templates, that result from DNA replication does not always apply. There are some selected interactions in gene silencing that involve DNA methylation. Inverted repeats could trigger methylation either by production of transcripts that can form dsRNA or by DNA–DNA interactions. Methylation can accumulate in the promoter or coding regions of a gene. Hypermethylation of a promoter correlates with transcriptional gene silencing (TGS). Increased levels of methylation in a coding region (the open reading frame [ORF]) may not have immediate consequences. Maintenance of TGS requires DNA DEMETHYLATION1 (DDM1), METHYLTRANSFERASE1 (MET1) and Morpheus molecule (MOM1) functions although mutations in each of these three genes have different effects on TGS and DNA methylation Methylation of DNA and its causes: Abnormal methylation of DNA is associated with the development and progression of certain types of cancer.Methylation occurs when DNA methyltransferase links a methyl group to a cytosine base. Methylation of promoter regions of genes results in transcriptional silencing; thus, methylation of tumor suppressor genes may result in unregulated cell division, abnormal cell growth, and the development of cancer. DNA Methylation and Gene Silencing CpG islands: The four nucleotide bases of DNA—cytosine (C), adenine (A), guanine (G), and thymine (T)—form a total of 16 possible dinucleotide pairs. One of these dinucleotides, in which a cytosine is adjacent to a guanosine in the 5′ direction (the CpG dinucleotide), occurs at a lower than expected frequency throughout most of the human genome but at a higher than expected frequency in small portions of DNA that are referred to as CpG islands. A characteristic of mammalian genomes is the occurrence of so-called “CpG islands”. The dinucleotide CpG is unusual because it is underrepresented within the genome, occurring at a frequency of less than 1%. Given that the average GC content of the human genome is around 40%, you would expect that the dinucleotide CpG would occur at a frequency of approximately 4%, a figure derived by multiplying the fraction of Cs and Gs within the genome (0.2 × 0.2). The explanation for this discrepancy is believed to be due to the fact that most CpG dinucleotides are methylated on the cytosine base, i.e. they contain 5-methylcytosine. Spontaneous deamination of 5-methylcytosine or cytosine generatesn thymine or uracil respectively. Any uracil residues generated by deamination will be repaired back to cytosine by the cell, since uracil should not be present in DNA. However, the product of 5-methylcytosine deamination, thymine, is a normal component of DNA and is therefore not removed. This means that over time methylCpG dinucleotides will slowly mutate to TpG, and so will be lost from the genome. The genome does contain “CpG islands” where the CpG dinucleotide content is approximately 4%, a frequency that matches that predicted from the GC content. These CpG islands contain CpG dinucleotides that normally contain unmethylated cytosines. These CpG islands are important because they occur at the 5? end of genes and have played an important role in identifying genes and predicting the number of genes within the genome. How DNA methylation causes cancer: These CpG islands are often concentrated near gene transcription start sites, the promoter regions where the transcription of DNA to RNA begins. In the normal cell, most of the CpG dinucleotides at gene promoter regions are unmethylated, whereas CpG islands found at other portions of the genome are generally methylated. The absence of CpG island methylation is a hallmark of an active transcription site that is capable of transcribing DNA to RNA. In cancer cells, this pattern of CpG methylation becomes disrupted. CpG islands in promoter regions of selected genes have an unusually. Where one allele of the gene is methylated, the second allele is deleted. Although the loss of both alleles as the result of mutation is uncommon, both alleles of genes are more often inactivated in association with DNA methylation. The Knudson model also describes the inactivation of a tumor-suppressing gene in inherited cancer when one tumor suppressor gene allele contains a germline mutation, and the loss of the second allele occurs in tumors through a chromosomal deletion. Again, methylation of the CpG-rich promoter region may also provide the second hit in inherited cancers. In this case, DNA methylation is observed on the retained, nonmutated allele only; the mutated allele is not abnormally methylated. Recent research has identified a growing list of cancer-related genes that develop significant DNA methylation at promoter CpG islands adjacent to transcription start sites. A review of tumor-related genes across the entire genome identified genes that are deactivated by methylation, by mutation, or by both mechanisms. Two major(common) sources of gene silencing: 1. DNA methylation 2. Mutation An analysis found that methylation is at least as common as mutation as a mechanism of gene silencing in currently identified tumor-related genes. It may even be the case that gene silencing by DNA methylation is a much more significant source of gene suppression than mutation. Many of the key gene silencing events occurs very early during the premalignant stages of tumor progression, and the process of epigenetic gene silencing continues through the entire progression of human cancer. The genes affected include those that regulate many developmental pathways, controlling processes such as cell migration, cell-substratum recognition, cell differentiation, and the balance between cell growth and apoptosis. How DNA methylation evolved: DNA methylation is thought to have evolved in bacteria as a defense against foreign DNA. For instance, the prokaryotic methylation–restriction systems consist of specific methylases that act at short palindromic sequences, and restriction enzymes that cleave these sequences if, and only if, they are unmethylated (as they are likely to be in the context of invading bacteriophage DNA). In eukaryotes, cytosine methylation has evolved into a mechanism that allows dividing cells to stably inherit states of gene activity. Occurrence of DNA methylation: DNA methylation is involved in a myriad of epigenetic regulatory processes found in the vast majority of eukaryotes, including plants, fungi and animals. DNA methylation is absent — probably lost — in several fungal and animal lineages that include the much-used model organisms Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans.
Key Concepts:
Demethylation at the 5′ end of the gene is necessary for transcription.
CpG islands surround the promoters of constitutively expressed genes where they are unmethylated.
They (CpG islands) are also found at the promoters of some tissue-regulated genes.
There are —29,000 CpG islands in the human genome.
Methylation of a CpG island prevents activation of a promoter within it. Repression is caused by proteins that bind to methylated CpG doublets.
Methylation of both DNA and histones is a feature of inactive chromatin.
The DNA and histones methylation events may be connected.
Acetylation of histones is associated with gene activation.
Methylation of DNA and of histones is associated with heterochromatin.
Active chromatin is acetylated on the tails of histones H3 and H4.
Inactive chromatin is methylated on 9Lys of histone H3.
Inactive chromatin is methylated on cytosines of CpG doublets.
Most methyl groups in DNA are found on cytosine on both strands of the CpG doublet.
Replication converts a fully methylated site to a hemi-methylated site.
Hemi-methylated sites are converted to fully methylated sites by a maintenance methylase.
DNA methylation is responsible for imprinting.
Paternal and maternal alleles may have different patterns of methylation at fertilization.
Methylation is usually associated with inactivation of the gene.
When genes are differentially imprinted, survival of the embryo may require that the functional allele is provided by the parent with the unmethylated allele.
Survival of heterozygotes for imprinted genes is different depending on the direction of the cross.
Imprinted genes occur in clusters and may depend on a local control site where de novo methylation occurs unless specifically prevented.
Imprinted genes are controlled by methylation of c/s-acting sites.
Methylation may be responsible for either inactivating or activating a gene.
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