DNA methylation
Encyclopedia
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group
to the 5 position of the cytosine
pyrimidine ring or the number 6 nitrogen of the adenine
purine ring (cytosine and adenine are two of the four bases of DNA
). This modification can be inherited through cell division
.
DNA methylation is a crucial part of normal organismal development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can "remember where they have been" or decrease gene expression; for example, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets. DNA methylation is typically removed during zygote
formation and re-established through successive cell divisions during development. However, the latest research shows that hydroxylation of methyl group occurs rather than complete removal of methyl groups in zygote. Some methylation modifications that regulate gene expression are inheritable and are referred to as epigenetic regulation.
In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements that have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin
structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer.
DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression and has been found in every vertebrate
examined. In adult somatic
tissues, DNA methylation typically occurs in a CpG
dinucleotide context; non-CpG methylation is prevalent in embryonic stem cell
s.
.
Between 60% and 90% of all CpGs are methylated in mammals. Methylated C residues spontaneously deaminate to form T residues over evolutionary time; hence CpG dinucleotides steadily mutate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency). (On the other hand, spontaneous deamination of unmethylated C residues gives rise to U residues, a mutation that is quickly recognized and repaired by the cell.)
Unmethylated CpGs are often grouped in clusters called CpG islands, which are present in the 5' regulatory regions of many genes
. In many disease processes, such as cancer
, gene promoter CpG island
s acquire abnormal hypermethylation, which results in transcriptional silencing that can be inherited by daughter cells following cell division. Alterations of DNA methylation have been recognized as an important component of cancer development. Hypomethylation, in general, arises earlier and is linked to chromosomal instability and loss of imprinting, whereas hypermethylation is associated with promoters and can arise secondary to gene (oncogene suppressor) silencing, but might be a target for epigenetic therapy.
DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins
to the gene, and second, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylase
s and other chromatin remodeling
proteins that can modify histone
s, thereby forming compact, inactive chromatin, termed heterochromatin
. This link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has been implicated in Rett syndrome
; and methyl-CpG-binding domain protein 2 (MBD2)
mediates the transcriptional silencing of hypermethylated genes in cancer.
Research has suggested that long-term memory storage in humans may be regulated by DNA methylation.
and a large body of evidence has demonstrated that aberrant DNA methylation is associated with unscheduled gene silencing, and the genes with high levels of 5-methylcytosine
in their promoter region are transcriptionally silent. DNA methylation is essential during embryonic development, and in somatic cells, patterns of DNA methylation are generally transmitted to daughter cells with a high fidelity. Aberrant DNA methylation patterns have been associated with a large number of human malignancies and found in two distinct forms: hypermethylation and hypomethylation compared to normal tissue. Hypermethylation is one of the major epigenetic modifications that repress transcription via promoter region of tumour suppressor genes. Hypermethylation typically occurs at CpG islands in the promoter region and is associated with gene inactivation. Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms.
Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the DNA methyltransferase
(DNMT), the replication machinery itself would produce daughter strands that are unmethylated and, over time, would lead to passive demethylation. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.
It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 (TRDMT1)
has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.
Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis
, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-Aza-2'-deoxycytidine (decitabine) is a nucleoside analog
that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome
of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation
. However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.
. DNA methylation in plants differs from that of mammals: while DNA methylation in mammals mainly occurs on the cytosine nucleotide in a CpG site
, in plants the cytosine can be methylated at CpG, CpHpG, and CpHpH sites, where H represents any nucleotide but guanine.
The principal Arabidopsis DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 protein is unique to the plant kingdom. There are currently two classes of DNA methyltransferases: 1) the de novo class, or enzymes that create new methylation marks on the DNA; and 2) a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughters strands after DNA replication. DRM2 is the only enzyme that has been implicated as a de novo DNA methyltransferase. DRM2 has also been shown, along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication. Other DNA methyltransferases are expressed in plants but have no known function (see the Chromatin Database).
It is not clear how the cell determines the locations of de novo DNA methylation, but evidence suggests that, for many (though not all) locations, RNA-directed DNA methylation
(RdDM) is involved. In RdDM, specific RNA transcripts are produced from a genomic DNA template, and this RNA forms secondary structures called double-stranded RNA molecules. The double-stranded RNAs, through either the small interfering RNA (siRNA
) or microRNA (miRNA
) pathways direct de-novo DNA methylation of the original genomic location that produced the RNA. This sort of mechanism is thought to be important in cellular defense against RNA viruses and/or transposons, both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, through an as yet poorly-understood mechanism, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect.
This value seems to vary both among species and among isolates of the same species. There is also evidence that DNA methylation may be involved in state-specific control of gene expression
in fungi.
Although brewers' yeast (Saccharomyces
) and fission yeast (Schizosaccharomyces
) have very little DNA methylation, the model filamentous fungus Neurospora crassa
has a well-characterized methylation system. Several genes control methylation in Neurospora and mutation of the DNA methyl transferase, dim-2, eliminates all DNA methylation but does not affect growth or sexual reproduction.
While the Neurospora genome has very little repeated DNA, half of the methylation occurs in repeated DNA including transposon
relics and centromeric DNA. The ability to evaluate other important phenomena in a DNA methylase-deficient genetic background makes Neurospora an important system in which to study DNA methylation.
or cytosine
methylation is part of the restriction modification system
of many bacteria, in which specific DNA sequences are methylated periodically throughout the genome. A methylase
is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzyme
s and cleaved. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage
.
E. coli DNA adenine methyltransferase (Dam) is an enzyme of ~32 kDa that does not belong to a restriction/modification system. The target recognition sequence for E. coli Dam is GATC, as the methylation occurs at the N6 position of the adenine in this sequence (G meATC). The three base pairs flanking each side of this site also influence DNA–Dam binding. Dam plays several key roles in bacterial processes, including mismatch repair, the timing of DNA replication, and gene expression. As a result of DNA replication, the status of GATC sites in the E. coli genome changes from fully methylated to hemimethylated. This is because adenine introduced into the new DNA strand is unmethylated. Re-methylation occurs within two to four seconds, during which time replication errors in the new strand are repaired. Methylation, or its absence, is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands. It has been shown that altering Dam activity in bacteria results in increased spontaneous mutation rate. Bacterial viability is compromised in dam mutants that also lack certain other DNA repair enzymes, providing further evidence for the role of Dam in DNA repair.
One region of the DNA that keeps its hemimethylated status for longer is the origin of replication
, which has an abundance of GATC sites. This is central to the bacterial mechanism for timing DNA replication. SeqA binds to the origin of replication, sequestering it and thus preventing methylation. Because hemimethylated origins of replication are inactive, this mechanism limits DNA replication to once per cell cycle.
Expression of certain genes, for example those coding for pilus
expression in E. coli, is regulated by the methylation of GATC sites in the promoter region of the gene operon. The cells' environmental conditions just after DNA replication determine whether Dam is blocked from methylating a region proximal to or distal from the promoter region. Once the pattern of methylation has been created, the pilus gene transcription is locked in the on or off position until the DNA is again replicated. In E. coli, these pilus operon
s have important roles in virulence in urinary tract infections. It has been proposed that inhibitors of Dam may function as antibiotics.
On the other hand, DNA cytosine methylase targets CCAGG and CCTGG sites to methylate cytosine at the C5 position (C meC(A/T)GG). The other methylase enzyme, EcoKI, causes mehtylation of adenine in the sequences AAC(N6A)GTGC and GCAC(N6A)GTT.
Most strains used by molecular biologists are derivatives of K-12, and possess both Dam and Dcm, but there are commercially available strains that possess dam-/dcm- activity. In fact, it is possible to unmethylate the DNA extracted from dam+/dcm+ strains by transforming into dam-/dcm- strains. This would help digest sequences that are not being recognized by methylation-sensitive restriction enzymes.
Methyl group
Methyl group is a functional group derived from methane, containing one carbon atom bonded to three hydrogen atoms —CH3. The group is often abbreviated Me. Such hydrocarbon groups occur in many organic compounds. The methyl group can be found in three forms: anion, cation and radical. The anion...
to the 5 position of the cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
pyrimidine ring or the number 6 nitrogen of the adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
purine ring (cytosine and adenine are two of the four bases of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
). This modification can be inherited through cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
.
DNA methylation is a crucial part of normal organismal development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can "remember where they have been" or decrease gene expression; for example, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets. DNA methylation is typically removed during zygote
Zygote
A zygote , or zygocyte, is the initial cell formed when two gamete cells are joined by means of sexual reproduction. In multicellular organisms, it is the earliest developmental stage of the embryo...
formation and re-established through successive cell divisions during development. However, the latest research shows that hydroxylation of methyl group occurs rather than complete removal of methyl groups in zygote. Some methylation modifications that regulate gene expression are inheritable and are referred to as epigenetic regulation.
In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements that have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer.
DNA methylation at the 5 position of cytosine has the specific effect of reducing gene expression and has been found in every vertebrate
Vertebrate
Vertebrates are animals that are members of the subphylum Vertebrata . Vertebrates are the largest group of chordates, with currently about 58,000 species described. Vertebrates include the jawless fishes, bony fishes, sharks and rays, amphibians, reptiles, mammals, and birds...
examined. In adult somatic
Somatic
The term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
tissues, DNA methylation typically occurs in a CpG
CpG site
CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. "CpG" is shorthand for "—C—phosphate—G—", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides...
dinucleotide context; non-CpG methylation is prevalent in embryonic stem cell
Stem cell
This article is about the cell type. For the medical therapy, see Stem Cell TreatmentsStem cells are biological cells found in all multicellular organisms, that can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells...
s.
In mammals
DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, suppression of repetitive elements, and carcinogenesisCarcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
.
Between 60% and 90% of all CpGs are methylated in mammals. Methylated C residues spontaneously deaminate to form T residues over evolutionary time; hence CpG dinucleotides steadily mutate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency). (On the other hand, spontaneous deamination of unmethylated C residues gives rise to U residues, a mutation that is quickly recognized and repaired by the cell.)
Unmethylated CpGs are often grouped in clusters called CpG islands, which are present in the 5' regulatory regions of many genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
. In many disease processes, such as cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
, gene promoter CpG island
CpG island
In genetics, CpG islands or CG islands are genomic regions that contain a high frequency of CpG sites but to date objective definitions for CpG islands are limited. In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length. They are in and near approximately 40% of promoters of...
s acquire abnormal hypermethylation, which results in transcriptional silencing that can be inherited by daughter cells following cell division. Alterations of DNA methylation have been recognized as an important component of cancer development. Hypomethylation, in general, arises earlier and is linked to chromosomal instability and loss of imprinting, whereas hypermethylation is associated with promoters and can arise secondary to gene (oncogene suppressor) silencing, but might be a target for epigenetic therapy.
DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
to the gene, and second, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylase
Histone deacetylase
Histone deacetylases are a class of enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on a histone. This is important because DNA is wrapped around histones, and DNA expression is regulated by acetylation and de-acetylation. Its action is opposite to that of histone...
s and other chromatin remodeling
Chromatin remodeling
Chromatin remodeling is the enzyme-assisted movement of nucleosomes on DNA.This is performed by chromatin remodeling complexes like SWI/SNF , RSC and Imitation SWI complexes ....
proteins that can modify histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
s, thereby forming compact, inactive chromatin, termed heterochromatin
Heterochromatin
Heterochromatin is a tightly packed form of DNA, which comes in different varieties. These varieties lie on a continuum between the two extremes of constitutive and facultative heterochromatin...
. This link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has been implicated in Rett syndrome
Rett syndrome
Rett syndrome is a neurodevelopmental disorder of the grey matter of the brain that almost exclusively affects females. The clinical features include small hands and feet and a deceleration of the rate of head growth . Repetitive hand movements, such as wringing and/or repeatedly putting hands into...
; and methyl-CpG-binding domain protein 2 (MBD2)
Methyl-CpG-binding domain protein 2
Methyl-CpG-binding domain protein 2 is a protein that in humans is encoded by the MBD2 gene.-Interactions:Methyl-CpG-binding domain protein 2 has been shown to interact with HDAC1, Histone deacetylase 2, SIN3A, MIZF, MBD3 and GATAD2B....
mediates the transcriptional silencing of hypermethylated genes in cancer.
Research has suggested that long-term memory storage in humans may be regulated by DNA methylation.
In cancer
DNA methylation is an important regulator of gene transcriptionTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
and a large body of evidence has demonstrated that aberrant DNA methylation is associated with unscheduled gene silencing, and the genes with high levels of 5-methylcytosine
5-Methylcytosine
5-Methylcytosine is a methylated form of the DNA base cytosine that may be involved in the regulation of gene transcription. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered .In the figure on the right, a methyl group, is...
in their promoter region are transcriptionally silent. DNA methylation is essential during embryonic development, and in somatic cells, patterns of DNA methylation are generally transmitted to daughter cells with a high fidelity. Aberrant DNA methylation patterns have been associated with a large number of human malignancies and found in two distinct forms: hypermethylation and hypomethylation compared to normal tissue. Hypermethylation is one of the major epigenetic modifications that repress transcription via promoter region of tumour suppressor genes. Hypermethylation typically occurs at CpG islands in the promoter region and is associated with gene inactivation. Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms.
DNA methyltransferases
In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities – maintenance methylation and de novo methylation.Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the DNA methyltransferase
DNA methyltransferase
In biochemistry, the DNA methyltransferase family of enzymescatalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions...
(DNMT), the replication machinery itself would produce daughter strands that are unmethylated and, over time, would lead to passive demethylation. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.
It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 (TRDMT1)
TRDMT1
tRNA -methyltransferase is an enzyme that in humans is encoded by the TRDMT1 gene.It has been shown that human DNMT2 does not methylate DNA but instead methylates cytosine 38 in the anticodon loop of aspartic acid transfer RNA .-Further reading:...
has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.
Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis
Carcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-Aza-2'-deoxycytidine (decitabine) is a nucleoside analog
Base analog
A base analog is a chemical that can substitute for a normal nucleobase in nucleic acids.A common example would be 5-bromouracil , the abnormal base found in the mutagenic nucleotide analog BrdU...
that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation
Translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of...
. However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.
In plants
Significant progress has been made in understanding DNA methylation in the model plant Arabidopsis thalianaArabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
. DNA methylation in plants differs from that of mammals: while DNA methylation in mammals mainly occurs on the cytosine nucleotide in a CpG site
CpG site
CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. "CpG" is shorthand for "—C—phosphate—G—", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides...
, in plants the cytosine can be methylated at CpG, CpHpG, and CpHpH sites, where H represents any nucleotide but guanine.
The principal Arabidopsis DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 protein is unique to the plant kingdom. There are currently two classes of DNA methyltransferases: 1) the de novo class, or enzymes that create new methylation marks on the DNA; and 2) a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughters strands after DNA replication. DRM2 is the only enzyme that has been implicated as a de novo DNA methyltransferase. DRM2 has also been shown, along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication. Other DNA methyltransferases are expressed in plants but have no known function (see the Chromatin Database).
It is not clear how the cell determines the locations of de novo DNA methylation, but evidence suggests that, for many (though not all) locations, RNA-directed DNA methylation
RNA-Directed DNA Methylation
RNA-directed DNA methylation is an epigenetic process first elucidated in plants whereby small double-stranded RNAs are processed to guide methylation to complementary DNA loci...
(RdDM) is involved. In RdDM, specific RNA transcripts are produced from a genomic DNA template, and this RNA forms secondary structures called double-stranded RNA molecules. The double-stranded RNAs, through either the small interfering RNA (siRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
) or microRNA (miRNA
Mirna
Mirna may refer to:geographical entities* Mirna , a river in Istria, Croatia* Mirna , a river in Slovenia, tributary of the river Sava* Mirna , a settlement in the municipality of Mirna in Southeastern Sloveniapeople...
) pathways direct de-novo DNA methylation of the original genomic location that produced the RNA. This sort of mechanism is thought to be important in cellular defense against RNA viruses and/or transposons, both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, through an as yet poorly-understood mechanism, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect.
In fungi
It can be seen that many fungi have low levels (0.1 to 0.5%) of cytosine methylation, whereas other fungi have as much as 5% of the genome methylated.This value seems to vary both among species and among isolates of the same species. There is also evidence that DNA methylation may be involved in state-specific control of gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
in fungi.
Although brewers' yeast (Saccharomyces
Saccharomyces
Saccharomyces is a genus in the kingdom of fungi that includes many species of yeast. Saccharomyces is from Greek σάκχαρ and μύκης and means sugar fungus. Many members of this genus are considered very important in food production. One example is Saccharomyces cerevisiae, which is used in making...
) and fission yeast (Schizosaccharomyces
Schizosaccharomyces
Schizosaccharomyces is a genus of fission yeasts. The most well-studied species is S. pombe. At present four Schizosaccharomyces species have been described . Like the distantly related Saccharomyces cerevisiae, S. pombe is a significant model organism in the study of eukaryotic cell biology...
) have very little DNA methylation, the model filamentous fungus Neurospora crassa
Neurospora crassa
Neurospora crassa is a type of red bread mold of the phylum Ascomycota. The genus name, meaning "nerve spore" refers to the characteristic striations on the spores. The first published account of this fungus was from an infestation of French bakeries in 1843. N...
has a well-characterized methylation system. Several genes control methylation in Neurospora and mutation of the DNA methyl transferase, dim-2, eliminates all DNA methylation but does not affect growth or sexual reproduction.
While the Neurospora genome has very little repeated DNA, half of the methylation occurs in repeated DNA including transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
relics and centromeric DNA. The ability to evaluate other important phenomena in a DNA methylase-deficient genetic background makes Neurospora an important system in which to study DNA methylation.
In bacteria
AdenineAdenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
or cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
methylation is part of the restriction modification system
Restriction modification system
The restriction modification system is used by bacteria, and perhaps other prokaryotic organisms to protect themselves from foreign DNA, such as the one borne by bacteriophages. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit the growth of viruses...
of many bacteria, in which specific DNA sequences are methylated periodically throughout the genome. A methylase
Methylase
A methylase is an enzyme that attaches a methyl group to a molecule.These are found in prokaryotes and eukaryotes. Bacteria use methylase to differentiate between foreign genetic material and their own, thus protecting their DNA from their own immune system...
is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s and cleaved. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
.
E. coli DNA adenine methyltransferase (Dam) is an enzyme of ~32 kDa that does not belong to a restriction/modification system. The target recognition sequence for E. coli Dam is GATC, as the methylation occurs at the N6 position of the adenine in this sequence (G meATC). The three base pairs flanking each side of this site also influence DNA–Dam binding. Dam plays several key roles in bacterial processes, including mismatch repair, the timing of DNA replication, and gene expression. As a result of DNA replication, the status of GATC sites in the E. coli genome changes from fully methylated to hemimethylated. This is because adenine introduced into the new DNA strand is unmethylated. Re-methylation occurs within two to four seconds, during which time replication errors in the new strand are repaired. Methylation, or its absence, is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands. It has been shown that altering Dam activity in bacteria results in increased spontaneous mutation rate. Bacterial viability is compromised in dam mutants that also lack certain other DNA repair enzymes, providing further evidence for the role of Dam in DNA repair.
One region of the DNA that keeps its hemimethylated status for longer is the origin of replication
Origin of replication
The origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
, which has an abundance of GATC sites. This is central to the bacterial mechanism for timing DNA replication. SeqA binds to the origin of replication, sequestering it and thus preventing methylation. Because hemimethylated origins of replication are inactive, this mechanism limits DNA replication to once per cell cycle.
Expression of certain genes, for example those coding for pilus
Pilus
right|thumb|350px|Schematic drawing of bacterial conjugation. 1- Donor cell produces pilus. 2- Pilus attaches to recipient cell, brings the two cells together. 3- The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell...
expression in E. coli, is regulated by the methylation of GATC sites in the promoter region of the gene operon. The cells' environmental conditions just after DNA replication determine whether Dam is blocked from methylating a region proximal to or distal from the promoter region. Once the pattern of methylation has been created, the pilus gene transcription is locked in the on or off position until the DNA is again replicated. In E. coli, these pilus operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...
s have important roles in virulence in urinary tract infections. It has been proposed that inhibitors of Dam may function as antibiotics.
On the other hand, DNA cytosine methylase targets CCAGG and CCTGG sites to methylate cytosine at the C5 position (C meC(A/T)GG). The other methylase enzyme, EcoKI, causes mehtylation of adenine in the sequences AAC(N6A)GTGC and GCAC(N6A)GTT.
Most strains used by molecular biologists are derivatives of K-12, and possess both Dam and Dcm, but there are commercially available strains that possess dam-/dcm- activity. In fact, it is possible to unmethylate the DNA extracted from dam+/dcm+ strains by transforming into dam-/dcm- strains. This would help digest sequences that are not being recognized by methylation-sensitive restriction enzymes.
Detection
DNA methylation can be detected by the following assays currently used in scientific research:- Methylation-Specific PCR (MSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR. However, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated.
- Whole genome bisulfite sequencing, also known as BS-Seq, which is a high-throughput genome-wide analysis of DNA methylation. It is based on aforementioned sodium bisulfite conversion of genomic DNA, which is then sequencing on a Next-generation sequencing platform. The sequences obtained are then re-aligned to the reference genome to determine methylation states of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil.
- The HELP assayHELP assayThe HELP Assay is an abbreviation for HpaII tiny fragment Enrichment by Ligation-mediated PCR. It is one of several techniques used for determining whether DNA has been methylated...
, which is based on restriction enzymes' differential ability to recognize and cleave methylated and unmethylated CpG DNA sites. - ChIP-on-chipChIP-on-chipChIP-on-chip is a technique that combines chromatin immunoprecipitation with microarray technology . Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo...
assays, which is based on the ability of commercially prepared antibodies to bind to DNA methylation-associated proteins like MeCP2. - Restriction landmark genomic scanningRestriction landmark genomic scanningRestriction Landmark Genomic Scanning is a genome analysis method that allows for rapid simultaneous visualization of thousands of landmarks, or restriction sites. Using a combination of restriction enzymes some of which are specific to DNA modifications, the technique can be used to visualize...
, a complicated and now rarely-used assay based upon restriction enzymes' differential recognition of methylated and unmethylated CpG sites; the assay is similar in concept to the HELP assay. - Methylated DNA immunoprecipitationMethylated DNA immunoprecipitationMethylated DNA immunoprecipitation is a large-scale technique that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine . This technique was first described by Weber M...
(MeDIP), analogous to chromatin immunoprecipitationChromatin immunoprecipitationChromatin Immunoprecipitation is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or...
, immunoprecipitationImmunoprecipitationImmunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins...
is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP-chip) or DNA sequencingDNA sequencingDNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
(MeDIP-seq). - PyrosequencingPyrosequencingPyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...
of bisulfite treated DNA. This is sequencing of an amplicon made by a normal forward primer but a biatenylated reverse primer to PCR the gene of choice. The Pyrosequencer then analyses the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user. If there is a mis-match, it is recorded and the percentage of DNA for which the mis-match is present is noted. This gives the user a percentage methylation per CpG island. - Molecular break light assay for DNA adenine methyltransferase activity – an assay that relies on the specificity of the restriction enzyme DpnI for fully methylated (adenine methylation) GATC sites in an oligonucleotide labeled with a fluorophore and quencher. The adenine methyltransferase methylates the oligonucleotide making it a substrate for DpnI. Cutting of the oligonucleotide by DpnI gives rise to a fluorescence increase.
- Methyl Sensitive Southern Blotting is similar to the HELP assay, although uses Southern blotting techniques to probe gene-specific differences in methylation using restriction digests. This technique is used to evaluate local methylation near the binding site for the probe.
See also
- Demethylating agentDemethylating agentDemethylating agents are compounds that can inhibit methylation, resulting in the expression of the previously hypermethylated silenced genes . Cytidine analogs such as 5-azacytidine and 5-azadeoxycytidine are the most commonly used demethylating agents...
- MethDBMethDBMethDB is a database for DNA methylation data....
DNA Methylation database - ReprogrammingReprogrammingReprogramming refers to erasure and remodeling of epigenetic marks, such as DNA methylation, during mammalian development. After fertilization some cells of the newly formed embryo migrate to the germinal ridge and will eventually become the germ cells...
- EpigeneticsEpigeneticsIn biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- -genetics...
, of which DNA methylation is a significant contributor - Genomic imprinting, an inherited repression of an allele, relying on DNA methylation
- 5-Hydroxymethylcytosine5-Hydroxymethylcytosine5-Hydroxymethylcytosine is a DNA pyrimidine nitrogen base. It is formed from the DNA base cytosine by adding a methyl group and then a hydroxy group. It is important in epigenetics, because the hydroxymethyl group on the cytosine can possibly switch a gene on and off. It was first seen in...