DNA METHYLATION is a process by which methyl groups are added to the
DNA molecule. Methylation can change the activity of a
without changing the sequence. When located in a gene promoter , DNA
methylation typically acts to repress gene transcription . DNA
methylation is essential for normal development and is associated with
a number of key processes including genomic imprinting , X-chromosome
inactivation , repression of transposable elements , aging and
Two of DNA's four bases, cytosine and adenine , can be methylated.
Cytosine methylation is widespread in both eukaryotes and prokaryotes,
even though the rate of cytosine
DNA methylation can differ greatly
between species: 14% of cytosines are methylated in Arabidopsis
thaliana , 4% in
Mus musculus , 2.3% in
Escherichia coli , 0.03% in
Drosophila , and virtually none (< 0.0002%) in yeast species such as
S. cerevisiae and S. pombe (but not N. crassa).
has been observed in bacterial, plant and recently in mammalian DNA,
but have received considerably less attention.
Methylation of cytosine to form
5-methylcytosine occurs at the same 5
position on the pyrimidine ring where the
DNA base thymine 's methyl
group is located, distinguishing thymine from the analogous RNA base
uracil which has no methyl group. The near-universal replacement of
uracil by thymine in DNA, but not RNA, may have evolved as an
error-control mechanism, to facilitate removal of uracils generated by
the spontaneous deamination of cytosine.
DNA methylation as well as
many of its contemporary
DNA methyltransferases has been thought to
evolve from early world primitive RNA methylation activity and is
supported by several lines of evidences.
In plants and other organisms,
DNA methylation is found in three
different sequence contexts: CG (or CpG ), CHG or CHH (where H
correspond to A,T or C). In mammals however,
DNA methylation is almost
exclusively found in CpG dinucleotides, with the cytosines on both
strand being usually methylated. Non-CpG methylation can however be
observed in embryonic stem cells , and has also been indicated in
neural development . Furthermore, non-CpG methylation has also been
observed in hematopoietic progenitor cells, and it occurred mainly in
a CpApC sequence context.
* 1 Conserved function of
* 1.1 CpG islands
* 1.2 Repression of CpG-dense promoters
* 1.3 Repression of transposable elements
* 1.4 Methylation of the gene body of highly transcribed genes
* 2 In mammals
* 2.1 During embryonic development
* 2.2 In cancer
* 2.3 In atherosclerosis
* 2.4 In aging
* 2.5 In exercise
* 2.6 In B-cell differentiation
* 2.7 In the brain
DNA methyltransferases (in mammals)
* 4 In plants
* 5 In fungi
* 6 In insects
* 7 In bacteria
* 7.1 Molecular cloning
* 8 Detection
Differentially methylated regions (DMRs)
DNA methylation marks
* 11 Computational prediction
* 12 See also
* 13 References
* 14 Further reading
* 15 External links
CONSERVED FUNCTION OF
DNA methylation landscape in mammals
DNA methylation landscape of vertebrates is very particular
compared to other organisms. In vertebrates, around 60-80% of CpG are
methylated in somatic cells and
DNA methylation appears as a default
state that has to be specifically excluded from defined locations.
By contrast, the genome of most plants, invertebrates, fungi or
protists show “mosaic” methylation patterns, where only specific
genomic elements are targeted, and they are characterized by the
alternation of methylated and unmethylated domains.
High CpG methylation in mammalian genomes has an evolutionary cost
because it increases the frequency of spontaneous mutations. Loss of
amino-groups occurs with a high frequency for cytosines, with
different consequences depending on their methylation. Methylated C
residues spontaneously deaminate to form T residues over time; hence
CpG dinucleotides steadily deaminate 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 change that is quickly recognized and
repaired by the cell.)
In mammals, the only exception for this global CpG depletion resides
in a specific category of GC- and CpG-rich sequences termed CpG
islands that are generally unmethylated and therefore retained the
expected CpG content. CpG islands are usually defined as regions with
1) a length greater than 200bp, 2) a G+C content greater than 50%, 3)
a ratio of observed to expected CpG greater than 0.6, although other
definitions are sometimes used. Excluding repeated sequences, there
are around 25,000 CpG islands in the human genome, 75% of which being
less than 850pb long. They are major regulatory units and around 50%
of CpG islands are located in gene promoter regions, while another 25%
lie in gene bodies, often serving as alternative promoters.
Reciprocally, around 60-70% of human genes have a
CpG island in their
promoter region. The majority of CpG islands are constitutively
unmethylated and enriched for permissive chromatin modification such
as H3K4 methylation. In somatic tissues, only 10% of CpG islands are
methylated, the majority of them being located in intergenic and
REPRESSION OF CPG-DENSE PROMOTERS
DNA methylation was probably present at some extent in very early
eukaryote ancestors. In virtually every organism analyzed, methylation
in promoter regions correlates negatively with gene expression.
CpG-dense promoters of actively transcribed genes are never
methylated, but reciprocally transcriptionally silent genes do not
necessarily carry a methylated promoter. In mouse and human, around
60-70% of genes have a
CpG island in their promoter region and most of
these CpG islands remain unmethylated independently of the
transcriptional activity of the gene, in both differentiated and
undifferentiated cell types. Of note, whereas
DNA methylation of CpG
islands is unambiguously linked with transcriptional repression, the
DNA methylation in CG-poor promoters remains unclear;
albeit there is little evidence that it could be functionally
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
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 deacetylases and
other chromatin remodeling proteins that can modify histones , thereby
forming compact, inactive chromatin, termed heterochromatin . This
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.
REPRESSION OF TRANSPOSABLE ELEMENTS
DNA methylation is a powerful transcriptional repressor, at least in
CpG dense contexts. Transcriptional repression of protein-coding genes
appears essentially limited to very specific classes of genes that
need to be silent permanently and in almost all tissues. While DNA
methylation does not have the flexibility required for the fine-tuning
of gene regulation, its stability is perfect to ensure the permanent
silencing of transposable elements .
Transposon control is one the
most ancient function of
DNA methylation that is shared by animals,
plants and multiple protists. It is even suggested that DNA
methylation evolved precisely for this purpose.
METHYLATION OF THE GENE BODY OF HIGHLY TRANSCRIBED GENES
A function that appears even more conserved than transposon silencing
is positively correlated with gene expression. In almost all species
DNA methylation is present,
DNA methylation is especially
enriched in the body of highly transcribed genes. The function of
gene body methylation is not well understood. A body of evidence
suggests that it could regulate splicing and suppress the activity of
intragenic transcriptional units (cryptic promoters or transposable
elements). Gene-body methylation appears closely tied to H3K36
methylation. In yeast and mammals, H3K36 methylation is highly
enriched in the body of highly transcribed genes. In yeast at least,
H3K36me3 recruits enzymes such as histone deacetylases to condense
chromatin and prevent the activation of cryptic start sites. In
mammals, DNMT3a and DNMT3b PWWP domain binds to H3K36me3 and the two
enzymes are recruited to the body of actively transcribed genes.
DNA methylation during mouse embryonic development.
E3.5-E6, etc, refer to days after fertilization. PGC: primordial germ
DURING EMBRYONIC DEVELOPMENT
DNA methylation reprogramming
DNA methylation patterns are largely erased and then re-established
between generations in mammals. Almost all of the methylations from
the parents are erased, first during gametogenesis , and again in
early embryogenesis , with demethylation and remethylation occurring
each time. Demethylation in early embryogenesis occurs in the
preimplantation period in two stages – initially in the zygote ,
then during the first few embryonic replication cycles of morula and
blastula . A wave of methylation then takes place during the
implantation stage of the embryo, with CpG islands protected from
methylation. This results in global repression and allows housekeeping
genes to be expressed in all cells. In the post-implantation stage,
methylation patterns are stage- and tissue-specific, with changes that
would define each individual cell type lasting stably over a long
DNA methylation is not necessary per se for transcriptional
silencing, it is thought nonetheless to represent a “locked” state
that definitely inactivates transcription. In particular, DNA
methylation appears critical for the maintenance of mono-allelic
silencing in the context of genomic imprinting and X chromosome
inactivation . In these cases, expressed and silent alleles differ
by their methylation status, and loss of
DNA methylation results in
loss of imprinting and re-expression of Xist in somatic cells. During
embryonic development, few genes change their methylation status, at
the important exception of many genes specifically expressed in the
DNA methylation appears absolutely required in
differentiated cells , as knockout of any of the three competent DNA
methyltransferase results in embryonic or post-partum lethality. By
DNA methylation is dispensable in undifferentiated cell
types, such as the inner cell mass of the blastocyst, primordial germ
cells or embryonic stem cells. Since
DNA methylation appears to
directly regulate only a limited number of genes, how precisely DNA
methylation absence causes the death of differentiated cells remain an
Due to the phenomenon of genomic imprinting , maternal and paternal
genomes are differentially marked and must be properly reprogrammed
every time they pass through the germline. Therefore, during
gametogenesis , primordial germ cells must have their original
DNA methylation patterns erased and re-established based on
the sex of the transmitting parent. After fertilization the paternal
and maternal genomes are once again demethylated and remethylated
(except for differentially methylated regions associated with
imprinted genes). This reprogramming is likely required for
totipotency of the newly formed embryo and erasure of acquired
DNA methylation in cancer and Regulation of
transcription in cancer
In many disease processes, such as cancer , gene promoter CpG islands
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 .
Global hypomethylation has also been implicated in the development
and progression of cancer through different mechanisms. Typically,
there is hypermethylation of tumor suppressor genes and
hypomethylation of oncogenes .
Generally, in progression to cancer, hundreds of genes are silenced
or activated . Although silencing of some genes in cancers occurs by
mutation, a large proportion of carcinogenic gene silencing is a
result of altered
DNA methylation (see
DNA methylation in cancer ).
DNA methylation causing silencing in cancer typically occurs at
multiple CpG sites in the CpG islands that are present in the
promoters of protein coding genes.
Altered expressions of microRNAs also silence or activate many genes
in progression to cancer (see microRNAs in cancer ). Altered microRNA
expression occurs through hyper/hypo-methylation of CpG sites in CpG
islands in promoters controlling transcription of the microRNAs .
DNA repair genes through methylation of CpG islands in
their promoters appears to be especially important in progression to
cancer (see methylation of
DNA repair genes in cancer ).
Epigenetic modifications such as
DNA methylation have been implicated
in cardiovascular disease, including atherosclerosis . In animal
models of atherosclerosis, vascular tissue as well as blood cells such
as mononuclear blood cells exhibit global hypomethylation with
gene-specific areas of hypermethylation.
DNA methylation polymorphisms
may be used as an early biomarker of atherosclerosis since they are
present before lesions are observed, which may provide an early tool
for detection and risk prevention.
Two of the cell types targeted for
DNA methylation polymorphisms are
monocytes and lymphocytes, which experience an overall
hypomethylation. One proposed mechanism behind this global
hypomethylation is elevated homocysteine levels causing
hyperhomocysteinemia , a known risk factor for cardiovascular disease.
High plasma levels of homocysteine inhibit
which causes hypomethylation. Hypomethylation of
DNA affects gene that
alter smooth muscle cell proliferation, cause endothelial cell
dysfunction, and increase inflammatory mediators, all of which are
critical in forming atherosclerotic lesions. High levels of
homocysteine also result in hypermethylation of CpG islands in the
promoter region of the estrogen receptor alpha (ERα) gene, causing
its down regulation. ERα protects against atherosclerosis due to its
action as a growth suppressor, causing the smooth muscle cells to
remain in a quiescent state. Hypermethylation of the ERα promoter
thus allows intimal smooth muscle cells to proliferate excessively and
contribute to the development of the atherosclerotic lesion.
Another gene that experiences a change in methylation status in
atherosclerosis is the monocarboxylate transporter (MCT3), which
produces a protein responsible for the transport of lactate and other
ketone bodies out of many cell types, including vascular smooth muscle
cells. In atherosclerosis patients, there is an increase in
methylation of the CpG islands in exon 2, which decreases MCT3 protein
expression. The down regulation of MCT3 impairs lactate transport, and
significantly increases smooth muscle cell proliferation, which
further contributes to the atherosclerotic lesion. An ex vivo
experiment using the demethylating agent
-deoxycytidine) was shown to induce MCT3 expression in a dose
dependant manner, as all hypermethylated sites in the exon 2 CpG
island became demethylated after treatment. This may serve as a novel
therapeutic agent to treat atherosclerosis, although no human studies
have been conducted thus far.
In humans and other mammals,
DNA methylation levels can be used to
accurately estimate the age of tissues and cell types, forming an
accurate epigenetic clock .
A longitudinal study of twin children showed that, between the ages
of 5 and 10, there was divergence of methylation patterns due to
environmental rather than genetic influences. There is a global loss
DNA methylation during aging.
In a study that analyzed the complete
DNA methylomes of CD4+ T cells
in a newborn, a 26 years old individual and a 103 years old individual
was observed that the loss of methylation is proportional to age.
Hypomethylated CpGs observed in the centenarian DNAs compared with the
neonates covered all genomic compartments (promoters, intergenic,
intronic and exonic regions). However, some genes become
hypermethylated with age, including genes for the estrogen receptor ,
p16 , and insulin-like growth factor 2 .
High intensity exercise has been shown to result in reduced DNA
methylation in skeletal muscle. Promoter methylation of PGC-1α and
PDK4 were immediately reduced after high intensity exercise, whereas
PPAR-γ methylation was not reduced until three hours after exercise.
By contrast, six months of exercise in previously sedentary middle-age
men resulted in increased methylation in adipose tissue . One study
showed a possible increase in global genomic
DNA methylation of white
blood cells with more physical activity in non-Hispanics.
IN B-CELL DIFFERENTIATION
A study that investigated the methylome of B cells along their
differentiation cycle, using whole-genome bisulfite sequencing (WGBS),
showed that there is a hypomethylation from the earliest stages to the
most differentiated stages. The largest methylation difference is
between the stages of germinal center B cells and memory B cells.
Furthermore, this study showed that there is a similarity between B
cell tumors and long-lived B cells in their
IN THE BRAIN
Research has suggested that long-term memory storage in humans may be
DNA METHYLTRANSFERASES (IN MAMMALS)
Possible pathways of cytosine methylation and demethylation.
Possible pathways of cytosine methylation and demethylation.
DNA methyltransferase (
DNA glycosylase (UNG)
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
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
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
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
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
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
Significant progress has been made in understanding
in the model plant
Arabidopsis thaliana .
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 not guanine. Overall, Arabidopsis
highly methylated, mass spectrometry analysis estimated 14% of
cytosines to be modified.
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
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)
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
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. Recently,
it was described that methylation of the
DNA is the main determinant
of embryogenic cultures formation from explants in woody plants and is
regarded the main mechanism that explains the poor response of mature
explants to somatic embryogenesis in the plants (Isah 2016).
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 in fungi. However, at a
detection limit of 250 attomoles by using ultra-high sensitive mass
DNA methylation was not confirmed in single cellular
yeast species such as
Saccharomyces cerevisiae or Schizosaccharomyces
pombe , indicating that yeasts do not possess this
Although brewers' yeast (
Saccharomyces ), fission yeast
Schizosaccharomyces ), and
Aspergillus flavus have no detectable
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
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
Epigenetics in insects
DNA methylation has been discovered in Honey Bees. DNA
methylation marks are mainly on the gene body, and current opinions on
the function of
DNA methylation is gene regulation via alternative
Drosophila melanogaster possess a very low level of
only that is however too low to be studied by methods such as
bisulphite sequencing. Takayama et al. developed a sensitive method
that allowed to find that the fly genome
DNA sequence patterns that
associate with methylation are very different from the patterns seen
in humans, or in other animal or plant species to date. Genome
methylation in D. melanogaster was found at specific short motifs
(concentrated in specific 5-base sequence motifs that are CA- and
CT-rich but depleted of guanine) and is independent of DNMT2 activity.
By using highly sensitive mass spectrometry approaches, Zhang et al.
have now demonstrated the presence of low (0.07%) but significant
levels of adenine methylation during the earliest stages of Drosophila
Adenine or cytosine methylation is part of the restriction
modification system of many bacteria , in which specific
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 enzymes and cleaved. Bacterial genomic
DNA is not recognized by these restriction enzymes. The methylation of
DNA acts as a sort of primitive immune system, allowing the
bacteria to protect themselves from infection by bacteriophage .
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
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
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
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
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 operons 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 methylation of adenines in the
sequences AAC(N6)GTGC and GCAC(N6)GTT.
Most strains used by molecular biologists are derivatives of E. coli
K-12, and possess both Dam and Dcm, but there are commercially
available strains that are dam-/dcm- (lack of activity of either
methylase). In fact, it is possible to unmethylate the
from dam+/dcm+ strains by transforming it into dam-/dcm- strains. This
would help digest sequences that are not being recognized by
methylation-sensitive restriction enzymes.
The restriction enzyme DpnI can recognize 5'-GmeATC-3' sites and
digest the methylated DNA. Being such a short motif, it occurs
frequently in sequences by chance, and as such its primary use for
researchers is to degrade template
DNA following PCRs (
lack methylation, as no methylases are present in the reaction).
Similarly, some commercially available restriction enzymes are
sensitive to methylation at their cognate restriction sites, and must
as mentioned previously be used on
DNA passed through a dam-/dcm-
strain to allow cutting.
DNA methylation can be detected by the following assays currently
used in scientific research:
Mass spectrometry is a very sensitive and reliable analytical
method to detect
DNA methylation. MS in general is however not
informative about the sequence context of the methylation, thus
limited in studying the function of this
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
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 sequenced 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.
HELP assay , which is based on restriction enzymes'
differential ability to recognize and cleave methylated and
PCR assay , which is based on new type of enzymes -
DNA endonucleases, which hydrolyze only
ChIP-on-chip assays, which is based on the ability of commercially
prepared antibodies to bind to
DNA methylation-associated proteins
Restriction landmark genomic scanning , 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.
DNA immunoprecipitation (MeDIP), analogous to chromatin
immunoprecipitation , immunoprecipitation is used to isolate
DNA fragments for input into
DNA detection methods such as
DNA microarrays (MeDIP-chip) or
DNA sequencing (MeDIP-seq).
Pyrosequencing of bisulfite treated DNA. This is sequencing of an
amplicon made by a normal forward primer but a biotinylated reverse
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
* MethylCpG Binding Proteins (MBPs) and fusion proteins containing
just the Methyl Binding Domain (MBD) are used to separate native DNA
into methylated and unmethylated fractions. The percentage methylation
of individual CpG islands can be determined by quantifying the amount
of the target in each fraction. Extremely sensitive detection can be
achieved in FFPE tissues with abscription-based detection.
High Resolution Melt Analysis (HRM or HRMA), is a post-PCR
analytical technique. The target
DNA is treated with sodium bisulfite,
which chemically converts unmethylated cytosines into uracils, while
methylated cytosines are preserved.
PCR amplification is then carried
out with primers designed to amplify both methylated and unmethylated
templates. After this amplification, highly methylated
contain a higher number of CpG sites compared to unmethylated
templates, which results in a different melting temperature that can
be used in quantitative methylation detection.
DNA methylation reconstruction, a method to reconstruct
DNA methylation from ancient
DNA samples. The method
is based on the natural degradation processes that occur in ancient
DNA: with time, methylated cytosines are degraded into thymines,
whereas unmethylated cytosines are degraded into uracils. This
asymmetry in degradation signals was used to reconstruct the full
methylation maps of the
Neanderthal and the
DIFFERENTIALLY METHYLATED REGIONS (DMRS)
Differentially methylated regions , are genomic regions with
different methylation statuses among multiple samples (tissues, cells,
individuals or others), are regarded as possible functional regions
involved in gene transcriptional regulation. The identification of
DMRs among multiple tissues (T-DMRs) provides a comprehensive survey
of epigenetic differences among human tissues. For example, these
methylated regions that are unique to a particular tissue allow
individuals to differentiate between tissue type, such as semen and
vaginal fluid. Current research conducted by Lee et al., showed DACT1
and USP49 positively identified semen by examining T-DMRs. DMRs
between cancer and normal samples (C-DMRs) demonstrate the aberrant
methylation in cancers. It is well known that
DNA methylation is
associated with cell differentiation and proliferation. Many DMRs
have been found in the development stages (D-DMRs) and in the
reprogrammed progress (R-DMRs). In addition, there are
intra-individual DMRs (Intra-DMRs) with longitudinal changes in global
DNA methylation along with the increase of age in a given individual.
There are also inter-individual DMRs (Inter-DMRs) with different
methylation patterns among multiple individuals.
QDMR (Quantitative Differentially Methylated Regions) is a
quantitative approach to quantify methylation difference and identify
DMRs from genome-wide methylation profiles by adapting Shannon entropy
. The platform-free and species-free nature of QDMR makes it
potentially applicable to various methylation data. This approach
provides an effective tool for the high-throughput identification of
the functional regions involved in epigenetic regulation. QDMR can be
used as an effective tool for the quantification of methylation
difference and identification of DMRs across multiple samples.
Gene-set analysis (a.k.a. pathway analysis; usually performed tools
such as DAVID, GoSeq or GSEA) has been shown to be severely biased
when applied to high-throughput methylation data (e.g. MeDIP-seq,
MeDIP-ChIP, HELP-seq etc.), and a wide range of studies have thus
mistakenly reported hyper-methylation of genes related to development
and differentiation; it has been suggested that this can be corrected
using sample label permutations or using a statistical model to
control for differences in the numberes of CpG probes / CpG sites that
target each gene.
DNA METHYLATION MARKS
DNA methylation marks , are genomic regions with specific methylation
pattern in a specific biological state such as tissue, cell type,
individual), are regarded as possible functional regions involved in
gene transcriptional regulation. Although various human cell types may
have the same genome, these cells have different methylomes. The
systematic identification and characterization of methylation marks
across cell types are crucial to understanding the complex regulatory
network for cell fate determination. Hongbo Liu et al. proposed an
entropy-based framework termed SMART to integrate the whole genome
bisulfite sequencing methylomes across 42 human tissues/cells and
identified 757,887 genome segments. Nearly 75% of the segments showed
uniform methylation across all cell types. From the remaining 25% of
the segments, they identified cell type-specific hypo/hypermethylation
marks that were specifically hypo/hypermethylated in a minority of
cell types using a statistical approach and presented an atlas of the
human methylation marks. Further analysis revealed that the cell
type-specific hypomethylation marks were enriched through
transcription factor binding sites in cell type-specific manner. In
particular, they observed that the cell type-specific hypomethylation
marks are associated with the cell type-specific super-enhancers that
drive the expression of cell identity genes. This framework provides a
complementary, functional annotation of the human genome and helps to
elucidate the critical features and functions of cell type-specific
The entropy-based Specific Methylation Analysis and Report Tool,
termed "SMART", which focuses on integrating a large number of DNA
methylomes for the de novo identification of cell type-specific
methylation marks. The latest version of SMART is focused on three
main functions including de novo identification of differentially
methylated regions (DMRs) by genome segmentation, identification of
DMRs from predefined regions of interest, and identification of
differentially methylated CpG sites. SMART is available at
DNA methylation can also be detected by computational models through
sophisticated algorithms and methods. Computational models can
facilitate the global profiling of
DNA methylation across chromosomes,
and often such models are faster and cheaper to perform than
biological assays. Such up-to-date computational models include
Bhasin, et al., Bock, et al., and Zheng, et al. Together with
biological assay, these methods greatly facilitate the
Differentially methylated regions
DNA methylation age
Epigenetics , of which
DNA methylation is a significant
Genomic imprinting , an inherited repression of an allele, relying
DNA Methylation database hosted on the UCSC Genome
DNA Methylation database
DNA methylation Reprogramming
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