DNA Methylation and Gene Expression

DNA methylation
DNA methylation is a biological process by which methyl groups are added to
the DNA molecule. Methylation can change the activity of a DNA segment
without changing the sequence. When located in a gene promoter, DNA
methylation typically acts to repress gene transcription. In mammals, 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 carcinogenesis.

Representation of a DNA molecule that is methylated. The two white spheres

represent methyl groups. They are bound to two cytosine nucleotide molecules that
make up the DNA sequence.

So far (2016) two nucleobases have been found on which natural, enzymatic
DNA methylation takes place: adenine and cytosine. The modified bases are
N6-methyladenine[, 5-methylcytosine and N4-methylcytosine.
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% to 8% in Physarum,[4]
7.6% in Mus musculus, 2.3% in Escherichia coli, 0.03% in Drosophila, 0.006%
in Dictyostelium[5] and virtually none (0.0002 to 0.0003%) in
Caenorhabditis[6] or fungi such as Saccharomyces cerevisiae and S. pombe
(but not N. crassa).[7][8]: 3699  Adenine methylation has been observed in
bacterial, plant, and recently in mammalian DNA,[9][10] but has 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; the same position distinguishes thymine from the analogous RNA
base uracil, which has no methyl group. Spontaneous deamination of 5-
methylcytosine converts it to thymine. This results in a T:G mismatch. Repair
mechanisms then correct it back to the original C:G pair; alternatively, they
may substitute A for G, turning the original C:G pair into a T:A pair, effectively
changing a base and introducing a mutation. This misincorporated base will
not be corrected during DNA replication as thymine is a DNA base. If the
mismatch is not repaired and the cell enters the cell cycle the strand carrying
the T will be complemented by an A in one of the daughter cells, such that the
mutation becomes permanent. The near-universal use of thymine exclusively
in DNA and uracil exclusively in RNA may have evolved as an error-control
mechanism, to facilitate the removal of uracils generated by the spontaneous
deamination of cytosine.[11] DNA methylation as well as many of its
contemporary DNA methyltransferases have been thought to evolve from
early world primitive RNA methylation activity and is supported by several
lines of evidence.[12]

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 strands being usually
methylated. Non-CpG methylation can however be observed in embryonic
stem cells,[13][14][15] and has also been indicated in neural development.[16]
Furthermore, non-CpG methylation has also been observed in hematopoietic
progenitor cells, and it occurred mainly in a CpApC sequence context.[17]

Conserved function of DNA methylation

The DNA methylation landscape of vertebrates is very particular compared to

other organisms. In mammals, around 75% of CpG dinucleotides are
methylated in somatic cells,[18] and DNA methylation appears as a default
state that has to be specifically excluded from defined locations.[15][19] 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.[20][21]

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).[22] (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.)

CpG islands

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.
[23] 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.[24]
Excluding repeated sequences, there are around 25,000 CpG islands in the
human genome, 75% of which being less than 850bp long.[22] 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.[25][26] 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
intragenic regions.
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.[20][27] 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.[28][29] Of note, whereas DNA
methylation of CpG islands is unambiguously linked with transcriptional
repression, the function of DNA methylation in CG-poor promoters remains
unclear; albeit there is little evidence that it could be functionally relevant.[30]

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,[31] 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 deacetylases and other chromatin remodeling proteins that
can modify histones, 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".

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.[32]
Transposon control is one of the most ancient functions of DNA methylation
that is shared by animals, plants and multiple protists.[33] It is even suggested
that DNA methylation evolved precisely for this purpose.[34]

Genome expansion

DNA methylation of transposable elements has been known to be related to

genome expansion. However, the evolutionary driver for genome expansion
remains unknown. There is a clear correlation between the size of the
genome and CpG, suggesting that the DNA methylation of transposable
elements led to a noticeable increase in the mass of DNA.[35]

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 where DNA
methylation is present, DNA methylation is especially enriched in the body of
highly transcribed genes.[20][27] The function of gene body methylation is not
well understood. A body of evidence suggests that it could regulate
splicing[36] and suppress the activity of intragenic transcriptional units (cryptic
promoters or transposable elements).[37] 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.[38] In mammals,
DNMT3a and DNMT3b PWWP domain binds to H3K36me3 and the two
enzymes are recruited to the body of actively transcribed genes.

The Role of 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.

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.

azacytidine Experiments Provide Early Clues to the Role of Methylation

in Gene Expression

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 (Table 1; Jones
& Taylor, 1980).

Table 1: Effect of Cytidine Analogs on Cell Differentiation and DNA Methylation

This straightforward experiment demonstrated that it was not the removal of

cytidine residues alone that resulted in changes in cell differentiation (because
Ara-C did not have an impact on differentiation); rather, only those analogs
that impacted methylation resulted in such changes. These experiments
opened the door for investigators to better understand exactly how
methylation impacts gene expression and cellular differentiation.

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.

The Role of Methylation in Gene Expression

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).

DNA Methylation and Histones

Although patterns of DNA methylation appear to be relatively stable in somatic

cells, patterns of histone methylation can change rapidly during the course of
the cell cycle. Despite this difference, several studies have indicated that DNA
methylation and histone methylation at certain positions are connected. For
instance, results of immunoprecipitation studies using human cells suggest
that DNA methylation and histone methylation work together during replication
to ensure that specific methylation patterns are passed on to progeny cells
(Sarraf & Stancheva, 2004). Indeed, evidence has been presented that in
some organisms, such as Neurospora crassa (Tamaru & Selker, 2001) and
Arabidopsis thaliana (Jackson et al., 2002), H3-K9 methylation (methylation of
a specific lysine residue in the histone H3) is required in order for DNA
methylation to take place. However, exceptions have been observed in which
the relationship is reversed. In one study, for example, H3 methylation was
reduced at a tumor suppressor gene in cells deficient in DNA
methyltransferase (Martin & Zhang, 2005).

In an interestingly coordinated process, proteins that bind to methylated DNA

also form complexes with the proteins involved in deacetylation of histones.
Therefore, when DNA is methylated, nearby histones are deacetylated,
resulting in compounded inhibitory effects on transcription. Likewise,
demethylated DNA does not attract deacetylating enzymes to the histones,
allowing them to remain acetylated and more mobile, thus promoting
transcription. In most cases, methylation of DNA is a fairly long-term, stable
conversion, but in some cases, such as in germ cells, when silencing of
imprinted genes must be reversed, demethylation can take place to allow for
"epigenetic reprogramming." The exact mechanisms for demethylation are not
entirely understood; however, it appears that this process may be mediated
by the removal of amino groups by DNA deaminases (Morgan et al., 2004).
After deamination, the DNA has a mismatch and is repaired, causing it to
become demethylated. In fact, studies using inhibitors of one DNMT enzyme
showed that this enzyme was involved in not only DNA methylation, but also
in the removal of amino groups.

DNA Methylation and Disease

Given the critical role of DNA methylation in gene expression and cell
differentiation, it seems obvious that errors in methylation could give rise to a
number of devastating consequences, including various diseases. Indeed,
medical scientists are currently studying the connections between methylation
abnormalities and diseases such as cancer, lupus, muscular dystrophy, and a
range of birth defects that appear to be caused by defective imprinting
mechanisms (Robertson, 2005). The results of these studies will be invaluable
for treating these disorders, as well as for understanding and preventing
complications that can arise during embryonic development due to
abnormalities in X-chromosome methylation and gene imprinting.

To date, a large amount of research on DNA methylation and disease has

focused on cancer and tumor suppressor genes. Tumor suppressor genes are
often silenced in cancer cells due to hypermethylation. In contrast, the
genomes of cancer cells have been shown to be hypomethylated overall
when compared to normal cells, with the exception of hypermethylation
events at genes involved in cell cycle regulation, tumor cell invasion, DNA
repair, and others events in which silencing propagates metastasis (Figure 1;
Robertson, 2005). In fact, in certain cancers, such as that of the colon,
hypermethylation is detectable early and might serve as a biomarker for the
disease.
The diagram shows a representative region of genomic DNA in a normal cell.
The region shown contains repeat-rich, hypermethylated pericentromeric
heterochromatin and an actively transcribed tumour suppressor gene (TSG)
associated with a hypomethylated CpG island (indicated in red). In tumour
cells, repeat-rich heterochromatin becomes hypomethylated and this
contributes to genomic instability, a hallmark of tumour cells, through
increased mitotic recombination events. De novo methylation of CpG islands
also occurs in cancer cells, and can result in the transcriptional silencing of
growth-regulatory genes. These changes in methylation are early events in
tumorigenesis.

Summary

Within the past thirty years, researchers have discovered numerous details
about the process of DNA methylation. For instance, scientists now know that
methylation plays a critical role in the regulation of gene expression, and they
have also determined that this process tends to occur at certain locations
within the genomes of different species. Furthermore, DNA methylation has
been shown to play a vital role in numerous cellular processes, and abnormal
patterns of methylation have been liked to several human diseases.
Nonetheless, as with other topics in the field of epigenetics, gaps remain in
our knowledge of DNA methylation. As new laboratory techniques are
developed and additional genomes are mapped, scientists will no doubt
continue to uncover many of the unknowns of how, when, and where DNA is
methylated, and for what purposes.