DNA methylation and Demethylation, Epigenetic Changes

1. DNA Methylation
Submitted By
Harish TS

2. Introduction
 Epigenetics is the study of heritable phenotype changes that do not involve alterations in the DNA sequence
 Epigenetic changes are modifications to DNA that regulate whether genes are turned on or off. These
modifications are attached to DNA and do not change the sequence of DNA building blocks. Within the
complete set of DNA in a cell (genome), all of the modifications that regulate the activity (expression) of the
genes is known as the epigenome.
 A common type of epigenetic modification is called DNA methylation. DNA methylation involves the
attachment of small chemical groups called methyl groups (each consisting of one carbon atom and three
hydrogen atoms) to DNA building blocks. When methyl groups are present on a gene, that gene is turned off
or silenced, and no protein is produced from that gene.

3. DNA Methylation
 DNA methylation is an epigenetic mechanism that occurs by the addition of a methyl (CH3)
group to DNA, thereby often modifying the function of the genes and affecting gene
expression.
 DNA methylation is the process through which a methyl group is added to DNA nucleotides.
The most common form of this occurs at the 5-carbon position of cytosine (5 methylcytosine or
5mC). DNA methylation can stably alter the gene expression of a cell, which may direct
processes like stem cell differentiation and genomic imprinting.
 The most widely characterized DNA methylation process is the covalent addition of the methyl
group at the 5-carbon of the cytosine ring resulting in 5-methylcytosine (5-mC), also
informally known as the “fifth base” of DNA.

4. • In human DNA, 5-methylcytosine is found in approximately 1.5% of genomic DNA.
• It is typically removed during zygote formation and then re-established in the embryo at
approximately the time of implantation.
• It is the basis of chromatin structure and usually is found in a CpG dinucleotide context.
• Research has shown that methylation plays a crucial role in the regulation of gene
expression and that these modifications tend to occur at specific locations within the
genomes of different species.
• It has been demonstrated as a vital contributor to a wide range of cellular processes, and
aberrant methylation patterns have been linked to several human diseases.

5.  The addition of methyl groups is controlled at several different levels in cells and is carried out by
a family of enzymes called DNA methyltransferases (DNMTs).
 Three DNMTs (DNMT1, DNMT3a and DNMT3b) are required for establishment and
maintenance of DNA methylation patterns.
 Two additional enzymes (DNMT2 and DNMT3L) may also have more specialized but related
functions. DNMT1 appears to be responsible for the maintenance of established patterns of DNA
methylation, while DNMT3a and 3b seem to mediate establishment of new or de novo DNA
methylation patterns.
 Diseased cells such as cancer cells may be different in that DNMT1 alone is not responsible for
maintaining normal gene hypermethylation (an increase in global DNA methylation) and both
DNMTs 1 and 3b may cooperate for this function.

6. DNA demethylation
 It is the removal of a methyl group from DNA. This mechanism is equally as important and
coupled with DNA methylation. The demethylation process is necessary for epigenetic
reprogramming of genes and is also directly involved in many important disease
mechanisms such as tumour progression.

7.  Demethylation of DNA can either be passive or active, or a combination of both.
 Passive DNA demethylation usually takes place on newly synthesized DNA strands via DNMT1
during replication rounds.
 Active DNA demethylation mainly occurs by the removal of 5-methylcytosine via the sequential
modification of cytosine bases that have been converted by TET enzyme-mediated oxidation.
 The ten-eleven translocation (TET) family of 5-mC hydroxylases includes TET1, TET2 and TET3.
These proteins may promote DNA demethylation by binding to CpG rich regions to prevent
unwanted DNA methyltransferase activity, and by converting 5-mC to 5-hmC, 5-hmC to 5-fC (5-
formylcytosine), and 5-fC to 5-caC (5-carboxylcytosine) through hydroxylase activity.
 The TET proteins have been shown to function in transcriptional activation and repression (TET1),
tumor suppression (TET2), and DNA methylation reprogramming processes (TET3).

8. Key Players in DNA Methylation
 Ten-eleven Translocation (TET) Enzymes: The discovery of Ten-eleven translocation
(TET) enzymes provide a mechanistic basis for a mostly hypothetical pathway, active
DNA demethylation. The enzymes are named for a common translocation in cancers.
 5-methylcytosine (5mC): Unless you’ve been living under a rock, or maybe under a
CpG island, then you’ve heard of 5-methylcytosine (5mC). 5mC is the normal cytosine
nucleotide in DNA that has been modified by the addition of a methyl group to its 5th
carbon. The role of this mark is so distinct that many consider 5mC to be the “5th
base” of DNA.
 5-hydroxymethylcytosine (5hmC): Has the potential to greatly deepen our
understanding of epigenetics of the brain and development. 5hmC is the first oxidative
product in the active demethylation of 5-methylcytosine (5mC). The three Ten-eleven
translocation (TET) enzymes oxidize each step in the demethylation of 5mC. 5mC is
first converted to 5hmC, then 5-formylcytosine (5fC), then 5-carboxylcytosine (5caC)

9.  5-formylcytosine (5fC): One of the oxidized derivatives of 5-methylcytosine
(5mC) demethylation. 5mC is oxidized to 5-hydroxymethylcytosine (5hmC)
which is then oxidized to 5fC.
 5-carboxylcytosine (5caC): The final oxidized derivative of 5-methylcytosine
(5mC). 5mC is oxidized to 5-hydroxymethylcytosine (5hmC) which is then
oxidized to 5-formylcytosine (5fC) then 5caC. Each of these oxidation steps are
catalyzed by the Ten-Eleven Translocation (TET) family of enzymes. 5fC can then
be further oxidized to 5-carboxylcytosine (5caC) by TET
 DNA Methyltransferases: DNMTs are the writers of the epigenome. DNMTs are a
highly conserved family of proteins present in nearly all life on earth. In
mammals, there are 3 major DNMTs: DNMT1, DNMT3a and DNMT3b.

10. The Role of Methylation in Gene
Expression
 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.
 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.

11. 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.
 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 . In fact, in certain cancers, such as that of the
colon, hypermethylation is detectable early and might serve as a biomarker for the disease.

12. THANK YOU