DNA METHYLATION

1
Epigenetics
• Study of heritable changes in gene expression that
occur without a change in a DNA sequence.
• Stable alteration in gene expression pattern.
• Dynamic process that plays a key role in normal cell
growth and differentiation.
• To date, the best understood epigenetic mechanisms
are
1. DNA methylation
2. Histone modifications

2
 DNA methylation
• DNA methylation is one of the most commonly
occurring epigenetic events taking place in the
mammalian genome.
• This change, though heritable, is reversible, making
it a therapeutic target.
• Methylation pattern is determined during
embryogenesis and passed over to differentiating
cells and tissues.

3
 DNA methylation
• DNA structure is
maintained from
generation to
generation.
• This structure is
modified by base
methylation in nearly all
cells and organisms.

4
 DNA methylation
• The DNA of most organisms is modified by a post-
replicative process which results in three types of
methylated bases in DNA:
C5-methylcytosine(5-mc)
N4-methylcytosine Wide spread in
N6-methyladenine. prokaryotes

• This Modification is called DNA methylation.

• DNA methylation is a covalent modification of DNA
that does not change the DNA sequence, but has an
influence on gene activity.

5
 DNA Methylation
• It occurs in the cells of fungi, plants, non-
vertebrates and vertebrates.
• In vertebrates, 3-6% of DNA cytosine is
methylated.
• No methylation in many insects and single-celled
eukaryotes.
• In plants, 30% of DNA cytosine is methylated.

6
DNA
methylation
•Addition of methyl group
to C-5 position of cytosine
residues.
•Most cytosine
methylation occurs in the
sequence context 5'CG3'
•Occurs almost exclusively
at cytosines that are
followed immediately by a
Guanine- CpG
Dinucleotide.

7
 Mechanism
• Methyl groups are
transferred from S-
adenosyl methionine in
a reaction catalysed by
a DNA methyl
transferases(DNMT) or
methylases.
• SAM is then converted
to SAH (S-adenosyl
homocysteine).

8
 Enzymes involved in DNA
methylation
• Enzymes involved-
DNA METHYLTRANSFERASES(DNMTs)
• DNMTs catalyze this reaction at different times during the cell
cycle.
• In Mammals,
1. DNMT1- Maintainance methylase
2. DNMT 2
3. DNMT3a and DNMT3b-‘de novo’methylases
4. DNMT3L

9
 Enzymes
DNMT1:
• maintains the pattern of DNA methylation after DNA
replication.
• requires a hemi-methylated DNA substrate and will
faithfully reproduce the pattern of DNA methylation on
the newly synthesized strand.
• DNA methylation- ‘an automatic semi conservative
mechanism’
DNMT3a and DNMT3b:
• Will add methyl groups to CG dinucleotides which are
previously unmethylated on both the strands.
• Re-establish the methylation pattern.

10
11
 PRE- Genome undergoes
IMPLANTATION Demethylation

AFTER
IMPLANTATION OF New Methylation
EMBRYO AND patterns are set by de-
DURING novo methylation.
CARCINOGENESIS

Methylation patterns
must be maintained.
DURING REPLICATION Therefore, DNMT1,
methylates the
hemimethylated DNA
after strand synthesis.

12
 Mammalian Genome
• The human genome is not methylated uniformly and
contains regions of unmethylated segments
interspersed by methylated regions.
• In contrast to the rest of the genome, smaller regions
of DNA, called CpG islands, ranging from 0.5 to 5 kb
and occurring on average every 100 kb, have
distinctive properties. These regions are
unmethylated normally.
• Approximately half of all the genes in humans have
CpG islands, and these are present on both
housekeeping genes and genes with tissue-specific
patterns of expression.

13
CpG Dinucleotides
• Occur at low abundance throughout the human
genome.
• Tend to concentrate in regions known as CpG
islands (found in 50% of promoter regions of
genes).
• Typically methylated in non-promoter regions and
unmethylated in promoter regions.
• Methylation within the promoter region correlates
with transcriptional silencing.
• Methylation of CpG islands is believed to
dysregulate gene transcription through the
inhibition of transcription factor binding either
directly or via altered histone acetylation.
14
 CpG ISLANDS

PROMOTER NON-
REGIONS PROMOTER
REGIONS

Non- Methylated Methylated Non-

methylated methylated

Silence
parasitic
Binding of TF Inhibition of
genetic Binding of TF
TF binding
elements

Transcription
Transcriptional Genomic Transcription
silencing stability

15
16
 Role of DNA methylation
• Plays a role in long term silencing of gene.
• Plays a role in silencing of repetitive elements ( eg:
transposons).
• Plays a role in X-chromosome inactivation.
• In the establishment and maintenance of imprinted
genes.
• Suppresses the expression of viral genes and other
deletorious elements that have been incorporated
into the genome of the host over time.
• In Carcinogenesis.

17
18
19
METHYLATION IMBALANCE may contribute to
TUMOR PROGRESSION

GLOBAL DNA
HYPOMETHYLATION HYPERMETHYLATION

Observed in neoplastic Inactivation of tumor-

cells suppressor genes: p16,
BRCA1

May induce neoplastic Inactivation of DNA repair

transformation genes: MLH1, MGMT

Genomic instability,
Abnormal chromosomal
structures and
Activating oncogenes.
20
A CpG island hypermethylation profile of human cancer

21
 Global Hypomethylation
• There will be significant decrease in 5-meC.
• Occurs in numerous solid tumors and in some
haematological malignances.
eg: Chronic lymphocytic leukaemia (CLL),
Chronic myelogenous leukaemia (CML),
Acute Myelogenous leukaemia (AML).
• Occurs in early stages of chest tumors, colorectal
cancer and chronic lymphocytic leukaemia

22
 Exceptions
• There are some examples where a CpG island in
a promoter is unmethylated while the gene is still
kept silent.
eg: The CpG island in human α-globin gene
promoter is unmethylated in both erythroid and
non-erythroid tissues (Bird et al., 1987).
Reason: role of histone modifications in gene
silencing.

23
 RISK FACTORS
• Carcinogens: Chronic exposure of human bronchial
epithelial cells to tobacco-derived carcinogens drives
hypermethylation of several tumor suppressor
genes.
• The reactive oxygen species (ROS) associated with
chronic inflammation is another source of DNA
damage.
• Cigarette smoke: causes hypomethylation.
• Aging.

24
Detection of DNA methylation

• Sodium bisulfite conversion (SBC)

• SBC LC-MS-MS
• cDNA microarray
• Restriction landmark genomic sequencing
• CpG island microarray

25
 1. Bisulfite conversion (SBC)
• Offers highest degree of
resolution of the methylation
status of a given sample,
allowing to determine the
positional CpG genotype for
individual samples.
• Involves the chemical
modification of DNA by
bisulfite treatment, where
sodium bisulfite deaminates
cytosine to uracil.
• Methylated cytosine is
resistant to this conversion.
26
Major Advance: Conversion of unmethylated cystosines
to uracil using sodium bisulfite

Sequencing: unemethylated cytosines read as

thymidine in sense strand; adenine in the
anti-sense strand.
Other technologies evolved from here.
27
 1. SBC contd..
• After bisulfite modification, there are a number of
methods available to study CpG island methylation.
• These include
➢ sequencing,
➢ methylation-specific polymerase chain reaction,
➢ combined bisulfite restriction analyses,
➢ methylation-sensitive single nucleotide primer
extension, and
➢ methylation-sensitive single-strand conformational
polymorphism.

28
 1. SBC contd..
• To be useful as a routine diagnostic tool, the actual
methylation detection method has to be sensitive,
quick, easy, and reproducible.
• After bisulfite modification, PCR is performed using
two sets of primers designed to amplify either
methylated or unmethylated alleles.
• Of the various techniques available, methylation-
specific polymerase chain reaction (MSP) seems to
be most useful at present.

29
Methylation specific PCR

30
31
DNA methylation inhibitors (clinical
approach)

• Agents targetted against DNMTs

5-azacytidine
2'deoxy-5-azacytidine (also known as Decitabine)

32
 References
• Wentao Gao et al, Carcinogenesis vol.29 no.10 pp.1901–
1910, 2008
• J.A. McKay*1, E.A. Williams† and J.C. Mathers*, Biochemical
Society Transactions (2004) Volume 32, part 6
• Robin Holliday, Biochemistry (Moscow), Vol. 70, No. 5, 2005,
pp. 500-504.
• Ibáñez de Cáceres and P. Cairns, Clin Transl Oncol (2007)
9:429-437
• Melissa Conerly1 and William M. Grady2,3,*, Disease Models
& Mechanisms 3, 290-297 (2010)
• Steven s. smith*, Proc. Nati. Acad. Sci. USA Vol. 89, pp.
4744-4748, May 1992,Biochemistry.

33
 References
• Manel Esteller, Human Molecular Genetics, 2007, Vol. 16,
Review Issue 1 R50–R59.
• Yaping Li et al, Journal of Dermatological Science 54 (2009)
143–149.

34
35