Dna methylation

1. DNA METHYLATION
Guided by:Presented by:
SUSHMA MARLA
M.PHARM (First Sem)
DEPT. OF BIOTECH.
PROF. KALPANA JOSHI
PROF. & HEAD
BIOTECH. DEPT.
SCOE. PUNE.
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2. EpigeneticsEpigenetics
•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
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3. DNA methylationDNA 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.
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4. DNA methylationDNA methylation
•DNA structure is
maintained from
generation to
generation.
•This structure is
modified by base
methylation in nearly all
cells and organisms.
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5. DNA methylationDNA 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
N6-methyladenine.
•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.
Wide spread in
prokaryotes
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6. DNA MethylationDNA 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.
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7. DNA DNA
methylationmethylation
•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.
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8. MechanismMechanism
•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).
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9. Enzymes involved in DNA Enzymes involved in DNA
methylationmethylation
•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
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10. EnzymesEnzymes
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.
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12. PRE-
IMPLANTATION
Genome undergoes
Demethylation
AFTER
IMPLANTATION OF
EMBRYO AND
DURING
CARCINOGENESIS
New Methylation
patterns are set by de-
novo methylation.
DURING REPLICATION
Methylation patterns
must be maintained.
Therefore, DNMT1,
methylates the
hemimethylated DNA
after strand synthesis.
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13. Mammalian GenomeMammalian 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.
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14. CpG DinucleotidesCpG Dinucleotides
•Occur at low abundance throughout the human
genome.
•Tend to concentrate in regions known as CpG CpG
islands 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.
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15. CpG ISLANDS
PROMOTER
REGIONS
NON-
PROMOTER
REGIONS
Non-
methylated
Methylated
Binding of TF
Transcription
Inhibition of
TF binding
Transcriptional
silencing
Methylated Non-
methylated
Silence
parasitic
genetic
elements
Genomic
stability
Binding of TF
Transcription
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17. Role of DNA methylationRole 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.
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20. METHYLATION IMBALANCE may contribute to
TUMOR PROGRESSION
GLOBAL
HYPOMETHYLATION
DNA
HYPERMETHYLATION
Observed in neoplastic
cells
May induce neoplastic
transformation
Genomic instability,
Abnormal chromosomal
structures and
Activating oncogenes.
Inactivation of tumor-
suppressor genes: p16,
BRCA1
Inactivation of DNA repair
genes: MLH1, MGMT
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21. A CpG island hypermethylation profile of human cancer
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22. Global HypomethylationGlobal 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
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23. ExceptionsExceptions
•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.
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24. •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.
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RISK FACTORSRISK FACTORS

25. Detection of DNA methylationDetection of DNA methylation
•Sodium bisulfite conversion (SBC)
•SBC LC-MS-MS
•cDNA microarray
•Restriction landmark genomic sequencing
•CpG island microarray
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26. 1. Bisulfite conversion (SBC)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.
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27. 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.
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28. •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.
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1. SBC contd..1. SBC contd..

29. 1. SBC contd..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.
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Methylation specific PCR

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32. DNA methylation inhibitors (clinical DNA methylation inhibitors (clinical
approach)approach)
•Agents targetted against DNMTs
5-azacytidine
2'deoxy-5-azacytidine (also known as Decitabine)
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33. ReferencesReferences
•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.
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34. ReferencesReferences
•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.
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