Epigenetics

Objectives:

Concept of Epigenomics
Components of the epigenetic code
Epigenetics in normal physiology
Epigenetics in Cancer causation
Epigenetics in diseases
Methods to study the Epigenome
Therapeutic targets of Epigenome

DNA (gene)
Transcription
RNA processing (splicing etc)

mRNA
Translation

Protein
Folding
Post translational modifications

Structural or Functional Activity

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Promoters, enhancers, silencers etc.

Epigenomics

Epi (Greek for above)

-genetics refers to
changes in the
phenotype or gene
expression caused by
mechanisms other than
changes in the
underlying DNA
sequence.
DNA methylation
Histone modifications

Epigenetic chromatin regulation

A. Modification at the DNA level
1. Cytosine methylation
B. Histone modification - the histone code
1. Histone acetylation
2. Histone methylation
3. Histone phosphorylation
4. Histone ubiquitination

The five nucleotides that make up

the DNA

DNA methylation
Covalent addition of methyl group to 5th position of
cytosine with CpG dinucleotides located in the
promoter region of genes

DNA methylation
CpG Cytosine phosphate Guanine
Strongly represented in repetitive sequence
associated with retroviral-derived sequence
Can be methylated to generate 5-methylcytosine
Spontaneously deaminates to form thymine
Poorly recognized by DNA repair systems thus:
CGTG mutation is propagated
CpG levels are less frequent than predicted
1/16
May contribute to relative inactivity of retroelements

CpG dinucleotides
are palindromic

5 CpG 3
3 GpC 5
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DNA methylation
3 human DNA
methyltransferases

DNMT1
DNMT3A
DNMT3B

maintenance methyltransferases
de novo methyltransferases highly expressed at embryo
implantation when waves of de novo methylation are
occurring in the genome

daughter strand

daughter strand

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Maintenance of methylation

Brandeis, M., Ariel, M. & Cedar, H. (1993) Bioessays 15, 709-713.

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DNA methylation
CpGisland methylation how does it affect transcription?

methylated-DNA binding proteins (MECP2, methyl CpG

binding protein 2 ) bind to DNA
this recruits a complex of histone deacetylases and SIN3A
induces a closed chromatin structure gene silencing
in contrast to usual deacetylation-related silencing, when
methylation is involved, its (almost) irreversible

gene

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Imprinting is maintained by DNA methylation

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Transcription factors sensitive

to methylation:

E2F
CREB
AP2
NF-KB
c -myc.

Roles of DNA methylation

Transcriptional silencing
Protecting the genome from
transposition
Genomic imprinting
X inactivation
Tissue specific gene expression

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Genomic imprinting
Difference in gene expression that depends on whether the
gene allele originated from the mother or the father

Neoplastic Transformation
It is a complex multi-event and multi-stage process

The process can be divided into two requisite

sequences:
1- Neoplastic conversion 2- Neoplastic
development

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Neoplastic Conversion
Chemical Carcinogen

Progression

-DNA Reactive
-Epigenetic effect
-DNA methylation
-Histone deacetylation

Promotion

DNA alteration
Genetic and epigenetic Levels

Neoplastic cell

Neoplastic
Development
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Figure 2 DNA methylation in normal and cancer cells

Copyright (2003) Massachusetts Medical Society. All rights reserved. Adapted with permission 2005.

Baylin SB (2005) DNA methylation and gene silencing in cancer

Nat Clin Pract Oncol 2: S4S11 doi:10.1038/ncponc0354

Genes promote hypermethylation in human cancers

The list of genes that are found to be

inactivated by DNA methylation events
includes genes involved in:
A- Signal transduction cascade pathways.
B- Cell cycle regulation. C-Angiogenesis.
D-Apoptosis.
E- DNA repair.

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P 15/P16 Methylation in cancer

-

Aberrant methylation of cyclin dependent kinase

inhibitor P16INK4a has been frequently detected in
many human cancers.

Hematological malignancies
squamous cell carcinoma.

The differential levels of methylated P16 and P15 in

plasma might be useful markers in screening high risk
population for an early detection of cancer.

and

head

and

neck

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Lung cancer

It has been shown that a panel of markers, for

aberrant methylation that detects lung cancer at
the early stages of development has been
observed.
This panel includes the following genes:
-P 16
-APC
-G-ST
-E-cadherin

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Breast cancer
BRCA gene is a breast cancer susceptibility gene,
that is tumor suppressor gene responsible for both
normal development and carcinogenesis in breast.
BRCA1, reveals multi functional protein involved in
DNA repair. Cell cycle regulation, transcription and
apoptosis
Aberrant methylation of BRCA1 CPG island Promoter
is associated with decreased BRCA1 mRNA in
sporadic breast cancer cells.
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Methylation based cancer screening

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Epigenetic chromatin regulation

A. Modification at the DNA level
1. cytosine methylation
B. Histone modification - the histone code
1. Histone acetylation
2. Histone methylation
3. Histone phosphorylation
4. Histone ubiquitination

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Histone modifications

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Mechanism of histone
acetylation
Acetylation of lysine residues of histone proteins

Removal of positive charge of the histones

Decreased affinity between histones and DNA

Easier access of transcription factor to promoter region

Histone acetylation
Mechanism:
Acetylation of H3 or H4 leads to unfolding and increased accessibility of
chromatin to enable transcription.
Histones are acetylated by HAT (histone acetylases) which are parts of
many chromatin remodeling and transcription complexes.

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Role of histone de-acetylation

Deacetylated histones are tightly packed and less
accessible to transcription factors.
Histones are deacetylated by HDAC (histone deacetylase) proteins.

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Histone phosphorylation (H3)

1. Histones are phosphorylated during
mitosis.
2. Histones are also phosphorylated by signal
transduction pathways like the ERK
pathway in response to external signals. It
is not known how (and if) this
phosphorylation contributes to gene
expression.
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Epigenetic chromatin regulation

A. Modification at the DNA level
1. cytosine methylation
B. Histone modification - the histone code
1. Histone acetylation
2. Histone methylation
3. Histone phosphorylation
4. Histone ubiquitination

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Histone ubiquitylation
Addition and removal of Ub (a LARGE moiety) to
histone tails Functions largely unknown in
vertebrates
ubiquitylation

H2A K119: repression

H2B K120: activation
H3 and H4: DNA repair
(CUL4)
de-ubiquitylation Recrutiment of other proteins in yeast
H2A
H2B

Dub (PCAF)
Ubp8 (SAGA)

Functions: transcription elongation, polycomb repression

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Epigenetic diseases:

Methods to Study The Epigenome

Bisulphite sequencing
Methylation sensitive-High resolution
melting (MS-HRM)
Microarray-based genome-wide analysis
Chromatin immunoprecipitation (ChIP) on
Chip assays

Methylation analysis study - Bisulphite sequencing

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Methylation sensitive-High resolution melting

(MS-HRM)

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Microarray-based genome-wide analysis

Methylated DNA
immunoprecipitation (MeDIP)
-requires immunoprecipitation
of DNA using
antimethylcytosine antibody
followed by hybridization to
DNA microarrays.
requires large amounts
of genomic DNA and antibody
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Study of histone modifications

Histone modifications are studied using the chromatin
immunoprecipitation (ChIP) assay.
ChIP on chip is the high throughput form of the ChIP
assay wherein the immunoprecipitated DNA, instead of
being subject to the usual PCR, is hybridized to a
microarray chip with printed oligonucleotides
corresponding to various regions of the genome.
This helps to study the localization of a specific histone
modification to various parts of the genome.

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Chromatin immunoprecipitation (ChIP) on Chip assays

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Different classes of Drugs

DNMT INHIBITORS

HDAC INHIBITORS

1.Nucleoside analogue
inhibitors

1.Hydroxamates
2.Cyclic tetrapeptides

2.Non nucleoside
analogue inhibitors
3.Antisense
oligonucleotides

3.Aliphatic acids
4.Benzamides

DNMT Inhibitor: Decitabine

Decitabine
deoxycytidine kinase
Decitabine triphosphate

Incorporated into DNA

Binds with DNMT and traps the enzyme

HDAC inhibitors: Vorinostat

 Intrinsic and acquired drug resistance remain the

most unpredictable factors affecting chemotherapy.
DNA hypermethylation has been found to be
associated with drug resistance acquired during
cancer chemotherapy and therefore, re-expression of
methylation-silenced genes resulted in increased
sensitivity to existing chemotherapy.

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SUMMARY:
Types of DNA modification and Histone modifications
Mechanism of their actions and maintenance
Their effects on transcription, growth and development,
differentiation, tumorigenesis
Markers of various tumors
Experimental procedures of their study
Therapeutic possibilities
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REFERENCES:
Harper's Illustrated Biochemistry, Twenty-Eighth
Edition.
Lehningers Principles of Biochemistry, Fifth Edition.
Stryers Biochemistry, Seventh Edition.
Epigenetics and gene expression. Gibney ER, Nolan CM.
Heredity (Edinb). 2010 Jul;105(1):4-13
When food meets man: the contribution of epigenetics to
health. De Fabiani E, Mitro N, Gilardi F, Galmozzi A,
Caruso D, Crestani M. Nutrients. 2010 May;2(5):551-71.
Environmental epigenetics. Bollati V, Baccarelli A.
Heredity (Edinb). 2010 Jul;105(1):105-12. Epub 2010
Feb 24.

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