Epigenetics final

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Epigenetics

Dr. Nilesh Chandra

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

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DNA (gene)

mRNA

Protein

TranscriptionRNA processing (splicing etc)

Translation

Folding

Post translational modifications

Structural or Functional Activity

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Promoters, enhancers, silencers etc.

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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

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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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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

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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:

• CG→TG mutation is propagated• CpG levels are less frequent than predicted 1/16• May contribute to relative inactivity of retro-elements

CpG dinucleotides are palindromic

5’ CpG 3’3’ GpC 5’

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DNA methylation

3 human DNA methyltransferases

• DNMT1• DNMT3A• DNMT3B

◄daughter strand

◄daughter strand

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

maintenance methyltransferases

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Maintenance of methylation

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

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DNA methylation

CpG–island 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, it’s (almost) irreversible

gene

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Imprinting is maintained by DNA methylation

Transcription factors sensitive to methylation:

E2F CREB AP2 NF-KB c -myc.

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Roles of DNA methylation

Transcriptional silencing Protecting the genome from

transposition Genomic imprinting X inactivation Tissue specific gene expression

Genomic imprinting

“Difference in gene expression that depends on whether the gene allele originated from the mother or the father”

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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 Promotion

-Epigenetic effect

-DNA methylation

-Histone deacetylation Neoplastic cell

Genetic and epigenetic Levels

DNA alteration NeoplasticDevelopment

Baylin SB (2005) DNA methylation and gene silencing in cancerNat Clin Pract Oncol 2: S4–S11 doi:10.1038/ncponc0354

Figure 2 DNA methylation in normal and cancer cells

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

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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 and head and neck 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.

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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

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

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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 de-acetylase) 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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Addition and removal of Ub (a LARGE moiety) to histone tails – Functions largely unknown in vertebrates

H2A K119: repression H2B K120: activation H3 and H4: DNA repair (CUL4)

ubiquitylation

H2A Dub (PCAF)H2B Ubp8 (SAGA)

de-ubiquitylation – Recrutiment of other proteins in yeast

Histone ubiquitylation

Functions: transcription elongation, polycomb repression

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

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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.

4141

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Chromatin immunoprecipitation (ChIP) on Chip assays

Different classes of Drugs

DNMT INHIBITORS

1.Nucleoside analogue inhibitors

2.Non nucleoside analogue inhibitors

3.Antisense oligonucleotides

HDAC INHIBITORS

1.Hydroxamates 2.Cyclic tetrapeptides

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

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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

REFERENCES:

Harper's Illustrated Biochemistry, Twenty-Eighth Edition.

Lehninger’s Principles of Biochemistry, Fifth Edition.

Stryer’s 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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