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    Epigenetics

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    Virendra Joshi

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    © All Rights Reserved
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    Epigenetics

    Uploaded by

    Virendra Joshi
    965 views49 pages

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    epigenetics.ppt

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    965 views49 pages

    Epigenetics

    Uploaded by

    Virendra Joshi

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
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    You are on page 1of 49
    At a glance
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    The key takeaways are the different types of epigenetic modifications like DNA methylation and histone modifications, how they affect gene expression and are involved in various cellular processes like imprinting and X-inactivation.

    The different components of the epigenetic code include DNA methylation, histone modifications like acetylation, methylation, phosphorylation and ubiquitination.

    DNA methylation can affect gene transcription by recruiting methyl-DNA binding proteins that induce chromatin closure and gene silencing. It also involves recruitment of histone deacetylases. The methylation is self-perpetuating and leads to near-irreversible gene silencing.

    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


    3

    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
    9

    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

    10

    Maintenance of methylation

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


    11

    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

    12

    Imprinting is maintained by DNA methylation

    13

    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

    15

    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

    17

    Neoplastic Conversion
    Chemical Carcinogen

    Progression

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

    Promotion

    DNA alteration
    Genetic and epigenetic Levels

    Neoplastic cell

    Neoplastic
    Development
    18

    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.

    20

    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

    21

    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

    22

    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.
    23

    Methylation based cancer screening

    24

    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

    25

    26

    Histone modifications

    27

    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.

    29

    30

    Role of histone de-acetylation


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

    31

    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.
    32

    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

    33

    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

    34

    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

    37

    Methylation sensitive-High resolution melting


    (MS-HRM)

    38

    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
    39

    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.

    40
    40

    41
    41

    Chromatin immunoprecipitation (ChIP) on Chip assays

    42

    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.

    46

    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
    47

    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.

    THANK YOU

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