Transcript Epigenetics

Epigenetics
Epigenetics

Epi- (Greek: over, above, outer)
 The study of mitotically (separating chromasomes in a
cell) and/or meiotically heritable changes in gene
function that cannot be explained by changes in DNA
sequence (96 Russo) (classical)
 11_Graff (www.cs.uml.edu/~kim/580/11_Graff.pdf)
 Structural adaptation of chromosomal regions so as to
register, signal, or perpetuate altered activity states
(recent)
 Study of changes in gene expression or cellular
phenotype (simple)
Meiosis
Chromatin
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A complex of macromolecules in cells
 Consists of DNA, protein, RNA
 Primary functions
 Package DNA
 Reinforce DNA macromolecule for mitosis
 Prevent DNA damage
 Control gene expression and DNA replication
 Primary protein component of chromatin
 histones
Chromosome Length
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3.4A per base
3 Billion bases
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1.8 meters of DNA
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0.09 nm of chromatin after being
wound on histones
Five families of histones
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H1/H5, H2A, H2B, H3, and H4
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A nucleosome is the basic unit of DNA packaging in
eukaryotes, consisting of a segment of DNA wound in
sequence around eight histone protein cores. This structure
is often compared to thread wrapped around a spool.
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Nucleosomes form the fundamental repeating units of
eukaryotic chromatin, which is used to pack the large
eukaryotic genomes into the nucleus while still ensuring
appropriate access to it (in mammalian cells approximately
2 m of linear DNA have to be packed into a nucleus of
roughly 10 µm diameter).
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A nucleosome core has about 146 bp of DNA
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Typical exon of around 140 nt
Three Major Levels of Epigenetic Changes
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3.
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Chemical modifications at the level of nucleotides
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Including DNA methylation and RNA interference
Modifications at the level of histones that encompass posttranslational
modifications (PTMs) of histone proteins and the incorporation of
histone variants
Nucleosome remodeling
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ATP (Adenosine Triphosphate)-dependent processes that regulate
the accessibility of nucleosomal DNA (ATP: stores energy)
=> Regulation of the accessibility of the chromatin structure to the
transcription machinery
1. a. DNA Modification: Methylation
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Covalent addition of a methyl group from methyl donor SAM (Sadenosylmethionine) to a cytosine base
 Occurs mainly at 5’ end of cytosine in CpG, CpHpG and CpHpH,
where H is A,T, or C
This reaction is catalyzed by a family of DNMT (DNA methyltransferase)
 DNMT1 is the main enzyme in mammals
Methylation patterns change over evolution
 In invertebrate animals, mosaic methylation, with stable methylated
domains interspersed with methylation-free regions
 In vertebrate genomes, globally methylated, with exception of CpG
islands
Methylation dynamically change among different cells, and even in a
single cell
The reaction catalyzed by DNA
methyltransferases (DNMTs). DNMTs are
the key enzymes for DNA methylation and
catalyze the
transfer of a methyl group from SAM to
cytosine, thus forming 5-methyl-cytosine
and SAH. Methylation of CpG sequences
might induce chromatin conformational
modifications and inhibit the access of the
transcriptional machinery to gene promoter
regions, thus altering gene expression
levels. Therefore, promoter rmethylation of
CpG islands is commonly associated with
gene silencing and promoter demethylation
with gene expression, though several
exceptions to this rule are known.
Methylation Inheritance
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Both C & its complementary G are methylated (fully methylated)
After replication, rapidly acted on by DNMT1 to regenerate two identical
fully methylated double helices
Epigenetic info is inherited in the form of DNA methylation patterns
DNA Methylation Binding
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Methyl-binding proteins (MBPs) bind to methylated DNA, typically in
promoters (e.g. MeCP2– Methyl CpG binding Protein 2)
Binding recruits other protein complexes that lead to transcription
repression
1.b. RNAi
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Epigenetic alterations of DNA can also be produced by double-stranded
RNA (dsRNA) and protein components of RNAi machinery
 Small RNAs produced by cleavage of dsRNA are thought to serve as
sequence-specific facilitators to guide other enzymes of epigenetic
machinery into place
 D.melanogaster (fruit fly)
 Members of RNAi machinery such as genes piwi and homeless
are mutated, centromeric heterochromatin formation is inhibited
 Fission yeast (Schizosaccharomyces pombe)
 Deletions of genes involved in RNAi machinery, such as
argonaute, result in reduced hetrochromatin formation and
reduced methylation on H3K9 (marker of gene repression)
 Mammals
 Short-interfering RNA (siRNA) induce methylation alongside H3
methylation, resulting in decreased gene expression
2. a. Histone Modification
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Histone
 Basic proteins regulating compaction of chromatin
 Consist of
 a loosely structure NH2-terminal tail
 out acting as regulatory substrates for nucleosomal stability
 These substrates establish condensed/uncondensed
states of the chromatin
 a globular histone core (nucleosome)
 Octamer of four core histones H2A, H2B, H3, and H4 in
duplicates
 Around the core, 147 bp wrap around in 10-nm-thick
primary structure
2. a. Histone Modification
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Histone
 Nucleosomes are linked together by linker histone H1
 Post Translational Modifications (PTMs) can occur on all histones
 Majority occurs on NH2-terminal tail
 PTM types
 Acetylation on lysine (K)
 Methylation
 Phosporylation
 Ubiquitnation
 sumoylation
DNA-gray
H2A – blue
H2B – yellow
H3 – green
H4 - red
PTM
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Their accessibility of DNA will change
Current research emphasis is on
 Role of modification in the transformation process from normal to
cancer cells
 Study imbalances in net expression of tumor suppressor vs.
oncogenes or overall genomic imbalances
Needs substrate specificity and residue-specific alteration
Acetylation
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Positively charged AA (lysine(K) and arginine (R) are neutralized by
acetyl group, leading to decreased affinity between histone tail and
negatively charged DNA
HAT (Histone Acetyltransferase) regulates acetylation of histones
 In most cancers, HAT genes are muted and HAT includes
chromosomal translocation of respective HAT
 But HDACs (Histone deacetylase) are frequently overexpressed
Histones prefer being methylated or phosphorylated at R, at acetylated
at K
Acetylation of K initiates active gene expression
Acetylation plays a role in nucleosome assembly and maintenance of
chromatin state affecting DNA repair, etc.
The unmodified side chain of posttranslationally modifiable residues is first presented, followed by
representations of the residue on which the posttranslational modification has occurred at respective sites. Abbreviations are as follows
R, arginine; Y, tyrosine; S, serine; T, threonine; -ac, acetylation; -me, monomethylation; -me2, dimethylation; -me3, trimethylation; -ph
The color code is as follows: yellow, carbon; blue, nitrogen; pink, polar hydrogen; red, oxygen; orange, phosphorus; green, methyl gro
of posttranslational modifications.
Red – acetyl group
Yellow - methylation
Green – methylation of R
HAT (Histone Acetyltransferease)
HDAC (Histone Deacetylase)
HMT (Histone Methyltransferase)
HDM (Histone Demethylase)
Effects of acetylation on protein
functions. Acetylation of
proteins affects many different
functions, some of which are
listed. The
double up-arrows indicate
increase and the double downarrows indicate decrease with
respect to the particular
function.
Some of the genes affected by
acetylation under specific
protein functions are listed [60].
Methylation
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K and R can be mono-, di- or tri-methylated forms
 Monomethylated H3K4 is found in expressed and repressed genes
 Trimethylated H3K4 is exclusively in silenced genes
Location – H3K9me in coding region for expression, in promoter, repression
Some times, same K is acetylated or methylated
 example: K4 and K9 residues of histone H3
Methylation is regulated by
 HMT (histone methyltransferase), specific to K and R
 And HDM (Histone Demethylase)
Methylation 2
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Common pattern in many cancers
 Loss of H4K16 acetylation and H4K20 tri-methylation
When tumor suppressor genes are down-regulated by hypermethylation,
oncogenes may be stimulated by acetylation or hypomethylation
 Example: hypermethylation of H3K79 promotes leukemogenesis
Tumor-specific epigenetic abnormalities can stem from altered
modifications of the histone residues, and/or altered expression of the
enzymes that catalyze the modifications
Methylation 3
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Histone lysine resideus are methylated by methyltransferases and
utilizes S-adnosyl methionine (SAM) in catalyzing the transfer of methyl
group to specific histone residues
Methyltransferases are specific based on target residues
 PKMT (Protein lysine methyltransferase) for lysine
 PRMT (Protein Arginine MT) for R
 PRMT primarily catalyze mono- and di-methylation of histone R
2,8,17, 26 of H3, and R 3 of H4
 H3K27 methylation is mediated by a PKMT called EZH2, which is
over-expressed in many tumors and considered to be responsible
for cancer aggressiveness
 Leukemogenesis is promoted by aberrant recruitment of H3K79
Role of Methylation
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Gene silencing (exceptions are found)
Maintaining cellular functions and development of autoimmunity and
aging
Aberrant methylation
 may be associated with disorder of gene expression
 Irregular memory function in development by heritability
Reversible process
 Demethylation by enzymes such as DNA glycolaes
2.b. Histone Variants
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Results from sequential and structural variation of core histones
 Replacement of large groups of AAs in histone tails and globular
central domains
 Only a few AA substitutions
Four core Histones are incorporated into nucleosomal structure
exclusively during replication
Histone variants can be integrated into specific regions of genome
throughout cell cycle
3. Nucleosomal Remodeling
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Chromatin structure is changed from net energy input
 Nucleosome remodeling is carried out by enzymes that are
catalytically dependent on ATP as energy source
Gene regulation
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J.Su, et al., “Revealing epigenetic patterns in gene regulation …,” Mol.
Biol Rep, 2012
Chromatin components mainly include
 Histone modifications
 Histone variants
 DNA-binding proteins and associate complexes
In mammalian genomes, chromatin components and DNA methylation
are associated with chromatin regulation, and influence gene
transcription
How they regulate chromatin structure and gene expression has
implications for understanding development, aging and disease
Most histone modifications occur at the flexible N-terminal tails
Gene regulation
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Histone acetylation – gene activation
Histone methylation – gene activation and repression
example.
 Enrichment of H3K9ac and H3K4me3 in promoters and CpG
islands are associated with gene repression
 Histone variant H2A.Z and RNA pol-II are preferentially deposited in
promoters, yielding gene activation
Gene Expression
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How chromatin components and DNA methylation affect gene
expression? Independently or synergistically ?
41 chromatin components
16,003 promoters are examined
Genes divided into high- and low-expression according to 50% present
and absent – 7,911 high- and 8,092 low-expression genes
Modification intensities of HGP (High-expression gene promoter) & LGP
are plotted
Gene Expression
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Modification intensities except H3K9me2 and H4K20me3 are
significantly distinct between HGPs and LGPs
Chromatin Structure Alteration
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Alteration of chromatin fiber structure is critical to control cellular
processes and regulate the expression fidelity of genes in particular cell
types
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Such regulation is carried out by a combination of several factors,
posttranslational histone tail modification, chromatin remodeling
enzymes.
One factor contributing this process comes from architectural proteins
such as H1 and members of HMG (high-mobility-group) superfamily
 HMG superfamily – HMGA, HMGB, HMGN
 HMGN – unique in its ability to bind directly to the nucleosome core
particles
 HMGN – associated with generation and maintenance of open
chromatin regions
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HMGN1 is enriched in
transcriptionally active
domains.
Transcriptionally inactive
chromatin is marked by
H3K27me3.
Active chromatin marks
such as H3K4me3 and
H3K9ac, is seen close to the
gene
Actrively transcribed with
RNA Pol II across promoter
Expression Level
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BT Wilhelm, et al., “Differential patterns of intronic and exonic DNA
regions …,” Genome Bio, 2011
Splicing is initiated together with transcription in a chromatin
 Maybe possible to have a functional relationship between splicing
and local chromatin environment
 Different markings in introns and exons may influence splicing
 Highly transcribed genes tend to be efficiently spliced
Data
 FAIRE (Formaldehyde-assisted isolation of regulatory elements) –
histone H3 occupancy and protein free area
FAIRE (red) – genefree
Histone H3 (blue)
H3K36me3 (green)
Pol II (black)
Alzheimer’s Disease
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AD
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Neurodegeneration in brain regions including temporal and paretal
lobes and restricted regions in frontal oortes and cingulate gyrus
Exracelluar amyloid deposits (senile plques, SP) and the presence of
neurofibrilliary tangels (NFT) composed of intraneuronal aggregates
of hyperphosphorylated tau protein
Primary component of SP is about 40 bp amyloid β (Aβ), resulting
from proteolytic processing of its precursor, amyloid precursor
protein (APP)
APP is processed by β- and γ-secretase (presenilin and other protein
complex) to produce Aβ: Aβ40, Aβ42
A high Aβ42/Aβ40 => AD
AD
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1% early AD
 50% due to mutations in APP, PSEN1, PSEN2
 50% may involve other
LOAD (Late-onset AD) over age 65
 ALZGene database (alzgene.org) lists over 1000 genes
 Most like genes
 APOE (apolipoprotein E)
 BIN1 (bridging integrator 1)
 CLU (clusterin)
 Found to have decreased folate values and increased plasma
homocysteine levels (hyperhomocysteinemia)
One-carbon metabolism
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Folate
 Essential nutrients required for one-carbon biosynthetic and
epigenetic process
 Derived entirely from dietary sources, mainly from green vegetables,
fruits, cereals, and meat
 After intestinal absorption, folate metabolism requires reduction
and methylation into the liver to form 5-methylterahydrofolate (5MTHF), release into blood and cellular uptake
 5-MTHF is used for synthesis of DNA and RNA precursors or for
conversion of homcyctein (Hcy) to methionine, which is used to form
S-adenoylmethionine (SAM)
 B6 and B12 participate in one-carbon metabolism
 Folic acid is used for
 DNA methylation process
 Synthesis of nucleic acid precursors
One-carbon (Folate) Metabolism
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MTR (methionine
synthase) transfers a
methyl group from 5methulTHF to methionine
and tetrahydrofolate
(THF).
Met is converted to SAM
(S-adenosylmethionine).
SAM transfers mythyl
group and is converted to
SAH (Sadenosylhomocycteine)
One-carbon (Folate) Metabolism
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DNMT (DNA methyltransferase) is the key enzyme for DNA
methylation
DNA methylation is dependent on its potential measured by
SAM/SAH level
High SAH level inhibit DNMTs (DNA methyltransferases)
High Hcy levels are found in AD patients
Integrative Genomics
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RD Hawkins, et al., “Next-generation genomics: an
integrative approach,” Nature, 2010
Genomic Data Sets Available
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Sequence variation data from individual genomes
Transcriptome
Epigenomic data – methylation in MethDB
Interactome – RNA-protein, protein-protein
Integrative Genomics
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Can address questions related to fundamental mechanisms
of genome function and disease
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How might particular risk-associated SNPs affect cellualr function,
leading to a disease ?
What functional sequences exist in human genome ?
How are key development pathways regulated by epigenetic
mechanisms ?
Annotating functional features of genome
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Regulation elements are not fully understood
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Enhancers, insulators
From characteristics of known Res, identify novel elements
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Chromatin signature of enhancers are used to find new enhancers
Inferring function of genetic variants
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SNPs in non-coding regions
are still poorly defined
SNVs (single-nucleotide
variants) in transcription
factor binding sites or
chromatin-marked
regulatory elements may be
used to determine
regulatory SNPs
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SNP at Pol II bonding regions
cause variability of gene
expression levels