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 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

• 3.4A per base• 3 Billion bases

• 1.8 meters of DNA• 0.09 nm of chromatin after

being wound on histones• Five families of histones

• H1/H5, H2A, H2B, H3, and H4

Chromosome Length

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.

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).

A nucleosome core has about 146 bp of DNA

Typical exon of around 140 nt

Three Major Levels of Epigenetic Changes1. Chemical modifications at the level of nucleotides

• Including DNA methylation and RNA interference2. Modifications at the level of histones that encompass

posttranslational modifications (PTMs) of histone proteins and the incorporation of histone variants

3. Nucleosome remodeling• 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 Covalent addition of a methyl group from methyl donor SAM

(S-adenosylmethionine) 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 thetransfer of a methyl group from SAM to cytosine, thus forming 5-methyl-cytosine and SAH. Methylation of CpG sequencesmight induce chromatin conformational modifications and inhibit the access of the transcriptional machinery to gene promoterregions, thus altering gene expression levels. Therefore, promoter rmethylation of CpG islands is commonly associated withgene silencing and promoter demethylation with gene expression, though several exceptions to this rule are known.

Methylation Inheritance

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 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 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 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 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-grayH2A – blueH2B – yellowH3 – greenH4 - red

PTM

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

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 byrepresentations of the residue on which the posttranslational modification has occurred at respective sites. Abbreviations are as follows: K, lysine;R, arginine; Y, tyrosine; S, serine; T, threonine; -ac, acetylation; -me, monomethylation; -me2, dimethylation; -me3, trimethylation; -ph, phosphorylation.The color code is as follows: yellow, carbon; blue, nitrogen; pink, polar hydrogen; red, oxygen; orange, phosphorus; green, methyl groupsof posttranslational modifications.

Red – acetyl group

Yellow - methylation

Green – methylation of R

HAT (Histone Acetyltransferease) HMT (Histone Methyltransferase)HDAC (Histone Deacetylase) HDM (Histone Demethylase)

Effects of acetylation on protein functions. Acetylation of proteins affects many different functions, some of which are listed. Thedouble up-arrows indicate increase and the double down-arrows indicate decrease with respect to the particular function.Some of the genes affected by acetylation under specific protein functions are listed [60].

Methylation

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

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 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

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

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

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 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 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 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

Modification intensities except H3K9me2 and H4K20me3 are significantly distinct between HGPs and LGPs

Chromatin Structure Alteration Alteration of chromatin fiber structure is critical to control

cellular processes and regulate the expression fidelity of genes in particular cell types

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

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 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) – gene-free

Histone H3 (blue)

H3K36me3 (green)

Pol II (black)

Alzheimer’s Disease AD

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 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 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 (5-MTHF), 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 MTR (methionine

synthase) transfers a methyl group from 5-methulTHF to methionine and tetrahydrofolate (THF).

Met is converted to SAM (S-adenosylmethionine).

SAM transfers mythyl group and is converted to SAH (S-adenosylhomocycteine)

One-carbon (Folate) Metabolism

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

RD Hawkins, et al., “Next-generation genomics: an integrative approach,” Nature, 2010

Genomic Data Sets Available Sequence variation data from individual genomes Transcriptome Epigenomic data – methylation in MethDB Interactome – RNA-protein, protein-protein

Integrative Genomics

Can address questions related to fundamental mechanisms of genome function and disease 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

Regulation elements are not fully understood Enhancers, insulators

From characteristics of known Res, identify novel elements Chromatin signature of enhancers are used to find new

enhancers

Inferring function of genetic variants 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 SNP at Pol II bonding

regions cause variability of gene expression levels