Epigenomics Bio5488 Ting Wang 2.15, 2.17/2016
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Epigenomics
Bio5488
Ting Wang
2.15, 2.17/2016
Outline
• Epigenetic phenomenon– A tale of two mice.
– Where is the queen bee?
– The Monarch!
• What is epigenetics/epigenomics?
• Epigenetic mechanisms
• Epigenome in health, in disease and with environment
• Modern epigenomic technology and resources
A tale of two mice
Yellow mouse
• High risk of cancer,
diabetes, obesity;
• Reduced lifespan
Agouti mouse
• Low risk of cancer,
diabetes, obesity;
• Prolonged lifespanMaternal supplements
with Zinc, methionine,
betaine, choline, folate,
B12
Morgan, Whitelaw,
1999
Waterland, Jirtle,
2004
Where is the Queen?
Larvae
Queen Worker
Figure 1. CpG (o/e) bias of protein-coding regions in the honey bee genome. Since the profiles for both queens and workers are virtuallyidentical, only the queen profile is shown.doi:10.1371/journal.pbio.1000506.g001
Figure 2. Comparison of CpG methylat ion profiles in dif ferentially methylated genes generated by two technologies, Solexagenome-wide shotgun sequencing and 454 sequencing of PCR produced amplicons. The ‘‘heat maps’’ represent the 454 sequencing ofPCRamplified segments, whereas the bars illustrate the Solexa reads. The eight nuclear genes for this experiment were chosen from the list of DMGsshown in Tables 3 and S2, taking into account the availability of convenient CpG-containing regions for primer design. Six genes are shown in thisfigure and the others in Figure 3. Gene annotations: GB18602 - membrane protein; GB18207 - cadherin; GB15132 - TAP42 (TORsignaling); GB14848 -clathrin assembly protein; GB15356 - syd, chromosome segregation; GB11061 - seryl-tRNA synthetase.doi:10.1371/journal.pbio.1000506.g002
Brain Methylomes in Honey Bees
PLoS Biology | www.plosbiology.org 4 November 2010 | Volume 8 | Issue 11 | e1000506
Workers are more methylatedInhibition of Dnmt3 phenocopy Royal Jelly
The King of Butterflies
The Monarch
Same Genome
Different Epigenome
Different Phenotype
What is Epigenetics/Epigenomics?
• A mitotically or meiotically heritable state of different gene activity
and expression (phenotype) that is independent of differences in
DNA sequence (genotype) – based on Conrad Waddington, 1942
• The sum of the alterations to the chromatin template that
collectively establish and propagate different patterns of gene
expression (transcription) and silencing from the same genome.
• Epigenetic changes influence the phenotype without altering the
genotype.
• While epigenetics often refers to the study of single genes or
sets of genes, epigenomics refers to more global analyses of
epigenetic changes across the entire genome.
The EpigenomeThe complete number, location, and types of
epigenetic modifications that occur in a given cell.
Conrad Hal Waddington
(1905–1975)
Developmental biologist
Paleontologist
Geneticist
Embryologist
Philosopher
Founder for systems biology
Epigenetic Landscape
Inheritance, broad definition of
epigenetics
Epigenetics Mechanisms
Gene Expression
RNA Interference
Histone Modifications
Nucleosome PositioningDNA Methylation
• DNA methylation
– Normal cells: role in gene expression and chromosome stability
– Cancer cells: consequences of aberrant hypo- and hyper-methylation
• Histone modification
– Normal cells - the histone code
– Cancer cells - consequences of altered histone modifying enzymes
• Interaction between DNA methylation, histone modifications and small RNAs
• Cell/tissue type specificity
• Gene/Environment interaction, disease susceptibility
Epigenetic mechanisms
What is DNA Methylation?
The 5th base
The mystery of DNA demethylation
DNA methylation is not distributed evenly
in the mammalian genome
• In human somatic cells, 60%-80% of all CpGs
(~1% of total DNA bases) are methylated
– Most methylation is found in “repetitive” elements
• “CpG islands”, GC-rich regions that possess a high density of CpGs, remain methylation-free
– The promoter regions of ~70% of genes are embedded in CpG islands
DNA Methylation is Heritable
me C
me C
G
G
SAM
SAH
DNMT
5’
3’
3’
5’
me C G
G C me
TT G
C
A
me
One-carbon
donors
DNMT: DNA methyltransferaseSAM: S-adenosyl-methionineSAH: S-adenosyl-L-homocysteine
Two classes of DNA methyltransferases
(DNMTs)
Jones and Liang, 2009
Nature Review Genetics
DNMT1 maintenance lethal
DNMT2 remains mystery, possibly a RNA methyl-transferase
DNMT1o maternal imprints lethal*
DNMT3a de novo lethal
DNMT3b de novo lethal
DNMT3L maternal imprints lethal *
(indirect)
function knock-out
Two classes of DNA methyltransferases
(DNMTs)
Normal pattern and function of
DNA Methylation
Function of DNA Methylation in
Mammalian System
• Host defense - endogenous parasitic sequence
(repeats, etc.)
• Imprinting
• X chromosome inactivation
• Heterochromatin maintenance, chromosome
stability, telomere length
• Gene expression controls
DNA methylation changes during
developmental epigenetic reprogramming
Normal Patterns of DNA Methylation
1 2 1 2 3
CpG islands and gene expression
Pericentromeric regions - stability
Transcription
Methylated CpG
Unmethylated CpG
DNA methylation pattern across a gene
structure
Lister et. al, Nature 2009
Methylation and Gene Expression
Gene
Gene
Transcription
No TranscriptionMethylated
CpG dinucleotides
Unmethylated
Mechanisms of gene silencing by
methylation
Direct mechanism:
Inhibition of transcription factor binding (eg.CTCF, UBF)
Not a universal mechanism since not all transcription factor binding sites contain CG dinucleotides
Indirect mechanism:
Inhibition mediated by methyl-CpG binding proteins MeCP1/ MeCP2
Recruitment of corepressor complexes including histone deacetylases(HDAC)
Change in chromatin conformation
MBD
me
Protein Mutant phenotype
MeCP2 Rett syndrome
MBD1
MBD2 behaviour abnormalities
MBD3 lethal
MBD4 increased mutation frequency
(a glycosylase for mismatch repair)
Methylated DNA binding proteins
DNA methylation in cancer
Point mutations
Deletions
CpG island hypermethylation
Chromosomal instability
Gene amplification
Genomic hypomethylation
Translocations
Copy numberchanges
Tumor Suppressor Gene Inactivation in
Human Cancer
Normal Tumor
Methylation
x
Mutation
And
Deletion
Homozygous
Deletion
CH3
Knudsen’s 2-hit hypothesis, 1971
Aberrant DNA Methylation in Cancer
1 2 1 2 3
CpG islands hypermethylation
Pericentromeric hypomethylation
STOP
Methylated CpG
Unmethylated CpG
Robertson, Nature Reviews Genetics, Vol6, 597
Different regions of the genome are
hypermethylated or hypomethylated in cancer
Hypomethylation and Cancer
• The overall level of 5-methylcytosine is decreased in a proportion of human tumors. (Gama-Sosa et al. 1983)
• Hypomethylation of c-Ha-ras and c-Ki-ras, two cellular oncogenes, was identified in primary colon tumors. (Feinberg and Vogelstein 1983)
• Loss of IGF2 imprinting increases colorectal cancer risk (Cui et al. 2003)
• Consequences of Hypomethylation?
– Chromosome instability (ICF syndrome)
– Transcriptional activation of retrotransposons
– Transcriptional activation of oncogenes
Methylation promotes mutation
Jones & Baylin (2002)
DirectIndirect
DNA methylation and other
diseases
-- Imprinting Disorder:
• Beckwith-Wiedemann syndrom (BWS)
• Prader-Willi syndrome (PWS)
• Transient neonatal diabetes mellitus (TNDM)
-- Repeat-instability diseases
• Fragile X syndrome (FRAXA)
• Facioscapulohumeral muscular dystroph
-- Defects of the methylation machinery
• Systemic lupus erythemtosus (SLE)
• Immunodeficiency, centromeric instability and
facial anomalies (ICF) syndrome
Imprinting Diseases:
Angelman and Prader-Willi Syndromes
Angelman Syndrome
• “Happy puppet”
• Severe mental retardation
• Absence of speech
• Happy disposition
• Excessive laughing
• Hyperactive, with jerky repetitive motions
• Red cheeks, large jaw and mouth
Prader Willi Syndrome
• Small hands and feet
• Underactive gonads,
tiny external genitals
• Short stature
• Mentally retarded
• Slow-moving
• Compulsive overaters
• Obese
Imprinting Diseases:
Angelman and Prader-Willi Syndromes
Chr15 deletion, ~4Mb
Loss of maternal contribution
UBE3A
Angelman Syndrome
Loss of paternal contribution
SNRPN
Prader-Willi Syndrome
Genomic imprinting
• Imprinting is unique to mammals and flowering plants. In mammals, about 1% of genes are imprinted.
• For imprinted genes, one allele is expressed and the other is silent.
• This is typically controlled epigenetically. The expressed alleles are unmethylated and associated with loosely packed chromatin.
• Imprinted genes bypass epigenetic reprogramming.
• Imprinting is required for normal development
Why imprinting?• The Genetic Conflict Hypothesis
– Many imprinted genes are involved in growth and metabolism.
– Paternal imprinting favors the production of larger offspring, and maternal imprinting favors smaller offspring
• Imprinted genes are under greater selective pressure.– No back up!
– Any variation in single gene is expressed
– Closely related species have different imprinting patterns
• Liger and Tigon
• Imprinted genes are sensitive to environmental signals.
What we don’t know about imprinting
• What targets a gene for imprinting?
– Why are some genes expressed from both alleles and other expressed from only one allele?
• How are the imprints imposed?
– Do males and females have different mechanisms for imprinting genes?
Rett Syndrome
• X-linked trait
• Mainly girls affected
• Normal at birth
• At 6-18 months, begin
losing purposeful
movement
• Persistent wringing of
hands
• Loss of speech, gait
• Mental retardation
ensues
Nature Genetics (1999) 23:127-128
Rett’s is due to defect in MeCP2
• Methyl-cytosine binding protein 2 (MeCP2) binds methylated DNA and recruits binding of a histonedeacetylase
• Normal role is tightening chromatin packing, leading to gene silencing
Mouse model for Rett’s
• Mice have a gene that is homologous to MeCP2
• Knocking out the gene in mouse gives a phenotype similar to human Rett’s
• This model offers good experimental system for studying the human disease
Male mice with MeCP2 knockout
develop normally for a while (middle),
but at 6 weeks of age, they begin to
develop neurological symptoms, such
as hindlimb clasping (right).
Natu
re G
enetics (
2001)
27:3
32
-336
Why is the phenotype neurological?
• The phenotype suggests that the targets are genes in the brain
• Normal neurological differentiation requires silencing of MeCP2 gene target(s)
• The target(s) of MeCP2 are not known
DNA Methylation with age,
environment
Differences in DNA methylation patterns between
identical twins increase with aging
Fraga et alPNAS 102:10604, 2005
Maternal Bisphenol A (BPA) Exposure
• Monomer that makes up polycarbonate plastic
• Endocrine active compound
• Found in commonly used products
• Present in 95% of humans tested
• Some animal studies reveal negative health outcomes
p-value =0.0074
Yellow SlightlyMottled
Mottled HeavilyMottled
PseudoAgouti
Perc
ent
of
Offsp
ring
0
5
10
15
20
25
30
35
Control Diet (n=60)
BPA Diet (n=73)
Maternal BPA Exposure Results: Coat Color Distribution
OHOH
HO
O
O
OHOH
HO
OOHOH
HO
O
O
OHOH
HO
OOHOH
HO
O
O
OHOH
HO
OOHOH
HO
O
O
Maternal Genistein Supplementation
• Plant phytoestrogen
• Found in soy and soy products
• Selective estrogen receptor modulator
• Worldwide exposure varies by diet
• Chemoprevention and decreased adipocyte deposition
Maternal Genistein SupplementationResults: Coat Color Distribution
0
10
20
30
40
Yellow SlightlyMottled
Mottled HeavilyMottled
PseudoAgouti
Perc
ent
of
Offsp
ring
Control (n=52)
Genistein Diet (n=44)
p-value =0.0005
Dolinoy, D et al (2006) Environ Health Perspect
Maternal Nutritional SupplementationResults: Genistein – Coat Color
Control Diet (n=60)
BPA Diet (n=73)
Perc
ent
of
Offsp
ring
0
5
10
15
20
25
30
35
Genistein Supplemented BPA Diet (n=39)
Yellow Slightly Mottled Heavily Pseudo-Mottled Mottled agouti
p-value=0.96 (control versus genistein supplementation)
DNA methylation
+
Histone modification
Chromatin
- 2 each of histones:
H2A,H2B, H3 and H4
Chromatin
DNA plus Protein in cells with nuclei
146 bp of DNA
Nucleosome
The Nucleosome core particle
Nucleosome
H3
H4
http://www.nature.com/nsmb/journal/v14/n11/images/nsmb1337-F1.gif
Post-translational Histone Modifications
Post-translational Histone Modifications
H3 tail Modifications:
Active
HDACsHATs
=Acetylation
=Methylation
KMTases
Repressive
Li e. al. (2007) Cell 128, 707
Histone Modifications in Relation to Gene Transcription
Histone Modifications in Relation to Gene Transcription
SRPK1 SLC26A8
MAPK14
Bisulfite-Seq
H3K27ac
H3K4me1
H3K4me3
H3K36me3
H3K27me3
H3K9me3
RefSeq genes
Predicting non-coding RNA?
• From sequence?
– Not clear which properties can be exploited
– Sequence features such as promoters are too
weak
• Histone modifications + conservation worked
DNA methylation mediated repression
Repression independent of DNA methylation
H3K9 methylation
“condensed” chromatin
H3K27 methylation mediated repression
1. H3K27 methylation
1. DNA methylation
Mechanisms of Epigenetic Crosstalk
“Epigenetic cancer therapy”
DNA-methylation and HDAC inhibitors
in clinical trials
Summary
• Dnmt1, Dnmt3A, Dnmt3b - the mammalian DNMTs
• Chromatin structure is influenced by covalent
modification of histone tails
• Multiple chromatin modification pathways involved in silencing
of genes which may show “crosstalk” with DNA methylation
How to detect epigenetic marks?
Restriction Landmark Genome Scanning (RLGS)
NotI
NotI
NotI
NotI
NotI
NotI
UnmethylatedPartial
MethylationHomozygous
Methylation
EcoRV, 1-D, HinfI, 2-D
1st-D
2nd-D
1st-D
2nd-D
1st-D
2nd-D
NotINotI
NotI
NotI digest endlabeling
Scaling to high coverage, high resolution
• Enrichment based methods– MeDIP-seq
– MBD-seq/MethylMiner
• Restriction enzyme based methods – MRE-seq
– HELP, Methyl-MAPS, Methyl-seq
• Bisulfite based methods– MethylC-seq
– RRBS, bisulfite padlock
• Direct reading of modified nucleotides– SMRT
– Nanopore sequencing
Enriching for methylated DNA
targets
CCGG
GGCC
CCGG
GGCC
Me
Me
Me MeMe
Me
MeMe
Sonicate gDNA
Size selection, end repair
Adapter ligation
Denaturation
Size selection
IP
MeDIP-seq and MBD-seq
5’ CpG islands
are unmethylated
3’ CpG island is
partially methylated
Methylated
Unmethylated
Typical MeDIP data on a genome browser
Taking advantage of methylation
dependent restriction enzymes
MRE-seq
CCGG
GGCC
CCGG
GGCC
Me
Me
CCGG
GGCC
C CGG
GGC C
Me
MeDigest gDNA (here with Hpa II)
Combine parallel digest
Size selection, end repair
Adapter ligation
Size selection
5’ CpG islands
are unmethylated
3’ CpG island is
partially methylated
Methylated
Unmethylated
Typical MeDIP/MRE data on a genomebrowser
Scale
chr3:
CpG Islands
CpG sites
5 kb
37033000 37034000 37035000 37036000 37037000 37038000 37039000 37040000 37041000EPM2AIP1
MLH1
MLH1
MLH1
MLH1
hMLH1
MLH1
MLH1
MLH1
Endo Normal Medip Density
Endo Normal MRE CpG
Endo 1099 Medip Density
Endo 1099 MRE CpG
Endo 1113 Medip Density
Endo 1113 MRE CpG
Endo 1647 Medip Density
Endo 1647 MRE CpG
Endo 2242 Medip Density
Endo 2242 MRE CpG
Endo 2249 Medip Density
Endo 2249 MRE CpG
Endo 2263 Medip Density
Endo 2263 MRE CpG
MLH1 Promoter
Normal
Type II
Type I
Allele-specific methylation at imprinted genes
Chr 7:
SNURF/SNURP
MeDIP-seq
MRE-seq
Chr 7:
MEST
MeDIP-seq
MRE-seq
Chr 18:
SMAD4
MeDIP-seq
MRE-seq
imprinted
imprinted
not
imprinted
DMR
DMR
Gold standard: bisulfite
sequencing
Bisulfite sequencing
Methylation call
ACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCCGGGTTTATTTGCTTTTTTATGGGCTGGGTTTATTTGCTTTTTTATGGGCTGGGTTTATTTGCTTTCCTATGGGCCGGGCTTATTTGCTTTCCTATGGGCCGGGCTTATTTGCTTTCCTATGGGC
3/5 0/5
60% methylated 0% methylated
CpG islands
98% of the genome
1 CpG/100bp
majority methylated
<2% of the genome
1 CpG/10bp short stretches (~1000bp)
majority unmethylated
RRBS: Reduced RepresentationsAllow Enrichment of CpG Dinucleotides
Direct detection of modified
nucleotides
Single-molecule, real-time (SMRT) sequencing
Nanopore sequencing
Modern DNA MethylomicsD
ete
ctio
n R
eagent
Sequencin
g P
latfo
rm
Microarray Massively Parallel Beads Arrays
Sequencing
Methylation-sensitive
Restriction Digest
Bisulfite
(C to T;mC to C)
5-meC Antibody
Immunoprecipitation
Methylated DNA
Affinity Column
Methylation-sensitive
Restriction Digest
MRE-seqMicroarray Massively Parallel Beads Arrays
Sequencing
Methylation-sensitive
Restriction Digest
Bisulfite
(C to T;mC to C)
5-meC Antibody
Immunoprecipitation
Methylated DNA
Affinity Column
5-meC Antibody
Immunoprecipitation
Methylated DNA
Affinity Column
MeDIP-seqMBD-seq
(MethylMiner)Microarray Massively Parallel Beads Arrays
Sequencing
Methylation-sensitive
Restriction Digest
Bisulfite
(C to T;mC to C)
5-meC Antibody
Immunoprecipitation
Methylated DNA
Affinity Column
Bisulfite
(C to T;mC to C)
Microarray Massively Parallel Beads Arrays
Sequencing
Methylation-sensitive
Restriction Digest
Bisulfite
(C to T;mC to C)
5-meC Antibody
Immunoprecipitation
Methylated DNA
Affinity Column
MethylC-seq
RRBS
Direct sequencing
(Pol. kinetics)
SMRT
Nanopore
CpG coverageCpG island
coverage
Resolution
(bp)
Illumina
Lanes
Genome
total28 M 28 K NA NA
BS
shot gun26 M 27 K 1
207 (2009)
10 (now)
RRBS 0.2-1M 15 K 1 1
MRE-seq
and
MeDIP-seq
25 M 27 K 1 and 200 8 (2009)
1 (now)
Golden-
Gate1,500 800 1 NA
Infinium27,500
480,000 (2012)12,000 1 NA
Comparison of Methylome coverage
Predicting single CpG methylation level with
Conditional Random Field (methylCRF)
95
Theoretical MRE
fragments
MeDIP
MRE
Infinium
RRBS
Lister BS
GIS BS
methylCRF
Technologies for Interrogating Chromatin States
ChIP-chip
Antibody specific
to one type of
histone
modification
Histone Modifications
ChIP-seq
Deep sequencing
Chromatin-IP Sequencing
K4me1K4me2K4me3K27me3 “repressive”
“active”
A. Align reads
B. Infer positions of ChIP fragments
C. Count fragments at each genomic position
K4me3
K27me3
Silent developmental geneTranscribed gene
K9me3
K20me3
Constitutive heterochromatin
FoxP1 Olig1
Olig1
K4me3
K27me3
‘Poised’ developmental gene
Histone methylation and transcriptional state
Nucleosome Positioning from Histone ChIP-seq
• Barski et al, Cell 2007– Nucleosome resolution ChIP-seq of 21 histone
marks in CD4+ T-cells
– Total 185.7 M
25 nt tags sequenced
– Analysis not at
nucleosome resolution
to map nucleosomes
at specific regions
Antibody for
MNase digest
Combine Tags From All ChIP-Seq
Extend Tags 3’ to 150 nt
Check Tag Count Across Genome
Take the middle 75 nt
DNase I hypersensitivity ~ Regulatory DNA
Inaccessible Inaccessible
Accessible
Precise delineation of the accessible regulatory DNA compartment
Digital DNaseI profiling
Digital DNaseI profiling: direct access to
regulatory sequences
ATAC-seq(assay for transposase-accessible chromatin)
Epigenetic annotation of GWAS hits
ChromHMM
Transcriptio
n Start SiteEnhance
rDNA
Observed
chromatin
marks.
Called based
on a Poisson
distribution
Most likely
Hidden State
Transcribed Region
1 6 53 4 6 6 6 6 5
1:
3:
4:
5:
6:
5
High Probability Chromatin Marks in State
2:
0.8
0.9
0.9
0.80.7
0.9
200bp
interval
s
All probabilities are
learned from the
data
2
K4me3 K36me3 K36me3 K36me3 K36me3K4me1 K4me3 K4me1
K27ac
0.8
K4me1
K36me3
K27ac
K4me1K4me3
K4me3
K4me1 K4me1
ChromHMM
110
Pro
mo
ter
Tran
scri
be
dA
ctiv
e in
terg
en
icR
epet
itiv
eR
epre
sse
d
Chromatin Marks from (Barski et al, Cell 2007; Wang et al Nature Genetics, 2008); DNAseI hypersensitivity from (Boyle et al, Cell 2008); Expression Data from (Su et al, PNAS 2004); Lamina data from (Guelen et al; Naature 2008)
Application of ChromHMM to 41 chromatin marks in CD4+ T-cells (Barski’07, Wang’08)
3D chromatin (HiC-seq)
Other interesting topics
• RNA component
– RNAi, miRNA, X inactivation, HOTAIR,
PiwiRNA
• Reprogramming
• Cloning
• Population epigenetics
• Evolution of DNA methylation
• Evolution of epigenome
Engaging today’s epigenomic
technologies and resources
WUSTLhttp://VizHub.wustl.edu
Accessing the community resource
Multiple Sclerosis:
• an autoimmune disease that affects the brain and
spinal cord (central nervous system).
• a collaborative GWAS involving 9,772 cases of
European descent collected by 23 research groups
working in 15 different countries
Example – got my GWAS hits! Now what?
Related Documents
