epigenetic dysregulation talk at ngsasia 2016
TRANSCRIPT
AltunaAkalin
Epigene.cdysregula.onincancer
@AltunaAkalin
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Outline
• Epigene.cdysregula.oninacutemyeloidleukemia(AML)– Whatisepigene.cs– WhatisAML– Muta.onscausingepigene.cdysregula.onandtheireffects
• Strategiesforanalyzingbisulfite-seqdata– Align+Processrawdata– Findinteres.ngregions(changesinmethyla.on)– Annotate
Epigene.cs
Epigene.csisthestudyofheritablechangesotherthanDNAsequencethataffectgeneac.vity.• DNAMethyla.on(andotherbasemodifica.ons)
• HistoneModifica.ons
• RNA-Associatedcontrol
Me OH
H3K4me3 H3K4me3
ncRNA
X1000
X1000
X1000
- Nofamilyhistory- Nopriorblooddisease- Highrelapseratesforcertainsubtypes
AcuteMyeloidLeukemia(AML)
Fromcancer.gov
• A malignant disease of the bone marrow where precursor cells arrested in early stage of development
Howdonormalcellstransformtoacutemyeloidleukemia?
Growthadvantagebyalteringsignaling
pathways
Altera.onsindifferen.a.oncontrol
genes
ClassImuta.ons ClassIImuta.ons
Howdonormalcellstransformtoacutemyeloidleukemia?
Growthadvantagebyalteringsignaling
pathways
Altera.onsindifferen.a.oncontrol
genes
ClassImuta.ons ClassIImuta.ons
Altera.onsinhydroxymethyla.onIDH1,IDH2&TET2
ClassIII?
Altera.onsinepigene.cmodifiers
MLL,DNMT3A
ClassIV?
(SeeShihetal.2012Nat.Rev.Cancer)
Twomainsub-sec.ons
① ComparisonofclassIII(hydroxymethyla.ondisrup.on)mutantsagainstclassIV(altera.onofepigene.cmodifiers)mutants
② Focusonmuta.onsthatimpair
hydroxymethyla.onpathway
① ComparisonofclassIIImutantsagainstclassIVmutants
IDH mutant MLL rearrangement
Known to induce DNA hypermethylation via altering hydroxymethlation pathway
fusion protein disrupting H3K4 methyltransferase domain
AML subtypes
ClassIII ClassIV
RRBS (Reduced Representation Bisulfite Sequencing)
MspIdiges.on
Sizeselec.on(40-220bp)
bisulfitetreatment
PCRandsequenceA Cm G Cm G A C A C A
A C G C G A T A T A Bisulfite treatment and sequencing
Sizeselec.on(70-320bp)
eRRBS
Lessstar.ngmaterial1000ng->5ng
CG....CG……..CG
70%
10% 10%Methyla.onscoreperChWp://code.google.com/p/methylkit/
Akalinetal.(2012)GenomeBiology
methylKit
| | | | | | | | | |
MLL
Differentially methylated CpGs (DMCs)
Hypo-methylated DMCs
Hyper-methylated DMCs
IDH
Akalin,GarreW-Bakelmanetal.(2012).PLoSgene)cs,8(6),e1002781
② Focusonmuta.onsthatimpairhydroxymethyla.onpathway
Demethyla.on
DNMTs
5mC
5hmCC
TETs
TETs
Intermediates
Conversionof5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC)
IDH1/2
TET2
5hmC5mC
α-KG
α-KG
isocitrate
Me OH
IDH1/2muta.onsdisrupt5hmCproduc.on
IDH1/2
TET2
5hmC5mC
isocitrate
2HG
2HG
Mutated
TET2muta.onsdisrupt5hmCproduc.on
IDH1/2
TET2
5hmC5mC
α-KG
α-KG
isocitrate
Mutated
Muta.onsdisrup.ng5hmCproduc.oninAML
• IDH1/2,TET2andWT1muta.onsaremutuallyexclusiveinAML=>hintsthattheyareonrelatedpathway
Howthesemuta8ons
affectgenome-wide5hmCprofiles?
DoesWT1mutanthas
thesameeffecton5hmC?
WT1muta.oncouldalsocausemethyla.ondysregula.on
Rampal,Akalin,Madzoetal.(2014)CellRep.
IDH1/2
TET2
5hmC5mC
α-KG
α-KG
isocitrate
5hmCprofiling
5mCprofiling
WT1
Mutated
MutatedMutated
“AnewpathtoleukemiawithWIT”
Sardina&Graf.(2015)Mol.Cell
StrategiesforanalyzingBS-seqdata
SequencingQualityCheck&removeadapters
Aligntothegenome:Bismark,
BSMAPetc.
Methyla.onCalling:usescoverageandqualityscores
Differen.alMethyla.on
Visualiza.on&Clustering/PCA:methylKitorRbase
func.ons
Methyla.onsegmenta.on
ResultsasTables,BEDfiles,summarysta.s.cs
Checkconversion
rates
Annota.onforDMRs/DMCs
CGGGATGAAGGCCCCCGAGCTCCCCGAGAGCAGCGTCAGGGCACGGATGC
BisulfiteReads
Genome
TGGGATGAAGGTTTTCGAGTTTTTCGAGAGTAGCGTTAGGGTGGGATGAAGGTTTTCGAGTTTTTCGAGAGTAGCGTTAGGGTATGGGATGAAGGTTTTCGAGTTTTTTGAGAGTAGCGTTAGGGTACTGGGATGAAGGTTTTTGAGTTTTTTGAGAGTAGCGTTAGGGTACGTGGGATGAAGGTTTTTGAGTTTTTCGAGAGTAGCGTTAGGGTACGG GGATGAAGGTTTTTGAGTTTTTCGAGAGTAGCGTTAGGGTACGGATGT GATGAAGGTTTTCGAGTTTTTCGAGAGTAGCGTTAGGGTACGGATGT TGAAGGTTTTCGAGTTTTTCGAGAGTAGCGTTAGGGTACGGATGT
Methylationproportion
1.0
0.5
0.0
Akalinetal.(2012)GenomeBiologyAkalinetal.(2012)PLoSGene.csRampal,Akalinetal.(2014)CellReports
Differen.almethyla.on
Baubec&Akalin,2016,Springer-Over-dispersioncorrec8on
- Differen.allymethylatedregionscouldbeassociatedwithregulatorychanges
- Abnormalmethyla.onishallmarkofcancer
Segmenta.onofDNAmethyla.onprofiles
Dipsinmethyla.onprofilemarksregulatoryregions
Stadler,2011,Nature
0 200 400 600 800 1000
0.0
0.2
0.4
0.6
0.8
1.0
CpGs over a locus
Met
hyla
tion
prop
ortio
n
0.0 0.2 0.4 0.6 0.8 1.0
01
23
methylation
dens
ity
0.0 0.2 0.4 0.6 0.8 1.0
01
23
4
methylation
dens
ity
Hypo M.State
Full M.StateMethylation
States
Hypo methylated regionsFully methylated regions
Emission distribution for Hypo M. state Emission distribution for Fully M. state
0.6
0.4
0.2 0.8
a) b)
0 200 400 600 800 1000
0.0
0.2
0.4
0.6
0.8
1.0
CpGs over a locus
Met
hyla
tion
prop
ortio
n
0.0 0.2 0.4 0.6 0.8 1.0
01
23
methylation
dens
ity
0.0 0.2 0.4 0.6 0.8 1.0
01
23
4
methylation
dens
ity
Hypo M.State
Full M.StateMethylation
States
Hypo methylated regionsFully methylated regions
Emission distribution for Hypo M. state Emission distribution for Fully M. state
0.6
0.4
0.2 0.8
a) b)
HMMproducesthemostprobablestateforeachCpG
Change-pointanalysisformethylomesegmenta.on
• Findchange-points• Clustersegmentsusingmixturemodelling
hWp://code.google.com/p/methylkit/alsoBioCAkalinetal.(2012)GenomeBiology
Annota.onandintegra.onwithothergenome-widedatasets
BAMBigWigBEDGFFTab txtGRanges
BEDGFFTab txtGRanges
Summarize
Annotation
Genomic Intervals
Annotate
Visualize & Cluster
Base-pairs/ bins1 2 3 4 ... n
ScoreMatrix/ScoreMatrixList object
region 1
region 2
region 3
region 4
...
region m
IntergenicIntronExonPromoter40.9
11.6
21.825.7
−1000 −500 0 500 1000
0.0
0.2
0.4
0.6
0.8
1.0
base-pairs around anchor
read
per
milli
on TF4TF3TF2TF1
−100
0
−50
0
0
500
100
0
0 0.5 1 1.5 2
TF 4
−100
0
−50
0
0
500
100
0
0 0.5 1 1.5 2 2.5
TF 3
−100
0
−50
0
0
500
100
0
0 0.5 1 1.5 2 2.5
TF 2
−100
0
−50
0
0
500
100
0
0 0.5 1 1.5 2 2.5
TF 1
−1000 −500 0 500 1000
base-pairs around anchor
TF1TF2TF3TF4
0.07
20.
340.
60.
861.
1
meta-region plots meta-region heatmaps
heatmaps for genomic interval sets
Piecharts for annotation
hWp://bioinforma.cs.mdc-berlin.de/genoma.on/alsoBioC Akalin,Frankeetal.(2014)Bioinforma.cs
Acknowledgements
- RossLevine
-DirkSchübeler
- Maria E. Figueroa
- Ari Melnick - Christopher Mason
- Vedran Franke - Kasia Wreczycka - Alex Godschan
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