AltunaAkalin

Epigene.cdysregula.onincancer

@AltunaAkalin

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•  ~1,600staffmembersandguestscien.stsworkattheMDC•  MDChasbeenranked14thintheThompsonReuterslistofthe

world’sbestresearchins.tutesformolecularbiologyandgene.cs•  Aspecialemphasisonsystemsbiology,theBerlinIns.tutefor

MedicalSystemsBiology(BIMSB)hasbeenestablished

BerlinIns.tuteforMedicalSystemsBiology

Bioinforma.csPlaNormatMDC/BIMSB

-Generegula.on-Comp.(Epi)genomics:diseaseanddev.

Data-intensivecomputa.onalmethodstoinferbiologicalknowledge

Research

Collabora.on

Training

Scien.ficITSupport

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-Maintaindatabases- Maintainscien.ficsoVware

hWp://bioinforma.cs.mdc-berlin.de

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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Funding: