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Comprehensive functional testing of ChIP-seq binding sites with ChIP- reporter assays Tim Reddy Duke University ENCODE Users Mee7ng June 9, 2016

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Page 1: Comprehensive functional testing of ChIP-seq binding sites with

Comprehensive functional testing of ChIP-seq binding sites with ChIP-

reporter assays

TimReddyDukeUniversityENCODEUsersMee7ngJune9,2016

Page 2: Comprehensive functional testing of ChIP-seq binding sites with

ChIP-seqhasrevealedtensofthousandsofTFbindingsitesinthehumangenome,farin

excessoftheregulatedgenes.

A549cells(lungepithelialcellline),3h,100nMDexamethasone

0

5000

10000~10,000

297 151

GR Binding Sites(ChIP-seq)

UpregulatedGenes

DownregulatedGenes

Page 3: Comprehensive functional testing of ChIP-seq binding sites with

Majorques7onpostChIP-seq:WhatarealloftheseTFbindingsitesdoing?

A549cells(lungepithelialcellline),3h,100nMDexamethasone

0

5000

10000~10,000

297 151

GR Binding Sites(ChIP-seq)

UpregulatedGenes

DownregulatedGenes

Page 4: Comprehensive functional testing of ChIP-seq binding sites with

Ourmodelsystem:Cor7sol(glucocor7coidsteroidhormone)

Suppressesimmunesystemandreducesinflamma7on

Increasesbloodpressureandbloodsugar

Synthe7cGC’susedclinicallytotreatPsoriasis,Crohn’sDisease,RheumatoidArthri7s

Majorcomponentofresponsetostressresponseandmetabolism

Page 5: Comprehensive functional testing of ChIP-seq binding sites with

Ourmodelsystem:Cor7sol(glucocor7coidsteroidhormone)

Suppressesimmunesystemandreducesinflamma7on

Increasesbloodpressureandbloodsugar

Synthe7cGC’susedclinicallytotreatPsoriasis,Crohn’sDisease,RheumatoidArthri7s

Majorcomponentofresponsetostressresponseandmetabolism

Page 6: Comprehensive functional testing of ChIP-seq binding sites with

Cortisol Production (HPA axis)

Adrenocor7cotropicHormone(ACTH)

Cor7sol

Hypothalamus

Pituitary

Adrenal Glands

Kidneys

Corticotrophin-releasing Hormone (CRH)

Page 7: Comprehensive functional testing of ChIP-seq binding sites with

The Glucocorticoid Receptor (GR)

Page 8: Comprehensive functional testing of ChIP-seq binding sites with

GRbinds>10,000sitesinthehumangenomeandregulateshundredsofgenes.

A549cells(lungepithelialcellline),3h,100nMDexamethasone

0

5000

10000~10,000

297 151

GR Binding Sites(ChIP-seq)

UpregulatedGenes

DownregulatedGenes

Page 9: Comprehensive functional testing of ChIP-seq binding sites with

Reporterassaystoquan7fytheac7vityofGCresponseelements

Luciferase }

Luciferase

GRE

GRE

+DEX

+EtOH

DEX=Dexamethasone,asynthe7cglucocor7coid

Page 10: Comprehensive functional testing of ChIP-seq binding sites with

GFP ACTGATCGAC

Usinghigh-throughputsequencingtomakereporterassayshighthroughput

Patwardhanetal,2012,Sharonetal,2012,Kheradapour,2013,Kwasnieskietal,2012and2014,Melnikovetal,2012and2013,

GFP GTACGTGCA

GFP CAGTCAGTA

GFP ACTATATCA

GFP ACTTAGTA

GRE1

GRE2

GRE3

...

GREN

Page 11: Comprehensive functional testing of ChIP-seq binding sites with

GFP

Regulatoryelementslocatedinthe3’UTRofthereportergene.

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

Page 12: Comprehensive functional testing of ChIP-seq binding sites with

GFP

Fromthatposi7on,theelementsregulatetheirownexpression.

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

Page 13: Comprehensive functional testing of ChIP-seq binding sites with

GFP

High-throughputsequencingofthecloningsiteintheexpressedreportergenecanthenbeusedtoquan7fyregulatoryelementac7vity

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

Page 14: Comprehensive functional testing of ChIP-seq binding sites with

GFP

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

•  Canassaymillionsoffragmentsatonce•  Elementscanbehundredstothousandsofbp•  Allowsdirectliga7onofcapturedDNAintohigh-

throughputreporterassays

Page 15: Comprehensive functional testing of ChIP-seq binding sites with

Chroma7nImmunoprecipita7on+STARR-seqtoquan7fytheac7vityof

allTFbindingsites

Page 16: Comprehensive functional testing of ChIP-seq binding sites with

Chroma7nImmunoprecipita7on+STARR-seqtoquan7fytheac7vityof

allTFbindingsites

Treatwithdrugorcontrol

Page 17: Comprehensive functional testing of ChIP-seq binding sites with

GRChIP-reporterresults

v

•  Assayed>12,000GRbindingsites•  10%wereDEX-responsive•  95%ofregulatoryelementsincreasedreporterexpression•  Validatedwithstandarddual-luciferase(r=0.77)

Page 18: Comprehensive functional testing of ChIP-seq binding sites with

Possibleinterpreta7ons:

Onepossibility:Only10%oftheGRbindingsiteshaveac7vity.Alterna<ve:OtherGRbindingsitesrequireaddi7onalgenomiccontext.

Page 19: Comprehensive functional testing of ChIP-seq binding sites with

SomewaysthatGRbindsthegenomeDirectbindingtoaGlucocor7coidResponseElement(GRE)

TetheredbindingviaotherTFssuchasAP-1

Coopera7vebindingtoacompositeregulatoryelement

Page 20: Comprehensive functional testing of ChIP-seq binding sites with

PresenceofaGREexplainstheresponseCo-bindingTFmo7fsdonot

Page 21: Comprehensive functional testing of ChIP-seq binding sites with

DEX-responsivesitesalsohaveepigene7cstatechangesthatreflectac7vityinthegenome

DEX-responsivenon-responsive

GRE GRE GRE GRE

DEX

EtOH

DEX

EtOH

Page 22: Comprehensive functional testing of ChIP-seq binding sites with

DirectbindingexplainsDEX-responsivereporterac7vity

DirectbindingtoaGlucocor7coidResponseElement(GRE)

TetheredbindingviaotherTFssuchasAP-1

Coopera7vebindingtoacompositeregulatoryelement

Page 23: Comprehensive functional testing of ChIP-seq binding sites with

Theproblem

•  ThesetofGC-responsivegenesvariesdrama7callybetweencelltypes

•  Thosedifferencescanlargelybeahributedtochangesinco-factors,par7cularlyAP-1

•  IfGRbindingatAP-1sitesdoesnothaveac8vity,thenwestruggletoexplaincell-type-specificdifferencesinGC-responses

(e.g.Biddieetal,2011;Johnetal,2011;Gertzetal,2013)

Page 24: Comprehensive functional testing of ChIP-seq binding sites with

Ourmodel

•  AP-1sitesmodulatetheac7vityofdirectGRbindingsitesinthegenome

•  However,theAP-1sitesarenotsufficientforDEX-responsiveregulatoryac7vity

•  Inthismodel,weexpectGRandAP-1toco-clusterinthegenome

Page 25: Comprehensive functional testing of ChIP-seq binding sites with

GRbindsthegenomeinclusters

Page 26: Comprehensive functional testing of ChIP-seq binding sites with

Morenumerousclustershaveasmallerfrac7onofDEX-responsivesites

(i.e.fewdirectsitescannucleatelargeclusters)

Page 27: Comprehensive functional testing of ChIP-seq binding sites with

AP-1sites,representedbyJunD,thatgainGRareclosertodirectGRbindingsites

Page 28: Comprehensive functional testing of ChIP-seq binding sites with

ClustersofGRbindingaredepletedforinterveningCTCF

Page 29: Comprehensive functional testing of ChIP-seq binding sites with

Conclusion

•  GRbidingatnon-responsivesitelikelyreflectsloopinginterac7onswithdistaldirectGRbindingsites

•  DoesthisalterDEX-responsiveregulatoryac7vity,poten7allyexplainingcell-typespecificity?

Page 30: Comprehensive functional testing of ChIP-seq binding sites with

AddingAP-1sitesamplifiesGC-responseby>10-fold

Page 31: Comprehensive functional testing of ChIP-seq binding sites with

Thatamplifica7onisdistantdependent

Page 32: Comprehensive functional testing of ChIP-seq binding sites with

Summaryofourmodel:GRbindingclustersreflectextensive

GR:AP-1tetheringinterac7ons.

Page 33: Comprehensive functional testing of ChIP-seq binding sites with

Summary•  Wedevelopedahigh-throughputapproachtomeasuretheac7vityofeveryGRbindingsiteinareporterassay

•  Theresultsrevealafunc7onaldiversityofGRbindingsites,andsuggestthatinterac7onsbetweendirectandtetheredGRbindingsitesarethebasisforcell-typespecificGCresponses

Page 34: Comprehensive functional testing of ChIP-seq binding sites with

Ongoingwork:GenomicsofGeneRegula7on

ExpandinguponthesefindingswithextensiveandcoordinatedChIP-seq,DNase-seq,RNA-seq,STARR-seq,Hi-CMatchedwithBayesianmodelsofregulatorynetworks,andepigenomeandgenomeedi7ngtotestpredic7ons

A549

Barcoded tubes

RNA-seq

12 x 3 reps=36 TOTAL

12 x 3 reps=36 TOTAL

44 x 12 x 3=1584 TOTAL

12 x 3 reps=36 TOTAL

5 x12 x3 reps=180 TOTAL

Experiment-specific processing pipelineQC metrics to ensure high quality data

Submit data to GEOUse data for network modeling

ChIP-seq42 TFs, 2 hist mods

ChIA-PETGR DNase-seq STARR-seq

5 exps.

10 2 3 4 5 6 7 8 9 10 12

Hours after treatmentAdd DEX

1,872 Exps

Page 35: Comprehensive functional testing of ChIP-seq binding sites with

RNA-seqandDNase-seqreleasedviaENCODEDCC

•  127mepoints,coordinatedtothesecond•  Atleastfourreplicatesofeach7mepoint•  ChIP-seq/Hi-C/STARR-seqdataforthcoming

Page 36: Comprehensive functional testing of ChIP-seq binding sites with

Lookingforpostdocstojointhelab

•  Lookingforbothexperimentalandcomputa7onalscien7sts

•  GenomicsandGene7csprojectsfeaturinghigh-throughputreporterassays,genomeandepigenomeedi7ng,networkmodeling,experimentaldesign,andspecificdiseasestudies.

Page 37: Comprehensive functional testing of ChIP-seq binding sites with

AcknowledgementsReddy Lab:Karl Guo, Ph.D.Chris Vockley (Looking for a postdoc)Anthony D’Ippolito Bill MajorosIan McDowellGraham JohnsonLinda HongSarah LeichterLuke BarteltGersbach Lab:Charlie GersbachTyler KlannJosh Black Isaac HiltonDewran KocakPratiksha ThakoreAmi KabadiLauren Polstein

NorthwesternBill LoweGeoff HayesDenise ScholtensBrian LaydenAnton LudvikMichael Nodzenski

Funding:

PennCasey Brown

PrincetonBarbara Engelhardt

Duke:Greg CrawfordGreg WrayAlex HarteminkBrigid Hogan

R01DK097534(Lowe)R01DK099820(Lowe)

U01HG007900(Reddy)

R01DA036865(Gersbach)

PredoctoralFellowshipsF31HD085666(Guo)

F31HL129743(Vockley)


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Statistical analysis of genomic binding sites using high-throughput ChIP-seq dataetheses.whiterose.ac.uk/12475/1/IBRAHIM_NAFISAH.pdf · 2016. 4. 5. · analysis of ChIP-Seq data,

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From ChIP-chip to ChIP-Seq: the study of mammalian transcription factor binding sites and epigenetics I519 Introduction to Bioinformatics, Fall, 2012

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