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

reporter assays

TimReddyDukeUniversityENCODEUsersMee7ngJune9,2016

ChIP-seqhasrevealedtensofthousandsofTFbindingsitesinthehumangenome,farin

excessoftheregulatedgenes.

A549cells(lungepithelialcellline),3h,100nMDexamethasone

0

5000

10000~10,000

297 151

GR Binding Sites(ChIP-seq)

UpregulatedGenes

DownregulatedGenes

Majorques7onpostChIP-seq:WhatarealloftheseTFbindingsitesdoing?

A549cells(lungepithelialcellline),3h,100nMDexamethasone

0

5000

10000~10,000

297 151

GR Binding Sites(ChIP-seq)

UpregulatedGenes

DownregulatedGenes

Ourmodelsystem:Cor7sol(glucocor7coidsteroidhormone)

Suppressesimmunesystemandreducesinflamma7on

Increasesbloodpressureandbloodsugar

Synthe7cGC’susedclinicallytotreatPsoriasis,Crohn’sDisease,RheumatoidArthri7s

Majorcomponentofresponsetostressresponseandmetabolism

Ourmodelsystem:Cor7sol(glucocor7coidsteroidhormone)

Suppressesimmunesystemandreducesinflamma7on

Increasesbloodpressureandbloodsugar

Synthe7cGC’susedclinicallytotreatPsoriasis,Crohn’sDisease,RheumatoidArthri7s

Majorcomponentofresponsetostressresponseandmetabolism

Cortisol Production (HPA axis)

Adrenocor7cotropicHormone(ACTH)

Cor7sol

Hypothalamus

Pituitary

Adrenal Glands

Kidneys

Corticotrophin-releasing Hormone (CRH)

The Glucocorticoid Receptor (GR)

GRbinds>10,000sitesinthehumangenomeandregulateshundredsofgenes.

A549cells(lungepithelialcellline),3h,100nMDexamethasone

0

5000

10000~10,000

297 151

GR Binding Sites(ChIP-seq)

UpregulatedGenes

DownregulatedGenes

Reporterassaystoquan7fytheac7vityofGCresponseelements

Luciferase }

Luciferase

GRE

GRE

+DEX

+EtOH

DEX=Dexamethasone,asynthe7cglucocor7coid

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

GFP

Regulatoryelementslocatedinthe3’UTRofthereportergene.

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

GFP

Fromthatposi7on,theelementsregulatetheirownexpression.

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

GFP

High-throughputsequencingofthecloningsiteintheexpressedreportergenecanthenbeusedtoquan7fyregulatoryelementac7vity

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

GFP

STARR-seq:Arnoldetal,Science,2013

STARR-seqreporterassays

•  Canassaymillionsoffragmentsatonce•  Elementscanbehundredstothousandsofbp•  Allowsdirectliga7onofcapturedDNAintohigh-

throughputreporterassays

Chroma7nImmunoprecipita7on+STARR-seqtoquan7fytheac7vityof

allTFbindingsites

Chroma7nImmunoprecipita7on+STARR-seqtoquan7fytheac7vityof

allTFbindingsites

Treatwithdrugorcontrol

GRChIP-reporterresults

v

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

Possibleinterpreta7ons:

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

SomewaysthatGRbindsthegenomeDirectbindingtoaGlucocor7coidResponseElement(GRE)

TetheredbindingviaotherTFssuchasAP-1

Coopera7vebindingtoacompositeregulatoryelement

PresenceofaGREexplainstheresponseCo-bindingTFmo7fsdonot

DEX-responsivesitesalsohaveepigene7cstatechangesthatreflectac7vityinthegenome

DEX-responsivenon-responsive

GRE GRE GRE GRE

DEX

EtOH

DEX

EtOH

DirectbindingexplainsDEX-responsivereporterac7vity

DirectbindingtoaGlucocor7coidResponseElement(GRE)

TetheredbindingviaotherTFssuchasAP-1

Coopera7vebindingtoacompositeregulatoryelement

Theproblem

•  ThesetofGC-responsivegenesvariesdrama7callybetweencelltypes

•  Thosedifferencescanlargelybeahributedtochangesinco-factors,par7cularlyAP-1

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

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

Ourmodel

•  AP-1sitesmodulatetheac7vityofdirectGRbindingsitesinthegenome

•  However,theAP-1sitesarenotsufficientforDEX-responsiveregulatoryac7vity

•  Inthismodel,weexpectGRandAP-1toco-clusterinthegenome

GRbindsthegenomeinclusters

Morenumerousclustershaveasmallerfrac7onofDEX-responsivesites

(i.e.fewdirectsitescannucleatelargeclusters)

AP-1sites,representedbyJunD,thatgainGRareclosertodirectGRbindingsites

ClustersofGRbindingaredepletedforinterveningCTCF

Conclusion

•  GRbidingatnon-responsivesitelikelyreflectsloopinginterac7onswithdistaldirectGRbindingsites

•  DoesthisalterDEX-responsiveregulatoryac7vity,poten7allyexplainingcell-typespecificity?

AddingAP-1sitesamplifiesGC-responseby>10-fold

Thatamplifica7onisdistantdependent

Summaryofourmodel:GRbindingclustersreflectextensive

GR:AP-1tetheringinterac7ons.

Summary•  Wedevelopedahigh-throughputapproachtomeasuretheac7vityofeveryGRbindingsiteinareporterassay

•  Theresultsrevealafunc7onaldiversityofGRbindingsites,andsuggestthatinterac7onsbetweendirectandtetheredGRbindingsitesarethebasisforcell-typespecificGCresponses

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

RNA-seqandDNase-seqreleasedviaENCODEDCC

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

Lookingforpostdocstojointhelab

•  Lookingforbothexperimentalandcomputa7onalscien7sts

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

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)