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)