Comprehensive functional testing of ChIP-seq binding sites with ...
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Comprehensive functional testing of ChIP-seq binding sites with ChIP-
reporter assays
TimReddyDukeUniversityENCODEUsersMee7ngJune9,2016
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ChIP-seqhasrevealedtensofthousandsofTFbindingsitesinthehumangenome,farin
excessoftheregulatedgenes.
A549cells(lungepithelialcellline),3h,100nMDexamethasone
0
5000
10000~10,000
297 151
GR Binding Sites(ChIP-seq)
UpregulatedGenes
DownregulatedGenes
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Majorques7onpostChIP-seq:WhatarealloftheseTFbindingsitesdoing?
A549cells(lungepithelialcellline),3h,100nMDexamethasone
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5000
10000~10,000
297 151
GR Binding Sites(ChIP-seq)
UpregulatedGenes
DownregulatedGenes
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Ourmodelsystem:Cor7sol(glucocor7coidsteroidhormone)
Suppressesimmunesystemandreducesinflamma7on
Increasesbloodpressureandbloodsugar
Synthe7cGC’susedclinicallytotreatPsoriasis,Crohn’sDisease,RheumatoidArthri7s
Majorcomponentofresponsetostressresponseandmetabolism
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Ourmodelsystem:Cor7sol(glucocor7coidsteroidhormone)
Suppressesimmunesystemandreducesinflamma7on
Increasesbloodpressureandbloodsugar
Synthe7cGC’susedclinicallytotreatPsoriasis,Crohn’sDisease,RheumatoidArthri7s
Majorcomponentofresponsetostressresponseandmetabolism
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Cortisol Production (HPA axis)
Adrenocor7cotropicHormone(ACTH)
Cor7sol
Hypothalamus
Pituitary
Adrenal Glands
Kidneys
Corticotrophin-releasing Hormone (CRH)
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The Glucocorticoid Receptor (GR)
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GRbinds>10,000sitesinthehumangenomeandregulateshundredsofgenes.
A549cells(lungepithelialcellline),3h,100nMDexamethasone
0
5000
10000~10,000
297 151
GR Binding Sites(ChIP-seq)
UpregulatedGenes
DownregulatedGenes
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Reporterassaystoquan7fytheac7vityofGCresponseelements
Luciferase }
Luciferase
GRE
GRE
+DEX
+EtOH
DEX=Dexamethasone,asynthe7cglucocor7coid
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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
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GFP
Regulatoryelementslocatedinthe3’UTRofthereportergene.
STARR-seq:Arnoldetal,Science,2013
STARR-seqreporterassays
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GFP
Fromthatposi7on,theelementsregulatetheirownexpression.
STARR-seq:Arnoldetal,Science,2013
STARR-seqreporterassays
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GFP
High-throughputsequencingofthecloningsiteintheexpressedreportergenecanthenbeusedtoquan7fyregulatoryelementac7vity
STARR-seq:Arnoldetal,Science,2013
STARR-seqreporterassays
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GFP
STARR-seq:Arnoldetal,Science,2013
STARR-seqreporterassays
• Canassaymillionsoffragmentsatonce• Elementscanbehundredstothousandsofbp• Allowsdirectliga7onofcapturedDNAintohigh-
throughputreporterassays
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Chroma7nImmunoprecipita7on+STARR-seqtoquan7fytheac7vityof
allTFbindingsites
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Chroma7nImmunoprecipita7on+STARR-seqtoquan7fytheac7vityof
allTFbindingsites
Treatwithdrugorcontrol
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GRChIP-reporterresults
v
• Assayed>12,000GRbindingsites• 10%wereDEX-responsive• 95%ofregulatoryelementsincreasedreporterexpression• Validatedwithstandarddual-luciferase(r=0.77)
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Possibleinterpreta7ons:
Onepossibility:Only10%oftheGRbindingsiteshaveac7vity.Alterna<ve:OtherGRbindingsitesrequireaddi7onalgenomiccontext.
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SomewaysthatGRbindsthegenomeDirectbindingtoaGlucocor7coidResponseElement(GRE)
TetheredbindingviaotherTFssuchasAP-1
Coopera7vebindingtoacompositeregulatoryelement
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PresenceofaGREexplainstheresponseCo-bindingTFmo7fsdonot
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DEX-responsivesitesalsohaveepigene7cstatechangesthatreflectac7vityinthegenome
DEX-responsivenon-responsive
GRE GRE GRE GRE
DEX
EtOH
DEX
EtOH
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DirectbindingexplainsDEX-responsivereporterac7vity
DirectbindingtoaGlucocor7coidResponseElement(GRE)
TetheredbindingviaotherTFssuchasAP-1
Coopera7vebindingtoacompositeregulatoryelement
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Theproblem
• ThesetofGC-responsivegenesvariesdrama7callybetweencelltypes
• Thosedifferencescanlargelybeahributedtochangesinco-factors,par7cularlyAP-1
• IfGRbindingatAP-1sitesdoesnothaveac8vity,thenwestruggletoexplaincell-type-specificdifferencesinGC-responses
(e.g.Biddieetal,2011;Johnetal,2011;Gertzetal,2013)
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Ourmodel
• AP-1sitesmodulatetheac7vityofdirectGRbindingsitesinthegenome
• However,theAP-1sitesarenotsufficientforDEX-responsiveregulatoryac7vity
• Inthismodel,weexpectGRandAP-1toco-clusterinthegenome
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GRbindsthegenomeinclusters
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Morenumerousclustershaveasmallerfrac7onofDEX-responsivesites
(i.e.fewdirectsitescannucleatelargeclusters)
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AP-1sites,representedbyJunD,thatgainGRareclosertodirectGRbindingsites
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ClustersofGRbindingaredepletedforinterveningCTCF
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Conclusion
• GRbidingatnon-responsivesitelikelyreflectsloopinginterac7onswithdistaldirectGRbindingsites
• DoesthisalterDEX-responsiveregulatoryac7vity,poten7allyexplainingcell-typespecificity?
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AddingAP-1sitesamplifiesGC-responseby>10-fold
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Thatamplifica7onisdistantdependent
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Summaryofourmodel:GRbindingclustersreflectextensive
GR:AP-1tetheringinterac7ons.
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Summary• Wedevelopedahigh-throughputapproachtomeasuretheac7vityofeveryGRbindingsiteinareporterassay
• Theresultsrevealafunc7onaldiversityofGRbindingsites,andsuggestthatinterac7onsbetweendirectandtetheredGRbindingsitesarethebasisforcell-typespecificGCresponses
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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
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RNA-seqandDNase-seqreleasedviaENCODEDCC
• 127mepoints,coordinatedtothesecond• Atleastfourreplicatesofeach7mepoint• ChIP-seq/Hi-C/STARR-seqdataforthcoming
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Lookingforpostdocstojointhelab
• Lookingforbothexperimentalandcomputa7onalscien7sts
• GenomicsandGene7csprojectsfeaturinghigh-throughputreporterassays,genomeandepigenomeedi7ng,networkmodeling,experimentaldesign,andspecificdiseasestudies.
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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)