Andersson, R. et al. An atlas of active enhancers across human cell

13 Pages·2014·1.74 MB·English

by  Robin Andersson

ARTICLE
doi:10.1038/nature12787
An atlas of active enhancers across
human cell types and tissues
RobinAndersson1*,ClaudiaGebhard2,3*,IreneMiguel-Escalada4,IlkaHoof1,JetteBornholdt1,MetteBoyd1,YunChen1,
XiaobeiZhao1,5,ChristianSchmidl2,TakahiroSuzuki6,7,EvgeniaNtini8,ErikArner6,7,EivindValen1,9,KangLi1,
LuciaSchwarzfischer2,DagmarGlatz2,JohannaRaithel2,BeritLilje1,NicolasRapin1,10,FrederikOtzenBagger1,10,
MetteJørgensen1,PeterRefsingAndersen8,NicolasBertin6,7,OwenRackham6,7,A.MaxwellBurroughs6,7,J.KennethBaillie11,
YuriIshizu6,7,YuriShimizu6,7,ErinaFuruhata6,7,ShioriMaeda6,7,YutakaNegishi6,7,ChristopherJ.Mungall12,
TerrenceF.Meehan13,TimoLassmann6,7,MasayoshiItoh6,7,14,HideyaKawaji6,14,NaotoKondo6,14,JunKawai6,14,
AndreasLennartsson15,CarstenO.Daub6,7,15,PeterHeutink16,DavidA.Hume11,TorbenHeickJensen8,HarukazuSuzuki6,7,
YoshihideHayashizaki6,14,FerencMu¨ller4,TheFANTOMConsortium{,AlistairR.R.Forrest6,7,PieroCarninci6,7,MichaelRehli2,3
&AlbinSandelin1
Enhancerscontrolthecorrecttemporalandcell-type-specificactivationofgeneexpressioninmulticellulareukaryotes.
Knowingtheirproperties,regulatoryactivityandtargetsiscrucialtounderstandtheregulationofdifferentiationand
homeostasis.HereweusetheFANTOM5panelofsamples,coveringthemajorityofhumantissuesandcelltypes,toproduce
anatlasofactive,invivo-transcribedenhancers.WeshowthatenhancerssharepropertieswithCpG-poormessengerRNA
promotersbutproducebidirectional,exosome-sensitive,relativelyshortunsplicedRNAs,thegenerationofwhichisstrongly
relatedtoenhanceractivity.Theatlasisusedtocompareregulatoryprogramsbetweendifferentcellsatunprecedented
depth,toidentifydisease-associatedregulatorysinglenucleotidepolymorphisms,andtoclassifycell-type-specificand
ubiquitousenhancers.Wefurtherexploretheutilityofenhancerredundancy,whichexplainsgeneexpressionstrength
ratherthanexpressionpatterns.TheonlineFANTOM5enhanceratlasrepresentsauniqueresourceforstudiesoncell-
type-specificenhancersandgeneregulation.
Preciseregulationofgeneexpressionintimeandspaceisrequiredfor samplesfromhuman6,weidentify43,011enhancercandidatesandchar-
development,differentiationandhomeostasis1.Sequenceelementswithin acterizetheiractivityacrossthemajorityofhumancelltypesandtissues.
ornearcorepromoterregionscontributetoregulation2,butpromoter- Theresultingcatalogueoftranscribedenhancersenablesclassification
distalregulatoryregionslikeenhancersareessentialinthecontrolof ofubiquitousandcell-type-specificenhancers,modellingofphysical
cell-typespecificity1.Enhancerswereoriginallydefinedasremoteele- interactionsbetweenmultipleenhancersandTSSs,andidentificationof
mentsthatincreasetranscriptionindependentlyoftheirorientation, potential disease-associated regulatory single nucleotide polymorph-
positionanddistancetoapromoter3.Theywereonlyrecentlyfoundto isms(SNPs).
initiateRNApolymeraseII(RNAPII)transcription,producingso-called
eRNAs4.Genomiclocationsofenhancerscanbedetectedbymapping BidirectionalcappedRNAsidentifyactiveenhancers
ofchromatinmarksandtranscriptionfactorbindingsitesfromchro- TheFANTOM5projecthasgeneratedaCAGE-basedtranscription
matinimmunoprecipitation(ChIP)assaysandDNaseIhypersensitive startsite(TSS)atlasacrossabroadpanelofprimarycells,tissuesand
sites (DHSs) (reviewed in ref. 1), but there has been no systematic celllinescoveringthevastmajorityofhumancelltypes6.Withinthat
analysisofenhancerusageinthelargevarietyofcelltypesandtissues dataset,well-studiedenhancersoftenhaveCAGEpeaksdelineating
presentinthehumanbody. nucleosome-deficient regions (NDRs) (Supplementary Fig. 1). To
Usingcapanalysisofgeneexpression5(CAGE),weshowthatenhan- determinewhetherthisisageneralenhancerfeature,FANTOM5CAGE
ceractivitycanbedetectedthroughthepresenceofbalancedbidirec- (SupplementaryTable1)wassuperimposedonactive(H3K27ac-marked)
tionalcappedtranscripts,enablingtheidentificationofenhancersfrom enhancersdefinedbyHeLa-S3ENCODEChIP-seqdata7.CAGEtags
smallprimarycellpopulations.BasedupontheFANTOM5CAGEexpress- showeda bimodal distributionflankingthe centralP300 peak,with
ionatlasencompassing432primarycell,135tissueand241cellline divergenttranscriptionfromtheenhancer(Fig.1a).Similarpatterns
1TheBioinformaticsCentre,DepartmentofBiology&BiotechResearchandInnovationCentre,UniversityofCopenhagen,OleMaaloesVej5,DK-2200Copenhagen,Denmark.2DepartmentofInternal
MedicineIII,UniversityHospitalRegensburg,Franz-Josef-Strauss-Allee11,93042Regensburg,Germany.3RegensburgCentreforInterventionalImmunology(RCI),D-93042Regensburg,Germany.
4SchoolofClinicalandExperimentalMedicine,CollegeofMedicalandDentalSciences,UniversityofBirmingham,Edgbaston,BirminghamB152TT,UK.5LinebergerComprehensiveCancerCenter,
UniversityofNorthCarolina,ChapelHill,NorthCarolina27599,USA.6RIKENOMICSScienceCentre,RIKENYokohamaInstitute,1-7-22Suehiro-cho,Tsurumi-ku,YokohamaCity,Kanagawa230-0045,
Japan.7RIKENCenterforLifeScienceTechnologies(DivisionofGenomicTechnologies),RIKENYokohamaInstitute,1-7-22Suehiro-cho,Tsurumi-ku,YokohamaCity,Kanagawa230-0045,Japan.8Centre
formRNPBiogenesisandMetabolism,DepartmentofMolecularBiologyandGenetics,C.F.MøllersAlle3,Building1130,DK-8000Aarhus,Denmark.9DepartmentofMolecularandCellularBiology,
HarvardUniversity,Cambridge,Massachusetts02138,USA.10TheFinsenLaboratory,RigshospitaletandDanishStemCellCentre(DanStem),UniversityofCopenhagen,OleMaaloesVej5,DK-2200,
Denmark.11RoslinInstitute,EdinburghUniversity,EasterBush,Midlothian,EdinburghEH259RG,UK.12GenomicsDivision,LawrenceBerkeleyNationalLaboratory,1CyclotronRoadMS64-121,Berkeley,
California94720,USA.13EMBLOutstation-Hinxton,EuropeanBioinformaticsInstitute,WellcomeTrustGenomeCampus,Hinxton,CambridgeCB101SD,UK.14RIKENPreventiveMedicineandDiagnosis
InnovationProgram,RIKENYokohamaInstitute,1-7-22Suehiro-cho,Tsurumi-ku,YokohamaCity,Kanagawa230-0045,Japan.15DepartmentofBiosciencesandNutrition,KarolinskaInstitutet,
Ha¨lsova¨gen7,SE-4183Huddinge,Stockholm,Sweden.16DepartmentofClinicalGenetics,VUUniversityMedicalCenter,vanderBoechorststraat7,1081BTAmsterdam,Netherlands.
*Theseauthorscontributedequallytothiswork.
{AlistofauthorsandaffiliationsappearsintheSupplementaryInformation.
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RESEARCH ARTICLE
Fraction of enhancersa 0000....00001234–500CAGE –Position 0relativeC toAGE + 500 Densityb 024– 1st0r0a%nd CdeRTBnSeehahfifSrlSaonanemnecqdcaeetdrin - + s1t0r0an%d P cFraction success (< 0.05) 000...257505 3High CAGE4 2Mid CAGE9 2Low CAGE5 Untranscribed DHS5 Untranscribed ENCODE8strong enhancer Untranscribed chromatin-4defined enhancer
0.00
P300 peak centre (bp) Directionality Prediction category
Figure1|BidirectionalcappedRNAsisasignaturefeatureofactive accordingtoFANTOM5-pooledCAGEtagsmappedwithin6300bpof
enhancers. a,Enhancersidentifiedbyco-occurrenceofH3K27acand 22,486TSSsofRefSeqprotein-codinggenesandcentrepositionsof10,138
H3K4me1ChIP-seqdata7,centredonP300bindingsites,inHeLacellswere HeLaenhancersdefinedasabove.c,Successratesof184invitroenhancer
overlaidwithHeLaCAGEdata(uniquepositionsofCAGEtag59ends, assaysinHeLacells.Verticalaxisshowsthefractionofactiveenhancers
smoothedbya5-bpwindow),revealingabidirectionaltranscriptionpattern. (successdefinedbyStudent’st-test,P,0.05versusrandomregions;alsosee
Horizontalaxisshowsthe6500bpregionaroundenhancermidpoints. SupplementaryFig.9).Numbersofsuccessfulassaysareshownonthe
b,Densityplotillustratingthedifferenceindirectionalityoftranscription respectivebar.Seemaintextfordetails.
wereobservedinothercelllines(SupplementaryFig.2a).Enhancer- (asdefinedbyCAGEtagfrequencyinHeLacells)andexaminedtheir
associatedreverseandforwardstrandtranscriptioninitiationevents activityusingenhancerreporterassayscomparedtorandomlyselected
were,onaverage,separatedby180basepairs(bp)andcorrespondedto untranscribedlociwithregulatorypotentialinHeLa-S3cells:15DHSs10,
nucleosomeboundaries(SupplementaryFigs3and4).Asaclass,active 26ENCODE-predicted‘strongenhancers’7and20enhancersdefined
HeLa-S3 enhancers had 231-fold more CAGE tags than polycomb- asinFig.1a(SupplementaryTables2and3).Whereas67.4–73.9%of
repressedenhancers,indicatingthattranscriptionisamarkerforactive theCAGE-definedenhancersshowedsignificantreporteractivity,only
usage.Indeed,ENCODE-predictedenhancers7withsignificantreporter 20–33.3%oftheuntranscribedcandidateregulatoryregionswereactive
activity8hadgreaterCAGEexpressionlevelsthanthoselackingreporter (Fig.1candSupplementaryFig.9a).Thesametrendwasobservedin
activity(P,4310222,Mann–WhitneyUtest).Alenientthreshold HepG2cells(SupplementaryFig.10a,b).Correspondingpromoter-less
on enhancer expression increased the validation rate of ENCODE constructs showed that the enhancer transcription read-through is
enhancersfrom27%to57%(SupplementaryFig.5). negligible(SupplementaryFig.9b,c).ManyCAGE-definedenhancers
Although capped RNAs of protein-coding gene promoters were overlappedpredictedENCODE‘strongenhancers’or‘TSS’states(25%
stronglybiasedtowardsthesensedirection,similarlevelsofcapped and62%,respectively,forHeLa-S3),buttherewasnosubstantialdiffer-
RNAinbothdirectionsweredetectedatenhancers(Fig.1bandSup- enceinvalidationratesbetweentheseclasses(SupplementaryFig.10c,d).
plementaryFig.2b,c).Thus,bidirectionalcappedRNAsisasignature Insummary,activeCAGE-definedenhancersweremuchmorelikelyto
featureofactiveenhancers.Onthisbasis,weidentified43,011enhan- bevalidatedinfunctionalassaysthanuntranscribedcandidateenhan-
cercandidatesacross808humanCAGElibraries(seeSupplementary cersdefinedbyhistonemodificationsorDHSs.
TextandSupplementaryFigs6–8).Interestingly,thecandidateswere
depleted of CpG islands (CGI) and repeats (with the exception of InitiationandfateofenhancerRNAsversusmRNAs
neuralstemcells,seeref.9). RNA-seqdatafrommatchingprimarycellsandtissuesshowedthat
Toconfirmtheactivityofnewlyidentifiedcandidateenhancers,we ,95%ofRNAsoriginatingfromenhancerswereunsplicedandtyp-
randomlyselected46strong,41moderateand36lowactivityenhancers icallyshort(median346nucleotides)—astrikingdifferencetomRNAs
a b c d e
Density0001....0482EEGGnnEEhhNN1aaCCnn0OOcc0LeeDDerr EEnRtr 1gamtNrktnahARsn N(cslorcA1igpr0i1tpk0, t gs, ecg1nae0olne0mo)kmicic Average predicted sites per kb 00110011........05050505 0 5T′TSSDSista5n0c0e (bp) 1,000FrequencyEnhancer0000′ CAGE 5 ends....6420 5TA0T Abp INR AACCCGGTT De novo motifsSRF (0.90)OCT4 (0.98)MEF2A (0.95) GATA2 (0.96)TEAD1 (0.87) Motif.0662 MITF (0.91)CEBPAP1: (0.94)AP1ETS: CEBPA (0.93) SPI1 (0.95) JUN (0.99) RUNX1 (0.97) SIX4 (0.82) JUND (0.95)GATA2 (0.73)ATF3 (0.88) RELA (0.95) GC-Box (0.95) NRF1 (0.97)ELF1 (0.96)Motif.0497 (0.76)YY1 (0.98) RFX (0.94)nCMotif enrichmentGabove backgroundI-prom22222o210––12terAverage fold changeSKIV2L2–/control123231......–000555R7o5erl0a etinvhea dnics0etar nmceid tpoo 7Tin5St0S
nnCCCGGGI TII TTSSSSSS s easnennstiesseense CEGnGehnIa oTnmScSiec r aenxtpiseecntasetion HigDhN2a4s be pI cleavagLeow nECCCnGGhGIIa--IpTn-TSrcoSSemrSoter ERERnneehhffSSaaeennqqcc eeTTrrSS –+SS ss sattrrenaantnnissddeense
(per nucleotide)
Figure2|FeaturesdistinguishingenhancerTSSsfrommRNATSSs. peaks(arrowat11indicatespositionofthemainenhancerCAGEpeaks;
a,DensitiesofthegenomicandprocessedRNAlengthsoftranscriptsstarting directionoftranscriptiongoeslefttoright)revealdistinctcleavagepatterns
fromenhancerTSSsandmRNATSSsusingassembledRNA-seqreadsfrom atsequencesresemblingtheINRandTATAelements.d,Denovomotif
13pooledFANTOM5libraries.b,FrequenciesofRNAprocessingmotifs enrichmentanalysesaroundenhancersandnon-enhancerFANTOM5
(59splicemotif(59SS,upperpanel)andthetranscriptionterminationsite CAGE-definedTSSs(CAGETSSsmatchingannotatedTSSsarereferredto
hexamer(TTS,lowerpanel))aroundenhancerandmRNATSSs.Verticalaxis as‘promoters’),contingentonCGIoverlap.Topenriched/depletedmotifsare
showstheaveragenumberofpredictedsitesperkbwithinacertainwindow shownalongwiththeirbest-knownmotifmatchname.Enrichmentversus
sizefromtheTSS(horizontalaxis)inwhichthemotifsearchwasdone.Dashed randombackgroundispresentedasaheatmap.e,Verticalaxisshowsaverage
linesindicateexpectedhitdensityfromrandomgenomicbackground.The HeLaCAGEexpressionfoldchangeversuscontrolatenhancersandRefSeq
windowalwaysstartsatthegeneorenhancerCAGEsummitsandexpands TSSsafterexosomedepletion.Horizontalaxisshowspositionrelativetothe
inthesensedirection.nCGI,non-CGI.c,Averagenucleotidefrequencies TSSorthecentreoftheenhancer.Translucentcoloursindicatethe95%
(toppanel)andDNaseIcleavagepatterns(lowerpanel)ofenhancerCAGE confidenceintervalofthemean.
456 | NATURE | VOL 507 | 27 MARCH 2014
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ARTICLE RESEARCH
(19%unspliced,median1,256nucleotides)(Fig.2aandSupplementary Anatlasoftranscribedenhancersacrosshumancells
Fig.11a–c).UnlikeTSSsofmRNAs,whichareenrichedforpredicted The FANTOM5 CAGE library collection6 enables the dissection of
59splicesitesbutdepletedofdownstreampolyadenylationsignals11,12, enhancerusageacrosscelltypesandtissuescomprehensivelysampled
enhancersshowednoevidenceofassociateddownstreamRNApro- acrossthehumanbody.Clusteringbasedonenhancerexpressionclearly
cessingmotifs,andthusresembleantisensepromoterupstreamtran- groupedfunctionallyrelatedsamplestogether(Fig.3candSupplemen-
scripts(PROMPTs)11(Fig.2bandSupplementaryFig.11d).MostCAGE- tary Figs 18 and 19).Although fetal and adult tissue often grouped
definedenhancersgaverisetonuclear(.80%)andnon-polyadenylated together, two large fetal-specific clusters were identified: one brain-
(,90%) RNAs13 (Supplementary Fig. 11e). Based on RNA-seq, few specific (pink) and one with diverse tissues (green). The fetal-brain
enhancerRNAsoverlapexonsofknownprotein-codinggenesorlarge cluster isassociatedwith enhancersthatare located close to known
intergenicnoncodingRNAs(9and1outof4,208enhancersdetected,
neuraldevelopmentalgenes,includingNEUROG2,SCRT2,POU3F2
respectively),indicatingthattheyarenotasubstantialsourceofalter-
andMEF2C(SupplementaryFig.18b),forwhichgeneexpressionpat-
nativepromotersforknowngenes(asinref.14).
ternscorrelatewithenhancerRNAabundanceacrosslibraries,suggest-
TSS-associated,uncappedsmallRNAs(TSSa-RNAs),attributedto
ingregulatoryinteraction(seebelow).Theresultscorroboratethefunc-
RNAPII protection and found immediately downstream of mRNA
tionalrelevanceoftheseenhancersfortissue-specificgeneexpression
TSSs15,16,weredetectableinthesamepositionsdownstreamofenhan-
andindicatethattheyareanimportantpartoftheregulatoryprograms
cerTSSs(SupplementaryFig.12),indicatingthatRNAPIIinitiationat
ofcellulardifferentiationandorganogenesis.
enhancerandmRNATSSsissimilar.Indeed,CAGE-definedenhancer
Toconfirmthatcandidateenhancerscandrivetissue-specificgene
TSSs resembled the proximal position-specific sequence patterns of
expressioninvivo,fiveevolutionarilyconservedCAGE-definedhuman
non-CGIRefSeqTSSs(Fig.2candSupplementaryFig.13a).Furthermore,
enhancers (including the POU3F2 and MEF2C-proximal enhancers
denovomotifanalysisrevealedsequencesignaturesinCAGE-defined
enhancerscloselyresemblingnon-CGIpromoters(Fig.2dandSup-
plementaryFig.13b). a
CD19 CD4 CD8 CD56 CD14
BecauseofthesimilaritywithPROMPTs,wereasonedthatcapped
enhancerRNAsmightberapidlydegradedbytheexosome.Indeed, ers
c
smallinterferingRNA-mediateddepletionoftheSKIV2L2(alsoknown n
a
h
asMTR4)co-factoroftheexosomecomplexresultedinamedian3.14- en
d
foldincreaseofcappedenhancer-RNAabundance(Fig.2eandSup- e
n
fi
plementaryFig.14a,b),butonlyanegligibleincreaseatmRNATSSs. de
ThisincreasingtrendissimilartothatofPROMPTregionsupstream GE-
A
of TSSs, although the increase of enhancer RNAs was significantly C
higher(P,4.6310267,Mann–WhitneyUtest;Fig.2eandSupplemen-
tabtahtureteynCiFnhAigapG.nr1cEo4ember,xsocpit)ser.erTasslhssoiuoonsnlp,ytrohetfehseeebnniahdtnaiartnteicscpeetirnroossnmeianRlocttNroearnAnst,sricaosrslidpasetnugiodgrngaSdeaKsletaIdeVc.dt2FivpLuir2tryet-vhodieboerspumelsrelovytree1ed7d,, -1 Crep 1reACpGlAiErep 2cGa-1kbEterep 3s+1kb-1kb-+1kb4-1kb+1kb-10–1 kbpRDoeslN0aitatiiosv+1 kbnee -6 HLiogwh CAGE DNase I H3K4me1 H3K27ac
cellswasproportional(SupplementaryFig.14d),indicatingthatvir-
tnuuamllybearlloidfednettifeicetdabelnehbaindcierresctpiorondaulcCeAeGxoEsopmeaek-sseninscitrievaesRedN1A.7s.-Tfohlde b CCeAlGl-tEy peen-hsapnecceifirsc c C AGE tissuelibraries
uponSKIV2L2depletionandnovelenhancercandidateshadonaverage CD19 CD4 CD56 CD14
sHimeLilaarc,ebllust(wSeuapkpelre,mchernotmaraytiFnimg.o1d4iefi)c.ationsignalscomparedtocontrol gnal 0.01 CD19
CAGEidentifiescell-specificenhancerusage DNase I si 00..0002 CD4C
tTcieoorntuesasatngwdehitenrtihvpielvircoCa,tACehGCIEPA-eGsxepEqre(asHnsai3olKyns2ce7asancwiadenreendtpHifey3rcKfoe4lrlmm-tyee1pd)e,i-nDspNfeicvAiefimcpeernitmhhyaalnray-- ChIP-seq or 000...000001 CD56ell type
broloaoddmcaeplelptyigpeenso,manidcsc.oormg/p,aSruedpptloempuenbltiasrhyedTaDbHleS4d)a.tCaA(hGttEp-:d//ewfiwnewd. Mean -10H003..00K034me1HH3D3KKN42amH7s3eae1c KI27ac CD14 Whole blood Heart
enhancerswerestronglysupportedbyproximalH3K4me1/H3K27ac –1 0 1 –1 0 1 Cerebellum Lymphatic organs
peaks (71%) and DHSs (87%) from the same cell type. Conversely, enhancDeisr tmanidcpeo tion t (kb) FBeratainl brain FReetparlo tdisuscuteivse organs
H3K4me1andH3K27acsupportedonly24%ofDHSsdistaltopro-
Figure3|CAGEexpressionidentifiescell-type-specificenhancerusage.
moters and exons andonly 4% ofDHSs overlappedCAGE-defined
a,RelationshipbetweenCAGEandhistonemodificationsinbloodcells.Rows
enhancers(SupplementaryFig.15),indicatingthataminorityofpro-
representCAGE-definedenhancersthatareorderedbasedonhierarchical
moter-distalDHSsidentifyenhancers.Fromtheoppositeperspective,
clusteringofCAGEexpression.ColumnsfortheCAGEtags(pink)represent
only11%ofH3K4me1/H3K27aclocioverlappedCAGE-definedenhan- theexpressionintensityforthreebiologicalreplicates.DNaseIhypersensitivity
cers and untranscribed loci showed weaker ChIP-seq signals than andH3K27acandH3K4me1ChIP-seqsignals61kbaroundtheenhancer
transcribedones(SupplementaryFig.16).Moreover,therewasaclear midpointsareshowningreen,blueandorange,respectively.b,Meansignalof
correlationbetweenCAGE,DNaseIhypersensitivity,H3K4me1and DNase-seqaswellasChIP-seqforH3K27acandH3K4me1(verticalaxes)per
H3K27acforCAGE-definedenhancersexpressedinbloodcells(Fig.3a). celltype(rows)in61kbregions(horizontalaxes)aroundenhancermidpoints,
Accordingly,cell-type-specificenhancerexpressioncorrespondstocell- forenhancerswithblood-celltype-specificCAGEexpression(columns).
c,Dendrogramresultingfromagglomerativehierarchicalclusteringoftissue
type-specifichistonemodifications(Fig.3b).Themajorityofselected
samplesbasedontheirenhancerexpression:eachleafofthetreerepresentsone
cell-type-specificenhancerscouldbevalidatedincorrespondingcell
CAGEtissuesample(foralabelledtreeandthecorrespondingresultson
linesandwereassociatedwithcell-type-specificDNAdemethylation
primarycellsamples,seeSupplementaryFigs18and19).Sub-treesdominated
(Supplementary Text, Supplementary Fig. 17 and Supplementary byonetissue/organtypeormorphologyarehighlighted.Someoftheenhancers
Tables5–8,seealsoref.18).Thus,bidirectionalCAGEpairsarerobust responsibleforthefetal-specificsubgroupinthelargerbrainsub-treeare
predictorsforcell-type-specificenhanceractivity. validatedinvivo(Fig.4).
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RESEARCH ARTICLE
identifiedabove)weretestedviaTol2-mediatedtransgenesisinzebra- withthecut-offsused.Noteworthyexceptionsincludebloodandimmune
fishembryos.Weobservedtissue-specificenhanceractivitywith3of5 cells,testis,thymusandspleen,whichhavehighenhancer/generatios.
fragments,whichcorrespondedtothehumanenhancertissueexpress- Conversely,smoothandskeletalmuscleandskin,boneandepithelia-
ion (Fig. 4). None of three control fragments without CAGE signal relatedcellshavelowratios.Differentialexosomeactivitybetweencell
activated the GATA2 promoter (Supplementary Table 9). Although typesmightaffecttheseresults,buttherewasnocorrelationbetween
thesamplesizeisnothighenoughtoreliablyestimatethevalidation SKIV2L2 mRNA expression and the number of enhancers detected
ratesinzebrafish,thecorrelationbetweentheenhancerusageprofiles (SupplementaryFig.22e,f).
inzebrafishtothosedefinedinhumanbyCAGEisnotable. Asexpected,consensusmotifsofknownkeyregulatorsareover-
Wegroupedtheprimarycellandtissuesamplesintolarger,mutu- represented in corresponding facet-specific enhancers, for instance
allyexclusivecelltypeandorgan/tissuegroups(referredtoasfacets), ETS, C/EBP and NF-kB in monocyte-specific enhancers, RFX and
respectively, with similar function or morphology (Supplementary SOXinneurons,andHNF1andHNF4ainhepatocytes(Supplemen-
Tables10and11).Figure5summarizeshowmanyenhancerswere taryFig.23).Notably,theAP1motifappearstobeenrichedacrossall
detectedineachfacetandthedegreeoffacet-specificCAGEexpress- facets, perhaps associated with a general role for AP1 in regulating
ion (see also Supplementary Fig. 21). From the data we can draw openchromatin19.
severalconclusions:
First,themajorityofdetectedenhancerswithinanyfacetarenot Expressionclusteringrevealsubiquitousenhancers
restrictedtothatfacet.Exceptions,wherefacetsuseahigherfraction Hierarchical clustering of enhancers by facet expression revealed a
of specific enhancers, include immune cells, neurons, neural stem smallsubsetofenhancers(200or247,definedbyprimarycellortissue
cellsandhepatocytesamongstthecell-typefacets,andbrain,blood, facets, respectively) expressed in the large majority of facets (Sup-
liverandtestisamongsttheorgan/tissuefacets. plementary Text, Supplementary Figs 24 and 25, and Supplemen-
Second, despite their apparent promiscuity, enhancers are more taryTables12and13).Comparedtootherenhancers,theseubiquitous
generallydetectedinamuchsmallersubsetofsamplesthanmRNA (u-)enhancersare8timesmorelikelytooverlapCGIsandtheyare
transcripts(SupplementaryFigs21and22a,b),consistentwithcell- twiceasconserved(SupplementaryFig.26a–c).U-enhancersoverlap
linestudies7andthehigherspecificityofncRNAsingeneral13.Facets typicalchromatinenhancermarksbuthavehigherH3K4me3signal
inwhichwedetectmanyenhancerstypicallyalsohaveahigherfrac- (SupplementaryFig.26d).Althoughtheyproducesignificantlylonger
tionoffacet-specificenhancers(SupplementaryFig.22c,d). ncRNAsthanotherenhancers(median530nucleotides,P,1.531028,
Third, the number of detected expressed enhancers and mRNA Mann–WhitneyUtest),thetranscriptsremainpredominantly(,78%)
transcripts is correlated (Supplementary Fig. 21b), but the number unsplicedandsignificantlyshorter(P,4.2310218,Mann–Whitney
ofdetectedexpressedgenetranscripts(.1tagpermillionmappedreads Utest)thanmRNAs(Supplementary Figs27and28),donotshare
(TPM))is19–34foldlargerthanthenumberofdetectedenhancers exonswithknowngenes,andareexosome-sensitive(Supplementary
Fig.14b).Therefore,itisunlikelythatthesearenovelmRNApromo-
a mid Human enhancer ters. They are also highly enriched for P300 and cohesin ChIP-seq
fb expression (TPM)
0 1 peaks20andRNAPII-mediatedChIA-PETsignal21comparedtoother
hin sp
E1 100 μm Neuron enhancers (Supplementary Fig. 26d). These results indicate that
CR u-enhancerscompriseasmallbutdistinctsubsetofenhancers,which
Astrocyte
probably has specific regulatory functions used by virtually every
Brain nt 100 μm Brain humancell.
b 0 3
Neural stem cell LinkingenhancerusagewithTSSexpression
fp Neuron
E2 100 μm Astrocyte A major challenge is to link enhancers to their target genes21,22.
CR fp Spinal cord Uniquely,FANTOM5CAGEallowsfordirectcomparisonbetween
Brain transcriptional activity of the enhancer and of putative target gene
100 μm TSSsacrossadiversesetofhumancells.Basedonpairwiseexpression
c iv iv dv 0 8 correlation,nearlyhalf(40%)oftheinferredTSS-associatedenhan-
Salivary gland
Blood vessel of cers(Methods)werelinkedwiththenearestTSS,and64%ofenhan-
epithelial cell
CRE3 100 μm da KepidEhitennhpedeyaolitatihcl e csleiianlllu cseolild of caessroschiaatvioenast(l1e0a,s2t6o0n;e15c.o3r%re)laatreedsuTpSpSowrteitdhibny5C0h0IAki-lPobEaTse(sR.NSeAvPerIIa-l
ysl Urothelial cell mediated) interaction data21, and the supported fraction increases
ysl mu 100 μm eRpeisthpeirlaiatol crye ll withthecorrelationthreshold(SupplementaryFig.29a).Thefraction
Figure4|Invivovalidationinzebrafishoftissue-specificenhancers. ofsupportedassociationsis4.8-foldhigherthanthatofassociations
ValidationsofinvivoactivityofCAGE-definedhumanenhancersCRE1,CRE2 predictedfromDNaseIhypersensitivitycorrelations10(20.6%versus
andCRE3inzebrafishembryosatlong-pecstage.Eachpanelshows,fromleftto 4.3%,atthesamecorrelationthreshold),indicatingthattranscription
right,representativeyellowfluorescentprotein(YFP)andbrightfieldimagesof
isabetterpredictorofregulatorytargetsthanchromatinaccessibility.
embryosinjectedwiththehumanenhancergata2promoterreportergene
Conserved sequence motifs and ChIP-seq peaks also co-occurred
construct(left),YFPzoom-ins(middle)andCAGEexpressioninTPMin
significantly in associated enhancer-promoter pairs (Benjamini–
humantissues/celltypesfortheenhancer(right).Muscle(mu)andyolk
syncytiallayer(ysl)activitiesarebackgroundexpressioncomingfromthegata2 Hochbergfalsediscoveryrate(FDR),0.05,binomialtest),suggest-
promoter-containingreporterconstruct.Allimagesarelateral,headtotheleft. ing an additive or synergistic cooperation between enhancers and
Notethecorrespondencebetweenzebrafishandhumanenhancerusage/ promotersatRNAPIIfoci.
expression.SupplementaryFig.20showsUCSCbrowserimagesofeach Onaverage,aRefSeqTSSwasassociatedwith4.9enhancersandan
selectedenhancer.a,CRE1,,230kbupstreamoftheMEFC2gene,drives enhancerwith2.4TSSsandweobserveddifferentregulatoryarchi-
highlyrobustexpressioninthebrain(brain)andneuraltube(nt).Rightpanel
tecturesaroundgenes(SupplementaryFig.30).Forexample,atthe
giveszoom-inoverlayimageshowingexpressionintheforebrain(fb),midbrain
beta-globinlocustheCAGEexpressionpatternsoffourlocuscontrol
(mid),hindbrain(hin)andspinalcord(sp).b,CRE2,5kbupstreamofthe
POU3F2gene,isactiveinthefloorplate(fp).c,CRE3,10kbupstreamofthe regionhypersensitivesitesarehighlycorrelated(Pearson’srbetween
SOX7geneTSS,showsspecificexpressioninthevasculature(including 0.88and0.98)withtheexpressionofknowntargetgenes23,24HBG2
intersegmentalvessels(iv),dorsalvein(dv)anddorsalaorta(da). andHBD,andtosomeextentHBG1.
458 | NATURE | VOL 507 | 27 MARCH 2014
©2014Macmillan Publishers Limited. All rights reserved
ARTICLE RESEARCH
8 Figure5|Enhancerusageand
ncers perAGE tags) 6 8 sudppeetpececirftiepcdiatenyneilhngaingvrceoesrustphpseeonrfumcmeillbllieso.rnoTfhe
og(enhamillion C 4 CofArGelEatetadgcsewllitthypineeliabcrhargireos.uTph(feacet)
l
2 expressionspecificityofthe
Expression specificitybiquitousExclusive00001.....24680 0F.0raction o0f. 3e5nhancer0s.7 etfiecnrhnaoaecrhechraatpeicnassohppcnneoeefrcoalnsicfbdfeiieeisctnxlios(tpghycwropoer.llwsaCuosnnmteosgdlaenoosesun(n)arrohosthhwarseganhsaatc)otn.eawmF/rrtseoitashripsneuien
U0.0 facetsandgenes,seeSupplementary
KeratocyteTrabecular meshwork cellEnteric smooth muscle cellSmooth muscle cell of tracheaSmooth muscle cell of prostateCiliated epithelial cellFibroblast of periodontiumTendon cellFibroblast of choroid plexusPericyte cellCorneal epithelial cellMyoblastEpithelial cell of oesophagusFibroblast of gingivaFibroblast of lymphatic vesselHair follicle cellCardiac myocyteOsteoblastHepatic stellate cellLens epithelial cellCardiac fibroblastFibroblast of tunica adventitia of arteryAmniotic epithelial cellSkeletal muscle cellAstrocyteChondrocyteRetinal pigment epithelial cellFat cellKeratinocytePlacental epithelial cellMesothelial cellAcinar cellEpithelial cell of MalassezStromal cellSkin fibroblastUrothelial cellPre-adipocyteMammary epithelial cellGingival epithelial cellEndothelial cell of hepatic sinusoidEpithelial cell of prostateEndothelial cell of lymphatic vesselRespiratory epithelial cellSensory epithelial cellMelanocyteBlood vessel endothelial cellKidney epithelial cellVascular-associated smooth muscle cellMesenchymal cellMacrophageNeuronReticulocyteHepatocyteDendritic cellMast cellT cellMonocyteLymphocyte of B lineageNatural killer cellNeuronal stem cellNeutrophil Fig.21.
Theseobservationscallforcomputationalmodelsofenhancerregu- Modelshrinkageshowedthatinmostcases,onlyonetothreeenhancers
lation,inwhichmultipleenhancersmayworkinconcerttoenhance arenecessarytoexplaintheexpressionvarianceobservedinthelinked
theexpressionofagene.Tothisend,wefocusedon2,206RefSeqTSSs gene,andgenerallyproximalenhancersaremorepredictivethandistal
for which the joint expression of nearby enhancers (the closest ten ones(Fig.6a,SupplementaryFig.29b–dandSupplementaryText).One
enhancerswithin500kb)ishighlypredictiveofthegeneexpression. hypothesisexplainingthefunctionofmultipleenhancersdrivingthe
a b d
0.8 Basophil, NK cell, cardiac NK cell, B cell, whole Tonsil, B cell,
Proportional contribution Rr22/)to full model (0.4 log(gene expression)2(max TPM)167890 mnceyeuo sbltclr,mIoyo nmwtpaflelahae,l smiliwlC nGd,td mthrhiciresosoaaeaesehvlilttaerlanoe,i ns sf’sbrToseeey’l l,ocl icoeldlel, bamsmooapnshoti lcc, yeTtl elc,, e ll sebdnleosnooddryr,i tseicpp licetheeelnl,l,i a Thyroid glanddwenhsdoplreilte ibcel noc,oe ldl,
0.0 disease
1 3 5 7 9 0 2–3 4–5 6–7 >7 lympChhorcoyntiicc
Enhancer Number of highly leukemia
(ranked by proximity) correlated enhancers 22 23 24 25 26 27
Enhancer over-representation (odds ratio)
Odds ratio c 1120505 6 (0.308) 12 (0.859) 9 (0.713) 4 (0.381) 9 (0.989) 11 (1.418) 6 (0.891) 4 (0.616) 6 (0.964) 13 (2.374)SNmP17 (3.192)asi no6 (1.248)lyv einr6 (1.329)- reenp20 (4.489)hr7 (2.308)aens40 (11.092)ce13 (5.704)enrt6 (1.758)sed 75 (22.37) 15 (4.837) 8 (2.698) 61 (21.325)24 (10.965)11 (4.084) 19 (7.965) 12 (5.121) 11 (4.805) 16 (8.135) 19 (9.675) 64 (32.637)32 (16.781)24 (12.916) 5 (1.193)S9 (1.109)NP4 (0.618)ms oa5 (0.919)ivnelyr-4 (0.824) rine pe5 (1.284)rxeosn6 (1.727)esnte6 (1.791)d 14 (4.183) 12 (3.835) 17 (6.972) 13 (6.101) 26 (15.149) 4 (0.404) 4 (0.437) 4 (0.45) 7 (0.825)S4 (0.587)NmPa5 (0.754)sin olyv8 (1.304) ei6 (1.418)nr -4 (0.729)prerop9 (1.692)mreoste5 (0.996)enr16 (3.977)tse5 (1.046)d 13 (3.066) 17 (4.175)36 (17.826)37 (9.165) 11 (3.296)27 (13.168)11 (3.462)33 (14.423)19 (7.416) 11 (4.35)Enhancers50 (29.853)Exons19 (7.849)17 (7.483)Promoters37 (17.372)20 (8.932)Random67 (34.835)
0
Thyrotoxic hypokalaemic periodic paralysisBirth weightHodgkin’s lymphomaEpirubicin-induced leukopenia Prostate-specific antigen levelsSystemic sclerosisCorneal astigmatismNodular sclerosis Hodgkin lymphomaPsoriatic arthritisInflammatory bowel diseaseLeprosyBasal cell carcinomaChronic lymphocytic leukaemiaEndometriosisCoeliac diseaseSystemic lupus erythematosusIgA nephropathyCoeliac disease and rheumatoid arthritisHair morphologyMultiple sclerosisCutaneous neviPrimary biliary cirrhosisAortic root sizeC-reactive proteinSystolic blood pressure Mean platelet volume Crohn’s diseasePlatelet countsSoluble levels of adhesion moleculesEwing sarcomaSoluble ICAM-1HaemoglobinRed blood cell traits Primary sclerosing cholangitisHaematological parametersOther erythrocyte phenotypesFibrinogenCreatinine levelsWarfarin maintenance doseAge-related macular degene rationChronic kidney diseaseInflammatory bowel disease (early onset)Cystatin CPhosphorus levelsDehydroepiandrosterone sulphate levelsVitamin D insufficiencyKeloidEye colour traitsGraves’ diseaseTelomere lengthAlopecia areataMajor mood disordersType 1 diabetes autoantibodiesLiver enzyme levels (alkaline phosphatase)AsthmaType 1 diabetesHaematological and biochemical traitsPsoriasisRheumatoid arthritisAttention deficit hyperactivity disorder Metabolic traitsUlcerative colitisHeight
Figure6|LinkingenhancerstoTSSsanddisease-associatedSNPs. a,The withinenhancers,exonsandmRNApromoters.Observedandexpected
proportionalcontribution(seeMethods)ofthe10mostproximalenhancers overlapsareshownabovebars.Theverticalaxisgivesenrichmentoddsratios.
within500kbofaTSSinamodelexplaininggeneexpressionvariance(vertical ThehorizontalaxisshowsGWAStraitsordiseases.d,Diseaseswith
axis)asafunctionofenhancerexpression.xaxisindicatesthepositionofthe GWAS-associatedSNPsover-representedinenhancersofcertainexpression
enhancerrelativetotheTSS:1theclosest,etc.Barsindicateinterquartileranges facets.Thehorizontalaxisgivestheoddsratioasinpanelc,brokenupby
anddotsmedians.b,Relationshipbetweenthenumberofhighlycorrelated expressionfacets:eachpointrepresentstheoddsratioofGWASSNP
(‘redundant’)enhancersperlocus(horizontalaxis)andthemaximalexpression enrichmentforadisease(verticalaxis)inaspecificexpressionfacet.Summary
(TPM)oftheassociatedTSSinthesamemodeloverallCAGElibraries(vertical annotationsofpointcloudsareshown.SeealsoSupplementaryFig.31.
axis).Errorbarsasina.c,GWASSNPsetspreferentiallyoverrepresented
27 MARCH 2014 | VOL 507 | NATURE | 459
©2014Macmillan Publishers Limited. All rights reserved
RESEARCH ARTICLE
sameexpressionpatternisthattheymightconferhighertranscriptional METHODSSUMMARY
outputofagene25,26.Indeed,thenumberofhighlycorrelated(redund- Single-molecule HeliScopeCAGE data was generated as described elsewhere6.
ant)enhancersclosetoTSSs(SupplementaryMethods)increasedwith SequencingandprocessingofribosomalRNA-depletedRNAs,shortRNAsand
theobservedmaximalTSSexpressionoveralllibraries(Fig.6b),imply- H3K27ac or H3K4me1 ChIPs as well as the processing of publicly available
ingthattheseenhancersareredundantintermsoftranscriptionpatterns DNase-seqdataaredescribedintheMethods.
butadditiveintermsofexpressionstrength.Expressionredundancyis Putativeenhancerswereidentifiedfrombidirectionallytranscribedlocihaving
alsocommoningenomicclustersofcloselyspacedenhancers(24%of divergent CAGE tag clusters separated by at most 400bp (described in Sup-
plementaryFig.6a).Werequiredlocitobedivergentlytranscribedinatleast
815identifiedgenomicclusters,SupplementaryTable15).Theseare
oneFANTOM5sample,definedbyCAGEtag59endswithin200bpdivergent
associatedwithTSSsofgenesinvolvedinimmuneanddefenceresponses
strand-specificwindowsimmediatelyflankingthelocimidpoints.Theexpression
and,assuggestedbyapreviousstudy27,haveahigherexpressionthan
ofeachenhancerineachFANTOM5samplewasquantifiedasthenormalized
otherenhancer-associatedgenes(eightfoldincreaseonaverage).
sumofstrand-specificsumsofCAGEtagsinthesewindows.Asample-setwide
directionalityscore,D,foreachlocusoveraggregatednormalizedreverse,R,and
Disease-associatedSNPsareenrichedinenhancers forward,F,strandwindow-expressionvaluesacrossallsamples,D5(F2R)/
Manydisease-associatedSNPsarelocatedoutsideofprotein-coding (F1R),werethenusedtofilterputativeenhancerstohavelow,non-promoter-
exons and a large proportion of human genes display expression like,directionalityscores(jDj,0.8).Furtherfilteringensuredenhancerstobe
polymorphism28.UsingtheNHGRIgenome-wideassociationstudies locateddistanttoTSSsandexonsofprotein-andnoncodinggenes.
(GWAS)catalogue29andextendingthecompilationofleadSNPswith MotifenrichmentanalysesweredoneusingHOMER36.Regulatorytargetsof
enhancerswerepredictedbycorrelationtestsusingthesample-setwideexpress-
proxySNPsinstronglinkagedisequilibrium(similartorefs30,31),
ionprofilesofallenhancer-promoterpairswithin500kb.Theregulatoryeffects
weidentifieddiseases/traitswhoseassociatedSNPsoverlappedenhan-
ofmultipleenhancersweremodelledusinglinearregressionfollowedbylasso-
cers, promoters, exons and random regions significantly more than
basedmodel-shrinkage37.
expectedbychance(Fisher’sexacttestP,0.01,SupplementaryTable16).
EnhanceractivitywastestedinvivoinzebrafishembryosusingTol2-mediated
Disease-associatedSNPswereover-representedinregulatoryregions transgenesis38.Expressionpatternsweredocumentedat48hpostfertilization
toagreaterextentthaninexons(Fig.6c).Formanytraitswhereenriched using .200 eggs per construct. Large-scale in vitro validations on randomly
disease-associatedSNPswerewithinenhancers,enhanceractivitywas selectedenhancerswereperformedusingfirefly/Renillaluciferasereporterplas-
detectedinpathologicallyrelevantcelltypes(Fig.6dandSupplementary mids with enhancer sequences cloned upstream of an EF1a basal promoter
Figs 31 and 32). Examples include Graves’ disease-associated SNPs separatedbyasyntheticpolyAsignal/transcriptionalpausesiteinamodified
enriched in enhancers that are expressed predominantly in thyroid pGL4.10(Promega)vector(SupplementaryFig.9d).Fulldetailsareprovided
tissue,andsimilarlylymphocytesforchroniclymphocyticleukaemia. intheMethods.
Asaproofofconcept,wevalidatedtheimpactoftwodisease-associated OnlineContentAnyadditionalMethods,ExtendedDatadisplayitemsandSource
regulatorySNPswithinenhancers(SupplementaryFig.33). Dataareavailableintheonlineversionofthepaper;referencesuniquetothese
sectionsappearonlyintheonlinepaper.
Conclusions
Received4January;accepted16October2013.
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RESEARCH ARTICLE
METHODS definedforeachbidirectionallocusasthemidpositionbetweentherightmost
CAGEdata.SinglemoleculeHeliScopeCAGE39datawasgeneratedasdescribed reversestrandTCandleftmostforwardstrandTCincludedinthemergedbidir-
elsewhere6.Weusedasetof432primarycell,135tissueand241celllinesamples ectionalpair.Eachbidirectionallocuswasfurtherassociatedwithtwo200bp
thatpassedqualitycontrolmeasuresof.500,000Q20Delve(T.L.etal.,manu- regionsimmediatelyflankingthecentreposition,one(left)forreversestrand
transcriptionandone (right)forforwardstrandtranscription,ina divergent
scriptinpreparation)mappedCAGEtags,RNAintegrityandreproducibility(for
manner.Themergedbidirectionalpairswerefurtherrequiredtobebidirection-
furtherdetails,seeref.6).
allytranscribed(CAGEtagssupportingbothwindowsflankingthecentre)inat
Proofofconceptanalysis.WedefinedsilentandactiveenhancersfromENCODE
leastoneindividualsample,andtohaveagreateraggregateofreverseCAGEtags
HeLa-S3,GM12878andK562broadpeaks,downloadedfromtheUCSCENCODE
(overall FANTOM5samples) thanforwardCAGEtagsin the200bp region
repository, according to the co-existence of histone modifications H3K4me1,
associatedwithreversestrandtranscription,andviceversa.Thesefilteringsteps
H3K27acandH3K27me3.Activeenhancersweredefinedasco-localizedH3K4me1
resultedin78,555bidirectionallytranscribedloci.
andH3K27acpeakswith noH3K27me3peak,whereas silentenhancerswere
considered loci with H3K4me1 andH3K27me3 peaks but no H3K27ac peak. Expressionquantificationofbidirectionallytranscribedlociandpredictionof
LociwerefilteredtobelocateddistanttoTSSs(500bp)andexons(200bp)ofprotein- enhancers.Wequantifiedtheexpressionofbidirectionallociforeachstrandand
coding genes, multi-exonic noncoding genes and mRNAs (from ENSEMBL, 200bpflankingwindowineachofthe432primarycell,135tissueand241cellline
GENCODE(v10),RefSeqandUCSC,downloadedJanuary12,2012),andother samplesseparatelybycountingtheCAGEtagswhose59endswerelocatedwithin
lncRNAsfromagene-centricsetderivedfromliterature40aswellasmanually thesewindows.Theexpressionvaluesofbothflankingwindowswerenormalized
annotated sense-antisensepairs(coding-noncoding andnoncoding-noncoding byconvertingtagcountstotagspermillionmappedreads(TPM)andfurther
sense-antisensepairs)with59expressedsequencetag(EST)andcomplementary normalizationbetweensampleswasdoneusingtherelativelogexpression(RLE)
DNAsupport,and59ESTswithnolocuswithprotein-codingcapacity.Trans- normalizationprocedureinedgeR41.ThenumberofCAGEtagsalignedonChrM
criptionaldifferencesbetweenactiveandsilentenhancersetsweredeterminedby wassubtractedfromthetotalnumberofalignedCAGEtagsineachlibrarybefore
comparingtheaveragenumberofFANTOM5CAGEtag59endsfromthesame normalization.Thenormalizedexpressionvaluesfrombothwindowswereused
ENCODEcelllines(pooledtriplicates)inawindow6300bparoundtheH3K4me1 tocalculateasample-setwidedirectionalityscore,D,foreachenhancerover
peakmidpoints. aggregatednormalizedreverse,R,andforward,F,strandexpressionvaluesacross
The active enhancer sets of HeLa-S3, GM12878 and K562 cells were then allsamples(SupplementaryFig.6a);D5(F2R)/(F1R).Drangesbetween21
centredonproximal(within200bp)P300ENCODEbindingsitepeaks(joint and1andspecifiesthebiasinexpressiontoreverseandforwardstrand,respect-
P300andGATA1peaksforK562)toderivecentrepositions.FANTOM5CAGE ively(D50means50%reverseand50%forwardstrandexpression,whilejDj
datafromthesameENCODEcelllines(pooledtriplicates)werethenoverlaid closeto1indicatesunidirectionaltranscription).Adirectionalityscorecalculated
overthesecentredenhancerregionsandtheabsence(0)andpresence(1)of(one frompooleddataisagoodestimateofsampledirectionality(Supplementary
ormore)CAGEtag59endsin10bpnon-overlappingwindowsweredetermined Fig.6b).Eachbidirectionallocuswasassignedoneexpressionvalueforeach
andanaverageprofilewascalculatedtoassesstheaveragebidirectionalpatternof samplebysummingthenormalizedexpressionofthetwoflankingwindows.
transcriptionatchromatin-derivedenhancers. Bidirectionallociwerefurtherfilteredtohavelow,non-promoter-like,direc-
PooledCAGEdatafromallFANTOM5libraries(describedabove)werefur- tionalityscores(jDj,0.8)andtobelocateddistanttoTSSsandexonsofprotein-
theroverlaidwiththeseregionsandadirectionalityscorebasedontheaggregate and noncodinggenes (see‘Proofof conceptanalysis’above fordetails).This
ofCAGEtagsfallingwithin6300bpfromthecentrepositionswerecalculatedto resultedinafinalsetof43,011putativeenhancers.
determinepotentialstrandbias.Forcomparison,werepeatedthesamecalcula- Wefurthertestedwhethertheexpressionlevelforeachsampleandcandidate
tionsforgenomicregions6300bparoundTSSsofRefSeqproteincodinggenes. enhancerwassignificantlygreaterthanthegenomicbackground(seeconstruc-
Directionalitywascalculatedas(F-R)/(F1R),whereFandRisthesumofCAGE tionofrandomgenomicbackgroundregionsbelow).APvaluewascalculatedfor
tagsalignedontheforwardandreversestrand,respectively.Directionalitycloseto eachenhancerexpressionvalueforeachprimarycell,tissueandcelllinesample
-1or1indicatesaunidirectionalbehaviourwhile0indicatesperfectlybalanced bycountingthefractionofrandomgenomicregionswithgreaterexpressionlevel
bidirectionaltranscription. inthesamesample.EnhancerswithPvalueslessthan0.001andBenjamini–
Positional cross correlations were calculated between reverse and forward HochbergadjustedFDR,0.05wasconsideredtranscribedinthatsample.This
CAGEtag59endsatChIP-seq-derivedactiveHeLa-S3andGM12878enhancer analysis yielded binary expression values, which were used for constructing
centrepositions(asdeterminedbyP300peaks)6300bp(maximumlag300)to enhancersetsassociatedwitheachsample.Intotal,38,554enhancersweretran-
identifytheirmostlikelyseparation.Crosscorrelationswerealsocalculatedin300bp scribed at a significant expression level in at least one primary cell or tissue
windows(maximumlag150)flankingtheenhancercentresbetweenCAGE59ends sample.Below,werefertothissetasthe‘robustset’ofenhancersandindicate
andENCODEH2A.Zsignals(fromthesamecellline)forHeLa-S3andGM12878 wheneveritwasused.Forallanalyses,weusethewhole(‘permissive’)setof
aswellasbetweenCAGE59endsand59endsofENCODEGM12878micrococcal 43,011enhancersifnototherwisementioned.
nuclease-digested nucleosome sequencing (MNase-seq) reads (9 pooled repli- Constructionofrandomgenomicbackgroundregions.Werandomlysampled
cates).Inthelatteranalysis,correlationsweremadeusingreadsonthesamestrand. 100,000genomicregionsof401bpthatweredistaltoTSSsandexonsofknown
Pooled,uniqueCAGEtags(inwhichonlyoneCAGEtagperbpwascounted)were genes(sameasthefilteringproceduredescribedaboveforbidirectionallytran-
consideredinallcorrelationanalysesandenhancerswereweighedaccordingtothe scribedloci).Thesewerefurtherfilteredtonotoverlapwithoursetof43,011
aggregatedsignalbeforesubsequentaveragingoverlagsnottomakeanylibraryor predictedenhancers,whichyielded98,942randomgenomicregionswhoseexpress-
enhancerhaveanundueinfluence. ionlevelswerequantifiedandnormalizedinthesamemannerasdescribedfor
ReporteractivityofENCODEenhancersinrelationtotranscriptionalstatus. bidirectionalloci(above).
Weusedpublished8resultsonamassivelyparallelreporterassaymeasuringthe CorrelationbetweenENCODEepigenomicdataandCAGE-definedenhan-
activityofENCODE-predictedenhancersinHepG2andK562cells.Allresultson cers.UsingtheUCSCENCODErepositorydata(downloadedandpooled26
non-scrambledsequenceswereconsidered,regardlessofthelevelofconservation. March2012),weassessedthesignalofRNAPolymeraseII(RNAPII),thepooled
198outof738testedK562enhancersand307outof1,136testedHepG2enhancers transcriptionfactorsupertrack(allTFs),CCCTC-bindingfactor(CTCF),E1A
hadsignificantenhancerreporteractivity(asdeterminedbytheoriginalpublica- binding protein P300, DNase I hypersensitive sites (DHSs) and two histone
tion).Wedeterminedtheexpressionin401bpwindowscentredonmidpointsof marks:H3K4me1andH3K27acaroundenhancers,TSSsandrandomgenomic
ENCODE-predictedenhancersusingFANTOM5CAGEfromthesamecelllines. sites.
Wefurthercalculatedthefalsediscoveryrateafteraminimumexpressionthresh- Largescaleenhancerreportervalidations.Werandomlyselected125CAGE-
oldintheinterval[0,0.5]TPM,asthefractionofnon-significantenhancersamong definedenhancerswithsignificantlyhigherexpressionthanrandomgenomic
thosefulfillingtheexpressioncutoff. regions in at least two out of three HeLa-S3 replicates. These were grouped
Identification of bidirectionally transcribedloci. Bidirectionally transcribed according to Hela-S3 expression tertiles: low (36), mid-level (41) and strong
lociweredefinedfromasetof1,714,047forwardand1,597,186reversestrand (46).Thesecouldbesplitupfurtheraccordingtooverlap(midposition)with
CAGEtagclusters(TCs)supportedbyatleasttwoCAGEtagsinatleastone combined ENCODE (release January 2011) segmentations of Segway42 and
sample(TCsdefinedinref.6).OnlyTCsnotoverlappingantisenseTCswere ChromHMM43chromatinstateprediction:25,27and14strongly,mid-leveland
used.Weidentified1,261,036divergent(reverse-forward)TCpairsseparatedby lowlyexpressedCAGEenhanceroverlappedENCODEstate‘E’(‘strongenhancer’)
atmost400bpandmergedallsuchpairscontainingthesameTC,whileatthe whereas21,16and22strongly,mid-levelandlowlyexpressedCAGEenhancer
sametimeavoidingoverlappingforwardandreversestrandtranscribedregions overlappedENCODEstate‘TSS’.
(prioritizationbyexpressionranking),whichresultedin200,171bidirectional Wefurtherrandomlyselected26and15untranscribed(negligibleamountof
loci (procedure illustrated in Supplementary Fig. 6a). A centre position was overlappingFANTOM5HeLa-S3CAGEtags)500bpregionscentredonmid
©2014Macmillan Publishers Limited. All rights reserved
ARTICLE RESEARCH
positions of HeLa-S3 E states and HeLa-S3 ENCODEDHSs. Two literature- differingbetweenthestructurallyrelatedbidirectionallytranscribedenhancer
derived44HeLa-S3positiveenhancersand4randomregions(see‘Construction TSSsandnonCGI-associatedpromoterTSSs,weextracted600bpregionsdown-
ofrandomgenomicbackgroundregions’)wereusedforcomparison.Forcom- streamofeachTSSandperformedcomparativedenovomotifsearchesusing
parison,wealsorandomlyselected20manuallydefineduntranscribedHeLa-S3 HOMER.Here,weanalysedonesetusingtheothersetasbackground(corrected
chromatin-definedactiveenhancers(see‘Proofofconceptanalysis’). forregionsize,matchedforGCcontentandauto-normalized)tocalculatemotif
PCR primers for the amplification of enhancer and control regions were enrichmentonlyonthegivenstrand.Thetopmotifenricheddownstreamof
designedusingthePerlPrimertool45,andpurchasedfromOperonLtdPrimers nCGIpromoterswasthe59-splicesitemotif.Genomicdistributionsoftheenriched
includedBamHIorSalIrestrictionsitesforcloningandsequencesarelistedinSup- splicesitemotif,aswellastheAATAAAterminationsignalweregeneratedusing
plementaryTables2and3.Controlfragmentsrangedbetween420and1,452bp. HOMER.
Enhancerfragmentsusuallyincludeda500bpwindowaroundthemid-pointof RNA-seqsamplesandlibrarypreparation.Priortopreparationofsequencing
our predicted enhancers and depending on the availability of unique primer libraries, rRNA was removed by poly(A)1 selection (CD191 B-cells, CD81
sequences,enhancerfragmentsrangedbetween470and840bp. T-cells,500ng)orrRNAdepletion(fetalheart,1mg).Poly(A)1selectionwas
WeinsertedanEF1abasalpromoterfragmentintoHindIIIandNheIsitesof donetwicebyusingDynabeadsOligo(dT) (LifeTechnologies)accordingtothe
25
themultiplecloningsiteinthepromoter-lesspGL4.10(Promega)toconstructthe manufacturer’smanual.rRNAdepletionwasdonebyusingRibo-ZerorRNA
pGL4.10EF1avector.WenextremovedtheBamHIandSalIcontainingfragment removalkit (Epicentre, Illumina)accordingto themanual.ThetreatedRNA
locateddownstreamoftheSV40latepoly(A)signal,andre-insertedthefragment wasdissolvedin20mlwater.
attheSpeIsitethatislocatedupstreamofthesyntheticpoly(A)signal/transcrip- ThepretreatedRNAwasthenfragmentedbyheatingat70uCfor3.5minin
tionalpausesitetogeneratemodifiedversionsofpGL4.10EF1aandpGL4.10(see fragmentationbuffer(Ambion),followedbyimmediatechillingoniceandaddi-
SupplementaryFig.9d). tionof1mlofStopsolution.FragmentedRNAwaspurifiedwiththeRNeasy
EnhancerandcontrolregionswerePCR-amplifiedusingKODpluspolymer- MinElute kit (Qiagen) following the instructions of the manufacturer except
ase(Toyobo)fromHEK-293TgDNA,digestedwithBamHIandSalI(Takara 675mlof100%ethanolisusedinsteptwo,insteadof500ml.PurifiedRNAwas
Bio),andpurifiedusingtheE-GelSizeSelectsystem(LifeTechnologies).Fivemlof dephosphorylated in phosphatase buffer (New England Biolabs) with 5 U of
purifiedPCRproductswereligatedwith100ngoftheBamHI-andSalI-digested Antarctic phosphatase (New England Biolabs) and 40 U of RNaseOut (Life
modifiedpGL4.10EF1aandpGL4.10plasmidsusingLigation-high(Toyobo), Technologies)at37uCfor30minfollowedby5minat65uC.Afterchillingon
andtransformedintoDH5acompetentcells(Toyobo).Correctinsertionofthe ice RNA was phosphorylated by addition of the following reagents; 5ml of
PCR products into the plasmids was checked by colony PCR. Vectors were 103PNK buffer, 20 U of T4 polynucleotide kinase (New England Biolabs),
purifiedusingtheQIAGENPlasmidPlus96MiniprepKit(Qiagen). 5mlof10mMATP(Epicentre,Illumina), 40UofRNaseOut,17mlofwater.
HeLa-S3cells(JCRBCellBank)wereculturedinMEM(WAKO)supplemen- Thereactionwasincubatedat37uCfor60min.PhosphorylatedRNAwaspuri-
tedwith10%FBS(NichireiBioscienceInc.,lotno. 7G0031),100Unitsml21 fiedwiththeRNeasyMinElutekit(Qiagen)asdescribedabove.PurifiedRNAwas
penicillinand100mgml21streptomycin(bothLifeTechnologies).HepG2Cells concentrated to 6ml by vacuum centrifugation on a SpeedVac (Eppendorf).
(RIKENBRC)wereculturedinDMEM(LifeTechnologies)supplementedwith Onemlof2mMpre-adenylated39DNAadaptor,59-App/ATCTCGTATGCCGT
10%FBS(NichireiBioscienceInc.,LotNo.7G0031),andMEM(WAKO)sup- CTTCTGCTTG-39 was added to the concentrated RNA. After incubation at
plementedwith10%FBS(NichireiBioscienceInc.,lotno.7G0031),100Units 70uCfor2minfollowedbychillingonicefor2min,thefollowingreagentswere
penicillin and 100mgml21 streptomycin (Life Technologies). Cell lines were addedtoligatetheadaptoratthe39endoftheRNA;1mlof103T4RNAligase2
seededinto96wellplatesatadensityof7.53103cellsperwellonedaybefore truncatedbuffer,0.8mlof100mMMgCl,20UofRNaseOUTand200UofRNA
2
transfection.Fireflyluciferasereporterplasmids(190ng)and10ngofpGL4.73 ligase2truncated(NewEnglandBiolabs).Aftertheincubationat20uCfor60min,
Renillaluciferaseplasmid(Promega)wereco-transfectedintoHepG2orHeLa-S3 1mlofheat-denatured5mM59RNAadaptor,59-GUUCAGAGUUCUACAGU
cellsusingLipofectamine(LifeTechnologies)accordingtothemanufacturer’s CCGACGAUCGAAA-39wasligatedwith39adaptorligationproductswith20U
instruction.Eachtransfectionwasindependentlyperformedthreetimes.After24h, ofT4RNAligase1(NewEnglandBiolabs)and1mlof10mMATP(NewEngland
theluciferaseactivitiesweremeasuredbyGloMax96Microplateluminometer Biolabs)at20uCfor60min.4mlofadaptorligatedRNAwasmixedwith1mlof
(Promega)usingtheDual-gloluciferaseassaysystem(Promega)accordingtothe 20mMRTPrimer,59-CAAGCAGAAGACGGCATACGA-39,followedbyincuba-
manufacturer’sinstruction. tionat70uCfor2min,andimmediatelykeptonice.Thereversetranscription
Sequencemotif analysis on global CAGE enhancer and promoter sets. To reactionwasdonewith2ml53PrimeScriptbuffer,1mlof10mMdNTP,20Uof
comparemotifsignaturescharacterizingbidirectionallytranscribedenhancers RNaseOUTand200UofPrimeScriptReverseTranscriptase(TakaraBIO)at44uC
(permissive set) with those of CAGE-defined promoters, we used the set of for30min.ThecDNAproductwasamplifiedbyPCRwith10mlof53HFbuffer,
184,827robusthumanCAGEclustersdefinedbyref.6separatedinto61,322 1.25mlof10mMeachdNTPmix,2mlof10mMFWDprimer,59-AATGATACG
CGIand123,505nonCGI-associatedclusters.Wemadefurthersubsetsofthese GCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-39,2mlofRTprimer
CAGEclusters,contingentontheiroverlapwithannotatedTSSsfromRefseqand and1UofPhusionHigh-FidelityDNAPolymerase(NewEnglandBiolabs).PCR
Gencode.WemergedoverlappingextendedCAGEclusters(2300,150;based wascarriedoutinatotalvolumeof50mlwiththefollowingthermalprogram;
ontherobustclusterset;averagesizenonCGI:422bp;averagesizeCGI:544bp) 98uCfor30s,12PCRcyclesof10sat98uC,30sat60uC,and15sat72uC,
contingentonCGIstatusandsubtractedCAGEclusterregionsthatoverlapped followedbyat72uCfor5minandthenkeptat4uC.RemainingPCRprimerswere
withextendedenhancers(midposition6200bp). removedtwicebyusing1.2volumesofAMPureXPbeads(BeckmanCoulter).The
Thiscreatedfivesetsofregionsrepresentingnon-overlappingbidirectional resultinglibrarieswerecheckedforsizeandconcentrationbyBioAnalyzer(Agilent)
enhancers,nonCGIpromotersandCGIpromoters(annotatedandfullsetsfor usingtheHigh-SensitivityDNAKit(Agilent).Qualifiedsequencinglibrarieswere
thetwolatterones).MotifenrichmentwasanalysedusingHOMER36version3,a loadedontheHiSeq2000(Illumina)usingthecustomsequencingprimer,59-CG
suiteoftoolsformotifdiscoveryandnext-generationsequencinganalysis(http:// ACAGGTTCAGAGTTCTACAGTCCGACGATCGAAA-39.
biowhat.ucsd.edu/homer/).Sequencesofthethreeregionsets(enhancers,nonCGI AllRNA-seqsamplesprofiledinthisstudywerealsoprofiledintheFANTOM5
andCGIpromoters)werecomparedtoequalnumbersofrandomlyselectedgeno- promoteromemanuscriptandaredescribedindetailthere6.Brieflyallhuman
micfragments of the average region size, matched for GC content andauto- samplesusedintheprojectwereeitherexemptedmaterial(availableinpublic
normalized to remove bias from lower-order oligo sequences. After masking collectionsorcommerciallyavailable),orprovidedunderinformedconsent.All
repeats,motifenrichmentwascalculatedusingthecumulativebinomialdistri- non-exempt material is covered under RIKEN Yokohama Ethics applications
butionbyconsideringthetotalnumberoftargetandbackgroundsequenceregions (H17-34andH21-14).ForthesamplesprofiledbyRNA-seq,thehumanfetalheart
containingatleastoneinstanceofthemotif.Onehundredmotifsweresearchedfor RNAwaspurchasedfromClontech(Catalogueno.636583).CD191B-cellsand
arangeofmotiflengths(7–14bp)resultinginasetof800denovomotifsperset. CD81T-cellswereisolatedusingthepluriBeadsystem(huCD4/CD8cascadeand
Afterfilteringredundantmotifs,thetop50motifsresultingfromeachsearchwere huCD19single;PluriSelect).RNAwasthenextractedusingthemiRNeasykit(Qiagen).
combined, remapped and ranked according to enrichment (depletion) in the RNA-seqmappingandtranscriptassembly.Single-end100bplongreadsfrom
enhancerset.Inparallel,wealsousedHOMERtocalculatetheenrichmentof librariesoriginatingfromthesimilarcellsources(allsix‘‘CD191Bcells’’librar-
ChIP-seqderivedknowntranscriptionfactormotifs.Motifcollectionsincluding ies,allsix‘‘CD81Tcells’’librariesandone‘‘Fetalheart’’library)wereprocessed
searchparametersaredepositedinawebdatabaseathttp://enhancer.binf.ku.dk. togetherviatheMoiraipipeline(Hasegawa,Y.etal.,manuscriptinpreparation).
Histograms of PhastCons scores were generated using the annotation tool in TheprocessingstepsimplementedwithintheMoiraipipelineincluded(1)raw
HOMER. sequencedreadsPolyAtailand‘‘CTGTAGGCACCATCAAT’’adaptorclipping
AnalysisofsplicesiteandterminationsignalsdownstreamofCAGEenhancer usingFASTQ/AClipperfromFASTX-Toolkit(http://hannonlab.cshl.edu/fastx_
TSSsandpromoterTSSs.ToidentifymotifsdownstreamofTSSspotentially toolkit/),(2)removalofsequencedreadscontaining‘‘N’’andsequencessimilarto
©2014Macmillan Publishers Limited. All rights reserved
RESEARCH ARTICLE
ribosomalRNAusingrRNAdustversion1.02(T.L.etal.,manuscriptinprepara- AssessmentofdegradationratesofRNAsemanatingfromCAGEenhancers
tion),and(3)mappingtheresultingreadsagainstthehg19humangenomeusing andpromoters.Bidirectionallytranscribedlociwereidentifiedinthesameway
TopHat46(version1.4.1)usingbothTopHatdenovojunctionfindingmodeand aswithpooledFANTOM5CAGElibraries(see‘Identificationofbidirectionally
knownexon-exonjunctionsextractedfromGENCODEV10,withalltheother transcribedloci’and‘Expressionquantificationofbidirectionallytranscribedloci
parameterssettotheirdefaultvalues.MappedreadsflaggedasPCRduplicates andpredictionofenhancers’above)fromtagclusters(asdefinedin51)derived
wereremovedandtheremainingTopHatalignedreadswerethenassembledusing frompooledHeLaCAGE(mocktreatedcontrol,SKIV2L2)libraries.From5,892
Cufflinks47(version1.3.0)withCufflinksparameterssettotheirdefaultvalues. bidirectionallocidistanttoTSSsandexons,4,196werepredictedtobeenhancers
AssessmentoflengthsofRNAsemanatingfromenhancersandpromoters.All basedonbalanceddirectionalityoftranscriptionofwhich3,896hadsignificantly
Cufflinksassembledtranscripts,whose59ends,regardlessofstrand,werelocated greaterexpressionthanrandomgenomicregionsinatleastonelibrary.These
withintheouterboundariesofCAGEenhancersor,onthesamestrand,within were then overlapped with the whole set of FANTOM5 CAGE enhancers to
200bp(upstreamordownstream)ofaGENCODE(v10)protein-codingTSS estimatethefractionofunseentranscribedenhancers.
wereconsideredforfurtheranalysis.FortheseCufflinkstranscriptswecalculated TheexpressionfoldchangeofHeLadepletedofSKIV2L2comparedtomock-
their(intron-less)RNAlength,(possiblyintron-containing)genomiclengthas treated control were assessed and compared between expressed HeLa CAGE
thegenomicdistancebetweentheir59and39ends,aswellastheirnumberofexons. enhancers,promotersofRefSeqprotein-codinggenesingeneralandbrokenup
ExonsofCufflinkstranscriptswith59endsinenhancerswerefurthercheckedfor intoCpGandnon-CpGpromoters,andubiquitousFANTOM5CAGEenhancers.
atleast50%(reciprocal)overlapwithexonsofGENCODE(v10)known,level1, We further calculated the average footprints of H3K4me1, H3K4me2 and
protein-codinggenesandlincRNAs.Werepeatedthesameanalysisspecificallyfor H3K27ac from ENCODE (Broad, Bernstein) signal files in 601bp windows
u-enhancers. centredonmidpointsofenhancersidentifiedinHeLacellsandthosethatwere
SmallRNAlibrarypreparationandmapping.ShortRNA-seqsequencinglib- novelinSKIV2L22.
rarieswerepreparedas24-plexusingtheTruSeqSmallRNASamplePrepKit Purificationofbloodcelltypes.Peripheralbloodmononuclearcellswereiso-
(Illumina)followingthemanufacturer’smanual.Allstartingsourceswere1mg
latedfromleukapheresisproductsofhealthyvolunteersbydensitygradientcent-
oftotalRNA.ThepreparedsequencinglibrarieswereloadedonaHiSeq2000
rifugationoverFicoll/Hypaque(BiochromAG).Collectionofbloodcellsfrom
(Illumina).AllsamplesprofiledinthisstudywerealsoprofiledintheFANTOM5
healthydonorswasperformedincompliancewiththeHelsinkiDeclaration.All
promoteromepaper6andaredescribedindetailthere.Briefly,allhumansamples
donorssignedaninformedconsent.Theleukapheresisprocedureandsubsequent
usedintheprojectwereeitherexemptedmaterial(availableinpubliccollections
purificationofperipheralbloodcellswasapprovedbythelocalethicalcommittee
orcommerciallyavailable),orprovidedunderinformedconsent.Allnon-exempt
(referencenumber92-1782and09/066c).CD41cellswereenrichedusingmag-
materialiscoveredunderRIKENYokohamaEthicsapplications(H17-34and
neticallylabelledhumanCD4MicroBeads(MiltenyiBiotec)andtheMidi-MACS
H21-14).ForthesamplesprofiledbysRNA-seq,thehumanfetalheartRNAwas
system(MiltenyiBiotec).TheCD41fractionwasstainedwithCD4FITC(fluor-
purchasedfromClontech(catalogueno.636583).CD191B-cellsandCD81T-cells
esceinisothiocyanate;BectonDickinson,catalogueno.345768),CD25phycoery-
were isolated using the pluriBead system (huCD4/CD8 cascade and huCD19
thrin(PE;BectonDickinson,catalogueno.341011)andCD45RAallophycocyanin
single;PluriSelect).RNAwasthenextractedusingthemiRNeasykit(Qiagen).
(APC)andCD31CD41CD252TcellsweresortedonaFACS-Ariahigh-speed
ShortRNAswereprofiledusingtheTruseqprotocolfromIllumina,usingan
cellsorter(BDBiosciences).CD81cellswereenrichedusingmagneticallylabelled
8-plex.The8-plexwasfirstsplitbybarcodeandtheresultingFASTQsequences
humanCD8MicroBeads(Miltenyi).TheCD81fractionwasstainedwithCD3
trimmedofthe39adaptorsequence.SequenceswithlowqualitybaseNwere
FITC(BectonDickinson,catalogueno345763)andCD8APC(BectonDickinson,
removed. Ribsomal RNA sequences were then removed using the rRNAdust
catalogueno.345775)andsortedforCD31CD81Tcells.CD191andCD561
program.RemainingreadswerethenmappedusingBWAversionis0.5.9(r16)
cellswereenrichedfromtheCD82fractionusingmagneticallylabelledhuman
andmultimapperswererandomlyassigned.
CD19andCD56MicroBeads(Miltenyi).EnrichedcellswerestainedwithCD3
AnalysisofsmallRNAsatenhancerTSSsandpromoterTSSs.59and39endsof
FITC(BectonDickinson,catalogueno.345763),CD19PE(BectonDickinson,
mappedsRNAsaswellaspooledCAGE59endswereoverlaidwindowsof601bp
catalogueno.345777)andCD56APC(BectonDickinson,catalogueno.341027)
centredonforwardstrandsummitsofenhancer-definingCAGEtagclustersand
andsortedintoCD31CD191BcellsandCD31CD561naturalkiller(NK)cells.
sensestrandsummitsinpromotersofRefSeqprotein-codinggenes.Theaverage
Purificationofbloodmonocytesisdescribedelsewhere52.
cross-correlationbetweenCAGE59endsandsRNA39endswerecalculatedin
Generation of ChIP data for blood cells. Chromatin was obtained from
thesewindowsallowingamaxlagof300.Forfootprintplots,readsmappingto
CD41CD252Tcells,CD81Tcells,CD191Bcells,andCD561NKcellsof
thesamegenomiclocationswereonlycountedoncenottomakeanylibraryor
genomicregionhaveanundueinfluence. two healthy male donors each. Chromatin immunoprecipitation (ChIP) for
HeLacellsculturingandSKIV2L2depletion.HeLacellsweregrowninDMEM H3K4me1 and H3K27ac and library construction were done essentially as
msiRedNiAum-mseudpiaptleedmkennotecdkdwoiwthn1o0f%eitfheetarlEbGovFiPn(ecosnetrruoml),aatn3d7SuKCIVan2Ld25(%MTCRO42). edreesncrciebseedquelesnecweh(eGreR52C.hS3e7q/uhegn1c9e)tuasginsgwBeorewmtiea5p0paneddotonltyhuenciuqrureelnytmhaupmpaendtraegfs-
wereperformedusing22nMofsiRNAandLipofectamin2000(Invitrogen)as wereusedfordownstreamanalyses.H3K4me1andH3K27acChIP-seqdatafor
transfectingagent.Asecondhitof22nMsiRNAwasgivenafter48h.Cellswere CD141monocyteswasgeneratedelsewhere52.ComplementaryDNasehyper-
collected an additional 48 h after the second hit, and protein depletion was sensitivitysequencingdatawasobtainedfromtheEpigeneticsRoadmapproject
verified by western blotting analysis as described elsewhere48. The following (http://www.roadmapepigenomics.org)andmappedasabove.BloodcellChIP-
siRNAsequenceswereused: seq data have been deposited with the NCBI GEO database (accession code
egfpGACGUAAACGGCCACAAGU[dT][dT] GSE40668)andUCSCGenomeBrowsertrackhubdataoftheentirebloodcell
egfp_asACUUGUGGCCGUUUACGUC[dT][dT] data set can be found at http://www.ag-rehli.de/NGSdata.htm. Also see Sup-
SKIV2L2CAAUUAAGGCUCUGAGUAA[dT][dT] plementaryTable4.
SKIV2L2_asUUACUCAGAGCCUUAAUUG[dT][dT] ClusteringofbloodcellCAGEandepigeneticsdata.CAGEsamplescorres-
HeLaCAGElibrarypreparationsanddataprocessing.CAGElibrarieswere pondingto CD41, CD81, B cells,NK cellsand monocytes wereselectedin
preparedfrom5mgoftotalRNApurifiedfrom23106HeLacellsusingthe triplicatesfromamongthesetofprimarycellsamples.Basedonthetotalsetof
Purelinkminikit(Ambion)with1%2-mercaptoethanol(Sigma)andon-column 43,011permissiveenhancers,asubsetof6,609blood-expressedenhancerswas
DNaseItreatment(Ambion)asrecommendedbymanufacturer.CAGElibraries definedasbeingsignificantlyexpressedabovegenomicbackground(described
werepreparedasdescribedpreviously49.Priortosequencingfourlibrarieswith above)inatleasttwoofthetriplicatesamplesforatleastonebloodcelltype.This
differentbarcodeswerepooledandappliedtothesamesequencinglane.The subset of enhancers was clustered for heat map visualization using complete
librariesweresequencedonaHiSeq2000instrument(Illumina).Tocompensate linkageagglomerativehierarchicalclusteringbasedonenhancerusagepercell
forthelowcomplexityin59endoftheCAGElibraries30%Phi-Xspike-inwere type(binarymatrix)andManhattandistance.
addedtoeachsequencinglaneasrecommendedbyIllumina.CAGEreadswere Enhancersweredefinedasbeingspecificallyexpressedinonebloodcelltypeif
assignedtotheirrespectiveoriginatingsampleaccordingtoidenticallymatching havingapairwiselog2foldchange.1.5withrespecttotheotherfourbloodcell
barcodes.Assignedreadsweretrimmedtoremovelinkersequencesandsubse- types.Thefoldchangewascalculatedbasedonthemeanexpressionovertrip-
quentlyfilteredforaminimumsequencingqualityof30in50%ofthebasesusing licate samples per cell type. Footprints for DNase I hypersensitivity (DHS),
theFASTX-Toolkit(http://hannonlab.cshl.edu/fastx_toolkit/).Mappingtothe H3K4me1andH3K27acwerecalculatedpercell-type-specificenhancersetand
humangenome(hg19)wasperformedusingBowtie50(version0.12.7),allowing celltypebyextensionofreadsto200bpandoverlapaggregationforawindowof
formultiplegoodalignmentsandsubsequentlyfilteringforuniquelymapping 61kbaroundenhancermidpointasthemeanTPMsignaloverallenhancersin
reads.ReadsthatmappedtounplacedchromosomepatchesorchrMwerediscarded. thatspecificsubset.
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