Genome-wide studies of nuclear receptors in cell fate decisions.

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decisions.

Marco-Antonio Mendoza-Parra, Hinrich Gronemeyer

To cite this version:

Marco-Antonio Mendoza-Parra, Hinrich Gronemeyer. Genome-wide studies of nuclear receptors in cell

fate decisions.. Seminars in Cell and Developmental Biology, Elsevier, 2013, 24 (10-12), pp.706-15.

�10.1016/j.semcdb.2013.07.001�. �inserm-00854329�

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Pleasecitethisarticleinpressas:Mendoza-ParraM-A,GronemeyerH.Genome-widestudiesofnuclearreceptorsincellfatedecisions.Semin CellDevBiol(2013),http://dx.doi.org/10.1016/j.semcdb.2013.07.001

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SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx

ContentslistsavailableatScienceDirect

Seminars

in

Cell

&

Developmental

Biology

j ou rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b

Review

Genome-wide

studies

of

nuclear

receptors

in

cell

fate

decisions

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Marco-Antonio

Mendoza-Parra

∗

,

Hinrich

Gronemeyer

∗

DepartmentofCancerBiology,InstitutdeGénétiqueetdeBiologieMoléculaireetCellulaire(IGBMC)/CNRS/INSERM/UniversitédeStrasbourg,BP10142, 67404IllkirchCedex,France

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Articlehistory: Available online xxx Keywords: Nuclearreceptors Retinoicacidreceptor Functionalgenomics Systemsbiology

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Nuclearreceptors(NRs)areimportantmediatorsoftheinformationencodedinthechemicalstructure ofitscorrespondingligand,astheyinterpretsuchinformationinthecontextofthecellidentityand physiologicalstatusandconvertitintosequentialtranscriptionregulatoryevents.Atthecelllevelthis canresultintemporallycoordinatedprocessessuchascellfatetransitions,whichcomprisetheregulation ofaplethoraofgeneprogramsincludingamongothersregulationofcellproliferation,metabolismand speciﬁcfunctionalitiesthatareacquiredbythedifferentiatedcell.Whileboththeearlystepsofnuclear receptorfunctionandtheirimpactonanimal/organphysiologyisratherwellunderstood,littleisknown aboutthedynamicgenenetworksthatultimatelycauseaparticular(cell)physiologicalphenomenon inducedbythecognateNRligand/hormone.

Thankstoadvancesinmassiveparallelsequencingandbioinformaticsanalysesofgenome-widedata sets,timehascomeforthedevelopmentofNRsystemsbiology.Indeeditisnowpossibletointegrate globaltranscriptionfactorbinding,epigeneticchromatinhistoneandDNAmodiﬁcationpatternswith transcriptomesand3-dimensionalchromatinstructures,extractdecisionpointsintemporalstudiesand decipherthetemporalcontrolofgenenetworksthataretheultimategeneticreadoutsofNR ligand-inducedphysiologicalphenomena.Inthisreviewwewillsummarizethechronologyofthedevelopment ofincreasinglylargerdatasetsforNRaction,withaparticularfocusonstudiesperformedwiththe RAR/RXRnuclearreceptorfamily,anddiscussthepresentattemptstointegrateamultitudeof genome-widedatasetsintheultimatecontextofthetemporal3-dimensionalchromatinstructure.

Contents

1. Introduction... 00

2. Nuclearreceptorsinapost-genomicera... 00

2.1. AssessingtheglobalgeneexpressionsignaturesdrivenbyNRs... 00

2.2. Mappingthechromatinbindingsitesofnuclearreceptors... 00

3. NRgeneexpressionprogramsandtheirassociatedkeyfactorsinvolvedinsignalingdiversiﬁcation... 00

3.1. StudyingNR-drivencellfatetransitionsasdynamicgeneexpressionprograms... 00

3.2. Additionalmechanismsinvolvedincontrollingtheretinoids-drivendynamicdiversiﬁedgeneprograms... 00

3.2.1. MultipleRXR–RARheterodimersmediateRA-signaling... 00

3.2.2. Epigeneticmodiﬁcationsandco-regulatorsestablishregulatoryprinciplesaffectingRA-regulatedgeneprograms upstreamanddownstreamofRXR–RARheterodimeraction ... 00

3.2.3. Thethree-dimensionalchromatinorganizationanditsdynamicchangesdrivenbyNR-signaling... 00

4. FuturedirectionsinthestudyofNR-drivencellfatetransition ... 00

References... 00

夽 Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

∗ Correspondingauthors.Tel.:+33388653473;fax:+33388653437.

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1. Introduction

Nuclear receptors(NRs; 48 NRs exist in humans)constitute amajorclassoftranscriptionalregulatorsinmetazoansthatare believedtohaveevolvedpriortothedivergenceofvertebratesand invertebrates.Theﬁrstreceptor(estrogenreceptor,ER)was iden-tiﬁed1958byElwoodJensenbutonlyaftercloningseveralNRs inthe1980sitbecameapparentthatthesereceptorsforsteroids, thyroids,retinoicacidsandothersmallmoleculeligands,several ofwhichactinanintracrinefashion,constituteasuperfamilyof transcription factors(TFs) that includesreceptorsfor which no naturalligandisknownormaynotexist.NRsbindina ligand-dependent(e.g.,estrogen receptor)or ligand-independent (e.g., retinoicacidreceptor)mannertocis-actingDNAregulatory ele-ments,whichmaybepositionedproximaltopromoterregionsof targetgenesorregulategenesduetostructuralproximityinthe contextofchromatinarchitecture,actasactivatorsand/or repres-sorsoftranscription,andmayexertnon-genomicactivities.NRsare ofmajorsocial(the“pill”,NRantagonistsforabortion), pharmaceu-ticaland clinicalimportance(e.g.,endocrine-dependentcancers ormetabolicdiseases),asalargemajorityof physiological pro-cessesandpathologiesinvolve(aberrant)NRaction[forreviews see1,2–13].

Conceptually, ligand binding modulates the communication of the nuclear receptor with the intracellular environment, which entails essentiallyreceptor-protein and receptor-DNA or receptor-chromatininteractions.Duringthisprocess,receptorsare importantmediators ofthe information encoded inthe chemi-calstructureofagivenligand,astheyinterpretthisinformation inthe context ofcellular identityand cell-physiologicalstatus; thus transforming it into a dynamic chain of receptor-protein andreceptor-DNAinteractions.NRspresentamodularstructure mainlycharacterizedbyaDNA-binding(DBD)andligand-binding (LBD)domains,whose3Dstructuresinpresenceandabsenceof cognateDNAresponseelementsandvariousagonistsor antago-nists,respectively,havebeendetermined[14–19].TheLBDserves as dual input–output information processor, as ligand binding (otherinputsare,forexample,receptorphosphorylations)induces allostericchangesofreceptorsurfacesthatrepresentdockingsites for subunits of transcription and/or epigenetic machineries, or enzymecomplexes(output).FurthermoreNRsareactively regu-latedbypost-translationalmodiﬁcations(e.g.,phosphorylations; ubiquitinylations)whichmayhaveadirectorindirectroleintheir transcriptionalregulationfunction(reviewedin[20]).

Boththeearly stepsof nuclearreceptor function as wellas theirphysiologicalimpactareingeneralratherwellunderstood. Infact,duetoaplethoraofmolecularandstructuralbiology stud-ies,thesequenceofeventsthatfollowsthebindingofaligandis largelyknown,andweunderstandhowtheseeventscanbe mod-ulatedby liganddesign [3,21,22].Brieﬂy, bindingof anagonist totheNRligandbindingdomain(LBD)inducesanallosteric con-formationalreorganizationwhichaltersurfacesintheligand-free receptor(apoNR)towhichco-repressors(CoRs)bindresultingin dissociationofCoRcomplexes,whichcontainepigeneticenzymes (HDACs,histone deacetylases).Importantly,only some apoNRs, suchasRARsandTRs,recruitCoRcomplexesandthus,canactas transcriptionalrepressorswhenbindingtochromatininabsenceof ligand.OtherNRs,likeERandGRarebelievedtobindtochromatin onlyafterinteractionwiththeircorrespondingligands.

In addition to the molecular/structural insights, extensive mousegenetics providedimportant informationconcerningthe physiologicalrolesofseveralnuclearreceptors[23,24],andofsome of theirco-regulators [25,26].However, how a singlemolecule thatbinds toitscorrespondingreceptorregulatesa plethora of cell-speciﬁcdynamicnetworksofgenesandhowtheepigenome contributestotranscriptionalregulationthatultimatelyreadsout

asa(cell)physiologicalphenomenon,isstillaunknown(Fig.1).In thisreview,wesummarizetheeffortsperformedtopavetheway intothedevelopmentofsystemsbiologyofnuclearreceptoraction. Wewilladdressthechronologyofthedevelopmentofincreasingly largeromicsdatasetsforNRs action,withaparticularfocuson theRAR/RXRnuclearreceptors,anddiscussthepresentattempts tointegrateamultitudeofgenomewidedatasetsintheultimate contextofthe4-dimensionalstructureofchromatin.

2. Nuclearreceptorsinapost-genomicera

Thepublicationoftheﬁrstdraftofthehumangenomesequence in2001,followedbythoseofvariousothermodelorganisms,gave risetoanewwaytoaddressthemoleculargeneticsofthe homeo-stasisoflivingorganisms.Sincethen,anybiologicalphenomena andits(de)regulationcanbeexplored,inprinciple,ina “genome-wide”context.Indeed, thankstotheadvancesin genome-wide or “omics” approaches, it is now possible to assess the global transcriptionalactivitybyavarietyofapproaches(i.e.,by microar-rays;RNAsequencing,etc.),characterizethegenomiclocalization oftranscriptionfactorsorevaluateepigeneticchromatin modiﬁ-cationinahighresolutionmanner(ChIP-chip;ChIP-seqassays). Furthermore,newmethodologiesusingaproximity-basedligation approacharestartingtogiveinsightsintothe3-dimensional chro-matinstructuresprovidinganewwaytointerrogatethemolecular principlesregulatinglivingsystems, suchas thegenenetworks involvedincellfatedecisionsthataretriggeredbyinternalor exter-nalfactors(Fig.1).

2.1. AssessingtheglobalgeneexpressionsignaturesdrivenbyNRs ThedissectionofNRligand-inducedsignalinginvolvedin var-iousphysiologicalprocesseshasbeenearlyonevaluatedbyhigh throughputgenomicmethods.Importantly,thisnewwayto inter-rogatethemolecularhomeostasisofbiologicalsystemsgenerates highernumberofsignificanttargetsthanthoseidentifiedin pre-viousyears bystandard genetics/molecularbiology approaches, thus providing a more comprehensive view of the regulatory eventsduringNR-signaling.Indeed,in2002BalmerandBlomhoff summarizedmore than1191publishedarticlesonretinoic acid receptorsandclassified532genesasRAregulatedtargets[27]. Inthesameyear,twootherstudiesfocusedonRA-inducedcell differentiationintwo wellknownembryocarcinomacell(ECC) models(F9differentiatesintoparietalendoderm[28],whileP19 differentiatesintoneuronalcells[29])identifiedasimilarnumber ofdifferentiallyregulatedgenesbyincorporatingintheirassays one of the early versions of the microarray technology(cDNA PCR-spottedmicroarrays;reviewed in[30]).Furthermore,these twostudiesaswellasothersfocusedintheglobaltranscriptional regulation response driven by various other NRs (anextensive reviewconcerningtheuseofmicroarraysforthegenomicprofiling inaNR-mediated contexthasbeenpresentedin[31]),assessed thechangesintranscriptionalactivityoverdifferenttime-points asawaytoidentifygene-specificsignaturesaswellastemporal associations,pavingthewaytowardsaspatio-temporalviewof cell-fatetransitions(furtherdiscussedinSection3).

DespitetheimportantnumberofNR-regulatedgenesidentified inthesestudies,themajorlimitationofthis approachwasthat directNRtargetscouldnotbedistinguishedfromgenesthatwere indirectlyregulated.Topartiallycircumventthisproblem,theuse ofproteinsynthesisinhibitors,likecycloheximide,wasintroduced during the assays to avoid transcriptional regulation cascades progression.Inthiscontext,Harrisetal.inducedF9differentiation during6hinpresenceof ATRAandtheproteininhibitor cyclo-heximidewhichgaverisetotheidentificationof109significantly

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Fig.1. Schematicrepresentationoftheretinoicacidsignalingtransductionprocessassessedbyglobalapproaches.Fromtoptobottom:cellfatetransitionisaconsequence ofaninitialcue[all-transretinoicacid(ATRA)andrelatedretinoidligands]thatinitiatessignaltransductionthroughtranscriptionregulationprimarilymediatedthrough thecorrespondingRAR/RXRreceptors.Theinitialsignaltransductioncascadeisdiversiﬁedoverseveralsignaltransductionlayers,whichtogetherspecifytheassociated cellfatetransition.Boththeprimarysignalingresponse,aswellasthefurthersignalingtransductionlayerscanbeevaluatedthroughglobalapproaches(rightsideofthe panel).Methodslistedforassessingthe3D-chromatinorganizationcorrespondstoHi-C(Highresolutionchromatinconformationcapture[74])andChIA-PET(Chromatin InteractionAnalysisbyPaired-endtagssequencing[72]).

differentiallyregulatedgenes[28].Whiletheuseofsuchinhibitor appears as an elegant way to identify in a selective manner primary/directtargets,thestudyperformedintheRA-inducedF9 differentiationsystemdemonstrated thatonly22 ofthemwere presentintheATRAcontrolassay,suggestingthattheothergenes areartifactuallyinducedbycycloheximidetreatment.

Further global transcriptome studies performed in different modelsystemsincorporatedsystematicallyearlyandlate treat-menttimepoints,undertheassumptionofadirectcorrelationwith putative primary/direct and secondary/indirect NR-responsive genes[32].Otherstookadvantageoftheavailabilityofspeciﬁc lig-andsasawaytorestricttheanalysistoagivensetofdifferentially regulatedgenes.Forinstance,thesyntheticpan-RARagonistTTNPB hasbeenusedtodecreasethe‘contamination’withgenes respon-dingtopermissiveRXRheterodimers,asall-transRAisomerizesto theRXRligand9-cisRA[33,34].

Considering that NRs can be expressed as different iso-types/isoforms,evaluatingtheirspeciﬁctranscriptionalregulation cascades through global approaches became a crucial task for understandingthebiologicalroleofNRdiversity.Intheparticular caseoftheRAnuclearreceptors,RARsandRXRsareeachexpressed fromthethreeisotypicgenes(␣,␤and␥),whichexpressisoforms bydifferentialpromoterusageandsplicing[1].WhileallthreeRAR isotypeswereshowntobepresentinmodelsystemsliketheF9 ECCs,previousstudieshadalreadyprovidedevidenceforspeciﬁc rolesofsuchisotypes;forinstanceRXR␣/RAR␥heterodimer iso-typeisessentialforRA-inducedF9differentiation[35–37].

Inthiscontext,SuandGudasaimedatidentifyingthespeciﬁc role of RAR␥ by performing global gene expression proﬁling with wild-type and RAR␥−/− F9 cells in presence or absence of ATRA[38]. While theydemonstratedthat wildtype and the RAR␥−/−cellspresentedsimilarmorphologicalandproliferation

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expression differences were observed; this reveals limitations for theuse of RAR knockoutcells to decipher speciﬁc roles of RARisotypes.Similarobservationsmadeforglobaltranscription regulationstudiesperformedinRAR␣−/−F9cells[39]andin a gene-centricmannerforseveralRXRandRARisotypeknockouts [35,36]revealed“artifactual”ligandresponsesofspeciﬁcRXR–RAR heterodimersthussuggestingthatglobalstudieswithRXR–RAR knockoutcellsneedtobeinterpretedcarefully.

In summary,global approaches for assessing thechanges in geneexpressionregulationinthecontextofaliganded-NRfunction werewidelyusedinthepastprovidingaratherfastandaccurate waytoidentifygeneexpressionsignaturesinthestudiedsystems. Nevertheless,whilethesestudiesidentiﬁeddifferentiallyregulated genes,theydidnotprovideinsightsintothegeneprogramming functionsofligandedNR(isotype)s.

2.2. Mappingthechromatinbindingsitesofnuclearreceptors NRsignalingisbasedontheircapacityofregulating transcrip-tion; thus thedissectionof the effects of NRs onphysiological processesrequiresthecomprehensivemappingoftheirdynamic interactionswiththechromatintoidentifyregulatedtargetgenes. As the DBD is the primary determinant of DNA interaction specificityalargenumberofstudieshasbeendevotedtothe under-standingofthesequence-specificandstructure determinantsof NRDBD–DNAinteraction([40–44];foralistofNRbindingsites see[45]).InthecaseofRAreceptors,invitrobindingand trans-activationstudiesdemonstratedthatRXR/RARheterodimersbind preferentiallytoinverted(IR)ordirectrepeat(DR)sequencesof thehexamericmotif (A/G)G(G/T)TCA,oftenspaced by5, 2or 1 nucleotide(DR5, DR2, DR1) due tothe dimerization character-isticsoftheDNAbindingdomain[41–43,46].Thischaracteristic RA-ResponsiveElement(RARE),hasbeenshowntopresentmajor divergencewhencomparedwiththeRXR/RARbindingsites asso-ciatedtowell-knownRA-inducedgenes[47],indicatingthatthe consensusRAREs maycorrespondto highaffinitybinding sites but occurrarelyin natural RAtarget genes.Indeed, high affin-itybindingsitescanbeisolatedbyco-immunoprecipitationwith DNAbutthosesitesarenotusedforgeneregulation,mostlikely becausetheyarenotaccessibleinthecorrespondingchromatin [48].Furthermore,mappingthepotentialRXR/RARbindingsites bycomparingtheconsensusRAREsdoesnottakeinconsideration additionalmechanisms,liketheepigeneticmechanismsthat regu-lateaccessofRXR/RARheterodimers[49,50]andsteroidreceptor homodimers[51],and/or thesynergisticinteraction withother NR/TFs[52].

Forthesereasons,thecurrentmethodofchoicefor comprehen-siveandunbiasedmappingoftheprotein-chromatininteractions istheuseofchromatinimmunoprecipitationcombinedwithhigh throughputsequencing.Notethatthehybridizationof immunopre-cipitatedchromatintomicroarraychips(knownalsoasChIP-chip) represented the ﬁrst approaches for global mapping of NR-chromatininteraction.Infact,Delacroixetal.aimedatidentifying theRARbindingsitestodiscriminatebetweendirectandindirect RA-regulatedtargetsusing Taf4lox/− MEFs,which undergo mor-phologicalchangesuponRAtreatmentaccompaniedbyregulation of>1000genes[53].Afterintegrationof3xFlag-HAtaggedRAR␣ orRAR␥isotypestheyperformedChIPassaysandhybridizedthe IPedDNAtoAgilentpromotermicroarrays(ChIP-chip)[54]. Sur-prisingly,theyidentiﬁed∼300RAR-occupiedsitesofwhich<25% correspondedtodifferentiallyRA-regulatedgenes.Laterstudies performedin variousothersystems demonstrated thatonly an smallfractionof theRXR/RARbindingsites arelocated in pro-moterregions,thusexplaining,atleastpartially,thelowcorrelation

betweenRARoccupancyandgeneexpressionregulationobserved inthisstudy.

Infact,usingaconceptuallysimilarapproach,Huaetal.have integratedeGFP-taggedRAR␣orRAR␥intohumanMCF-7breast cancercellstocharacterizetheRARisotype-selectivelyregulated pathwaysimplicatedintheanti-proliferativeandapoptoticeffects of RA [55]. Importantly, the RAR-speciﬁc IPed chromatin was hybridizedtotilingarrayscontainingmorethan40million oligonu-cleotideprobesvirtuallyinterrogatingtheentirehumangenome. Undertheseconditions,theyfound>3000RAR␥and>7000RAR␣ bindingsitesrespectively,fromwhichmorethan85%ofthe iden-tiﬁedsiteswerelocatedinintronicorpromoter-distalintergenic regions.

In a similar manner a recent study withmouse embryonic stemcellsaimedatidentifyingtheRA-dependentgeneprograms involvedinneuronaldifferentiation[56],butincontrasttothe pre-viouslymentioned studies,thebindingofendogenous RARwas mappedbyusingapan-RARantibodyandcombiningChIPassays withmassiveparallelsequencing(ChIP-seq); thustheyavoided over-expressionand/or“artefactual”bindingoftaggedconstructs. This assay, performedbefore and after8hof ATRA treatment, revealedbothconstitutiveanddenovobindingsitesuponRA expo-sure,which werethen correlatedwithglobal microarray-based gene expression and RNA polymeraseII initiation and elonga-tion(assessedbyChIP-seq).Thefractionofdifferentiallyregulated genesassociatedwithRARbindingwasestimatedbyusinga5kb proximity criterion; this way only 15% of the identiﬁed bind-ing sites could be linked to a (transcriptionally active) coding region.Indeed,itisnowgenerallyacceptedthatdistalenhancers, which cannot be identiﬁed by simple binding site proximity, can regulateNR-responsive genes (furtherdiscussed in Section 3.2.3).

Ourownrecentstudyusedthewell-establishedF9modelto dissectthegeneregulatorypathwaysthatareresponsibleforthe RA-inducedendodermaldifferentiationbyintegratingtheglobal RAR bindingand gene regulation information from five differ-enttime-points duringthefirst48hafterRAexposure[57].For eachtime-pointthedifferentialtranscriptionalregulationhasbeen assessedaftertreatmentwithATRAor RAR␣,␤,␥-specific ago-nists.

GiventheessentialroleofRAR␥inF9celldifferentiation[37],we inferredtheRXR␣–RAR␥heterodimer-genomiclocationby map-pingbothheterodimercomponentsseparatelyatall5time-points (Fig.2A).Overall,RXR␣displayedmorebindingsitesthanRAR␥,as wasexpectedfromthepromiscuousheterodimerizationofRXR␣ withmultiplepartners.WhenevaluatingRXR␣and RAR␥ bind-ingsitesasheterodimercomponents,weidentiﬁedaconstitutive RXR␣–RAR␥bindingpopulationplusanotherpresentingahighly dynamicbehaviorduringATRAtreatment(Fig.2B).Infact,while theoverallnumbersofRXR␣–RAR␥bindingsitesdecreased dur-ingF9differentiation (∼2000 sites inthe absenceof treatment andlessthan1000sitesafter48hinpresenceofATRA)(Fig.2B andC),wedetectedsigniﬁcantamountsofdenovorecruited het-erodimersevenafter24or48hoftreatment,indicatingsustained andhighlydynamic interactionof theRXR␣–RAR␥heterodimer withchromatintargetsduringthiscellphysiologicalprocess. Unex-pectedly,theoveralldecreaseofRXR␣–RAR␥heterodimersbinding sitesdidnotcorrelatewiththeobservedamountsofRXR␣ bind-ingsites,thussuggestingthattheobserveddecreaseofthebinding sitesofRXR␣–RAR␥heterodimersmayresultfromanexchange withotherRXR␣heterodimersduringtheprocessofdifferentiation (Fig.2C).

ComparingRA-inducedgeneexpressionwiththereceptor bind-ing in a 10kb distance interval (Fig. 3A) we found that more than 50%of genesinduced duringthe ﬁrst24hof ATRA treat-mentshowedaRXR␣oranRXR␣–RAR␥bindingsitewithin10kb

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Please cite this article in press as: Mendoza-Parra M-A, Gronemeyer H. Genome-wide studies of nuclear receptors in cell fate decisions. Semin Cell Dev Biol (2013), http://dx.doi.org/10.1016/j.semcdb.2013.07.001

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Fig.2.RXR␣andRAR␥nuclearreceptorspresentahighlydynamicbindingtochromatinduringATRA-inducedF9differentiation.(A)TemporalrecruitmentofRAR␥andRXR␣inproximityoftheCyp26a1locus,asrevealed

byChIP-seq.Notethatbothreceptorsbindtotheidenticalchromatinlocusbutwithdifferentdynamics.(B)ThenumberglobalRXR␣–RAR␥bindingsites(deﬁnedbytheco-occurrenceofbothreceptors)areillustratedinthe

contextoftheirtemporalrecruitment,durationofoccupancyanddissociation.RXR␣–RAR␥co-occupiedsitespertimepointaresub-classiﬁedbasedontheirrecruitmentintervalsanddepictedbycolorcoding.(C)Schematic

modelillustratingthe(i)globalprogressivelossofRXR␣–RAR␥heterodimerasobservedinﬁgure(B);aswellas(ii)thatofRAR␥butnotofRXR␣fromchromatinbindingsitesobservedduringATRA-inducedF9differentiation;

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Fig.3.DifferentialgeneexpressioninducedbyretinoidsinF9embryonalcarcinomacells.(A)Schematicrepresentationillustratingthetranscriptionregulationactivity associatedtotheproximallocalizationofRXR␣–RARnuclearreceptors.NotethatgeneswereclassifiedasputativetargetgenesifatleastoneRXR␣orRXR␣–RAR␥binding sitewaslocatedinupto10kbdistance.(B)GenesexhibitingATRA-inducedorrepressedmRNAlevelsattheindicatedtimepointsduringF9celldifferentiation(induced genes≥1.8-fold;repressedgenes≤0.5-foldrelativetovehicle)wereclassifiedasputativetargetgenesfollowingthecriterionillustratedin(A).(C)RXR␣bindingsites classifiedintheirgenomiccontextdemonstratesthatmorethan70%ofthemarelocatedfarawayfromcodingregions(>10kbdistance;leftpanel),inadditiontotheirstrong preferenceforintergenicregions(rightpanel).(D)RXR␣–RAR␥ATRA-putativetargetgenesdescribedin(B)werefurtherclassifiedbasedontheirresponsetoRAR-specific agonists.TakeninconsiderationthatonlytheRAR␥agonist(BMS961)canreproducetheF9differentiationphenotypeobservedduringATRAtreatment,thecharacterized RXR␣–RAR␥ATRA-putativetargetgeneswerefurtherclassifiedas“dispensable”and“required”forinducingthedifferentiationphenotype.

proximity(Fig.3B).Incontrast,mostofthedown-regulatedgenes lacked such sites. Importantly, more than 70% of the mapped RXR␣ sites could not be associated to an annotated coding region(Fig.3C), suggesting that theymight regulate transcrip-tionthrough3-dimensionalchromatinstructuresormayregulate asyet non-annotated transcripts. Tofurther confirmthe direct transcriptionalregulationbythecharacterizedRXR␣–RAR␥ bind-ingsites,wecomparedthetranscriptionalresponsesinpresence of ATRA or RAR-specific agonists [57]. Importantly, 67% of the ATRA-inducedputativeRXR␣–RAR␥targetsdidrespondsimilarly tothedifferentiationcompetentRAR␥agonistBMS961. Surpris-ingly, also thetreatment with BMS753 (RAR␣-specific agonist) orBMS641(RAR␤-specificagonist) induceda responseofsome ATRA-RXR␣–RAR␥ targets,albeit only in a minor fraction(42% and6% of theATRA-inducedgenes, respectively).Thissuggests thatamongthecharacterizedATRA-dependentRXR␣–RAR␥ tar-gets(i)_∼30%ofthemaredispensableforinducingtheobserved celldifferentiationphenotype;and (ii)fromthe remaining70% onlyathirdofthemareindeedessentialfordrivingthe differen-tiationprocess(Fig.3D). Importantly,insidethislastpopulation comprisesseveralTFs,likeFoxa1,Foxp1,Hoxa5,Hoxb5,Rarbor RXR␥,indicatingthat RA-signaltransductioninvokesthe induc-tionof“downstream”transcriptionfactors,whichinturnregulate signalingbifurcationeventstoyieldthefinaldifferentiated pheno-type.

3. NRgeneexpressionprogramsandtheirassociatedkey

factorsinvolvedinsignalingdiversiﬁcation

3.1. StudyingNR-drivencellfatetransitionsasdynamicgene expressionprograms

Asmentioned above,the integrative analysisof global gene expression response to a given ligand and the corresponding NR-chromatin association can, in principle, identify an impor-tant proportion of the NR-mediated gene-regulatory events. Furthermore,theuseofdynamicbindingandtranscription infor-mation provided additional insight in molecular mechanisms occurringduringcell-fatetransition.Infact,thesestudiesrevealed (1) a highlydynamic target gene expression[28,29,57] and (2) similarly dynamic chromatin occupancyof pre-existing and de novo recruited RXR–RAR heterodimers, including heterodimer replacementorevenheterodimerpartnerswaps[57].Similar stud-iesperformedinothermodelsystemslike3T3-L1cellsintegrated theglobalchromatinlocalizationofRXRandPPAR␥NRswiththat assessedforRNApolymeraseIIduringtheinductionofadipocyte differentiation[58].LikeinthepreviouslydiscussedRA-induced F9differentiationstudy[57],thisintegrativeanalysisperformed in a temporal manner revealed a differential recruitment of PPARsandRXRduringadipogenesisandallowedtheclassiﬁcation genes by their relative transcriptional activity assessed from

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M.-A.Mendoza-Parra,H.Gronemeyer/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 7 RNA polymerase IIbinding and thepresence of RXR/PPAR␥ in

proximity.

Despitethesefindings,thegainofinformationbythistypeof integrativeomicsanalysesremainsrestrictedtodirectly charac-terizethecorrespondingNR-regulatedtargetsandtheirpotential temporal changes,which representsonlya smallfractionofall differentiallyregulatedgenes.NR-drivencell-fatetransitionsare expectedtotakeplacethroughasignaltransductionmechanism, inwhichthedirecttargetsareinthefrontlineofthesignaling process (‘initiator program’) and the downstream layers com-prisetemporallyspecified(‘executor’)geneprogramsthatresult inamplification,diversificationandspecificationofthesignaling atdifferentlevels[57,59],whichultimatelyleadstotheemergence ofaspecificcellphenotype/functionality.

Thefirstssignaltransductionlayersaremediatedmainly,albeit notexclusivelybytranscriptionfactors(TFs).Whilethe reconstruc-tionoftheexecutorprogramsmayprofitfromthecharacterization ofthecascadeofTFsthatpropagatethesignaltransductionand diversificationprocess,itisvirtuallyimpossibletodirectly charac-terizetheseeventsinagivensystembyapplyingseriesofChIP-seq assaystargetingallpotentiallyrelatedTFs.Nevertheless,the avail-abilityinpublicrepositoriesofTFsinteractomesformultiplecell modelsrepresentsanimportantresourceforinsilicodataset inte-gration;notein this respect theimportant contributionbythe ENCODEconsortium[60].

Infact,thedeconvolutionoftheRAsignalingpathwaysduring theF9induceddifferentiationhasbeenperformedbyintegrating TFtargetgeneannotations,includingtheidentifieddirectputative RXR␣–RAR␥targets,withtheATRA-inducedgeneprogramming [57].Thisanalysis,performedwiththeDynamicRegulatoryEvents Miner (DREM; [61]), predicted six distinct gene co-expression paths, which recapitulate the different subprograms generated duringtheRA-inducedsignaltransduction.Inadditiontoclassify thetemporalgeneexpressioninformationinco-expressionpaths, DREMevaluateswhetheragivenco-expressionpathisenriched for genesthat are annotated as targetsof a specific TF, whose action contributes tothe predictedbifurcation.In this manner, DREMpredicted3bifurcationpointsleadingtosignal diversifica-tionandassociatedtocandidateTFs.Asproof-of-principle,DREM associatedRXR␣–RAR␥withupregulatedsubprogramsvalidated bydifferentialgeneexpressionandthechromatin-bindingpattern ofRXR␣–RAR␥(Fig.4).Notably,DREMpredictedtranscription fac-torsoftheHomeoboxfamily(e.g.,Hoxa1,Hoxb2,Hoxb4,Hoxb5) andotherslikeRAR␣orFoxa2,tobeenrichedintheupregulated subprograms,whiletherepressedpathwasassociatewithTFslike Egr1[62]andSox2[63],previouslydescribedaspositively regulat-ingcellproliferationandstemcellpluripotency.

The predictedRA-induced co-expression paths were further evaluatedinthecontextofgeneco-citationinteractionsto con-struct the RA-driven RXR␣–RAR␥-mediated signaling network. This type of analysis provides a global view of the relevant genesinvolvedinsignaltransductionbyintegratinginformation extractedfromtheexistingliteratureonpreviouslyreported inter-actionsandprovidesacomprehensivewaytoassociatefunctional features tothepredictedsubprograms. Importantly,this analy-sisillustratesthecomplex temporalcoordination ofthevariety ofmolecularprocessesinvolvedinRA-induceddifferentiationand predictscriticalnodesassociatedwiththecellfatetransition initi-atedbyRA[57].

3.2. Additionalmechanismsinvolvedincontrollingthe retinoids-drivendynamicdiversiﬁedgeneprograms 3.2.1. MultipleRXR–RARheterodimersmediateRA-signaling

As mentioned above, the ﬁrst level of signal diversiﬁcation resultsfromthemultiplicityofRXR–RARcomplexesthatcanbe

formeddependingontheactualexpressionlevelsof6receptor isotypes(RXR␣;RXR␤;RXR␥RAR␣;RAR␤;RAR␥).Anenigmatic aspectcharacterizedin our studyconcerns thehighly dynamic bindingand the potential“heterodimercomponents swapping” ofRXR␣–RAR␥heterodimersduringtheRA-induced differentia-tionprocess.Whilethemethodologiesallowedhighlightingsuch phenomenon,itsbiologicalsigniﬁcanceremains elusive.Clearly, exploringtheroleofotherRXR/RARheterodimersduringthis pro-cesswillprovideinsightsintotheheterodimercross-functionalities asbasisfortheobserveddynamics.

SuchstudiesnecessitatereChIPassaystoprovidereliable infor-mationaboutco-occupancyoftheevaluatedheterodimerpartners at agiven chromatinsite. WhilereChIPassays werepreviously shown as a powerful method for evaluating simultaneous co-occupancy events in a locus-centric manner [64,65], their low yieldsarenotcompatiblewiththerequirementsforglobal ChIP-seqassays.Toovercomethisproblemwehaverecentlycombined reChIPswithlinearDNAampliﬁcation(LinDA-reChIP-seq)inorder todeﬁnetheglobalbindingpatternofco-occupiedRXR␣andRAR␥ chromatinsitestopredictheterodimerbindingpatterns[66–68]. Usingsuchstrategies,thecomplexityofRXR–RARheterodimers canbedecorticatedtowardthecontributionsofthedifferent com-binationsofreceptors.

3.2.2. Epigeneticmodiﬁcationsandco-regulatorsestablish regulatoryprinciplesaffectingRA-regulatedgeneprograms upstreamanddownstreamofRXR–RARheterodimeraction

In addition to theTF-driven decisions for signal diversifica-tionprocessduringRA-induceddifferentiation,severaladditional factorsandregulatoryparadigmsmayimpactonprogram execu-tion.Infact,epigeneticmodificationofchromatinanditsinterplay withRAregulationhasalreadybeendemonstratedingene-centric studieswithPolycombproteinsandH3K27me3[49,69,70].Other epigeneticmodificationsmayalsoregulateNR’srecruitmentand theepigenetic actionof co-activator/co-integrators recruitedby ligandedRXR/RARheterodimers mayexertpioneeringactivities specifyingdownstreamprograms.

A novelmechanism of signalingpathwaydiversification has beenreportedrecentlybyCeschinetal.[59].Studyingestrogen receptor(ER␣)signalinginbreastcancercellstheyanalyzedthe roleoftwoepigeneticfactors,thehistoneacetyltransferase(HAT) CBP and the methyltransferase CARM1/PRMT4. Both CBP and CARM1bindtotheSRC/p160co-activators andareco-recruited by agonist-bound ER␣ tochromatintargets. Based onprevious knowledge that CARM1 methylates CBP at specific arginine residues [71] and the observation that CBP methylation was exclusivelyCARM1-dependent,theymappednotonlythebinding siterepertoiresofER␣,SRC3,CBP,CARM1andacetylatedhistones (H3K18ac)butalsothemethylatedCBPspeciesusingantibodies thatrecognizeselectivelythemethylatedCBPresidues[59]. Inter-estingly,thefirstobservationwasthatmethylationatR2151was requiredforestrogen-dependentrecruitmentofCBPtochromatin. Themolecularbasis forthisrequirementisnotknownanditis unclearifthisisageneralphenomenonorrestrictedtocertaincell (types). Moreover, the subsequent multi-dimensional analysis, whichincludedatime-seriesoftranscriptomics,identifieddistinct “hubs”ofER␣targetgenes.Thesehubsdifferedbytherecruitment oftheparticularmethyl-CBPspecies(whichvaryinHATactivities), thusindicatingthatthecrosstalkbetweenco-recruitedepigenetic factorscanleadtopathwaydiversification.Itwillbeinteresting toassesswhetherthesedifferenthubs,aswellasthose formed bytheotherHATp300correspondtofunctionallyrelatedtarget genesandwhethera“methyl-HATcode”mayindeedexist.

Theabovestudiesshowthatcomprehensivemulti-dimensional omics-derivedinformationtogetherwiththebioinformaticstools todeﬁne dynamic gene regulatorynetworks by integrating NR

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8 M.-A.Mendoza-Parra,H.Gronemeyer/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 0 2 -1 1 48h 24h 6h 2h Ex p re ss ion le v el ( log 2) ATRA treatment (i)63 genes (iv)512 genes (ii)235 genes (v)456 genes (iii)357 genes (vi)248 genes • Embryonic morphogenesis •Positive regulation of transcription • Positive regulation of cell differentiation

•Steroid metabolic process • Cholesterol metabolic process

•Positive regulation of response to external stimulus • Cell surface receptor linked signalling pathway/cell ashesion

• Cell-cycle regulation

•Positive regulation of cell proliferation • Negative regulation of Cell differentiation • Actin cytoskeleton organization

Asso

ciated

GO ter

ms (enr

ichment

p-value<10

-2

₎

RXRα/RARγ

Hoxb4; Hoxa1 Myc

RARα; RARγ; Runx1; Hoxb4; Jun; Foxa2; Hoxb2; Hoxb5; Pbx1; Gata4; RXRα/RARβ

RXR_α/RARγ

Myc; Foxa2; Jun; Egr1; Sox2

Fig.4. DynamicregulatorymapofATRA-inducedtranscriptome.DREMco-expressionanalysis;color-codedpathssummarizecommoncharacteristics.Diamondsindicate predictedbifurcationpointswhichgiverisetothedifferentco-expressionpaths;transcriptionfactorswhosetargetgenesareover-enrichedinagivenpathareillustrated. Thenumberofgenesperco-expressionpath,aswellastheirrelevantGeneOntologytermsisdisplayedontheright.

chromatinbindingpatterns,epigenomesandtranscriptomeswill shedsigniﬁcantlightonthemolecularmechanisms,keyfactors anddecisionpointsthatdeﬁnedecisionsspecifyingcellfateand cellfunction.

3.2.3. Thethree-dimensionalchromatinorganizationandits dynamicchangesdrivenbyNR-signaling

Thedesignationof NRtargetgenes fromChIP-seqstudies is generallybased onlinearproximitycriteria. Howeverthelarge majorityofbindingsitesarelocatedinintergenicregionsandthus, onlya smallfractionofall identiﬁedbindingeventsare gener-allyconsideredinsuchanalyses.Thefunctionoftheseintergenic bindingsiteshasbecomemuchclearerfromrecentstudies interro-gatingthe3-dimensionalorganizationofchromatininthenucleus; forinstanceintheparticularcaseofER␣[72].Itisnowgenerally acceptedthatthechromatinarchitecture,i.e.,theorganizationof chromatinin“loops’,‘domains’andpossibly‘factories’with dedi-catedfunctionalities,correspondstoastructuralorganizationthat speciﬁesthephysicalinteractionbetweenpromotersanddistant regulatoryelements, sometimes with the involvement of non-codingRNAs.Indeed,theentirenucleushastobeconsideredasa regulatorynetworkofitsown[73].Importantly,thecombinationof proximityligation-mediatedassayswithmassiveparallel sequenc-ingprovidedthetechnologytoanalyzethisarchitectureglobally (Hi-C[74]; TCC[75]).Furthermore,theincorporationof a prior immunoprecipitationstepduringtheseassaysallowstostudysuch 3D-chromatinorganizationinassociationwithagivensignaling orregulatory/processing component(ER␣[72]; CTCF [76]; RNA polymeraseII[77]).Yet,thedynamicaspectofnuclear architec-tureinprocesses likeRA-induceddifferentiationorthechanges ofnucleararchitectureinrelatedpathologies,withitssubsequent consequencesonsignalingofthediseasedcell/organ,hasnotyet beenaddressed.Itisinterestingtonoteinthisrespectthatlinks betweenchromatinarchitectureandfeaturesofcancercellsare emerging[78,79].

4. FuturedirectionsinthestudyofNR-drivencellfate

transition

Howcanthestructuralinformationthatispresentinasimple chemical molecule, likeall-trans retinoic acid(ATRA), be ‘read’ toset-up the sequence of temporally controlled events, which

finally lead to the cell-physiological changes that characterize a differentiated cell? Our previous study performed with the embryocarcinomaF9cellmodelsystemprovidedforthefirsttime asystems biologyviewoftheATRA-inducedsignalingpathway diversificationthrough differentregulatory decisions character-izedatdifferenttime-pointsduringdifferentiation[57].Yetthe viewoftheretinoicacid(RA)-inducedsignaltransductionevents inferredfromthisstudyisfarfrombeingcomprehensive.Thisisin partduetothereducednumberofmoleculareventsthatcouldbe importedinthespatio-temporalomicsdataanalyses(discussed above), but also a consequence of technicalconstraints related tothecomplexityof thesystemwhichoperates withuptosix receptorsandmultipleheterodimers.

Therapiddevelopmentofnext-generationsequencing(NGS) technologiesposesmultiplechallengesforthebioinformatics anal-ysesoftheenormousamountsofdatathataregatheredbymassive parallelsequencing.Whileinthepastyearsseveralcomputational effortsaimingtoassessthelocalenrichmentconfidenceinsingle NGS-generatedprofileshavebeenreported,anumberofkeyissues concerningmethodologiesformulti-profilecomparisonsare lack-ingorareonlyincompletelyaddressed.Asdiscussedabove,the useofintegrativegenomicsapproachesmaybecomethe method-ologyofchoicefordecorticatingtheNR-drivensignaltransduction events;thustheimplementationofsuitedcomputational meth-odsfocusedonenhancingtheconfidenceinomicsdataassessment atthetimeoftheirintegrationrepresentsanessentialaspectto considerforthiskindofstudies.Importantly,futuredataset analy-sesofNR-drivendifferentiationstudieswillneedtointegratetwo majoradditionalelements:(i)thethree-dimensional chromatin structure revealed bymethodologies like Hi-C(High resolution chromatin conformation capture [74]) or ChIA-PET (Chromatin InteractionAnalysisbyPaired-endtagssequencing[72])and(ii)the temporalnatureoftheevaluatedeventsthroughouttheinduced (cell physiological) process. Importantly, such spatio-temporal analysis willprovide information aboutNR binding withinthe chromatin architecture, the chromatin modification status and nucleosomeoccupancy,and theobserved differential transcrip-tional/translational activity in a given physiological context. In addition,computationalmethodsforreconstructingthedynamic regulatorygenenetworksmaybeappliedwiththeaimof infer-ringtheATRA-inducedsignalingpathwaydiversificationthrough temporallydefinedregulatorydecisionsasillustratedinprevious

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ARTICLE IN PRESS

GModel

M.-A.Mendoza-Parra,H.Gronemeyer/SeminarsinCell&DevelopmentalBiologyxxx (2013) xxx–xxx 9 studies[57,61,80].Thesestudieshaveprovidedaninitialinsight

intotheenormouscomplexitythatwearefacingalreadyinmodel systems,suchasstemcells,whentryingtounderstandata molec-ularandmechanisticlevelthe4-dimensionalhierarchiesthatare initiatedbyasingleinducerandgoverncellfatetransition.

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