REGULATION OF GENE EXPRESSION BY ENHANCER RNAs -eRNAs · gene regulation. The mechanisms underlying...

REGULATIONOFGENEEXPRESSIONBYENHANCERRNAs- eRNAs

Central promoter elements control the activity of the basalapparatus of RNA Pol II at the promoter

• Binding ofTFIID/TBP to theTATAboxis thefirststep ininitiation.• Other basal transcription factors bind to thecomplex inadefined order,extending thelength oftheprotected region onDNA.•When RNApolymerase II binds to thecomplex,it initiates transcription.

eukaryotespromoterRegulatory

elements

BASALTRANSCRIPTION

FACTORS

The gene control region of a typical eucaryotic gene. The promoter is the DNA sequence where thegeneral transcription factors and the polymerase assemble. The regulatory sequences serve as bindingsites for gene regulatory proteins, whose presence on the DNA affects the rate of transcription initiation.These sequences can be located adjacent to the promoter, far upstream of it, or even within introns ordownstream of the gene. DNA looping is thought to allow gene regulatory proteins bound at any ofthese positions to interact with the proteins that assemble at the promoter. Whereas the generaltranscription factors that assemble at the promoter are similar for all polymerase II transcribed genes,the gene regulatory proteins and the locations of their binding sites relative to the promoter aredifferent foreach gene.

A complex interplay of regualtory sequences and transcription factors control the basal transcription complex

Additional regulatoryelementsincreasecomplexityofgeneregulation

Regulatorysequencesalsofarawayfromthepromoter (max1Mb)à Functionofregulatoryelements

- enhancer (1- Xelements):regulateonegeneinaparticularmomentand/oratdifferentcelltypesandcanrespondtosignals- silencingelements:mediatetherepressionofapromoter- insultors/bounday elements:sequenences thatdirectenhancerfunctiontoaparticulargene

promoterRegulatoryelements

à Themediator complex (<20protein subunits)comunicates betweenpromoterandenhancer elements (interconnects transcription factors)à Essential for transcroptional activation

TheMediatorcomplex

LOOPFORMATIONBRINGSENHANCERELEMENTSTOPROMOTERàEFFICIENTACTIVATOINOFTRANSCRIPTIONàLOOPISFORMEDBYCOHESINPROTEINS

ACTIVTYOFENHANCERELEMENTSISREGULATEDBYCHROMATINSTRUCTURE

HumanGenome:400.000enhancers(upto1.000.000)

Enhancersarecentralregulatorsofgeneexpressionanddevelopment

Enhancers arebound bydefined setsoftranscription factors that areexpressed inatissue/cell specificmanner

Enhancers

- Positivelydrivetargetgeneexpression- Functionalindependence ofgenomicdistanceandorientationrelativetotargetgene

promoter- HypersensitivitytoDNasedigestion- De-compactedchromatinstatus- Presenceofspecificsequences allowingthebindingoftranscriptionfactors- enrichedbindingoftranscriptionco-factors- Histoneacetylation(p300)

- Epigeneticsofenhancers:- HighratioofH3K4me1/H3K4me3- H3K27Ac- Histonevariants:H2AZ- TranscriptionofsmallRNAs(eRNAs)

- Generalfeaturesofenhancers:

- TopologyofDNA/Chromatin- Loopformation

H3K4me1/me3low,H3K27me3:INACTIVEENHANCER

H3K4me1/me3high,H3K27Ac,p300HAT:ACTIVEENHANCER

Mappingoftranscribedenhancers

- Selection ofDNAelements that carry epigenetic codeofenhancers (highH3K27Ac;highH3K4me1/meratio;peak forp300HAT

- CAGEsequencing ofHela cells

- Mapping CAGEtags inputativeenhancer regions

eRNA transcription:canbedirectional,divergent

on+and- strand

eRNAs:balanced +/- strand usagemRNAs:unbalanced -/+strand usage


eRNAs areshort(100-1000) eRNAs transcriptional startsite is welldefined andTATAandINRsite(initiationtrascription)aresensitve toDNasedigestionà Similar toclassic Pol IIpromoter


40.000– 65.000eRNAs inhumancells

Modelsforenhancerfunction

Tracking model

Loopingmodelà Supported bychromatinconformation techinques (4C/5C)

Tracking model: RNA polymerase II (RNAPII) and the associated transcriptional machinery track through the intervening DNA in-between enhancers and promoters

Looping model: Machinery is loaded at the enhancers and then reaches the promoter due to a physical interaction (that is, through looping)

Commonaim:Increasethe

concentrationoftranscriptional

activemachineriesat

cognatepromoters

4.Generegulationbyloopformation- enhancersandinsulators

InteractionbetweenEnhancerandPromoterà Loopformation

àcohesin caninteractwiththemediatorcomplexàStableloopformationà activatorproteinsarelocalizedatpromoter/enhancer(note:inchromosomearmsthemajoramountofcohesin isinvolvedinintrachromosomicloops;onlylittleamountholdssisterchromatidstogether)Incontrastatcentromereshighconcentrationofcohesin holdchromatidstogether

Associationofenhancersandpromotersatseveralgenesinembryonicstemcells(ESCs)— forexample,octamer-bindingprotein4(OCT4;alsoknownasPOUdomain,class5,transcriptionfactor1)ismediatedbyphysicalinteractionsbetweenMediatorandcohesin complexes.

Allchromatininteractionsarecreatedandmaintainedinahierarchyof3Dchromatinarchitectures,includingA/Bdomains,topologicallyassociateddomains.Thesefindingsindicatethatanyinter-relationshipsbetweenenhancersandpromoters,bothasregulatedandpotentiallyregulatorytranscriptionunits,havetobeconsideredinthecontextofahighlyorganized3Dgenome.

Atopologically associating domain(TAD)is aself-interacting genomicregion,meaning thatDNAsequences within aTADphysicallyinteract witheach other morefrequentlythanwithsequences outside theTAD.Thesethree-dimensional chromosomestructuresarepresent inanimals as well as someplants,fungi,andbacteria.TADs canrange insizefromthousands tomillions ofDNAbases.Thefunctions ofTADs arenot fully understood,but insomecases,disrupting TADs leads todisease because changing the3Dorganization ofthechromosomedisruptsgeneregulation.Themechanisms underlyingTADformation arealso complex andnotyetfully elucidated,though anumber ofproteincomplexes andDNAelements areassociatedwithTADboundaries.Thearrangementofchromosomes candetermine their properties.Chromosomesareorganised into two compartmentslabelled A("active")andB("inactive"),eachwithdistinctproperties.Moreover,entirechromosomes segregateinto distinct regionscalled chromosometerritories.

4.Generegulationbyloopformation- enhancers- TADs

B-domainA-domain

Doesthetumorsuppressor p53bindtoactiveenhancers?

Step1:Identifyp53bindingsites(anti-p53ChIP-seq)

Step2:Whatofthesesitesareassociatedwithmarksofactiveenhancerhistonemodifications(ENCODEDATA):H3K4me1high/H3K4me3low/highH3K27Ac

Step3:Howdoesp53regulatetheactivityofenhancers

Representativeenhancers,boundbyp53=p53BER(p53bindingenhancerregions)

p53binds toactiveenhancersinimmortalizedhumancells

p53bindingsite

p53bindingsite

ENHANCER:H3K4me1high/H3K4me3low/highH3K27Ac


anti-p53ChiPPCRamplification

ofp53BER2

ValidationofChIPseq +ENCODEdatabyChIPonpreciseregionsofp53BER

q-RT-PCR:quantitativereal-timePCR


Enhanceractivitydependsonp53bindingtotheenhancer

luciferaseCMV2kbBER luciferasereporter

- Luciferasereporterreducedwhenp53RNAi(blackbars)- Enhancerworksinbothorientations-Whenp53bindingsitedestroyedenhancerhasreducedactivity

Experiments incellswithp53expression

What arethegenes regulated byp53binding sitecontainingenhancers ??

CircularizedChromosomeConformationCapture(4C)

canbeseveralkbofdistance

3CanalyzesexcisedDNAfragmentsgeneratedfromformaldehydecross-linked,thenrestrictionenzymedigestedchromatintofindpointswhereselectedDNAregionsareconnectedthroughaproteincomplex.ThefrequencyandidentityofthesefragmentsarethendeterminedbysitespecificPCRormassiveparallelsequencing.

4CenablesidentificationofpreviouslyunknownDNAregionsthatinteractwithalocusofinterest,whichmakes4C especiallywellsuitedtodiscovernovelinteractionswithaspecificregionthatisbeinginvestigated

Blue:p53BER

yellow:unknownpromoterinteractingwithp53BER

3C(one-vs-one):Thechromosomeconformationcapture(3C)experimentquantifiesinteractionsbetweenasinglepairofgenomicloci.Forexample,3Ccanbeusedtotestacandidatepromoter-enhancerinteraction.LigatedfragmentsaredetectedusingPCRwithknownprimers.

4C(one-vs-all):Chromosomeconformationcapture-on-chip(4C)capturesinteractionsbetweenonelocusandallothergenomicloci.Itinvolvesasecondligationstep,tocreateself-circularizedDNAfragments,whichareusedtoperforminversePCR.InversePCRallowstheknownsequencetobeusedtoamplifytheunknownsequenceligatedtoit.Incontrastto3Cand5C,the4Ctechniquedoesnotrequirethepriorknowledgeofbothinteractingchromosomalregions.Resultsobtainedusing4Carehighlyreproduciblewithmostoftheinteractionsthataredetectedbetweenregionsproximaltooneanother.Onasinglemicroarray,approximatelyamillioninteractionscanbeanalyzed.

CircularizedChromosomeConformationCapture(4C)

NGS

p53BERinteractwithseveralgenes

NOTE:Sequencingidentifiesunknownsequencesligatedtothep53BER

p53BER:showshighestSequencingcounts(Y-axis)SequencesthatareLigatedoftenwithp53BERSequencesshowhighSequencecountsàincellsthesesequencesinteractwiththep53BER

IMPORTANT:Interactionisreducedwhenp53isknockeddown!!HOWEVER:interactionisstillpresent!!!

p53isimportant foractivatinggenesthatinteractwithp53BER

Nutlin isacompoundthatstabilizesp53protein=p53levels increases

p53increaseresultsinaincreaseofgenesthatinteractwithp53BERs

Genesregulatedbyp53BERs:DUSP4,IER5,PAPPA

p53BERassociatewithRNAPolymeraseIIQuestion:doesp53causethetranscriptionofp53BERs=eRNAs ???

EnhancerswitheRNAs:- Max2000nt- TranscribedbyRNAPol II- Notpolyadenylated- HighH3Kme1;lowH3K4me3;highH3K27Ac- eRNA lociboundbyRNAPol IIandp300/CBP

p53

p53peaksoverlapwithp300andRNAPol IIpeaks(ENCODEproject

p53peaksoverlapwithp300andRNAPol IIpeaks(ENCODEproject

Anti-RNAPol IIChIPconfirmsthatRNAPol IIislocatedonp53BER

QuantitativeRT-PCR:Increasingp53byNutlin treatmentIncreasesRNAfromp53BERs!!!à eRNAproduction

Anti-p53ChIPIncreasingp53byNutlin treatmentincreasesp53atp53BERs

p53stimulateseRNA expressionatp53BERs

Northernblot

1. Createvectorsthatcontainsp53BERelements.Thisregions1,2,3aretaggedtotheMS2RNA-stemloopmotif2. ExpressaMS2coatproteinthatisfusedwithaGAL4DNAbindingdomain3. Makealuciferasereporterthatcarries5repeatsoftheDNAsequenceboundbyMS2-GAL4

Re-buildingp53BERfunctioninvitro

Idea:p53BEReRNA connectstheGAL4domainwiththepromoterandstimulatestranscriptionofthereporter.

p53

Sequenceconservation

Re-buildingp53BERfunctioninvitro

RNAfromregion1activatesReporteractivity

Whenp53bindingsitesinRegion1aremutated

reporteractivityisreducedWhy:Region1BERelementis

transcriptionalinactiveduetolackofp53binding

White:cellstransfectedwithreporter,p53BER-MS2RNA,MS2-GAL4

Black:cellstransfectedwithreporter,MS2-GAL4àà p53BER-RNAcomponentisrequiredforthestimulation

ofthereporter

LOSSOFp53BEReRNA EXPERIMENTS

Anti-Pol IIChIPonNutlintreatedcells(morep53)à Knockdownofp53BEReRNAsresultinreducedabundanceofRNAPol IIatgenesthatinteractwiththep53BER(PAPPA,IER5)

Knockdownofp53BEReRNAsresultinreducedexpressionofgenesthatinteractwiththep53BER(PAPPA,IER5)

PUTTINGp53BEReRNA INAPHYSIOLOGICCONTEXT–p53DEPENDENTDNADAMAGERESPONSE

Gammairradiation DNAdamage DNAdamage

signalingActivationofp53,p21

CellcycleArrest

atG1/S;G2/M

Experiment:si-p53BEReRNA à DNAdamageà Nocodazole (arrestinM)–>countcells inG1/S/G2/Mphase(=FACS)Question:howmanycellscanstillarrestatG1/Scheckpoint

Result:si-p21;si-p53:lesscellsinG1=nocellcyclearrest,arrestinG2/Msi-p53BER:similarresult=si-p53BEReRNAsareimportantforp53

dependentcell cyclearrestsi-PAPPA:hassameeffectlikesi.p53BEReRNA

eRNA

FINALMODEL

Enhancerelementandpromoterofnearbygenesphysicallyinteract(loopformation)à 4Cdata!!!

Transcriptionfactorassociatedwithenhancerelement

Important:thedirectinteractionbetweenenhancerandnearbypromotersdoesnotdependonp53(transcriptionfactor)

TranscriptionfactorsboundtoenhancerelementsstimulateseRNAsproductionbyRNAPol II.

eRNAs activatethetranscriptionofpromotersattachedtoenhancerelementsàPol IIlocatedonenhancerandalsoonpromoter(inclosevicinity)

Activationofanenhancer.

Theassembly ofthetranscriptional apparatus atanenhancer is firstinitiated bythebindingofpioneer transcription factors (pTFs)170,whichbind theDNAinnucleosomes togenerateopenchromatin,allowing therecruitmentoflineage-determining transcription factors (dTFs)todictate theenhancer siteforactivation inaspecific cell lineage.Collaborativetranscriptionfactors (cTFs)andimportant cofactors (CoFs)arefurther recruited,such as histonemethyltransferases,which ‘write’mono- anddimethylation ontheH3tail at lysine4(H3K4me1andH3K4me2,respectively).Each red circle represents amethyl group.

Activationofanenhancer

These previous steps prepare the enhancer for further recruitment of other CoFs, such as histone acetyltransferases (HATs; for example, CREB-binding protein and p300 (CBP/p300)) that deposit histone acetylation marks (green circles), as well as general transcription factors (GTFs) andRNA polymerase II (RNAPII) holoenzymes to initiate bidirectional transcription. The acetylated histone tail recruits additional CoFs, such asbromodomain-containing protein 4 (BRD4), which probably works together with positive transcription elongation factor-b complex (pTEFb) (notshown) to promote transcriptional elongation of enhancer RNAs (eRNAs). The recruitment of chromatin looping factors (CLFs) facilitatesenhancer–promoter interactions.However, theorder of their recruitmentand roles in enhancer transcription is not fully understood

CLASSI-IIIofeRNASeRNA Fucntion??

ClassesofeRNAs

CLASSI:Thetranscriptionprocess andenhancer RNAs (eRNAs)arenon-functional andaremerely transcriptional noise

Aa)Enhancer transcriptionmerelyincreases thelocalconcentration oftranscriptionalmachinery toaugmentpromoteractivities.

Ab)Promoter- loadedmachinerypassively orrandomly collideswithenhancers duetotheirphysical proximity.

RNAproductionat enhancer is rather aby-product oftranscriptional noise (remember:virtually allparts ofthegenomearetranscribed (at least at low levels))Conservation ofenhancers is low;chromatin rather open,low RNAlevels à randomtranscription,eRNAs result fromarandomscanningbyRNAPolII

- eRNAdoes not have abiological function;objective:load RNAPol IItopromter- eRNAs arebyproduct byRNAPol IIthat might read Enhancers looped topromoter

CLASSII:Theactofenhancertranscriptionmediatesfunction

ClassesofeRNAs

Ba)Thetranscription ofsomeenhancers byRNAPIIcouldremodel theintervening chromatinbetween enhancers andpromotersandactivate targetgenes overalongrange;transcribing RNAPIIcould carry histonemodifiers suchas histone acetyltransferases(HATs)orhistonemethyltransferases (not shown)tomodify theenhancer region andtheintervening DNA.

Bb)Forother enhancers,especiallythose inintrons,their transcriptionmay interferewiththeoverlappinggenetranscription.

eRNAs arejustaby-product.TheACToftranscriptionbyRNAPolymerase IIisrelevant

eRNAs arefunctionallyrelevant

Evidence fromaseries ofexperiments:

- siRNAmediated knock-downofeRNAs:targetpromoterexpression islower

- RNA-tethering assays (MS2system)à eRNAs areastructural/functionalcomponentmediating activationofpromotersbyenhancers

- Multiplestudies showthat eRNA productionis important forenhanceractivity (à Generalconcept ofenhancer activity)

ClassIIIofeRNAs andeRNA function

CLASSIII:Genes onthesame chromatin fibre(cis),orpotentially onotherchromosomes (trans),areregulatedbyaneRNA (MAJORTYPE)

LOOPING -CHROMOSOMETOPOLOGY

Functional mechanisms ofeRNAsinclude:1.eRNAs interact withchromosomal loopingfactors(CLFs)topositively influenceenhancer–promoterloopingandgenetranscription;

RNAbinding protein (RBP)RNAprocessingfactor (RPF)Trancription factor (TF)Chromosomal loopingfactor (CLF)

1.2. 3.

ExamplesforaroleofeRNAs inlooping:-eRNA knock-downresultsinimpairedlooping(insomecases)-eRNAs werefoundtointeractwithcohesin-eRNAs werefoundtointeractwiththemediatorcomplexExamplesthatclaimthateRNAs tonotsupportlooping:-BlockingtheelongationofRNAtranscriptiondoesnotnegativelyimpactonlooping-Knock-downofeRNAs didnotimpactlooping(insomecases)



Functional mechanisms ofeRNAs include:2. eRNAs bind transcriptionfactors (TFs)tohelp ‘trap’themat enhancers

RNAbinding protein (RBP)RNAprocessingfactor (RPF)Trancription factor (TF)Chromosomal loopingfactor (CLF)

1.2. 3.

eRNAs trap transcription factors thatenhance thetranscription ofenhancers(YY1– eRNA interaction;YY1binding isincreased at enhancers




Functional mechanisms ofeRNAs include:3.eRNAs act as a‘decoys’or‘repellents’toinhibittranscriptional repressors (TxRs).Transroles could beachieved byeRNA translocation todistantsites (rightsideofpanel)orproximity-based regulation inwhich eRNAs andtargetgene(s)reside incertain transcriptionallyassociated territory (lightyellowarea).RNAbinding protein (RBP)

RNAprocessingfactor (RPF)Trancription factor (TF)Chromosomal loopingfactor (CLF)

1.2. 3.


EVIDENCETHATeRNAs CONTROLPAUSINGOFRNAPol IIATPROMOTERS

Arcgeneisessentialforbraindevelopmentandfunction.

RNAPol IIandTFs arereadyonpromoter.Afteracellstimulusthe

activationofthegeneisveryfastandefficient(=IEG:immediately

earlygene)Intheinactivestatus,RNAPol II is

locatedatthepromoterbuttheproteinNELF(negativeelongationfactor)pauses

theactivityofRNAPol II

Afterstimulus,ArcEnhancerproduceseRNAs

ThatbindtoNELFandcausethereleaseofNELFfromthepromoter;P-TEFBphosphorylatesRNAPolCTDSer-2

à IMMEDIATE BURSTOFTRANSCRIPTION


GenomicVariationofenhancersindisease

Transcriptionofenhancersthatarecharacterizedbylowconservationmyleadgenomicinstabilitythatcontributestotheformationofdisease relevantmutationsorthegenerationofnewgenes

TranscriptionalactivityatenhancersmayresultinnucleicacidstructuresincludingR-loops,althoughtheprevalenceofR-loopsatenhancersstillrequiresfurthergenome-wideelucidation.Variousfactors,asshownhere,arepresumablyinvolvedintheprocessofR-loop resolutionorenhancertranscription.Despitetheirbindingtoenhancers,mostDNAdamageresponse(DDR)factorsarestillmechanisticallyenigmaticinenhancertranscriptionorfunction.TheadditionalfactorsinthefigureincludeArgonaute1(AGO1),whichhasbeenshowntobindactiveenhancersdependingonenhancertranscription178,andactivation-inducedcytidinedeaminase (AID),themis-targetingofwhichintheBcellgenomehasrecentlybeenassociatedwithenhancertranscription.Questionmarksdenoteunclearfunctionalrolesormechanisms.Thetranscriptionprocessofenhancersmaybelinkedtoseveralimportantbiological,pathologicalorevolutionaryfunctions,asshowninthediagram.Exosomecomponent10(EXOSC10;alsoknownasRRP6)andEXOSC3(alsoknownasRRP40)denotethetwocomponentsoftheRNAexosome complex.


Heatmaps showH3K27acandH3K4me1signals for1,000candidateenhancers (rows)in12immunecell types (columns).Enhancers areclustered bythecell type-specificity oftheir H3K27acsignals.Adjacent heatmap showsaverageRNA-seq expression forthegenes nearesttotheenhancers ineach cluster.Greyscale (right)depicts theenrichmentofPICSautoimmunity SNPs ineach enhancer cluster(hypergeometricP values calculated based onthenumber ofPICSSNPs overlapping enhancers fromeach cluster,relativetorandomSNPsfromthesame loci).TheAP-1motif is over-represented inenhancers preferentiallymarked instimulated Tcells,compared tonaive Tcells.

PICS:Probabilistic Identification ofCausal SNPs

SNPs found inautoimmunegenesweremappedtoenhancer elements andtherespectiveenrichment was determined.à SNPés areenriched inenhancers.


Candidate causal SNPs displayed along with H3K27ac and RNA-seq signals at the PTGER4 locus. A subset of enhancers with disease variants (shaded) shows evidence of stimulus-dependent eRNA transcription.

SNPs associated withAutoimmunedisease

eRNA peaks inenhancer

expression ofclosest gene

In autoimmune disease it is estimated that immune enhancers overall account for 60% of candidate causal SNPs, whereas promoters account for another 8% of these variants


H3K27ac,DNaseI26andconservationsignals,andselected transcriptionfactorbindingintervalsareshowninaSMAD3introniclocus.rs17293632,anon-codingcandidatecausalSNPforCrohn’s disease,disruptsaconservedAP-1bindingmotifinanenhancermarkedbyH3K27acinCD141monocytes.SummingofChIP-seq readsoverlappingtheSNPintheheterozygousHeLacell lineshowsthatonlytheintactmotifbindsAP-1transcriptionfactors,JunandFos.

ASNPC-->Tin aenhancer ofdestroys thebindign siteforakey transcription factorAP1(heterodimer ofJun andFos)

Note:theTvariant is therespective SNP

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