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INTRODUCTION Progressintechnologiestoaddresslong-rangechromosomalinteractionsinvivohasextensivelyrevisedconceptsaboutdifferentaspectsoftranscriptionalregulation.Thesemethodsallowprobingphysicalproximitiesbetweenchromatinelementswithoutspecificallyidentifyingtheproteincomponentsthatmediatesuchinteractions.HerewedescribeadetailedprotocolforCombined3C-ChIP-Cloning(6C)technology,whichcombinesmultipletechniquestoidentifytheproteinsthatbridgedistantgenomicregions,whilesimultaneouslyidentifyingsuchphysicalproximities.Thismethodisalsousefulfordeterminingifacandidateproteinmightmediatelong-rangeinteractions,bothincisandintransinthenucleus.Wediscusshowthe6Ctechniquecanbeincorporatedwithothertechniquestodiscoverallthechromatinregionsinthenucleusthatinteractwithagivengeneorchromatinregionofinterestinaspecificprotein-dependentmanner.Suchinformationallowscomplete,cell-type-specificmappingofallthechromatininteractionsmediatedbyspecificproteins.The6Cassayadvancesourunderstandingofthethree-dimensionalaspectsofthehigher-orderfoldingofchromatinandprovidesanimportanttooltoexaminetheroleofspecificproteinsinnuclearorganization.Inadditiontoprovidingadetailedprotocolofthe6Ctechnique,wediscusshowthistechnologycanbeusedbyinvestigatorsworkingintheareaofchromatinBIOLOGy,withspecialinterestinchromatinlong-rangeinteractions. RELATEDINFORMATION The6Cassaycombinesthreedifferentmethodologies:chromosomeconformationcapture(3C)(Dekkeretal.2002),chromatinimmunoprecipitation(ChIP),andcloning(Fig.1).Thefirststepinvolvesconventional3Cmethodology:Thechromatiniscross-linked,digestedwithrestrictionenzymes,andligatedunderconditionsthatfavorintramolecularligation.Immediatelyafterligation,thechromatinisimmunoprecipitatedusinganantibodyagainsttheproteinofinterest(i.e.,thesUSPected"bridgingprotein"ortheproteinwhosemediatingphysicalproximitiestheinvestigatorwishestomap).Thereafter,thecross-linksarereversed,andtheDNAispurifiedfurther.Thefragmentsobtainedarethenclonedintoavectorharboringthesamerestrictionenzymesiteoverhangsthatweregeneratedintheenzymedigestionstepofthe3Cportionoftheprotocol.Theclonesarefurtherscreenedbydigestionwiththesamerestrictionenzyme.Ideally,clonesshowingmultipleinsertswillresultfromtheintramolecularligationandshouldrepresentphysicalproximitiesinvolvingtheproteintargetedintheimmunoprecipitationsteps.Theseclonesarechosenforsequencingtorevealtheidentityofthepartners. Forareviewofotherrecentmethodsdevelopedtoexaminelong-rangechromosomalinteractionsinvivo,seeSimonisetal.(2007).Foradditionaldetailsonthe6Cmethodpresentedhere,seeTiwarietal.(2008). MATERIALS Reagents Agarosegel(1%,UltraPure;Invitrogen) Antibody,specificfortheproteinofinterest Bovineserumalbumin(BSA;0.5%inPBS;NewEnglandBiolabs) Celltypeofinterest,grownunderappropriatecellcultureconditionswithappropriatemedium Cells,bacterial,competent,high-efficiency( XL10-Goldultracompetentcells(Stratagene)haveproducedoptimalresults. Cloningvector Thevectorusedshouldhaveenzymeoverhangssimilartothosegeneratedinthe3Cassay.Suchvectorscanbecustom-constructedorobtainedcommercially.Forexample,forcloningfragmentscontainingEcoRIends,usepBluescriptIIRIPredigestedVector(Stratagene). DNAladder,100-bpand/or1-kb(NewEnglandBiolabs) DNApolymerase(REDTaq;SigmaD4309)(forpolymerasechainreaction[PCR];seeStep45andDiscussion) dNTPset(100mM;Invitrogen)(forPCR;seeStep45andDiscussion) Ethanol(70%and100%) Fetalbovineserum(10%inPBS) Glycine(2M;FisherBP381) Glycogen(Roche) H2O,nuclease-free Preparethehigh-saltandlow-saltversionsofthisbufferseparately. Addtheappropriateantibiotic(seeStep51). Dilutethe10Xstockto1.15XforStep18. Magneticbeads,ProteinA-conjugated Magneticbeads,ProteinG-conjugated PCRbuffer(10X) PCRprimers,T3andT7 Phosphate-bufferedsaline(PBS;Gibco20012) Proteaseinhibitorcocktail(SigmaP8340) PureLinkHQ96MiniPlasmidDNAPurificationKit(Invitrogen) Thiskitisdesignedtoyieldhighamountsofhigh-qualityDNAfrom96differentplasmidsintheleastamountoftime,facilitatingthesimultaneousgrowthofseveralbacterialcoloniesandlarge-scalescreeningoftheplasmids.Itisparticularlyusefulforscreeningseveralplasmidsinordertoobtaincloneshavingmultipleinserts. Restrictionenzyme Theselectionofarestrictionenzymeisacriticalstepfor6Cmethodologythataffectsboththe3Cstepandthecloning.Italsodeterminestheresolutionofinteractionmapsthatcanbeobtainedusingthe6Cassayintermsofdefiningtheexactchromatinelementsthatareinvolvedinphysicalpairing.Enzymesthatproducecohesiveendsfrompalindromicrecognitionsitesarepreferredin3Cassays,andthesix-cutterssuchasEcoRI,HindIII,andBglIIarecommonlyused.Theseshouldcutevery4kbalongthegenomicDNAandofferagoodstartingpoint.Toobtainhigher-resolutionmaps,frequentcutterssuchasMseI(predictedtocutevery256bp)canbeused.Beforebeginninga6Cassay,useanaliquotoftherestrictionenzyme-digestednucleitotestafewregionsofthegenomefordigestionefficiencyeitherbySouthernorPCR-basedanalysis. Restrictionenzymebuffer(10X) Dilutethe10Xstockto1.14XforSteps12and13. T4DNAligase(400U/µL) Dilute10Xstockto1Xbeforeuse. Equipment Aluminumfoil Cellscraper Centrifuge,clinical Centrifuge,high-speed,refrigerated,equippedwithswingingbucketrotorfor14-mLtubes Dishes,cellculture DNAanalysissoftware(VectorNTI) DNAsequenceranalysissoftware(FinchTV) Gelelectrophoresistank,horizontal GelimagingsystemforquantifyingPCRproducts Heatblockpresetto65°C Hemocytometer Ice Incubator,humidified,equilibratedwith5%CO2,presetto37°C Magneticstandfor1.5-mLtubes(Invitrogen) Microcentrifuge,refrigerated Microscope,inverted PasteurPipettes Pipettorsandtips,5-to1000-µL Refrigeratororcoldroompresetto4°C Rotatingwheel/platform Shakingincubatorpresetto37°Cand65°C Sonicator Spectrophotometer Thermalcycler,automated Timer Tubes,microcentrifuge,1.5-mL Tubes,polypropylene,14-mL(e.g.,17-x100-mm)foruseinhigh-speedcentrifuge Tubes,polypropylene,50-mL Tubes,polypropylene,conical,15-mL(e.g.,17-x120-mm) Vacuumaspirator Vortexmixer Waterbath,variabletemperature METHOD PreparingNuclei RestrictionEnzymeDigestionandIntramolecularLigation EnrichmentofLigationFragmentsContainingtheProteinofInterestintheComplex Cloningof3C-LigatedImmunoprecipitatedFragments PlasmidPurification ScreeningforLigatedPartners TROUBLESHOOTING Problem:Thefragmentsaretoobig,and/ortherearemorethantwoinsertsintheplasmids. [Step54] Solution:Subclonethefragmentsintoanewrestrictionenzyme-digestedvectorbeforeproceedingwithsequencing. DISCUSSION The6C-capturedinteractionsshouldbevalidatedbyperformingindependent3Cassays(Tolhuisetal.2002),inwhichoneperforms3C-PCRsusingmultipleprimercombinationsfromtwodifferentremotesequences.TheprimersforthispurposearedesignedexactlyasdescribedbySplinteretal.(2004).Italsoisimportanttoestablish,byusingseparateassays,thattheinteractingregionscapturedinthe6Cassayaretrulyoccupiedbytheproteinofinterestincells.Tothisend,performChIPassays(Tiwarietal.2008)usingantibodiesspecificfortheproteinofinterest.TheimmunoprecipitatedDNAcanthenbeamplifiedusingprimersspanningtherestrictionenzymesitefoundtobeinvolvedinthe3Cligationwithotherpartner(s)ineachofthe6Cclones. Inearlyattemptsofthe6Cassay(Tiwarietal.2008),screeningtheclonesbyrestrictiondigestionidentifiedaverylowfrequencyofcloneshavingmultipleinserts(fiveoutof352).Anumberofreasonscouldaccountforthis.First,therestoftheclonesthathadasingleinsertprobablyrepresenteddistinctgenomicsitesthatwereboundbythespecificprotein(inthiscase,EZH2)butwerenotengagedinanylong-rangeassociations.Interestingly,thismightalsoreflectthefrequencywithwhichcertainprotein-dependentlong-rangeassociationstakeplaceinthenucleus.Second,thenumberofcloneswithmultipleinsertsmightalsobereducedbecauseofdifficultyincloning(i.e.,ligationandtransformation)orsequencingoflargefragmentsresultingfromtheligationfragmentsgeneratedbyasix-cutterrestrictionenzyme(e.g.,EcoRI).Third,intramolecularligationaftercross-linkinganddigestionwillleadtoafractionoftheDNAoccurringascircularDNAthatcannotbecloned;thismightfurtherreducethenumberofclonesinsuchanassay.Finally,thestudycouldalsohavemissedsomeinteractionsinvolvingpartnersthatbearDNAmethylationattheEcoRIsite(EcoRIisaDNAmethylation-sensitiveenzyme).Futureworkshoulddevelopstrategiestotackleeachoftheseissuestoimprovetheoverallefficiencyofthemethod. The6Ctechniquecanbecombinedwithotherrecentlypublishedtechniquestodiscoverallthechromatinregionsinthenucleusthatarebroughtinclosephysicalproximitytothegene(oranyotherchromatinregionofinterest)inaspecificprotein-dependentmanner(Fig.2).Followingimmunoprecipitationwiththeantibodyofinterest,thesamplescanbesubjectedto4Canalysis(Zhaoetal.2006)orreverse-cross-linked,purified,digestedwithafour-cutterenzymeofchoice,andprocessedfurtherforeither3C-chip(Simonisetal.2006)ortheACTassay(Lingetal.2006).The6Cprocedurecanalsobeusedtorevealwhethertwoknownchromatinregionsarebroughtintoclosephysicalproximitybyaspecificproteinbyfollowingtheamplificationcriteriausedintheoriginal3Cassay,subsequenttothereversalofcross-linkingandpurification(Dekkeretal.2002). 
Viewlargerversion(17K):Figure1.SummaryoftheCombined3C-ChIP-Cloning(6C)method. 
5-Bromo-4-chloro-3-indoyl-β-D-galactopyranoside(X-gal;25mg/mLinDMF[dimethylformamide])
Beadelutingbuffer
5x109cfu/µgDNA)CellularlysissolutionChIPdiluentDNAgelstain(SYBRSafe,10,000XconcentrateinDMSO[dimethylsulfoxide];Invitrogen)Formaldehydesolution(36.5%;Sigma-Aldrich33220)Immunecomplexwashbuffers(high-saltandlow-salt)Isopropyl-β-D-thiogalactopyranoside(IPTG;200mg/mL)LBagarLB-ampicillinagarplatesAlternatively,LB-kanamycinagarplatescanbeused(seeStep48).LB(Luria-Bertani)liquidmediumLigationbuffer(10X)Phenol:chloroform:isoamylalcohol,UltraPure(25:24:1v/v/v;Invitrogen)ProteinaseK(10mg/mLinTEbuffer,pH8.0;Invitrogen)RNaseA,DNase-free(10mg/mL;SigmaR6513)SDS(Sodiumdodecylsulfate;20%w/v;FisherBP166)Sodiumacetate(3M,pH5.2;FisherBP333)TEbuffer(pH8.0)TritonX-100(20%v/v;VWR)Trypsin(1X;Invitrogen25300)Liquidnitrogen(optional;seeStep11)UVlight
Viewlargerversion(18K):Figure2.FurtherapplicationsandthefutureofCombined3C-ChIP-Cloning(6C)methodology.
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