DNA Immunoprecipitation (DIP) for the Determination of DNABinding Specificity

INTRODUCTION

KnowledgeoftheDNA-bindingspecificityofatranscriptionfactoraidsinunderstandingthefunctionofthatfactorintheregulationofgenetranscription.OnepopularmethodofidentifyingthegenomicDNAsitesboundbyagivenproteininvivoistheChromatinImmunoPrecipitationwithmicroarrayanalysis(ChIP-chip)technique.However,thismethodrevealsabindingpatterninfluencedbyinvivophenomenathatmaymasktheactualDNA-bindingspecificityofthefactor,suchaschromatineffectsandcompetitiveorcooperativeprotein-proteininteractions.ChIP-chipalsorequiresadequateexpressionoftheproteininthecelltypechosentocreatetheextractusedforimmunoprecipitation.DNAImmunoprecipitation(DIP)isanalternativetechniquethatallowsonetotakeadvantageofthefavorablepropertiesofbothinvivoandtrADItionalinvitrotechniques(e.g.,electromobilityshiftassay[EMSA]andbindingsiteselection[SELEX]).DIPutilizesnakedgenomicDNAasabindingsubstrateforoneormorepurifiedrecombinantproteins.BecausegenomicDNAisusedasatemplateinDIPreactions,theresultsaredirectlycomparabletoChIP-chiporChIP-seqdata.DIPcanbecarriedoutinthepresenceofcofactorssuchasheterodimerpartners,competitors,orsmallmoleculebindinginhibitors.AfterDNAisisolatedbyDIP,itismostefficientlydetectedusingahighlyparallelgenomictechniquesuchasaDNAmicroarray(DIP-chip)orhigh-throughputsequencing(DIP-seq).Inthisprotocol,wedescribeaDIPofayeast(Saccharomycescerevisiae)proteinwithyeastgenomicDNA.

RELATEDINFORMATION

FormoreinformationabouttheDIP-chiptechnique,seeLiuetal.(2005,2006).

MATERIALS

Reagents

Amyloseresin(3mg/mLmaltose-bindingprotein[MBP]-bindingcapacity;NewEnglandBiolabs)

DIPbinding/washbuffer

DIPelutionbuffer

DNA(genomic,purifiedandshearedtoanaveragesizeof~600bp)

GenomicyeastDNAfromthestrainS288CwaspurifiedbyCsClgradientandshearedbysonication.

TheproteinandgenomicDNAusedforDIPneednotbefromthesamespecies,andgenomicDNAfromanyspecieswithasequencedgenomecanbeused.

Proteinofinterest(MBP-tagged,purified)

Reagentsfordetection(i.e.,aDNAmicroarraycorrespondingtothegenomicDNAused,orreagentsforhigh-throughputsequencing;seeStep11)

Equipment

DNApurificationcolumns(e.g.,Zymo-SpinI;ZymoResearch)

Equipmentfordetection(i.e.,DNAmicroarrayorhigh-throughputsequencingcapacity;seeStep11)

Ice

Microcentrifuge(capableof2000gformicrocentrifugetubes)

Microcentrifugetubes(1.5mL)

Rotator(Labquakee.g.,BarnsteadInternational400110orcomparableend-over-endrotator)ormicropipettor(seeStep4)

METHOD

ThisprotocolwasmodifiedfromthemethodbyLiuetal.(2005).

1.Topreparea100µLreaction,combinethepurifiedproteinofinterest(MBP-tagged)toafinalconcentrationof40nMandtheshearedgenomicDNAtoafinalconcentrationof0.3pMin100µLDIPbinding/washbuffer.ForyeastDIPs,0.3pMgenomicDNAisequivalentto200ngina100-µLreaction.Thesuggestedproteinconcentrationof40nMisbasedonatypicalDNA-bindingproteinK_d,andmaybevariedaccordingtotheproteinused.

2.Incubatefor30minatroomtemperature(~22°C).

3.Wash10µLofamyloseresinwith100µLDIPbinding/washbufferina1.5-mLmicrocentrifugetube.Centrifugeat2000gfor5mininamicrocentrifugeatroomtemperature.StoreoniceuntilStep4.

4.Addtheprotein/DNAreactionmixture(100µL)tothewashedamyloseresin.Rotateend-over-endonaLabquakerotatorfor15minatroomtemperature.ItisessentialthattheresinremainsinsUSPensionduringthisstep.Nutationisinsufficienttomaintainsuspension.Ifend-over-endrotationisunavailable,frequent(every30-60sec)pipettingmaybesubstituted.

5.Centrifugetheresinat2000gfor5min.

6.Washtheresinwith50µLDIPbinding/washbufferandcentrifugeat2000gfor5min.

7.RepeatStep6threeadditionaltimesforatotaloffourwashes.Removethesupernatantafterthefinalwash.

8.Elutetheproteinbyadding50µLDIPelutionbuffertotheresin.

9.Centrifugetheresinat2000gfor5minandcollectthesupernatant.

10.IsolatetheDNAfromthesupernatantwithaZymo-SpinIcolumn(orequivalentDNApurificationmethod)andeluteintothedesiredvolume.Astartingamountof200ngofyeastDNA(Step1)typicallyresultsinatotalyieldof16-30ngofisolatedDNA.Theelutionvolumemaybeadjustedtoachievethedesiredfinalconcentration.

11.AmplifyanddetecttheisolatedDNA.DNAisolatedbyDIPhasbeensuccessfullyamplifiedbyrandompriming,ligation-mediatedPCR,andWholeGenomeAmplification(WGA)(GenomeplexWGA2;Sigma-Aldrich).DetectionoptionsincludeDNAmicroarray(DIP-chip)orhigh-throughputsequencing(DIP-seq)methods.Followthesamplepreparationrecommendationsforthepreferreddetectiontechnique;detailedmethodsforthedifferentdetectionplatformsvarybymanufacturerandaretypicallywell-described.

TROUBLESHOOTING

Problem:Proteinrecoveryfails.

[Step8]

Solution:ConfirmthattheresinremainssuspendedinStep4.Iftheresinisnotsuspended,theproteinwillnotbindefficiently.

Problem:LittletonoenrichmentofDNAisachieved.

[Step11]

Solution:Considerthefollowing:

• Determinetheoptimalnumberofwashesfortheproteinofinterest.ThewashconditionswereestablishedusingMBP-Leu3DBD,butmayvarybasedontheK_doftheproteinused.Cross-linkingtheproteinandDNAcomplexwithformaldehydeorasimilarreversIBLecross-linkermayenhanceisolationofDNAwhenproteinswithalow-affinityforDNAareused,butthishasnotbeenrigorouslytested.
• ConfirmthattheproteiniscapableofbindingDNA,becausesomerecombinantproteinsfailtofoldproperly.Iftheconsensussequenceisalreadyknown,traditionalmethods(suchasEMSA)canbeusedtochecktheprotein’sABIlitytobindDNA.Asanalternativetousingrecombinantprotein,nativeproteinmaybepurifiedfromtheorganismofinterest.TreatmentofthepurifiedproteinwithDNasepriortoDIP-chipwilllikelyberequiredtoremoveresidualnativeDNA.

DISCUSSION

Traditionaltechniquesforstudyingtheinvitrobindingspecificityofpurifiedproteins,suchasEMSA(GarnerandRevzin1981;EllingtonandSzostak1990)andSELEX(Brenowitzetal.1986;TuerkandGold1990),bypassthedifficultiesassociatedwiththeinvivoChIP-chipmethod(seeIntroduction).However,EMSAtypicallyrequiressomepriorknowledgeofbindingspecificityandcannotassayawidediversityofDNAsequencessimultaneously,whileSELEXissubjecttoover-selectionofboundsequences,whichmaycausesignificantbutlower-affinityinteractionstobemissed.Morerecently,severalhigh-throughputtechniqueshavebeendeveloped;oneexampleisproteinbindingmicroarray(PBM),inwhichpurifiedproteinisdirectlyincubatedwithaDNAmicroarray.DetectionofDNAfragmentsboundinthereactionisachievedbyfluorescentlylabelingtheprotein.AlthoughthearraysusedcanbecomprisedofPCRproductsoroligonucleotidesrepresentingtheentiregenome,recentstudieshavefocusedonusing"universal"oligonucleotidesdesignedtorepresenteverypossiblek-mer(Mukherjeeetal.2004;BergerandBulyk2006;Bulyk2007).

BothPBMsandDIP-chipcanbeusedtodetermineinvitroconsensussequencesforproteinsbyassayingmillionsofartificialornaturalDNAsequencesinparallel.DIP-chiphasbeenshowntoperformaswellastraditionalassays(SELEXandEMSA)andtheChIP-chipmethodinidentifyingconsensussequences(Liuetal.2005).DuringDIP,theproteinandDNAareinsolutionduringthebindingreaction,allowingtheproteintosampletheDNAinthreedimensionsandeliminatinganyphysicalhindrancecausedbyhavingtheDNAtetheredtoasurface.DIPalsoallowscontrolledmanipulationofthebindingconditions,enablingquantitativeconclusionstobemadeabouttheeffectofreactionparametersonDNA-bindingspecificity.DIP-chipresultsaredirectlycomparabletoChIP-chipresults,becausethesamegenomicDNAtemplateisusedforbinding.ByaddingproteincofactorstoDIPreactionsandthencomparingthelociboundinvitroandinvivo,itmaybepossibletoquantifytherelativecontributionsofvariousfactors,suchaschromatinstructure(Liuetal.2006)andcofactor-assistedtargeting.