G3: Genes|Genomes|Genetics Early Online, published on January 15, 2016 as doi:10.1534/g3.115.025577
TheE2F-DP1transcriptionfactorcomplexregulatescentrioleduplicationinC.
elegans
JacquelineD.Goeres-Miller1,YanLiu1,ChristopherWilliams1,HaroldE.Smith2,KevinF.
O’Connell1,3
1LaboratoryofBiochemistryandGenetics,and2GenomicsCore,NationalInstituteof
DiabetesandDigestiveandKidneyDiseases,NationalInstitutesofHealth,Bethesda,MD
20892,USA
3Towhomcorrespondenceshouldbeaddressed.
ContactInformation:
email:[email protected]
phone:301-451-4557
fax:301-402-0240 1
© The Author(s) 2013. Published by the Genetics Society of America.
Runningtitle:TranscriptionalControlofCentrioleDuplication.
KeyWords:centrioleduplication,C.elegans,transcriptionalregulation,E2F/DP1,SAS-6
2
Abstract
Centriolesplaycriticalrolesintheorganizationofmicrotubule-basedstructuresfromthe
mitoticspindletocilia,andflagella.Inordertoproperlyexecutetheirvariousfunctions,
centriolesaresubjectedtostringentcopynumbercontrol.Centraltothiscontrol
mechanismisapreciseduplicationeventthattakesplaceduringSphaseofthecellcycle
andinvolvestheassemblyofasingledaughtercentrioleinassociationwitheachmother
centriole.Recentstudieshaverevealedthatpost-translationalcontrolofthemaster
regulatorPlk4/ZYG-1kinaseanditsdownstreameffectorSAS-6iskeytoensuring
productionofasingledaughtercentriole.Incontrast,relativelylittleisknownabouthow
centrioleduplicationisregulatedatatranscriptionallevel.Hereweshowthatthe
transcriptionfactorcomplexEFL-1-DPL-1bothpositivelyandnegativelycontrolscentriole
duplicationintheCaenorhabditiselegansembryo.Specifically,wefindthatdown
regulationofEFL-1-DPL-1canrestorecentrioleduplicationinazyg-1hypomorphicmutant
andthatsuppressionofthezyg-1mutantphenotypeisaccompaniedbyanincreaseinSAS6proteinlevels.Further,wefindevidencethatEFL-1-DPL-1promotesthetranscriptionof
zyg-1andothercentrioleduplicationgenes.Ourresultsprovideevidencethatinasingle
tissuetype,EFL-1-DPL-1setsthebalancebetweenpositiveandnegativeregulatorsof
centrioleassemblyandthusmaybepartofahomeostaticmechanismthatgoverns
centrioleassembly.
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Introduction:
Centriolesarecylindricalmicrotubule-basedorganellesthatdirectformationof
centrosomesandcilia(WineyandO'Toole2014).Dividingcellspossessoneortwopairsof
centrioleswitheachpaircontaininganewer(daughter)centrioleorientedorthogonallyto
anolder(mother)centriole.Inthiscellularcontext,centriolepairsareenvelopedbya
proteinaceousmatrixcalledthepericentriolarmaterialorPCM,therebyforming
centrosomes,thecell’sprimarymicrotubule-organizingcenter(MTOC).Centrosomes
mediateintracellulartransport,establishcellpolarity,andorganizethepolesofthemitotic
spindletoaidinthesegregationofchromosomes.Innon-cyclingcells,centriolesshedtheir
PCMandmovetotheplasmamembranewherethemothercentrioleservesasabasalbody
toorganizeciliaandflagella,structuresimportantforcellmotilityandcellsignaling.
Giventhecrucialrolesofcentriolesinbothcyclingandnon-cyclingcells,itisnot
surprisingthataberrationsincentriolenumberorstructurehavebeenlinkedtodisease.
Anexcessnumberofcentriolesisfoundinmanydifferenttypesoftumorcellswherethey
candisruptspindlestructureandchromosomesegregationleadingtoaneuploidy(Ganem
etal.2009).Excesscentriolescanalsointerferewithcilia-basedcellsignaling(Mahjoub
andStearns2012)andpromotecellmigrationandinvasivebehavior(Godinhoetal.2015).
Thusexcesscentriolescanimpactthegrowthofcellsinmultipleways.Beyondcancer,
defectsincentriolestructureornumberhavebeenlinkedtoseveralhumandiseases
includingautosomalrecessiveprimarymicrocephaly,primordialdwarfismandacollection
ofdisorderscalledciliopathies(Chavalietal.2014).
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Individingcells,centriolenumberismaintainedthroughapreciseduplicationevent
inwhicheachmothercentriolegivesrisetooneandonlyonedaughtercentrioleduringS
phase(Firat-KaralarandStearns2014).Aseachcentriolepairwillformaspindlepole
duringtheensuingMphase,stringentcontrolofcentrioleassemblyhelpsensurespindle
bipolarityandthefidelityofcelldivision.Forwardandreversegeneticstudiesinthe
nematodeCaenorhabditiseleganshaveledtotheidentificationofasetoffivecorefactors
thatarerequiredforcentrioleduplication(O'Connelletal.2001;Kirkhametal.2003;
LeidelandGönczy2003;Kempetal.2004;Pelletieretal.2004;Delattreetal.2004;
Dammermannetal.2004;Leideletal.2005;Kitagawa,Flückiger,etal.2011;Songetal.
2011).Functionalorthologsofeachofthesefactorshavesincebeenidentifiedinother
speciesincludingfliesandhumans,therebyestablishingthebroadevolutionary
conservationofthecentrioleduplicationpathway(Leideletal.2005;Habedancketal.
2005;Bettencourt-Diasetal.2005;Bastoetal.2006;Kleylein-Sohnetal.2007;RodriguesMartinsetal.2007;VladarandStearns2007;Zhuetal.2008;Kohlmaieretal.2009;
Stevensetal.2010;Arquintetal.2012;Vulprechtetal.2012).
CentrioleassemblyisinitiatedbytherecruitmentofPolo-likekinase4(Plk4)tothe
siteofcentrioleassembly(Dzhindzhevetal.2010;Cizmeciogluetal.2010;Hatchetal.
2010;Slevinetal.2012;Sonnenetal.2013;Kimetal.2013;Shimanovskayaetal.2014).
Invertebrates,thisstepisexecutedthroughadirectphysicalinteractionbetweenPlk4and
itscentriolereceptorsSPD-2andCep152.Asimplermechanismoperatesinwormswhere
SPD-2issolelyinvolvedinrecruitingthePlk4relativeZYG-1(Delattreetal.2006;Pelletier
etal.2006).ZYG-1/Plk4thenrecruitsthecoiled-coildomaincontainingproteinsSAS-6
andSAS-5/Stil.Themoleculardetailsofthisstepappearspecies-specificbutinvolvea
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directphysicalinteractionbetweenPlk4/ZYG-1andeitherSAS-5orSAS-6andsubsequent
phosphorylation(Lettmanetal.2013;Dzhindzhevetal.2014;Arquintetal.2015;Kratzet
al.2015;Moyeretal.2015).Attheassemblingcentriole,SAS-6dimersoligomerizetoform
thecentriolescaffold,anelegantcartwheelstructureinhumansandfliesorasimpler
centraltube-likestructureinworms(Kitagawa,Vakonakis,etal.2011;vanBreugeletal.
2011).Finally,thecoiled-coilcontainingproteinSAS-4isrecruitedtothenascentcentriole
andisrequiredfortheassemblyofthemicrotubulesoftheouterwall(Pelletieretal.2006;
Dammermannetal.2008;Schmidtetal.2009).
Whilemanyofthemoleculardetailsofcentrioleassemblyhavebeenelucidatedby
recentstructuralandbiochemicalstudies,manymysteriesregardingtheregulationofthis
processremain.Inparticular,itisnotclearhowamothergivesbirthtoasingledaughter
centrioleduringeachroundofduplication.Overexpression/over-activationofthecore
duplicationfactorsZYG-1/Plk4orSAS-6resultintheproductionofmultipledaughter
centrioles(Habedancketal.2005;Peeletal.2007;Kleylein-Sohnetal.2007;Bastoetal.
2008;Petersetal.2010),indicatingthatcarefulregulationofthelevelsand/oractivityof
thesefactorsplaysaroleinlimitingthenumberofdaughtersassembledduringeachround
ofduplication.Morerecently,anumberofstudieshaveshedlightontheimportanceof
post-translationalmechanismsinregulatingcentrioleduplication;boththelevelsof
Plk4/ZYG-1andSAS-6arestringentlycontrolledbyregulatedproteolysis(Strnadetal.
2007;Cunha-Ferreiraetal.2009;Rogersetal.2009;Puklowskietal.2011;Peeletal.2012;
Čajáneketal.2015).
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Littleisknownhoweverabouthowcentrioleduplicationiscontrolledatthelevelof
transcription.In1999,Meraldiandcolleaguesshowedthattheheterodimeric
transcriptionfactorE2F-DPplayedaroleinregulatingthere-duplicationofcentriolesinSphasearrestedCHOcells(Meraldietal.1999).Howevertherelevantgenestargetedby
E2Fwerenotidentified.Morerecently,severalisoformsoftheE2Ftranscriptionfactor
family(E2F4andE2F5)alongwiththeirbindingpartnerDPandacell-specificco-regulator
multicillin,werefoundtodirectlyactivatethetranscriptionofthecorecentriole
duplicationfactorsinmulticilliatecellstoupregulatecentriolebiogenesis(Maetal.2014).
Infact,activationofthistranscriptionalcomplexwasrequiredformulticilliatecell
differentiation.IncontrasttothepositiveroleforE2F4andE2F5inmulticilliatecells,a
negativeroleforE2F3wasdemonstratedinmouseembryonicfibroblasts(MEFs).
Specifically,inactivationofE2F3,butnototherisoformsofE2F,inMEFsresultedin
centrosomeamplification(Saavedraetal.2003).ThesestudiesshowthatE2F-DPmayplay
eitherapositiveornegativeroleinregulatingcentrioleduplication,withthenatureofthe
roleappearingtodependuponthecelltypeandthespecificisoformofE2F.Here,weshow
thatE2F-DPalsoplaysaroleinregulatingcentrioleduplicationinC.elegansembryos.
Remarkably,wefindthatE2F-DPplaysbothapositiveandanegativeroleinasinglecell
typeandproposethatE2F-DP1controlsthebalanceofpositiveandnegativeregulatorsof
centrioleassembly.
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Results:
Mutationofszy-10suppressesembryoniclethalityandrestorescentrioleduplication
inthezyg-1(it25)mutant
Theszy-10genewasinitiallyidentifiedasageneticsuppressorofthetemperaturesensitivezyg-1(it25)mutant(Kempetal.2007).Atthenon-permissivetemperatureof
24°C,embryoniccentrioleduplicationfailsinthezyg-1(it25)mutant.Asaresult,thepair
ofcentriolesderivedfromthespermseparateandestablishesabipolarmitoticspindle
duringthefirstembryonicdivision.Theabsenceofcentrioleduplicationduringthefirst
cellcycleresultsineachdaughtercellinheritingonlyasinglecentriolewhichgoesonto
organizeamonopolarspindleineachblastomereofthetwo-cellembryo(Figure1A).This
failureincentrioleduplicationalsoresultsinafullypenetrantembryoniclethalphenotype
at24°C.Incontrasttothezyg-1(it25)singlemutant,azyg-1(it25)szy-10(bs21)double
mutantisabletoproduceasignificantnumberofviableprogenyat24°C(Figure1B).At
theslightlyhighertemperatureof25C,verylittlesuppressionisobservedinthezyg1(it25)szy-10(bs21)doublemutant(Figure1B).Asthezyg-1(it25)mutantissignificantly
moreimpairedat25°C,thefailureofsuppressionatthehighertemperatureindicatesthat
theszy-10(bs21)mutationdoesnotbypasstherequirementforzyg-1incentriole
duplication.Rather,theszy-10(bs21)mutationlikelyeitherelevatestheresidualZYG-1
activityinthemutant,oralternatively,easestherequirementforZYG-1byfacilitating
executionofthepathwaydownstreamofZYG-1.
Toinvestigatewhetherszy-10suppresseszyg-1embryoniclethalitybyrestoring
centrioleduplication,time-lapsemicroscopywasperformedinzyg-1(it25)andzyg-1(it25)
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szy-10(bs21)mutantsexpressingGFP-labeledSPD-2asamarkerforthecentrosomesand
mCherry-labeledhistoneH2BasaproxyfortheDNA.Asexpected,centrioleduplication
invariablyfailedinzyg-1(it25)mutantsleadingtomonopolarspindlesatthetwo-cellstage
(Figures1Cand1D).However,inthezyg-1(it25)szy-10(bs21)doublemutant,all
centrosomessuccessfullyduplicatedtogeneratebipolarspindlesatthetwo-cellstage
(Figures1Cand1D).Theseresultsdemonstratethatszy-10(bs21)suppressesembryonic
lethalitybyrestoringcentrioleduplicationinthezyg-1(it25)mutant.
Todetermineifmutationofmaternalszy-10issufficientforsuppressionofthezyg1(it25)centrioleduplicationdefectorwhetherthereisapaternalcontribution,zyg-1(it25)
szy-10(bs21)hermaphroditeswerematedtozyg-1(it25)malesandcentrioleduplication
wasanalyzedintheresultingprogeny.Thecentrosomesduplicatedinthreeoutofthree
embryos,indicatingthatperturbationofmaternalSZY-10issufficientforsuppressionof
zyg-1(it25)(datanotshown).Thus,paternalexpressionofthemutantszy-10genedoesnot
appeartocontributetosuppressionofthezyg-1(it25)centrioleduplicationdefect..
Todeterminethemolecularidentityoftheszy-10gene,acombinationoftraditional
three-factormappingandwhole-genomesequencingwasemployed.Theszy-10genehad
previouslybeenmappedtoaregionbetweendpy-10andunc-4onchromosomeII(Kempet
al.2007).Whole-genomesequencingofthezyg-1(it25)szy-10(bs21)doublemutant
identifiedjustfourmutationsaffectingsplice-sitesoreffectingnon-synonymousaminoacid
changeswithinthisinterval(Figure2AandSupplementaryFigureS1A).Higher
resolutionmappingdefinitivelydemonstratedthatthemutationresponsiblefor
suppressionofthezyg-1(it25)phenotypewasamutationinthedpl-1gene.Specifically,
zyg-1(it25)dpy-10(e128)unc-4(e120)/zyg-1(it25)+szy-10(bs21)+hermaphroditeswere
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constructedandF1DpynonUncandUncnonDpyrecombinantswereisolated(Figure2A
andFigureS1B).Recombinantscontainingthemutantdpl-1alleleexhibitedsuppression
ofthezyg-1(it25)embryoniclethality(Figure2B)whilethepresenceoftheother
mutationsdidnotcorrelatewithsuppression.Conversely,recombinantsthathadlostthe
dpl-1mutationbutretainedtheotherthreemutationsgenesfailedtosuppresszyg-1(it25)
(SupplementaryFigureS1B).Takentogether,theseresultsindicatethatmutationofdpl1isnecessaryandsufficientforsuppressionofthezyg-1phenotype.Tofurtherconfirm
theidentifyofthegene,weusedmolecularcomplementation;specifically,wefoundthat
introductionofadpl-1-gfptransgeneintothezyg-1(it25)szy-10(bs21)strainresultedin
lossofsuppression(Figure2C).RNAi-mediateddepletionofthetransgeneusingRNAi
directedagainstgfpreversedtheseeffects,ensuringthatthelossofsuppressionwasdueto
thepresenceoftheextracopiesofdpl-1ratherthananyothergeneticvariancesintroduced
bythetransgenicstrain(Figure2D).Weconcludethatthebs21mutationisanalleleofthe
dpl-1geneandhereafterrefertothisgeneasdpl-1.
Thecomplementationofdpl-1(bs21)byawild-typedpl-1transgeneindicatedthat
dpl-1(bs21)isaloss-of-functionmutation.Consistentwiththisfinding,RNAi-mediated
depletionofdpl-1inthezyg-1(it25);gfp-spd-2;mCherry-h2bstrainrestoredcentriole
duplication(Figure2EandF).Whilecentrioleduplicationneveroccurredinzyg-1(it25)
animalstreatedwithcontrolRNAi,centrosomeduplicationoccurred~80%ofthetimein
zyg-1(it25)animalstreatedwithdpl-1RNAi(Figure2F).Theseresultsconfirmedthata
lossofDPL-1functionmediatedeitherbymutationorRNAisuppressesthezyg-1(it25)
centrioleduplicationdefectandembryoniclethalityandindicatesthatDPL-1isanegative
regulatorofcentrioleduplication.
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InhibitionoftheC.elegansE2F-DP1transcriptionfactorsuppresszyg-1(it25)defects
Thedpl-1(DP-like)geneencodesaconservedtranscriptionfactor,whichisrequired
fortheG1-to-S-phasecellcycletransitioninhighereukaryotes.DPheterodimerizeswitha
memberoftheE2FfamilyoftranscriptionfactorsandinsomecasestheE2F-DP1
heterodimerinteractswiththetumorsuppressorprotein,Retinoblastoma(Rb),toregulate
anumberofgenesinvolvedintheG1-Scellcycletransition.InC.elegans,thisfamilyof
transcriptionalregulatorsalsocontrolscellcycleprogression(ParkandKrause1999;
BoxemandvandenHeuvel2001;Fayetal.2002;BoxemandvandenHeuvel2002);
however,amorepredominantrolehasbeenelucidatedinthecontrolofdevelopmentallyregulatedprocessessuchasvulvaldevelopment,oocytematuration,andearly
embryogenesis(CeolandHorvitz2001;Pageetal.2001;ChiandReinke2006;Kirienko
andFay2007).
Todeterminewhetherthesuppressionofzyg-1(it25)centrioleduplicationdefects
wasduetoeffectsonthetranscriptionfactoractivitiesofDPL-1,wetestedwhetherlossof
itsheterodimerizationpartnerEFL-1wouldalsosuppressthezyg-1(it25)phenotype.
Becausenullmutantsofefl-1aresterile,weutilizedtheconditionalpartialloss-of-function
alleleefl-1(se1)totestforgeneticinteractionbetweenthesefactors.Theefl-1(se1)mutant
exhibitstwotemperature-sensitiveperiods.Shiftingthemutanttothenon-permissive
temperatureof26°CpriortotheL4stageresultsinsterility,whileshiftingaftertheL4
stageresultsinmaternal-effectembryoniclethality.Inordertodeterminewhetherlossof
efl-1functioncouldsuppresszyg-1(it25),weshiftedgravidadultstothenon-permissive
temperatureof25°Candallowedthemtolayeggsfor24hours.Theadultswerethen
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removedandtheembryosallowedtodevelopfor24hours.Undertheseexperimental
conditions,zyg-1(it25)mutantsexhibitedanaverageof92%embryoniclethality,whileefl1(se1)mutantsexhibitedanaverageof31%embryoniclethality(Figure3A).Thezyg1(it25);efl-1(se1)doublemutantsexhibitedanaverageof64%embryoniclethality.This
resultindicatesastrongpositiveepistaticinteraction,asfortwonon-interactingmutations
thefitnessofthedoublemutantshouldsimplybetheproductofthefitnessvaluesofeach
singlemutant(Beltraoetal.2010).Thus,ifzyg-1(it25)andefl-1(se1)werenon-interacting
mutations,wewouldexpectthedoublemutanttoexhibitafitnessof0.06(94percent
embryoniclethality):0.08(fitnessofzyg-1(it25))x0.69(fitnessofefl-1(se1))=.06.
Howeverthefitnessofthedoublemutantwas0.36(36%viabilitywithastandard
deviationof8.4%),morethanthreestandarddeviationsabovetheexpectedvalue.We
concludethatalossofeithermemberoftheEFL-1-DPL-1transcriptionfactorcomplex
suppresseszyg-1(it25)defects,implicatingeitheradirectorindirectrolefor
transcriptionalregulationbyEFL-1-DPL-1inthecontrolofcentrioleduplication.
FurthercorroboratingarolefortheEFL-1-DPL-1transcriptionalcomplexin
regulatingcentrioleduplication,weindependentlyidentifiedanalleleofefl-1asagenetic
suppressorofzyg-1(it25).Theszy-11genehadpreviouslybeenmappedtothevicinityof
unc-76onlinkagegroupV(Kempetal.2007).Weoutcrossedthezyg-1(it25);szy-11(bs22)
linetoaHawaiianpolymorphiclinecarryingthezyg-1(it25)mutationtensuccessivetimes.
Whole-genomesequencingofthisHawaiian-ingressedzyg-1(it25);szy-11(bs22)double
mutantidentifiedaregionontherightarmofLGVthatlackedHawaiianSNPs.Withinthis
region,sixopenreadingframescontainedprotein-codingmutations.(Figure3B).Oneof
thesemutationswaslocatedintheefl-1ORFandispredictedtoresultinanon-
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synonymouscodonchange.Toconfirmthemolecularidentityoftheszy-11(bs22)
suppressor,weusedCRISPRtechnologytorevertthemutationinefl-1tothewild-type
sequenceandexaminedthesuppressionofzyg-1(it25)embryoniclethality.At24°C,zyg1(it25);szy-11(bs22)hermaphroditesareabletoproduceavariablenumberofviable
offspring(Figure3C),However,reversionoftheefl-1mutationinthisstrainresultedinan
essentiallycompletelossofsuppressionatthenon-permissivetemperature,confirming
thatbs22isanalleleofefl-1.Curiously,introductionoftheefl-1(bs22)mutationintothe
originalzyg-1(it25)strainprovidedonlyveryweaksuppressionat24°C(Figure3C).
However,suppressionofzyg-1(it25)embryoniclethalitybytheCRISPR-engineeredefl1(bs22)mutationwasevidentatthelessrestrictivetemperatureof23.5°C(Figure3D).
Thus,whiletheefl-1(bs22)mutationprovidesmoderatesuppressionofzyg-1(it25),
evidentlyothergeneticelementspresentintheoriginalzyg-1(it25);szy-11(bs22)strain
alsocontributetosuppression.Nonetheless,theseresultsindicatethatbothDPL-1andits
heterodimericpartnerEFL-1negativelyregulatecentrioleduplication.
EFL-1-DPL-1directlyregulatestheexpressionofcentrioleduplicationfactors
TodeterminethemolecularmechanismbywhichlossoftheEFL-1-DPL-1
transcriptionalcomplexsuppresseslethalityinthezyg-1(it25)strain,weinvestigated
whetherthiscomplexdirectlymodulatesthelevelsofzyg-1and/orothercoreduplication
factors.Mostofthegenesencodingcoreduplicationfactors,suchasspd-2,zyg-1,sas-5,and
sas-6,containconsensusEFL-1-DPL-1bindingsiteswithintheirpromoters(TableS1).
Moreover,allofthesepromotersareknowntobeoccupiedbybothEFL-1andDPL-1invivo
(Kudronetal.2013).TodeterminewhetherEFL-1-DPL-1directlyregulatestheexpression
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ofzyg-1,wegeneratedatranscriptionalreporterstrain,inwhichthezyg-1promoterdrives
expressionofaGFP-labeledhistoneH2B.Thezyg-13’UTRwasusedtodirectthe
translationofthisreporterstrain(Figure4A).Wealsogeneratedaconstructcontaining
mutationsinallthreeoftheputativeEFL-1-DPL-1bindingsiteswithinthezyg-1promoter
(Figure4A).Single-copyinsertionofthereporterconstructswasachievedusingtheMosImediatedsinglecopyinsertion(MosSCI)methodoftransgenesis(Frøkjær-Jensenetal.
2008),andallowedfordirectcomparisonofzyg-1expressioninthewild-typeandEFL-1DPL-1-bindingmutant.Examinationofseveralindependentstrainsexpressingthewildtypezyg-1reporterrevealedthatzyg-1isproducedthroughoutthegermlineandwithin
earlyembryos.Expressionwasdetectedasearlyasthedistalgonad(Figure4B).IfEFL-1DPL-1isanegativeregulatorofzyg-1expression,wewouldexpectthatalossoftheEFL-1DPL-1-bindingsiteswithinthepromoterwouldresultinanincreaseinzyg-1expression.
However,wefoundthatmutationoftheseEFL-1-DPL-1bindingsitesresultedina
completelossofexpressionofthezyg-1reporter(Figure4B).Thisresultimplicatesa
positiveroleforEFL-1-DPL-1intheregulationofzyg-1expressionwithinthegermlineand
earlyembryo.Thusourresultisconsistentwithmicroarrayexperimentsshowingthat
EFL-1-DPL-1primarilypromotestheexpressionofgeneswithinthegermline(Chiand
Reinke2006).
TofurtherinvestigatetheroleforEFL-1-DPL-1inregulatingtheexpressionof
centrioleduplicationgenes,weusedquantitativereal-timePCRtomeasuretranscript
levelsinthewildtypeanddpl-1mutants.Specifically,weexaminedmessagelevelsofthe
coreduplicationfactors(spd-2,zyg-1,sas-5,andsas-6)inwild-typeanimalsorinanimals
carryingeitheroftwopartialloss-of-functiondpl-1mutations:(dpl-1(bs21)anddpl-
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1(n3643)).Consistentwithourfindingsusingthezyg-1reporterconstructs,wefoundthat
partiallossofdpl-1functionresultedinslighttomoderatedecreasesinthemessagelevels
oftheendogenouscentrioleduplicationgenes(Figure4C).Takentogether,theseresults
indicatethatEFL-1-DPL-1likelypromotestheexpressionofseveralcorecentriole
duplicationfactors.Therefore,suppressionbyloss-of-functionmutationsindpl-1andefl-1
isnotduetoanincreaseintheexpressionofcoreduplicationfactors,butinstead,islikely
mediatedindirectlythroughchangesintheexpressionofanas-of-yetunidentifiedfactor(s).
LossofEFL-1-DPL-1suppresseszyg-1throughanindirectmechanism
AlthoughlossofEFL-1-DPL-1activitydidnotresultinanincreasedlevelofanyof
thetranscriptsencodingcentrioleduplicationfactors,wedecidedtoexaminethesteadystateproteinlevelsofthesesamefactorsinanimalscompromisedforEFL-1-DPL-1
function.Specifically,embryonicextractswerepreparedfromwild-type,dpl-1(bs21),or
dpl-1(n3643)strainsandquantitativeimmunoblotanalysiswasperformedusing
antibodiesspecificforZYG-1,SPD-2,SAS-5orSAS-6.Sampleswerenormalizedagainst
tubulin,whichwasusedasaloadingcontrol.Surprisingly,whilesas-6messagelevelswere
notincreasedindpl-1mutants,thelevelofSAS-6proteinwasconsistentlyelevated3-4fold
(Figures5Aand5B).Incontrast,nosignificantchangesintheproteinlevelsofZYG-1,
SPD-2,orSAS-5weredetected(Figures5Aand5B).SAS-6isnormallyrecruitedto
nascentcentriolesbyZYG-1duringtheearlyeventsofcentrioleduplication(Delattreetal.
2006;Pelletieretal.2006;Lettmanetal.2013).ItispossiblethatSAS-6recruitmentisless
efficientinthezyg-1(it25)mutant,andthatoverexpressionofSAS-6amelioratesthisdefect.
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Thus,theelevatedlevelofSAS-6inthedpl-1mutantsprovidesapossiblemechanismby
whichthelossofdpl-1compensatesforcrippledzyg-1activity.
Takentogether,ourdatasuggestthatEFL-1-DPL-1regulatescentrioleduplicationin
partbydownregulatingthelevelofSAS-6protein.AsourresultsindicatethatEFL-1-DPL1promotestranscriptionofsas-6andothercomponentsoftheduplicationpathway,the
elevatedlevelofSAS-6proteininthedpl-1mutantislikelytheindirecteffectofaltered
expressionofsomeyet-to-be-identifiedfactor(s).Invertebrates,SAS-6levelsareregulated
bytheanaphasepromotingcomplex/cyclosome(APC/C),anE3ubiquitinligasethat
targetsvariouscellcycleproteinsfordestructionbytheproteasome(Strnadetal.2007).
Interestingly,Kudronetal.(2013)identifiedseveralAPC/Ccomponentsaspotential
targetsofEFL-1-DPL-1.ThusonepossiblemodeltoexplainourresultsisthattheEFL-1DPL-1transcriptionalcomplexnormallypromotesexpressionofoneormoreAPC/C
componentsthatinturnleadstodownregulationofSAS-6.
ConsistentwitharolefortheAPC/CinregulatingSAS-6,weidentifiedamat-3lossof-functionalleleamongourzyg-1suppressors.Themat-3geneencodestheconserved
APCsubunitAPC8/CDC23.Specifically,wefoundthattheszy-13(bs29)mutation,whichwe
hadinitiallymappedtochromosomeII(Kempetal.2007),actuallymappedclosetomat-3
onLGIII(Figure6A).Furthermore,thezyg-1(it25);szy-13(bs29)strainpossesseda
missensemutationwithinthemat-3openreadingframe;thismutationresultsinasingle
aminoacidsubstitution(Arg425Gln)withintheconservedTPRrepeatsofMAT-3.The
molecularidentityofmat-3wasconfirmedbycomplementationexperiments.First,we
showedthatthemat-3(or180)andszy-13(bs29)mutationsfailedtocomplementeachother
forsuppressionofzyg-1(it25)embryoniclethality(Figure6B).Second,wefoundthatthe
16
mat-3(or180)andszy-13(bs29)mutationsalsofailedtocomplementeachotherfor
suppressionofthezyg-1(it25)centrioleduplicationdefect.Specifically,wefoundthatboth
zyg-1(it25);szy-13(bs29)andzyg-1(it25);szy-13(bs29)+/+mat-3(or180)strainsduplicated
centrioles92%ofthetime(n=24eventsperstrain).Weconcludethatbs29isanalleleof
mat-3andthatthelossofAPC/Cfunctionpotentlysuppressesthezyg-1(it25)centriole
duplicationdefect.
GiventhatMAT-3isanessentialcomponentoftheAPC/C,andthattheAPC/Cis
knowntodownregulateSAS-6levelsinhumantissueculturecells(Strnadetal.2007),we
soughttoinvestigatewhetherMAT-3mightalsoperformasimilarfunctioninworms.
Embryonicextractsofzyg-1(it25)andzyg-1(it25);mat-3(bs29)mutantswereanalyzedby
quantitativeimmunoblottingusingSAS-6-specificantibodies.Contrarytoourexpectations,
wefoundthatthepresenceofthemat-3(bs29)mutationdidnotresultinanincreaseinthe
levelofSAS-6,suggestingthatC.elegansembryos,unlikehumansomaticcells,donot
downregulateSAS-6viatheAPC/C.OurresultsfurtherindicatethatwhileEFL-1-DPL-1
regulatescentrioleduplicationbypromotingtranscriptionofthecoreduplicationfactors,
otherrelevanttranscriptionaltargetsexist.
EmbryoslackingEFL-1-DPL-1activitydisplaycelldivisiondefects
GiventheroleofEFL-1-DPL-1inmodulatingexpressionofcentrioleduplication
factors,wesoughttoinvestigatewhetherlossofEFL-1-DPL-1-mediatedregulation,would
affectcentriolebiogenesisorfunction.Notably,mutationofdpl-1(bs21)resultsin50-80%
embryoniclethality((Kempetal.2007)anddatanotshown).Todeterminethecauseof
embryoniclethality,weexaminedearlydivisioneventsinadpl-1(bs21)strainexpressing
17
gfp-spd-2andmcherry-his-58usingtime-lapsemicroscopy.Outof33embryos,
approximatelyone-thirdoftheembryosexhibitedoneormoredefectsduringtheearly
embryonicdivisions,rangingfromcentrosome-nucleusattachmentdefectstodelaysinthe
timingofdivisionevents.Mostintriguingly,weobservedthegenerationofextra
centrosomesintwooftheembryos(SupplementaryFigureS2).Thelackofastronger
effectcouldeitherreflectthefactthatdpl-1(bs21)isahypomorphicalleleorthatlossof
EFL-1-DPL-1affectsexpressionofbothpositiveandnegativeregulatorsofcentriole
duplication.Nevertheless,thepresenceofexcesscentrosomesinthismutantisconsistent
withtheproposedroleforofEFL-1-DPL-1inlimitingexpressionofSAS-6.
Discussion:
Transcriptionalregulationofcentrioleduplicationfactors
Overthepastseveralyears,anumberofstudieshaverevealedthatcentriole
duplicationisregulatedinlargepartbycontrollinglevelsofthecorecentrioleassembly
factors(Strnadetal.2007;Cunha-Ferreiraetal.2009;Rogersetal.2009;Puklowskietal.
2011;Peeletal.2012;Čajáneketal.2015).Whileitisclearthatregulatedproteolysis
playsanimportantroleinachievingtheappropriatelevelsoftheseproteins,muchlessis
knownabouthowcontrolmightbeexertedattheleveloftranscription.Membersofthe
E2Ffamilyoftranscriptionfactorshavebeenimplicatedinthecontrolofcentriole
duplicationbuttheexactrole(positiveornegative)differsbetweencelltypesandE2F
familymembers(Meraldietal.1999;Saavedraetal.2003;Maetal.2014).Herewe
provideevidencethatwithinasinglebiologicalcontext—theC.elegansembryo—the
transcriptionalregulatorcomplexE2F-DP1canplaybothpositiveandnegativeroles.Our
18
findingthatpartialloss-of-functionmutationsindpl-1andefl-1suppressthecentriole
duplicationdefectandembryoniclethalityofzyg-1(it25)introducedtheintriguing
possibilitythatoneormorecentrioleduplicationfactorsmaybecontrolledatthelevelof
transcription.Consistentwiththis,fourofthesixgenesencodingcoreduplicationfactors
containputativeEFL-1-DPL-1bindingsitesintheirpromoters.Furthermore,thesesites
havebeenshowntobeoccupiedbyDPL-1invivo(Kudronetal.2013).Tooursurprise,
however,wefoundthatthemRNAlevelsofthesefactorswerenotincreasedineitherof
twodpl-1mutants,indicatingthattheyarenotnegativelyregulatedbytheEFL-1-DPL-1
heterodimer.Infact,mutationofdpl-1ledtoareproducibledecreaseinspd-2,zyg-1,sas-5
andsas-6RNAlevels(Figure4C).Thisresultisconsistentwiththefindingsof(Chiand
Reinke2006)whofoundthatDPL-1andEFL-1largelyactivatetranscriptionwithinthe
germline.Furthermore,ourfindingthatmutationoftheDPL-1-bindingssiteswithinthe
zyg-1promoterextinguishesvisibleexpressionofazyg-1promoter-driventransgene
(Figure4B)providesadditionalevidencethatEFL-1-DPL-1promotesexpressionof
centrioleduplicationgenes.ThisdirectpositiveroleforEFL-1-DPL-1inregulating
centrioleduplicationisthereforecounterintuitivewhenconsideringthatlossofdpl-1or
efl-1suppressesthezyg-1(it25)centrioleduplicationdefect.Conceptually,theonlyway
thisissuecouldbereconciledisifEFL-DPL-1independentlyfunctionstonegatively
regulatecentrioleassembly,andthatpartiallossofEFL-1-DPL-1affectsitsnegative
regulatoryrolemorethanitspositiveregulatoryrole.
EFL-DPL-1negativelyregulatesthelevelofSAS-6
19
AkeyfindingofourstudyisthatwhilelossofEFL-1-DPL-1activityresultsin
decreasedmessagelevelsofsas-6andotherduplicationfactors,italsoresultsina
significantincreaseinSAS-6proteinlevels.Thisfindingraisestwoquestions:first,how
doeslossofEFL-1-DPL-1resultinincreasedexpressionofSAS-6andsecond,howcanthe
elevatedlevelofSAS-6explaintheabilityofdpl-1orefl-1mutationstosuppressthezyg1(it25)centrioleduplicationdefect?SinceEFL-1-DPL-1seemstopositivelyregulate
transcriptionofsas-6,theincreasedlevelofSAS-6proteinindpl-1mutantscanonlybe
explainedbyanindirectmechanism.GiventhatEFL-1-DPL-1predominantlyactivates
transcriptionofgenesinthegermline(ChiandReinke2006),themostprobable
mechanisminvolvesEFL-1-DPL-1promotingtheexpressionofoneormoregenesthat
downregulateexpressionofSAS-6protein.Insuchamodel,thereductioninEFL-1-DPL-1
activitywouldaffectexpressionofthisnegativeregulatormorethantheexpressionofSAS6,thustippingthebalanceinfavorofmoreSAS-6protein.
AsourresultsindicatethatEFL-1-DPL-1setsthebalancebetweenpositiveand
negativeregulatorsofcentrioleduplication,onecouldenvisionEFL-1-DPL-1aspartofa
homeostaticcontrolmechanismthatensurestheproperlevelsofactivatorsandrepressors.
Suchamechanismwouldrequirethatthegenesencodingtheactivatorsandrepressors
varyintheirsensitivitytoEFL-1-DPL-1.ByadjustingtheactivityofEFL-1-DPL-1,thecell
couldvarytherelativelevelsofpositiveandnegativeregulatorstoensureproper
executionofcentrioleduplication.ExperimentalmanipulationofDPL-1orEFL-1levelsas
reportedintheliteraturecouldalsohavethesameeffect.Thatis,partialdepletionofEFL1-DPL-1mightresultinoverduplication—possiblybyincreasingSAS-6levels,asseenin
20
ourstudy.Incontrast,astrongorcompletelossofEFL-1-DPL-1mightleadtoablockin
duplicationasaresultofextinguishingtranscriptionofthecorecentrioleduplicationgenes.
Sowhatistheidentityofthenegativeregulator(s)whoseexpressiondependsupon
EFL-1-DPL-1function?Toaddressthisquestionwelookedatthetranscriptionaltargetsof
EFL-1-DPL-1asdeterminedbymicroarray-basedexpressionprofiling(ChiandReinke
2006)andbygenome-widepromoterbindingprofiles(Kudronetal.2013).Amongall
potentialtargetsinthelaterstudy,theAPC/Cgenesemb-27,emb-30,gfi-3/apc-5,andfzy-1
stoodoutasthemostlikelycandidates.TheAPC/CisknowntonegativelyregulateSAS-6
proteinlevelsinhumans(Strnadetal.2007).Perhapsevenmoresuggestive,weidentified
aloss-of-functionalleleoftheAPC/Cgenemat-3asasuppressorofzyg-1(it25).Our
analysishoweverindicatesthatMAT-3(andtheAPC/C)functionsindependentlyofEFL-1DPL-1toregulatecentriolebiogenesis.FuturestudieswilladdresswhethertheAPC/C
playsaroleincontrollingthelevelsofcentrioleduplicationfactorsinwormsasitdoesin
humansomaticcells.Alongtheselines,it’sinterestingtonotethatC.elegansSAS-6lacksa
KENbox,whichisthemotifinhumanSAS-6recognizedbytheAPC/Cco-activatorprotein
Cdh1.IntriguinglyC.elegansSAS-5doeshaveaKENboxandthusthemechanismofAPC/C
mediatedcontrolofcentrioleassemblyinwormsmightfunctionthroughthedown
regulationofSAS-5ratherthanSAS-6.Additionalworkwillbeneededtoidentifythe
relevanttargetsofbothEFL-1-DPL-1andtheAPC/Ccomplexinthecentrioleduplication
pathway.
Finally,howdoelevatedlevelsofSAS-6provideanexplanationforthesuppression
ofthezyg-1(it25)phenotype?Recently,ithasbeendemonstratedthatZYG-1recruitsSAS6tositesofcentrioleassemblythroughadirectphysicalinteraction(Lettmanetal.2013).
21
Thezyg-1(it25)mutationmightinterferewiththisrecruitmentasitresultsinanonsynonymouscodonchange(P442L)withinthesocalledcrypticpolo-box,adomain
requiredtotargetZYG-1tocentrioles(Shimanovskayaetal.2014).Thus,theZYG1(P442L)proteinmightbelessabundantatcentriolesthanwild-typeZYG-1,leadingtoless
effectiverecruitmentofSAS-6.IncreasingthelevelofSAS-6couldoffsetthereduced
efficiencyofthemutantZYG-1leadingtosufficientSAS-6recruitmentandsuccessful
centrioleassembly.Whilethisisthemostsimplisticinterpretationofourresults,wehave
notyetshownthattheelevatedlevelofSAS-6isresponsibleforsuppressingthecentriole
duplicationdefectofzyg-1it25)mutants.Thus,itremainspossiblethatsuppressionarising
fromlossofEFL-1-DPL-1activityinvolvesthealteredexpressionofotherrelevantfactors.
OverallourworkindicatesthatE2F-DP1playsacomplexroleinregulatingcentriole
duplicationandmayservetoestablishanequilibriumwheretherelativelevelsofpositive
andnegativeregulatorsensurethefaithfulduplicationofcentrioles.Additionalworkwill
beneededtouncoverthemolecularmechanismcontrollingSAS-6proteinlevelsaswellas
themechanismsbywhichotherEFL-1-DPL-1targetscontributetotheregulationof
centrioleduplication.
MaterialsandMethods:
Wormmaintenanceandstrains
Wormstrainswerecultivatedusingstandardpractices(Brenner1974)at20°Con
MYOBplatesseededwithOP50E.coli.Acompletelistofthestrainsusedinthisworkcan
befoundinsupplementaryTableS2.Suppressionofthezyg-1(it25)phenotypewas
22
assayedat24°C,or23.5°C,asindicated.Scatterplotsdisplayingsuppressiondatawere
generatedusingtheExceltemplatesprovidedbyWeissgerberetal.(2015)
TransgenicwormstrainsweremadeusingMos1-mediatedsinglecopyinsertion
(MosSCI)transformation(Frøkjaer-Jensen2008).pKO113,thezyg-1transcriptional
reporterconstruct,wasgeneratedusingGateway®cloningtechnology(ThermoFisher
Scientific,Inc.,WalthamMA)andcontainedthewild-typezyg-1promoter(1082
nucleotidesupstreamofthetranscriptionalstartsite),agfp-his-58fusiongene,andthe
zyg-13’UTRclonedintotheMosSCItargetingvectorpCFJ210(Frøkjær-Jensen,2012).An
identicalapproachwasusedtoconstructpKO114,exceptthattheQuikchangeIIsitedirectedmutagenesiskit(AgilentTechnologies,Inc.,SantaClara,CA)wasusedtomutate
thethreeEFL-1-DPL-1bindingsitesinthezyg-1promoterentryclonepriortoGateway
cloning.
Mutationidentification
Molecularidentificationofsuppressormutationswasaccomplishedbycombining
differentmappingstrategieswithwhole-genomesequencing.Thepreparationofgenomic
DNA,constructionofsequencinglibraries,andgenerationofsequencedatawere
essentiallyasdescribedpreviously(Wangetal.2014).Variantswereidentifiedusinga
pipelineofBFAST(Homeretal.2009),SAMTools(Lietal.2009)andANNOVAR(Wanget
al.2010).MappingplotsweregeneratedusingR[RCoreDevelopmentTeam,2015].
Candidatesuppressoralleleswerelimitedtohomozygous(minimumthreeindependent
reads,≥85%variantcall),nonsynonymousmutations,andfilteredtoremovevariants
23
commontothestrainbackground.
Fordpl-1(bs21),thesuppressormutationwasmappedbyclassicalthree-factor
mappingbetweendpy-10andunc-4onlinkagegroupII(Kempetal.2007).Thestrain
containingbs21(OC204)wassequenced,andcandidatesuppressorsinthedpy-10-unc-4
intervalwereidentified.Format-3(bs29),theoriginalmappositiononlinkagegroupII
(Kempetal.2007)wasfoundtobeincorrect(datanotshown).Thepositionofthis
suppressorwasreinvestigated,andthree-factormappingwithunc-45anddpy-1revealed
thatbs29wastightlylinkedtounc-45ontheleftarmofchromosomeIII.Thestrain
containingbs29(OC184)wassequencedtoidentifycandidatesuppressorsinthevicinityof
unc-45.Forefl-1(bs22),avariationoftheone-stepmethodforsimultaneousmappingand
sequencingwasemployed(Doitsidouetal.2010).Thebs22suppressormutationwas
introgressedintotheHawaiianCB4856backgroundbybackcrossingtensuccessivetimes
toaHawaiian-introgressedzyg-1(it25)strain.MappingplotsofHawaiianSNPsacrossthe
genomerevealed(inadditiontotheintervalflankingzyg-1onchromosomeII)gapson
chromosomeI(between2.0-3.0Mb)andchromosomeV(from15.0Mbtotherightend).
ThechromosomeIintervalencompassesaknownlocusofgeneticincompatibility(zeel1/peel-1at2.35Mb)betweentheN2wildtypeandHawaiianstrainbackgrounds(Seidelet
al.2008)andwasnotpursuedfurther.Candidatesuppressorswereidentifiedinthe
chromosomeVinterval.
Genomeediting
24
Forgenomeediting,weutilizedco-CRISPRtechnologyessentiallyasdescribed
(Arribereetal.2014).Specifically,wedesignedguideRNAs(gRNAs)usingtheCRISPR
Designtoolathttp://crispr.mit.edu.gRNAsequenceswereinsertedintotheexpression
plasmidpDD162(Dickinson2013)usingtheQ5SiteDirectedMutagenesisKit(New
EnglandBiolabs,Inc.,Ipswich,MA).AlloligosusedcanbefoundinTableS3.Allconstructs
weresequenceverified.Forrepairtemplates,weusedsinglestrandedoligomers(Paixetal.
2014)synthesizedbyIntegratedDNATechnologies,Inc.(Coralville,IA).Formicroinjection,
wepreparedamixtureoftwoefl-1gRNAexpressionplasmidsat50ng/mleach,purified
efl-1repairtemplateat30ng/ml,thedpy-10co-conversiongRNAexpressionplasmidat50
ng/ml,andthedpy-10repairtemplateat20ng/ml.
Afterinjection,P0hermaphroditesweretransferredtoindividualMYOBplatesat
20°CandallowtoproduceanF1generation.F1progenyexhibitingaRolorDpyphenotype
werepickedindividuallytoMYOBplatesandallowedtolayF3eggs.F2adultswerethen
pickindividuallytoaPCRtube,lysed,thenscreenedbyPCRfortheloss(wt>mut)orgain
(mut>wt)ofanNlaIVrestrictionsiteaffectedbythebs22mutation(underlinedresiduesin
theefl-1gRNAsequencesinTableS3).
RNAi
RNAiexperimentswerecarriedoutbyfeedingwormsE.coliexpressinginducible
dsRNAconstructsaspreviouslydescribed(Kamathetal.2003).L4larvaewereseeded
ontofreshRNAiplatesandtheeffectsofRNAiweremonitored12-24hoursfromtheL4
stage.TheL4440vector(SourceBioscience,Nottingham,UK)expressingdsRNAagainst
smd-1genewasusedasanegativecontrolforallRNAiexperiments.
25
AntibodiesandQuantitativeImmunoblotting
Embryoswereisolatedbywashingwormsofffour10-cmplatesandsuspending
theminahypochloritesolution(1.65%hypochlorite,1NNaOH)for~5minutes.Once
adultwormsweredissolved,embryoswererinsed3timesusingM9buffer,suspendedin
~50μl2xLDSSamplebuffer(LifeTechnologies,Inc.,Carlsbad,CA)andheatedto95°Cfor
5minutes.SampleswereresolvedonNuPageBis-Trisgels(LifeTechnologies,Inc.,
Carlsbad,CA)andtransferredtonitrocelluloseusingthei-Blottransfersystem
(ThermoFisherScientific,Waltham,MA).Blotswereprobedandanalyzedusingthe
OdysseyInfra-redImagingSystem(LI-CORBiosciences,Inc.,Lincoln,NE)aspreviously
described(Songetal.2011).
Thefollowingantibodieswereusedatadilutionof1:500–1:2000:DM1A,analpha-
tubulinspecificantibody(Sigma),α-SPD-2(Kempetal.2004),α-ZYG-1(Kempetal.2007),
αSAS-4(Songetal.2008).TheSAS-6antibodyisapolyclonalantibodyraisedinguinea
pigstoafull-lengthGlutathione-S-Transferase-SAS-6fusionprotein.Theantibodywas
producedbyPoconoRabbitFarmandLaboratory,Inc.(Canadensis,PA),andaffinity
purifiedagainstafull-lengthMaltose-BindingProtein-SAS-6fusionprotein.Antibodytoa
SAS-5derivedpeptide(N-CPAERERRIREKYARRK-C)wasraisedinrabbitsandaffinitypurifiedbyYenZymAntibodiesLLC,(SanFrancisco,CA)IRDyesecondaryantibodies(LICORBiosciences)wereusedat1:15,000andmembraneswereimagedusingtheOdyssey
InfraredImagingSystem(LI-CORBiosciences).Bandswerenormalizedtoanalpha-tubulin
loadingcontrolandquantitatedusingImage-Jsoftware(NIH,Bethesda,MD).Graphs
depicttheaveragenormalizedproteinlevelsfrom3independentexperiments.
26
qRT-PCR
ToisolateRNA,L4wormsweretransferredto25°Covernight.Approximately50-
100adultwormsweresuspendedin200μlM9bufferandRNAwasisolatedusingthe
ArcturusPicoPure™RNAIsolationKit(ThermoFisherScientific,Inc.,Waltham,MA),
accordingtomanufacturer’sinstructions.RNAwastreatedwithDNAaseandthendiluted
to20ng/μlusingRNAase-freeddH2O.qRT-PCRreactionsweresetupintriplicateusing
20ngoftemplateRNA,QuantiFast®SYBR®GreenRT-PCRKitfromQiagen(Valencia,CA)
and10μMprimers(seeTableS4forprimersequences).Anegativecontrollackingreverse
transcriptasewassetupforeachRNAtemplateandanon-templatecontrolwassetupfor
eachprimerset.QuantitativeRT-PCRwasperformedonaCFX96Touch™Real-TimePCR
DetectionSystem(Bio-RadLaboratories,Inc.,Hercules,CA).Amplificationreactionswere
carriedoutusingthefollowingprogram:10minutesat50°C,5minutesat95°Candthen40
cycles[10secondsat95°Cand30secondsat55°C].Ameltingcurvewasdeterminedatthe
endofeachPCRruntoverifytheformationofasingleamplicon.AverageCtvalueswere
determinedusingCFXManager3.0Softwareandthefoldchangewascalculatedusingthe
2ΔΔCtmethod.Foreachprimerset,theCtvalueswerenormalizedtotba-1RNAlevelsand
thencomparedtoawild-typecalibratorsample.TheerrorbarsindicatetheSEMofthe
triplicateset.
MicroscopyandLiveCellImaging
Centrioleduplicationwasmonitoredusing4Dtime-lapsemicroscopyonaNikon
spinningdiscconfocalmicroscopeaspreviouslydescribed(Petersetal.2010).Imagesfor
27
comparingtheexpressionofthezyg-1transcriptionalreporterswastakenonthesameday
usingthesamecamerasettings.
Acknowledgements:
WethankValerieReinkeforgenerouslyprovidingtheDPL-1-GFP-expressingC.
elegansstrainandAndyGoldenforsomeoftheCRISPRreagents.WealsothankGeraldine
SeydouxandAlexPaixforsharingprotocolsforCRISPRtechnology.Somestrainswere
providedbytheCGC,whichisfundedbyNIHOfficeofResearchInfrastructurePrograms
(P40OD010440).ThisworkwassupportedbytheIntramuralResearchProgramofthe
NationalInstitutesofHealthandtheNationalInstituteofDiabetesandDigestiveand
KidneyDiseases.
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Figure1.szy-10(bs21)suppressesembryoniclethalityandcentrioleduplicationdefectsof
thezyg-1(it25)mutant.(A)Inwild-typeembryos,sperm-derivedcentriolesduplicate
duringthefirstcellcycleresultingintheformationofbipolarspindlesatthetwo-cellstage.
Inzyg-1(it25)mutants,thesperm-derivedcentriolesfailtoduplicate,leadingtomonopolar
spindlesatthetwo-cellstage.(B)zyg-1(it25)orzyg-1(it25)szy-10(bs21)mutantswere
shiftedtothenon-permissivetemperature(24Cor25C)attheL4larvalstageand
embryoniclethalityoftheirself-progenywasquantified.(C)zyg-1(it25);mcherry-his58;
gfp-spd-2orzyg-1(it25)szy-10(bs21);mcherry-his58;gfp-spd-2animalswereshiftedtothe
non-permissivetemperatureof24CattheL4larvalstageandallowedtogrowfor24hours.
Centrioleduplicationintheirembryoswasmonitoredusingtime-lapsemicroscopy.
35
Representativeimagesofcontrolzyg-1(it25)orzyg-1(it25)szy-10(bs21)embryosatthe
two-cellstageareshown.(D)Quantificationofcentrioleduplicationinzyg-1(it25);
mcherry-his58;gfp-spd-2orzyg-1(it25)szy-10(bs21);mcherry-his58;gfp-spd-2reporter
strains.
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Figure2.Thebs21mutationisanalleleofthedpl-1gene.(A)Schematicofrecombination
mappingoftheszy-10(bs21)mutation.(B)Embryoniclethalityofthezyg-1(it25)dpy10(e128)orrecombinantscontainingmutationsinoneormoreoftheszy-10candidate
geneswasquantifiedatthenon-permissivetemperatureof24C.(C)Agfp-dpl-1transgene
complementsdpl-1(bs21)-mediatedsuppressionofthezyg-1(it25)embryoniclethal
phenotype.Thegraphplotstheembryoniclethalityat24°Cofzyg-1(it25)controlanimals
37
andzyg-1(it25)dpl-1(bs21)animalswithandwithoutadpl-1-gfptransgene.(D)zyg1(it25)dpl-1(bs21);dpl-1-gfpanimalsweretreatedwithcontrolorgfpRNAiandembryonic
lethalitywastestedat24°C.(E)zyg-1(it25);mcherry-his58;gfp-spd-2L4animalswere
treatedwithcontrolRNAiordpl-1RNAifor24hoursandthefrequencyofcentriole
duplicationwasmonitoredbytime-lapsemicroscopy.Representativeimagesoftwo-cell
embryostreatedwithcontrolRNAi(left)ordpl-1RNAi(right)areshown.(F)
Quantificationofcentrioleduplicationeventsinzyg-1(it25);mcherry-his58;gfp-spd-2
animalstreatedwithcontrolordpl-1RNAi.
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Figure3.Mutationofefl-1suppresseszyg-1(it25)embryoniclethality.(A)zyg-1(it25),zyg1(it25)efl-1(se1),andefl-1(se1)wereshiftedto25°Casgravidadults.Shownarethelevels
ofembryoniclethalityfortheensuing24hours.(B)szy-11(bs22)wasmappedinthe
vicinityofunc-76ontherightarmofLGV.Shownaretheidentitiesandmappositionsof
linkedmutationsresultinginnon-synonymouscodonchanges.(C)szy-11(bs22)-mediated
suppressionofzyg-1(it25)requirestheefl-1mutation.Theefl-1mutationwasrevertedto
thewild-typesequenceinthezyg-1(it25);szy-11(bs22)strainusingCRISPRtechnologyand
suppressionofzyg-1(it25)embryoniclethalitywasquantitated.Additionally,theefl-1
mutation(G716A)wasintroducedintothezyg-1(it25)mutantandsuppressionof
39
embryoniclethalitywasmeasuredat24°C.(D)Quantitationofembryoniclethalityofzyg1(it25)orzyg-1(it25);CRISPRefl-1(G716A)at23.5°C.
40
Figure4.RegulationoftranscriptionofcentrioleduplicationfactorsbyEFL-1-DPL-1.(A)
Schematicofthewild-typezyg-1transcriptionalreporter(top)andthecorresponding
versionwiththethreeEFL-1-DPL-1-bindingsitesmutated(bottom).MutationsintheEFL1-DPL-1-bindingsitesarepicturedintheboxatright.(B)Representativeimagesofthe
germlinesoftransgenicanimalsexpressingthewild-typezyg-1transcriptionalreporter
(top)oranimalsexpressingthetriplemutantbindingsitereporter(bottom).(C)Relative
levelsofcentrioleduplicationtranscriptsinwild-typeanddpl-1mutantanimals.RNAwas
isolatedfromwild-type,dpl-1(n3643),ordpl-1(bs21)adultsandquantitativereal-timePCR
41
wasusedtoanalyzeRNAlevels.Valuessignificantlydifferentfromcontrolswere
determinedusingaStudent’st-testandareindicatedwithone(p<0.05)ortwo(p<0.01)
asterisks.
42
43
Figure5.SAS-6levelsareincreasedindpl-1mutants.(A-D)Immunoblotanalysisof
extractsmadefromwild-type,dpl-1(bs21),ordpl-1(n3643)embryos.Shownare
representativeblotsprobedfor(A)ZYG-1.(B)SAS-6(C)SAS-5,and(D)SPD-2.Ineach
case,alpha-tubulinwasusedasaloadingcontrol.ThespecificitiesoftheZYG-1,SAS-5,and
SPD-2antibodiesaredemonstratedbytheabsenceofabandinextractsdepletedofthat
specificfactorbyRNAi.ThespecificityoftheSAS-6antibodyisshowninFigure6.
Asterisksdenotenon-specificbands.(E)Relativelevelsofeachfactorinthewildtypeand
thetwodpl-1mutants.Signalswerenormalizedtotheloadingcontrolandplottedrelative
tothewildtype.Eachmeasurementisfromtwoormoreindependentexperiments.
44
Figure6.LossofAPC/Cactivitysuppresseszyg-1(it25).(A)szy-13(bs29)wasmapped
relativetounc-45anddpy-1ontheleftarmofLGIII(mapattop).Wholegenome
sequencingofthisstrainrevealedjusttwogenesinthevicinitywithnon-synonymous
codonchanges(tableatbottom).(B)Themolecularidentityofszy-13(bs29)wasconfirmed
throughcomplementationanalysis.Suppressionofzyg-1(it25)embryoniclethalitywas
measuredinszy-13(bs29)homozygotes;szy-13(bs29)heterozygotes,orinszy
45
13(bs29)/mat-3(or180)trans-heterozygotesat24°C.(C)Immunoblotanalysisof
embryonicextractsobtainedfromzyg-1(it25),orzyg-1(it25);szy-13(bs29)mutantsusinga
SAS-6-specificantibody.Alpha-tubulin(TBA-1/2)wasusedasaloadingcontrol.SAS-6
signalwasnormalizedforloadingandrelativelevelswerequantitated.Asteriskdenotesa
non-specificband.
46