Paro, 2004).Inmouseembryos,defectsin metazoan development (FrancisandKingston,2001;Ringrose required forepigeneticrepressionofHoxgenetranscriptionduring *Author forcorrespondence (e-mail:[email protected]) Chapel Hill,NorthCarolina 27599,USA. lysine 119ofhistoneH2A(H2A-K119)(deNapolesetal.,2004; mediated chromatinmodificationderived fromubiquitylationat 1999; Levine etal.,2002;Suzuki2002).EvidenceforPRC1- Hashimoto etal.,1998;Hemenway etal.,1998;Satijnand Otte, Gunster etal.,1997;SatijnSchoorlemmer Genome Informatics)(Alkema etal.,1997a;Alkema etal.,1997b; –Mouse (RNF2 –MouseGenomeInformatics)/RING1B (RING1 –MouseGenomeInformatics)/MPH2,andRING1A (PHC1 –MouseGenomeInformatics),RAE28 (PCSK1 Informatics)/PC3 –MouseGenome (PCSK2 Mouse GenomeInformatics)/PC2 – –MouseGenomeInformatics),M33(CBX2 (PCGF2 MEL18 complex 1(PRC1)involves theparalogousPcGproteinsBMI1/ mammals (OtteandKwaks, 2003).ThePolycombrepressive function inchromatinremodelinghave beenidentifiedin posterior homeotictransformationofthecorrespondingvertebrae. anterior somitestoward amoreposteriorfate andmanifestsas al., 2003;Isonoet2005).Thisalterstheidentityofaffected 1998; delMarLorenteetal.,2000;Suzuki2002;Voncken et 1996; Coréetal.,1997;Takihara etal.,1997;Katoh-Fukui Akasaka etal.,1996;Schumachervan derLugtetal., to thewild-typeHoxexpression boundary(van derLugtetal.,1994; repression ofHoxgenetranscriptioninindividual somitesanterior Originally discovered in INTRODUCTION KEY WO regulation ofvertebralidentity. and geneticelementscooperativelyrecruitretainjuxtaposedPolycombcomplexesinmammalianHoxgeneclusters towardco- K27, weredetectedinupstreamregulatoryregionsof andBMI1complexes,alongwithYY1methylatedH3- during themaintenancephaseofHoxgenerepression.JuxtaposedEED complex 27 ofhistoneH3(H3-K27),amediatorPolycombcomplexrecruitmenttotargetgenes,stablyassociatedwiththeEED EzH2 andRing1B,respectively, inmid-gestationembryos,YY1engagedbothPolycombcomplexes.Strikingly, methylatedlysine andBMI1formedseparatebiochemicalentitieswith pathways, whichconvergeatthelevelofHoxgeneregulation.WhereasEED homeotic phenotypesandco-repressionofasimilarsetHoxgenes,geneticanalysisimplicated complexes, intheregulationofvertebralidentityduringmousedevelopment.Despiteasignificantoverlapdosage-sensitive interrogated thegenetic,biochemicalandmolecularinteractionofBMI1EED,pivotalconstituentsheterologousPolycomb composition andfunctionexhibitcooperativeinteractionorhierarchicalinterdependencyattargetloci.Thepresentstudy means ofpost-translationalmodificationhistonetails.Dependingonthecellularcontext,Polycombcomplexesdiverse Best knownasepigeneticrepressorsofdevelopmentalHoxgenetranscription,Polycombcomplexesalterchromatinstructureby Se Young Kim identity Juxtaposed Polycombcomplexesco-regulatevertebral Development 133,4957-4968(2006)doi:10.1242/dev.02677 Accepted 3October 2006 1 Houston, Texas 77030,USA. Department ofMolecularandHumanGenetics, BaylorCollegeofMedicine, At leasttwo PcGcomplexes withdiverse compositionand RDS: Polycomb, 1 , SuzanneW. Paylor 2 Department ofGenetics,UniversityNorth Carolina, eed, Bmi1 , Hoxgenes,Mousedevelopment,Chromatin, Histones,Epigenetics , Polycomb(PcG)proteinsare 1 PcG , Terry Magnuson function causede- Hoxc8 and 2 and ArminSchumacher Hoxa5 this epigeneticmark andrepressionofHoxgenes (Caoetal.,2002; histone H3-K27inthevicinity of PREs,resultinginPcbindingto et al.,2001).Furthermore,EZH2, inacomplex withEsc,methylated complex andPRC1inpreblastodermembryos (Poux ESC/EZ/PHO complexes derived fromthetransientassociation of the adjacent somitesandvertebrae, respectively (Beletal.,1998). expression andposteriorhomeotictransformationsacrossmultiple homologs, oftheirmurine synergistic interactionbetween mutantalleles compared withthesinglemutants(Campbelletal.,1995).Likewise, mutant allelessignificantlyenhancedthehomeoticphenotypes but notall,pairwisecombinationsof similar elementsawait characterizationinmammals. the functionofseveral PREshasbeendelineatedin mediated repressionoftarget (Pirrottaetal.,2003).Whereas hundred basepairsandarenecessarysufficient forPcG- Polycomb responseelements(PREs),whichencompassseveral Kuzmichev etal.,2005).PcGcomplexes bindto K27 orH1-K26(Kuzmichev etal.,2002;Kuzmichev etal.,2004; toH3- targeting thehistonemethyltransferaseactivity ofEZH2 differentially engageinPRC2-relatedcomplexes (PRC2/3/4), generated byalternatetranslationstartsiteusageof isoforms, al., 2002;Kuzmichev etal.,2004).Several EED 1998; van derVlagandOtte,1999;Caoetal.,2002;Kuzmichev et (Denisenko etal.,1998;Sewalt etal.,1998;van Lohuizenetal., RBAP46/RBAP48, andthehistonedeacetylaseHDAC1 the zincfingerproteinSUZ12,histone-bindingproteins PRC2, encompassesEED,thehistonemethyltransferaseEZH2, Wang, H.etal.,2004;Cao2005).AsecondPcGcomplex, Drosophila 1995; Bajuszetal.,2001).For example, Psc andPcengagein 1991; Paro andHogness,1991;Chengetal.,1994;Campbell 1985; Kennison andTamkun, 1988;Adleretal.,1989; Molecular supportforcooperative interactionbetweenPcG Studies in . Thecombineddatasuggestamodelwhereinepigenetic Bmi1 PRC1 (Shaoetal.,1999),andintercrossesbetween Drosophila and M33 revealed stronggeneticinteractioninmany, 1, * was evident fromectopicHoxgene , eed and RESEARCH ARTICLE PcG Bmi1 mutant alleles(Jürgens, in parallel Drosophila eed cis mRNA, -acting 4957 ,

DEVELOPMENT et al.,1994;van derLugtetal.,1996). homeotic transformationsalongthevertebral column(van derLugt expression andhighlypenetrant,dosage-sensitive posterior analysis implicated phenotypes andco-regulation ofHoxgenes,thepresentgenetic al., 1999;Richieet2002). hematopoietic defectsinthebonemarrow andthymus(Lessardet (Mager etal.,2003).Furthermore, Magnuson, 2006;Kalantryetal.,2006)andgenomicimprinting et al.,2002;Plath2003;Silva etal.,2003;Kalantryand and imprintedXchromosomeinactivation (Wang etal.,2001;Mak includedtheregulation ofrandom developmental functionsofEED (Schumacher etal.,1996;Wang etal.,2002).Additional along thevertebral column,aswellectopicHoxgeneexpression penetrant, dosage-sensitive posteriorhomeotic transformations acid proteinwithfive WDmotifsandislikely toadoptatoroidal al., 1999). hematopoietic abnormalities(van derLugtetal.,1994;Lessard between birthand20weeksofagedisplayedneurological of-function alleleof Napoles etal.,2004;Wang, H.etal.,2004;Cao2005).Aloss- RING1A, BMI1isrequiredforubiquitylationofH2A-K119(de Gunster etal.,1997;Satijn1997).Inconjunctionwith RAE28, RING1A,RING1BandM33(Alkema etal.,1997a; a 324aminoacidringfingerprotein,whichinteractsdirectlywith embryo. PRC2/3/4, respectively, inaxialHoxgenerepressionthemouse andEED,pivotal constituentsofPRC1 and interplay ofBMI1 remains elusive. between PRC1andPRC2/3/4intheregulation ofvertebral identity reported atthemolecularlevel. Thus,thefunctionalrelationship transient interactionbetweenthecorePcGcomplexes hasnotbeen a cell-cycle dependentmanner(Hernandez-Munozetal.,2005), withcomponentsofPRC2/3/4in exhibited co-localizationofBMI1 PRC2/3/4-methylated H3-K27.Althoughmammaliancelllines hierarchical recruitmentofPRC1totarget genesuponbindingto Fujimura etal.,2006),theseresultssupportedamodelof embryonic stemcells(Kuzmichev etal.,2002;Cao2005; conjunction withsimilarfindingsinmammaliancelllinesand Czermin etal.,2002;MüllerWang, L.etal.,2004).In 4958 (Montgomery etal.,2005).Homozygosityforthe defects in PRC2/3/4 functionwas evident fromglobal H3-K27methylation in al., 1998;van derVlagandOtte,1999).Apivotal roleforEED (Denisenko etal.,1998;Sewalt etal.,1998;van Lohuizenet EZH2 hypomorphic allele,disruptedEEDinteractionwithHDACs and represented bythe essential forproteininteraction,andtwo pointmutations, et al.,1998;Kuzmichev etal.,2004).Integrity oftheWDmotifsis Bomsztyk, 1997;Ngetal.,Schumacher1998;Sewalt propeller structure(Schumacheretal.,1996;Denisenko and l7Rn5 1995; Faust etal.,1998).Bycontrast,animalshomozygousforthe patterning defectsoftheprimitive streakatgastrulation(Faust etal., allele causedembryoniclethalityandanteroposterior(AP) but not BMI1,associatedwithmethylated H3-K27.Thecombined While bothcomplexes containYY1asaDNA-binding factor, EED, engaged inseparate,but juxtaposedcomplexes atHoxtarget loci. andBMI1 converged atthelevel ofHoxgeneregulation. EED Surprisingly, despiteasignificantoverlap inhomeotic eed The presentstudyinterrogatedthegeneticandmolecular 1989SB (embryonic ectodermdevelopment) encodesa535amino RESEARCH ARTICLE Bmi1 eed hypomorphic allelewereviableandmanifestedhighly (B cell-specificMo-MLV integration site1)encodes mutant embryonicandtrophoblaststemcells l7Rn5 eed Bmi1 and 3345SB Bmi1 demonstrated ectopicHoxgene null alleleandthe eed in parallelpathways, which Bmi1 mutant animalsexhibited -deficient micedied l7Rn5 l7Rn5 3345SB 1989SB null - YY1 antibody. Following incubationwith appropriatehorseradish 4°C with Laboratories) for2hoursatroomtemperature andincubatedovernight at 7.6, 137mmol/lNaCl,0.1%Tween-20) orReliaBlotreagent(Bethyl wereblocked with5%BSAinTBST(20mmol/lTris, pH Membranes PVDFmembrane(BioRad) forwesternblotanalysis. onto Elutionswereseparatedon SDS-PAGE gelsandtransferred beads. (Oncogene).Asacontrol,lysates wereincubatedwithfree beads withan or [ProFound MammalianCo-Immunoprecipitation Kit(Pierce)] beads previously (van derLugtetal.,1994). alkaline digestioninpotassiumhydroxidefor7-10daysasdescribed Blue 8GX(Sigma)and0.005%AlizarinRedSclearedby Whole-mount preparationsofP0skeletons werestainedwith0.015%Alcian Skeletal preparations l7Rn5 Mouse strainsandgenotyping MATERIALS ANDMETHODS Hox geneclusters. model ofPcGcomplex recruitmentandretentioninmammalian genetic, biochemicalandmolecularresultsformthebasisfora overnight withan (Roche)].Two milligramsofproteinlysatewereincubated Inhibitor 1 mmol/lEGTA, 1%Triton X-100 withCompleteMiniProtease lysis buffer [20mmol/lTris pH7.5,150mmol/lNaCl,1EDTA, removed. Theremainingtrunkswerepooled( E12.5 embryosweredissectedandeviscerated andlimbsheadswere Immunoprecipitation blotanalysis andwestern Hoxa5 generated fromplasmidsdescribedpreviously: reported (Wilkinson andNieto,1993).AntisensecRNA probeswere enhance signalintensity. extended upto4weekswithweeklychangesofthesubstratesolution Depending ontheexpression level, thealkalinephosphatasereactionwas a standardprotocolwithminormodifications(Neubüseretal.,1995). parasagittal 7 described recently(Moketal.,2004).mRNA insituhybridizationto on parasagittal5 Immunohistochemistry andstreptavidin-biotin immunoperoxidasedetection Immunohistochemistry andmRNAinsituhybridization l7Rn5 et al.,1996;RinchikandCarpenter, 1999).TheT transversion inthe respectively, generatedby vaginal plugwas consideredE0.5. 8.5 or12.5fromtimedmatings.Noonofthedayappearance the developmental studies,embryoswererecovered atembryonicday(E) 1994). AllmicewereonapartiallycongenicFVB/NJbackground(N5).For by genetargeting anddetectedbyPCRasreported(van derLugtetal., et al.,1992), CAC (reverse) createdadiagnostic GGCCATGGAAATGTTA (forward) and5 (Schumacher etal.,1996).PCRamplificationwithprimers5 were employed. Sense to nucleotides1529-2270ofGenBankAccessionnumberXM130022) Accession numberNM010466)anda741bp Hoxc8 1988) and mount preparationsofembryosundercoverslips. generally reachedcompletionwithin3days.Imageswerecapturedfrom flat- (Wilkinson andNieto,1993).Thealkalinephosphatasedetectionreactions controls. Digoxigenin-11-UTP-labeled antisensecRNA probeswerepreparedas Whole-mount insituhybridizationwas performedasdescribed 3345SB 3345SB (Colberg-Poley etal.,1985), probe (correspondingtonucleotides274-682ofGenBank Hoxd4 -EED, and allele disruptedadiagnostic Hoxb4 -Bmi1 antibodypreincubatedwithproteinG/Aagarose m sectionsfromE12.5embryoswas conducted accordingto l7Rn5 l7Rn5 (Gaunt etal.,1989).Inaddition,a408basepair(bp) m sectionsfromE12.5embryoswereperformedas (Ramirez-Solis etal.,1993), -BMI1, -Eed or 1989SB 1989SB eed N -ethyl- and represent allele. The -RING1B, -Bmi1 antibodycovalently coupledto Bmi1 N Mse -nitrosourea mutagenesis(Schumacher Hoxa7 eed I siteinthecontext oftheT cRNA probesserved asnegative Bmi1 Alu (Yu etal.,1995), null andhypomorphicalleles, -EZH2, -CTATGCATTCTCAGAA- Bmi1 null mutationwas generated I siteusedforgenotyping Development 133(24) n eed Hoxb6 =2-3) andsonicatedin 1040 probe (corresponding (Faust etal.,1995), r -H3M2K27, or C transitioninthe (Schughart etal., Hoxb3 1031 -GTT- (Sham r A -

DEVELOPMENT The following antibodieswere employed: Antibodies 5 TCG (reverse). 5 TGGGAGA (forward) and5 expression boundariesof regions, trunksweredissectedfurtherandcutrostraltotheanterior andBMI1bindingtoHoxregulatory and heads.For spatialanalysisofEED Kit (Upstate).Inputconsistedofeviscerated E12.5embryoswithoutlimbs Chromatin immunoprecipitation(ChIP)was performedwiththeChIPAssay Chromatin immunoprecipitation Biotechnology). weredetectedbychemiluminescence(ECLreagent,SantaCruz peroxidase-conjugated secondaryantibodies,(co-)immunoprecipitated Polycomb complexesfunctioninparallelpathways ACTTTCGGAACGG (reverse). et al.(Mokal.,2006). carboxy terminus,custom-generatedbyBethylLaboratories;seealsoMok and (rabbit polyclonal,Abcam;rabbitSantaCruzBiotechnology), polyclonal, Upstate), Abcam; rabbitpolyclonal,BethylLaboratories), polyclonal, Abcam), RING1B (mousemonoclonal,kindlyprovided byHaruhiko Koseki; rabbit polyclonal, Abcam;goatSantaCruzBiotechnology), custom-generated byBethylLaboratories;mousemonoclonal,Upstate;goat residues123-140,custom-generatedbyBethylLaboratories), against EED primers 5 (reverse). Thepromoter region of AACTCTCACCTC (forward) and5 amplification ofHoxpromotersequences. Phenol:chloroform-extracted chromatinwas employed forPCR agarose. Crosslinkingwas reversed withsodiumchloride at65°Covernight. complex was collectedwithsalmonspermDNA/ A(orproteinG) overnight withapproximately5 salmon spermDNA/protein A(orproteinG)agarosebeads,andincubated between 200and1000bp.Sonicatedsupernatantswerepreclearedwith crosslinking, tissueswerewashed andsonicatedtosheartheDNA tolengths were incubatedin1%formaldehydefor10minutesat37°C.Following three entiretrunks,orpoolsoffive totenanteriorandposteriorfragments -BMI1 antibody(rabbitpolyclonal,raisedagainstBMI1residues229-314, -TGGGAGAACCTGAGAGCAAAG (reverse), 5 -ACCCAACTCCCCCATTAGTG and5 -FPN1 (rabbitpolyclonal,raisedagainstresidues553-568attheFPN1 -ACAAGGGCGGAGGCTATTC (forward) and5 Hoxc8 : 5 EH (rabbitpolyclonal,Upstate;rabbit -EZH2 -H3M3K27 (rabbitpolyclonal,Upstate), -TGAGCACAATAGCCCACACG (forward) and Hoxa5 -GGAATCATTTCGTGGCTGTG (reverse), (pv3) or - g antibody. Thechromatin-antibody -GCTCTCTGCTCACTGTCGGTAG served asanegative controlusing Hoxa5: Hoxc8 ED(rabbitpolyclonal,raised -EED -TGTGCTTGATTTGTGGC- 5 (pv12). Poolsoftwo to -GCCCTGACTTACT- -CGTAACCGTCAG- -H3M2K27 (rabbit -AGGCA- -YY1 - (Richie etal.,2002). function incarcinogen-inducedT-cell lymphomadevelopment the homeoticpathways, whichcontrastedwithitsdominant-negative l7Rn5 caused lethalityatgastrulation(Faust etal.,1995), whereas offspring (datanotshown). segregated withtheexpected Mendelianratioandresultedinviable died invariably duringthefirst24hours.Allothergenotypes double homozygotessurvived toterm,but theseverely runtedpups l7Rn5 neuroectoderm exhibited coexpression of wild-type embryos(Fig.1A,E).Likewise, atE12.5,prevertebrae and Coexpression of RESULTS Hox geneexpression in difference inthepenetranceofhomeotictransformationsandectopic results alsosuggestedhaploinsufficiency ofthe encompassed the 1996). Consequently, resultsfrom genotypes forthisstudy. Homozygosityforthe of doubleheterozygotes,yieldedthevarious doublemutant 1B,F). Controlexperiments with coexpression of Peytavi etal.,1999).mRNA insituhybridizationstudiesdetected Rietzler etal.,1998;SchumacherSewalt etal.,1998; and Bomsztyk,1997;Gunsteretal.,Denisenko etal.,1998; Bmi1 and celllines,indicatedwidespreadmRNA expression of Previous reports,mostlybasedonnorthernanalysis ofadulttissues neuroectoderm effects, datafor homozygosity (datanotshown). Intheabsenceofallele-specific Crosses between eed protein expression inthenucleiofE12.5prevertebrae (Fig.1D,H). andBMI1 Immunohistochemistry confirmedEED revealed nosignalonsectionedE12.5embryos(Fig.1C,G). l7Rn5 1989SB 1989SB (Schumacher etal.,1996;Alkema etal.,1997a;Denisenko Bmi1 3345SB /+ homozygotes wereviableandfertile(Schumacheretal., , with double mutantmice or eed l7Rn5 l7Rn5 l7Rn5 Bmi1 and eed 3345SB eed 1989SB 1989SB Bmi1 heterozygotes, followed byintersecrosses heterozygotes, i.e. and and images were captured at20 50 BMI1 inE12.5prevertebrae ( detected nuclearexpression ofEEDand embryos ( specific signalonsectionedE12.5 and A,B,E,F). Control hybridizationwith well asneuroectoderm (arrows in and prevertebrae (arrowheads inB,F),as mRNA insomites(arrowheads inA,E) Note coexpression of and mRNA insituhybridizationwith and sectionedE12.5embryos( images offlat-mountE8.5embryos( in wild-typeembryos. i.1 Coexpression of Fig. 1. trans heterozygotes presentednosignificant allele. Twenty percentofthe in somitesandneuroectodermE8.5 l7Rn5 Bmi1 Bmi1 (A,E), and1000 Bmi1 eed with RESEARCH ARTICLE eed C sense cRNAprobes revealed no antisense cRNAprobes (E,F). homozygotes atE12.5andP0 1989SB , G in somitesand eed ). Immunohistochemistry Bmi1 and were combined.These and Bmi1 l7Rn5 heterozygosity or eed Bmi1 l7Rn5 l7Rn5 (D,H). Representative eed and 1989SB sense probes mRNA (Fig. D 3345SB 3345SB and , Bmi1 (B,C,F,G), B H eed , eed ). The F allele in eed ) Bmi1 ; /+ allele 4959 eed Bmi1 (A,B) A and , E or )

DEVELOPMENT allelic modifier. Synergistic interactionbetween phenotypic penetranceandexpressivity inthepresenceofanon- reflect acommon,sensitizedpathway(s) susceptibletochangesin single mutants(van derLugtetal.,1994;Schumacher etal.,1996) this hinderedtheevaluation ofpotentialsupra-additive increases Cao etal.,2005), Kuzmichev etal.,2002;MüllerWang, L.etal.,2004; methylation ofH3-K27(Caoetal.,2002;Czermin of soledependencePRC1recruitmentonPRC2/3/4-mediated to transformations, aswellexpression ofnovel phenotypes,similar manifest assupra-additive increasesinthepenetranceofaxial between means forquantitative characterizationofthefunctionalrelationship Analysis ofwhole-mountskeletal preparationsprovided apowerful regulation ofvertebralidentity eed 4960 overlapping, dosage-sensitive transformationsin 100% in 54% indoubleheterozygotes. example, theL6 additive effect ofthesingleheterozygousphenotypes(Fig.2A).For comprising allninegenotypesobtainedfrom single mutantphenotype. eed transformations in homozygotes (Fig.2A).Strikingly, thepenetranceofskeletal entire APaxis,whichrangedbetween70and100%in exhibited dosage-sensitive, posterior transformationsalongthe der Lugtetal.,1994;Schumacher1996),mutantskeletons heterozygous intercrosses.Inagreementwithprevious studies(van Bmi1 The penetranceofallhomeotictransformationsapproached Fig. 2Asummarizestheresultsof668skeletal preparations +/– and ;+/+ and+/+; RESEARCH ARTICLE ; M33 eed eed Bmi1 –/– and double mutants(Beletal.,1998).Alternatively, incase ; Bmi1 r Bmi1 S1 transformationwas detectedin25and27%of eed eed function additivelyinthe Bmi1 –/– ; ; Bmi1 Bmi1 in axialpatterning.Bygeneticcriteria,the double homozygotes(Fig.2A).Although +/– skeletons, respectively, comparedwith double heterozygotesreflectedastrictly double mutantswould exhibit the eed eed and eed ; Bmi1 eed Bmi1 and and double would Bmi1 Bmi1 eed in axial segments andwerephenotypicallyidenticaltothosedetected genotype, thetransformationsremainedrestrictedtoindividual ascertained. Regardless oftheseverity ofthedoublemutant hallmark ofsynergistic geneticinteraction,couldreadilybe in penetrance,changesphenotypicexpressivity, another cervical region, whichrepresentedafocalpointof arch ofC1in rostral tothefirstcervicalvertebra andbroadeningoftheneural (Bel etal.,1998).Furthermore,two ectopicossificationcenters synergy andmanifestedtransformationsacrossmultiplevertebrae between enhanced by defects (van derLugtetal.,1994)withphenotypic expression not eed To accountforthehaploinsufficiency of genes eed andBMI1intheregulation ofvertebral identity. of EED homeotic transformationsaremostconsistentwithparallelfunction a sensitizedPcGpathway(s), additive increasesinthepenetranceof mediated methylationofH3-K27unlikely. Rather, inthecontext of rendered soledependenceofPRC1recruitmentonPRC2/3/4- result insynergistic phenotypes.Thedoublemutantphenotypealso interaction betweenconstituentsofPRC1andPRC2/3/4didnot shown). compared withthesinglemutantsandwild-typecontrols(datanot yielded sectionsfor352mRNA insituhybridizations (Fig.2B, 1997), 237embryosencompassingallnine interferences ofHoxgenetranscription(Gouldetal.,1997;Mann, as welltodetectpotentialcross-andpara-regulatory Thus, bycontrasttointercrossesofPRC1mutantalleles,genetic eed ; Bmi1 and and M33 double mutantsdidnotpresentany novel phenotypes Bmi1 Bmi1 eed Bmi1 and mutant alleles(Fig.3C,D).Finally, unlike crosses single mutants(Fig.3C-G).Thisincludedthe regulate anoverlappingsetofHox homozygotes representedknown Bmi1 , theaxialandappendicularskeleton in Hox probe are shown belowthecolumns. ( expression. Thegenotypes andthenumber embryos exhibitingectopicHoxgene columns indicatethepercentage of genes tested( depicts thecolor-coding ofthefourHox prevertebrae ofE12.5embryos.Theinset ectopic Hoxgeneexpression in chart summarizesthepenetranceof shown atthebottomoffigure. ( skeletons analyzedpergenotypeare The genotypesandthenumber( for adescriptionofthetransformations. were combined.SeethelegendforFig.3 Unilateral andbilateraltransformations lumbar (L)orsacral(S)transformations. exhibiting thecervical(C),thoracic(T), represent thepercentage ofskeletons five transformationsassessed.Thecolumns The insetdepictsthecolor-coding ofthe homeotic transformationsinP0skeletons. summarizes thepenetranceofposterior eed ectopic Hoxgeneexpression in Homeotictransformationsand Fig. 2. n ) ofembryosanalyzedpergenotype and Bmi1 mutants. Hoxa5 eed Development 133(24) eed , b6 and ( A , ) Thechart Bmi1 c8 Bmi1 Bmi1 and M33 genotypes n b4) function, -specific ) of . The B Bmi1 ) The

DEVELOPMENT and datanotshown). Likewise, discretelevels of ( of E12.5embryos:pv3( level Hoxgeneexpression weredetectedintheprevertebrae (pv) Akasaka etal.,1996),thefollowing anteriorboundaries ofhigh- Kessel andGruss,1991;Shametal.,1992;Eid etal.,1993; Dressler andGruss,1989;Gauntetal.,1990; Fig. 3A,B).Inagreementwithprevious reports (Gaunt,1988; Polycomb complexesfunctioninparallelpathways Hoxb4 ), pv7( Hoxb6 ), pv12( Hoxa5 Hoxc8 ), pv10( ) andpv2( Hoxa7 ), pv1( Hoxd4 Hoxa5, Hoxb4 Hoxb3 (Fig.3A,B ) ), pv2 Therefore, EEDfunctionsasanegative regulator ofHoxgene appeared unaffected (Fig.2B,Fig.3A,Banddata notshown). in prevertebrae, whereas displayed ectopicexpression of shown). directly rostraltotheanteriorboundary(Fig.3A,Banddatanot and Compared withwild-typelittermates,E12.5 Hoxd4 expression werefrequentlydetectedintheprevertebra skeletal analysisin EctopicHoxgeneexpression and Fig. 3. 18 situ hybridizationwith type andmutantembryosfollowingmRNAin captured at20 toward asacralidentity(L6 transformation ofthesixthlumbarvertebra with theilialbonesrepresents ahomeotic fusionoftheribs(T7 sternal seventh thoracicvertebraisevidentfrom lackof (C7 the identityoffirstthoracicvertebra transforms theseventhcervicalvertebratoward (arrowheads). (E)Thepresence ofribs in bothrostral andlateralviewsofC1 also broadens theanteriorarch, whichisvisible (C1 represents aposteriorhomeotictransformation body, incompleteregression ofthebodyinC1 1985). AsallvertebraeposteriortoC1containa vertebral bodyduringembryogenesis(Verbout, result from anincompleteregression ofthe Bmi1 ( Genotypes are indicatedabovethepanels. magnification. ( prevertebra. Allimageswere captured at50 Hox geneexpression boundaryandthefirst antisense probes. Arrows denotetheanterior significantly broader ventralarch in al., 1994).(D)Thefirstcervicalvertebrareveals a constitute broadening oftheneuralarch (asterisk) ectopic ossificationcenters(arrowheads) and thoracic (F)andlumbarregions (G).(C)Two and vertebralabnormalitiesincervical(C-E), A , B r r Representative imagesofsectionedwild- ) magnification (G).pv, prevertebra. T1*). (F)Posteriortransformationofthe C2). (D,E)Therudimentaryvertebralbody Hoxa7 mutant skeletons(arrow), whichislikelyto Bmi1 Hoxa5 , Hoxb4 RESEARCH ARTICLE -specific defects(vanderLugtet C-G (C,D), 16 , ) Homeotictransformations Hoxb3 eed Hoxa5 and r eed Bmi1 , r Hoxd4 S1*). Imageswere Hoxb6 (E), 12 T8). (G)Fusion (A) and mutant embryos mutants. eed expression and Hoxb4 (F) and and Hoxc8 4961 (B)

DEVELOPMENT eed increased withtheseverity ofthegenotypeandreached100%in penetrance ofectopic expression inthesinglemutants.Asshown inFig.2B,the expressivity was unaffected andectopicexpression remained were selectedfortheanalysisin repression inresponsetocellulardifferentiation. Four Hoxprobes (MEFs) (Caoetal.,2005)andsuggestschangesinPcG-mediated probes. Notethatwild-type and the presence( Hoxa5 downregulation of l7Rn5 hybridization with expression boundary. Allimageswere captured at50 (s). Arrows denotethefirst somiteandtheanteriorHoxgene present thesameanterior Hox geneexpression boundariesinsomites Hoxa5 et al.,1998),+/+; expression. genes, EEDisrequiredformaintenancebut notinitiationofHox embryos atE12.5(Fig.2B).Thus,similarlyto Hox generepression,theexpression of (Wang etal.,2002).To ascertainapotentialroleforEEDinearly transcription, corroboratingaprevious studyin E11.5embryos 4962 Representative flat-mount images ofwild-type( Fig. 4.NormalHoxgeneexpression inE8.5eedmutantembryos. examined bywhole-mountinsituhybridizationE8.5 with ectopic at thelevel ofsomites5and8,respectively. Theseresultscontrasted presented thesameanterior embryos (Fig.4). In agreementwithprevious studies(van derLugtetal.,1996;Bel ; Bmi1 1989SB , RESEARCH ARTICLE and Hoxb6 / 1989SB double homozygotes.Bycontrast,thephenotypic Hoxc8 Hoxa5 Hoxa5 and mutant embryos( Bmi1 Hoxa5 in Hoxc8 Hoxb6 , Hoxb6 Bmi1 l7Rn5 and –/– (A,B) and embryos manifestedectopicexpression of Hoxa5 (Fig. 2B,Fig.3A,B).Thiscontrastedwith Hoxb6 -deficient murineembryonicfibroblasts as wellnormalexpression levels of Hoxa5 1989SB/1989SB , Hoxc8 l7Rn5 , eed B Hoxb6 expression in Hoxb6 , and D 1989SB ; ) orabsence( Bmi1 ) followingmRNAinsitu Hoxb6 (C,D) antisensecRNA / 1989SB and wild-typeembryos double mutantsbasedon and Hoxa5 A , expression boundary C Bmi1 mutant embryos Hoxc8 ) and Hoxb4 l7Rn5 and magnification. and other Hoxb6 1989SB/1989SB ) ofectopic eed expression mutant PcG was immunoprecipitated withEED(Fig.5).Immunoprecipitation 2004). InadditiontoEZH2andYY1,dimethylatedH3-K27co- EED isoformspreviously reportedin293cells(Kuzmichev etal., and 75kDA fromE12.5trunk(Fig.5),representingthreeofthefour terminus precipitatedthreedistinctisoformsofapproximately50 An antibodyraisedagainstresidues123-140oftheEEDamino embryos EED andBMI1formseparateprotein complexesin parallel pathways converging atthelevel ofHoxgeneregulation. immunoprecipitation without antibody;input,2 both EEDandBMI1.IP, immunoprecipitation; beads,mock either complex.YY1,asabandof 49kDA,co-immunoprecipitated with protein complex,andtrimethylated H3-K27didnotpulldownwith immunoprecipitation didnot detectEEDandBMI1inacommon BMI1 asabandofapproximately 38kDa.Notethatreciprocal co- be confirmedasaBMI1isoform.RING1Bco-immunoprecipitated with immunoprecipitation withouttheBMI1antibodyand,hence,couldnot band, slightlylargerthanthetriplet,wasoccasionallydetectedinmock E12.5 trunkrevealed three isoformsinthe39-41kDA range.Afourth 17 kDA,respectively. Immunoprecipitation ofBMI1(rightcolumn)from H3-K27, whichpresented amolecularweightofapproximately 100and expression. EEDco-immunoprecipitated withEZH2and dimethylated the EEDisoformsininputlane,indicatinglowlevelsof three isoformsofapproximately 50and75kDA.Note theabsenceof Immunoprecipitation ofEED(leftcolumn)from E12.5 trunkidentified EEDandBMI1engageinseparateprotein complexes. Fig. 5. phenotypes stronglysupportthenotionthat homeotic transformationstosinglesegments, andabsenceofnovel heterozygotes, confinementofectopicHoxgeneexpression and of thegenotypicseverity (Fig.2B,Fig.3B). Furthermore, ectopic expression boundary(Fig.2B,Fig.3Aanddatanotshown). confined toasingleprevertebra rostraltothewild-typeanterior histone 3-K27. H3M2K27, dimethylatedhistone3-K27, H3M3K27,trimethylated In conclusion,additive effects ingeneticallysensitizeddouble Hoxb4 expression was notdetected,regardless Development 133(24) eed g protein lysate; and Bmi1 govern

DEVELOPMENT nbothregions aandb,includingTGTCCA in (NCBI Build35)revealed clustersofputative YY1bindingsites Hox regulatory regions, inspectionofthemousegenomesequence BMI1, YY1localizedtobothregions. InsupportofYY1 bindingto juxtaposition ofheterologousPcGcomplexes. SimilartoEEDand regulatory regions of binding, ChIPdetectedtrimethylatedH3-K27throughoutthe transcription startsiteof and YY1alsoco-localizedapproximately1.5kbupstreamofthe (Shrivastava and Calame,1994;Mihalyetal.,1998).EED,BMI1 sequences exhibited significanttoleranceformultiple nucleotides the coreofYY1consensusbindingsite,whileflanking CCCCCA TTTCCA the binding alsoclusteredwithinasmallfragment1.5kbupstreamof close proximityofthecomplexes (datanotshown). EEDandBMI1 (Fig. 6A,B).Thebindingsitescouldnotbeseparated,indicating of the 3A). ChIPdetectedEEDandBMI1bindingimmediatelyupstream as downstream targets ofEEDandBMI1function(Fig.2B,Fig. Ectopic expression inmutantembryosrevealed complexes inHoxregulatory regions Juxtaposition ofEEDandBMI1chromatin separate proteincomplexes. reciprocal co-immunoprecipitationdetectedEEDandBMI1in to associatewitheithertheEEDorBMI1complex. Importantly, engaged intheEEDcomplex, trimethylatedH3-K27didnotappear against different epitopes.Strikingly, whiledimethylatedH3-K27 )immunoprecipitating bandsweredetectedbyatleasttwo antibodies contained YY1(Fig.5).Itshouldbeemphasizedthatall(co- H3-K27. SimilartotheEEDcomplex, theBMI1complex also was foundinacomplex withRING1B,but notdimethylated identified threeBMI1isoformsofapproximately39-41kDA. BMI1 Polycomb complexesfunctioninparallelpathways Hoxc8 Hoxc8 T TT transcription startsite(Fig.6A,B),suggestinglong-range TAG andTCCCCA CC (region a),aswellACACCA transcribed region nearputative promoterelements Hoxc8 Hoxa5 and T AAA (region b).CCAT represents Hoxa5 (Fig. 6B).InadditiontoPcG (Fig. 6B).Furthermore, Hoxc8 TT and AG and AG T Hoxa5 GGC, promoter. Conversely, ChIPdetectedYY1atthe control, EEDandBMI1complexes didnotlocalizetothe Hoxc8 the specificityofexperiments andyieldednoamplificationof 1, amembrane-boundironexporter (Moketal.,2004),confirmed determined conclusively. ChIPusinganantibodyagainstferroportin of thedimethylatedhistonedomaininotherregions couldnotbe dimethylated H3-K27localizedtoregion bof silencing of posterior regions ofE12.5trunk.Inagreementwithtranscriptional regions was evident fromChIPanalysisofdissectedanteriorand transcriptional activator (Gordonetal.,2006). finding isconsistentwithacontext-dependent roleofYY1asa which containedaputative YY1bindingsiteCACCCA mammalian celllines(FrancisandKingston,2001;Lundvan PcG complex interactionin While recentyearshave witnessedsignificant progress inelucidating DISCUSSION entities atHoxtarget loci. remodeling complexes exist asseparate,but juxtaposed,biochemical support amodelinwhichEED-andBMI1-containingchromatin complex withdimethylatedH3-K27.Inaggregate, theresults immunoprecipitation demonstratedphysicalassociationoftheEED proteinsinHoxregulatory regions. Bycontrast,co- three complex withEEDorBMI1,despiteco-localization ofthe ChiP resultsindicatesthattrimethylatedH3-K27didnotforma al., 2006). regions oftheAPaxis,consistentwitharecentstudy(Fujimuraet findings implicatePcGcomplexes inHoxgenerepressionanterior regions ofthetrunk,where 6C). Bycontrast,EEDandBMI1bindingwas absentfromposterior detected upstreamoftheselociinanteriorregions ofthetrunk(Fig. Spatial regulation ofEEDandBMI1bindingtoHoxregulatory The combinedinterpretationoftheco-immunoprecipitationand and Hoxa5 Hoxc8 genomic fragments(Fig.6B).Asanadditional did notassociatewiththe controls. Asanegativecontrol, EEDandBMI1 without antibodyservedasadditional negative of thechromatin usedfor ChIP, andmockChIP E12.5 trunk.Inallcases,inputencompassed 1% in dissectedanteriorandposterior regions of EED andBMI1bindingtoHoxregulatory regions control. ( antibody againstFpn1servedasanegative K27 localizedtoregion bofthe b) ofthe the proteins atthetwoupstream regions (aand BMI1, YY1andtrimethylatedH3-K27detected sites. ( site. RedasterisksindicateputativeYY1-binding upstream (b)ofthe located immediatelyupstream (a)and1.5kb demark twoupstream regions identifiedbyChIP transcription startsite,andthegreen lines shown. Thebluearrow depictsthe representation ofthe the H3-K27 were alsodetected1.5kbupstream of inconclusive. EED,BMI1,YY1andtrimethylated results forregion awere variableand,hence, complexes atHoxtargetloci. JuxtapositionofEEDandBMI1 Fig. 6. and Hoxa5 Hoxa5 Hoxc8 B ) ChIPusingantibodiesagainstEED, C Hoxc8 ) ChIPdetecteddifferential bindingof transcription startsite.ChIPusingan , EEDandBMI1bindingwas and Drosophila RESEARCH ARTICLE . WhiledimethylatedH3- Hoxa5 Hoxc8 Hoxc8 are transcribed.These Hoxc8 transcription start - development and upstream region is actin - actin Hoxc8 ( . Thepresence A promoter. ) Aschematic Hoxc8 T CGC. This promoter, locus, - 4963 actin

DEVELOPMENT and vertebral phenotypesamongthemurine Notably, thisrepresentedthe most significantoverlap inmolecular a similarsetofHoxgenesandcommonposteriortransformations. significantly enhancedthehomeoticphenotypesin mutant crosses.Indeed,allelesof BMI1 phenotypespredictedstronggeneticinteractionindouble et al.,2003;Isono2005).ThedistinctsimilaritiesinEEDand al., 1998;delMarLorenteet2000;Suzuki2002;Voncken et al.,1996;Coré1997;Takihara etal.,1997;Katoh-Fukui 1994; Akasakaetal.,1996;Schumachervan derLugt thus far, includingtheBMI1homologMEL18(van derLugtetal., 2001). BMI1/MEL18 andRAE28(Tomotsune etal.,2000;Akasaka in maintenancebut notinitiationofHoxgenerepression,similarto PRC1 andPRC2/3/4,thepresentstudydemonstratedEEDfunction mouse embryo.Consistentwithacommontemporalrequirementfor their functionalinterdependency inHoxgeneregulation inthe Lohuizen, 2004;RingroseandParo, 2004),littleisknown about 4964 devoid ofmaternal synergistic interactionsin Conceivably, compensatoryactivity byPcGparalogs couldmask to singlesegments, andnovel phenotypeswerenotdetected. andhomeotictransformationsremained confined regardless oftheseverity ofthe heterozygotes comparedwiththesinglemutants.Furthermore, penetrance ofhomeotictransformationsin the rendering compensatoryactivity ofMEL18unlikely. Furthermore, synergistically enhancedthedeficiency for importantly, thesame ligase activity ofRING1Binvitro(Caoetal.,2005).Most al., 2001).Inaddition,BMI1,but notMEL18,promotedtheE3 genes (van derLugtetal.,1994;Akasaka1996; development andco-regulated asimilar, but notidentical,setofHox MEL18 exerted onlypartiallyoverlapping functionsduringmouse MEL18 display70%proteinsequenceidentity. However, BMI1and mouse genomeappearsdevoid ofanEEDhomolog,BMI1and interaction between PcG complexes intheregulation ofvertebral identity, thegenetic rather thanasynergistic orstrictly hierarchicalinterplayofthecore mutants indistinguishablefromthe vertebral identityalsodiffers fromstudiesin synergistic phenotypesuponmutationaldisruption. inherent capacityofthemammalianPcGrepressorsystemtoexpress complexes. Previous studiesin developmental co-existence ofYY1inheterologousPcGcore immunoprecipitated withboth EEDandBMI1,suggesting Interestingly, thezincfingertranscriptionfactor YY1 co- consistent withformationof PRC2/3/4andPRC1atE12.5. BMI1 inseparatecomplexes withEZH2andRING1B, respectively, pathway convergence, co-immunoprecipitationdetectedEED and repression. with parallelpathways converging atthelevel ofHoxgene Genetically, thisshouldrenderthephenotypesof al., 2002;MülleretWang, L.etal.,2004;Cao2005). recruitment (Caoetal.,2002;CzerminKuzmichev et and mammaliancelllines,whichsuggesthierarchicalPcGcomplex proclivity ofYY1toassociate with constituentsofPRC1or as wellmammaliancelllines, revealed acontext-dependent Surprisingly, thepresentstudy revealed additive increasesinthe The geneticinteractionbetween Toward elucidationofthemolecularmechanismsgoverning PcG Bmi1 eed RESEARCH ARTICLE ; M33 and Bmi1 double mutantcrossesprovide testimony tothe esc eed single mutantsrevealed ectopicexpression of (Campbell etal.,1995). and Bmi1 eed Bmi1 ; Bmi1 Drosophila allele employed inthisstudy eed eed mutant allelesismostconsistent eed ; double mutants.Whereasthe Bmi1 and single mutants.Therefore, Bmi1 genotype, ectopicHox and M33 Drosophila PcG Xenopus in theregulation of eed (Bel etal.,1998), rspiaPsc Drosophila eed genes analyzed esc ; Bmi1 ; Bmi1 +/– embryos, embryos embryos double double 2005), itdoesnot permanentlyassociatewiththis complex atlater al., 2002;Mülleret Wang, L.etal.,2004; Caoetal., recruitment (Caoetal.,2002;Czermin etal.,2002;Kuzmichev et Thus, whilemethylatedH3-K27 playsapivotal roleinPRC1 immunoprecipitated withEED, but notBMI1,fromE12.5trunk. associate witheitherPcGcomplex, dimethylatedH3-K27co- Strikingly, whiletrimethylatedH3-K27didnotappeartostably EED andBMI1complex bindinginHoxregulatory regions. ChIP detectedbothdi-andtrimethylatedH3-K27inthevicinity of silent Hoxgenesinmouseembryos. equally large repressedchromatin domainsacrosstranscriptionally Conceivably, long-rangeassemblyofPcGcomplexes mightform nearly 60kbinlengthfrom active chromatindomaingoverned bydimethylatedH3-K4spanned organization ofthemammalian Hoxclusters,atranscriptionally (Bernstein etal.,2005).Asanexample fortheunusualchromatin clusters contrastwithpunctatechromatindomainsatreference loci large continuousstretchesof histonemodificationsacrosstheHox arrays ofindependentPcGcomplexes. Insupportofthisnotion, regions. Accordingly, multipleYY1-bindingsiteswould anchor complex formationandhistonemodificationsinHox regulatory Alternatively, thepresentresultscouldimplicatelong-rangePcG knowledge, formationofYY1multimershasnotbeenreported. harbor putative YY1-bindingsites.However, tothebestofour associated withEEDandBMI1atthe al., 2002).Insupportofasimilarmechanism,allDNA fragments several PLZF-bindingsitesin promoter. Indeed,PLZFdimers or trimersformloopsbetween regions couldsuggestloopingofPcGcomplexes fromthecore complexes 1-1.5kbupstreamofthe et al.,2001;Saurin2001).DetectionofEEDandBMI1 supporting aroleforPcGtargeting ofthecorepromoter(Breiling transcription machineryinteractdirectlywithconstituentsofPRC1, Fujimura etal.,2006).Furthermore,componentsofthegeneral MEFs andE12.5mouseembryos,respectively (Caoetal.,2005; both PcGcomplexes tothe Hoxc8 fragments immediatelyupstreamofthetranscribedregions of locus, awaits investigation. YY1 orPLZFinteractwithBMI1,dependingontheHoxtarget YY1 formacommoncomplex withBMI1or, alternatively, whether with BMI1invivo (Barnaetal.,2002).WhetherbothPLZFand acting elementsnearHoxtranscriptionunitsandinteractsdirectly (promyelocytic leukaemiazincfinger)alsobindstomultiple regulatory regions inthemouseembryo.Interestingly, PLZF clustered YY1-bindingsites,governs theirjuxtapositioninHox DNA bindingonheterologousPcGcomplexes, and,byvirtueof strongly supportsthenotionthatYY1bestows sequence-specific several putative YY1-bindingsiteswithinfewer than50bp.This with DNA fragmentsatthe Gilthorpe etal.,2002).Indeed,EED,BMI1andYY1associated (Brown etal.,1998;Fritsch1999;Mihaly etal.,1998; detected nearmammalianHoxgenesaswellin 2006). mutant miceandgeneticinteractionwithRING1A(Lorenteetal., a PcGproteinderived fromaxialhomeotictransformationsin 2003; Lorenteetal.,2006).DirectsupportformammalianYY1as Levine etal.,2002;MakAtchison 2003;Jinetal., PRC2/3/4 (Garcíaetal.,1999;Poux2001;Satijn Consistent withthepresenceofstableheterochromatindomains, Juxtaposed EEDandBMI1complexes associatedwithDNA Clusters ofputative YY1/PhoDNA-binding siteshave been and Hoxa5 . Thesefindingsareconsistentwithbindingof Hoxc8 Hoxc13 Hoxd11 and Hoxa1 and Hoxc8 Hoxa5 regulatory regions (Barnaet Hoxb8 Hoxc8 to the Development 133(24) and locus, whichharbored promoter regions in Hoxa5 and Drosophila Hoxa7 Hoxa5 transcribed locus. PREs locus Yy1 cis -

DEVELOPMENT epigenetic andgenetic elementsintherecruitment andretentionof 2006). Thus,themodelimplicates acooperative interplaybetween differentiation (Kalantry andMagnuson,2006;Kalantryetal., prevents reactivation ofsilencedtarget lociduringcellular demonstrated thatPRC2/3/4-mediated methylationofH3-K27 differentiation. In supportofthisnotion,two recentstudies maintenance phaseofHox generepressionandsomite stably re-associateswithdimethylatedH3-K27throughout the DNA binding.JuxtaposedPRC2/3/4,boundtoDNA viaYY1, regulatory regions would predominantlydepend onYY1-mediated governed byconformationalchanges,PRC1 retention atHox loci. UpondissociationfrommethylatedH3-K27,possibly interaction withtheH3-K27markinrecruitmentofPRC1totarget et al.,2006),representstrongcandidatesforcooperative noncoding RNA molecules(Bernsteinetal.,2006;Sanchez-Elsner unlikely. Rather, YY1bindingsites,andasrecentlyshown sole dependenceofPRC1recruitmentonmethylatedH3-K27 additive homeoticphenotypesin serves asabindingsiteforPRC1toHoxtarget loci.However, loci viaYY1andmethylatesH3-K27.Inturn,theH3-K27mark al., 1999;Tomotsune etal.,2000),PRC2/3/4bindstoHoxtarget between E9and10(Yu etal.,1995;Yu etal.,1998;Deschamps early maintenancephaseofaxialHoxgenerepression,presumably complex assemblyinmouseHoxgeneclusters(Fig.7). Duringthe Tolhuis etal.,2006). 2006; Bracken etal.,2006;LeeSchwartz etal.,2006; immunoprecipitation experiments inmouseembryos(Boyer etal., the complementationofChIPresultsbyco- and histonesitesattherecentlyidentifiedtarget locinecessitates Hence, definingthephysicalrelationshipbetweenPcGcomplexes association betweenPcGcomplexes andthismodifiedhistonesite. trimethylated H3-K27doesnotequatetoastablephysical regulatory regions, thecloseproximityofPcGcomplexes and differentiating somites.Therefore,atleastinthecontext ofHox interaction ofPRC2/3/4withdimethylatedH3-K27in Tolhuis etal.,2006),thepresentstudydemonstratesdirectphysical 2006; Bracken etal.,2006;LeeSchwartz etal.,2006; fibroblasts, aswellin target lociinmammalianembryonicstemcellsand stages ofHoxgenesilencing.Beyond co-localization at potential Polycomb complexesfunctioninparallelpathways Fig. 7. referred toas‘EarlyMaintenance’phase.Complexassemblyduringthe‘Latephasewasascertainedfrom E12.5embry 2000), thetransitionfrom initiationtomaintenanceofHoxgeneexpression shouldoccurbetweenE9andE10ofmousedevelopmen and PcGproteins. Basedonprevious PcGandtrxGmutantanalysis(Yu etal.,1995;Yu etal.,1998;Deschamps1999;Tom respectively. Clustered YY1bindingsitesare indicatedinred. Thepurplelinerepresents potentialcooperativeinteractionsb The aggregate findingsaredepictedinadynamicmodelofPcG Model ofPcGcomplexassemblyatHoxtargetloci.NucleosomesandmethylatedH3-K27are depictedasblueovalsandorangediamo Drosophila eed Kc andS2cells(Boyer etal., ; Bmi1 double mutantsrender the regulation ofvertebral identity. delineating amoleculardefinitionofPcGpathway convergence in juxtaposed PcGcomplexes inmammalianHoxgeneclusters, Adler, P. N.,Charlton,J.andBrunk,B. References Research InstituteandtheCurtisHankamerBasicResearch FundtoA.S. grant. Thisworkwassupportedbygrantsfrom theNIH,Merck Genome equipment. S.Y.K. received partialsupportfrom aNIGMSpredoctoral training hybridization, andGerard Karsentyforaccesstomicroscopes andimaging Koseki fortheRING1Bantibody, ThomasGuentherforhelpwithmRNAinsitu Krumlauf kindlyprovided theplasmidsforHoxprobes. We thankHaruhiko Deschamps, DenisDuboule,PeterGruss,StanleyKorsmeyerandRobb Bmi1 The authorsare gratefultoMaartenvanLohuizenforgenerously providing the Adler, P. N.,Martin,E.C.,Charlton,J.andJones,K. Akasaka, T., Kanno,M.,Balling,R.,Mieza,M.A.,Taniguchi, M.andKoseki, Bel, S.,Coré, N.,Djabali,M.,Kieboom,K.,vanderLugt,Alkema, M.J. Barna, M., Merghoub,T.,Barna, Costoya,J.A.,Ruggero, D.,Branford, M.,Bergia, Akasaka, T., vanLohuizen,M.,derLugt,N.,Mizutani-Koseki, Y., Kanno, Bajusz, I.,Sipos,L.,Gyorgypal,Z.,Carrington,E.A.,Jones,R.S.,Gausz, J. Alkema, M.J.,Jacobs,Voncken, J.W., Jenkins,N.A.,Copeland,G., Alkema, M.J.,Bronk, M., Verhoeven, E.,Otte,A.,van’t Veer, A. L.J.,Berns, Atchison, L.,Ghias,A.,Wilkinson,F., Bonini,N.andAtchison,M.L. suppressor 2ofzeste Posterior SexCombs consequences andgeneticinteractionsofanullmutationinthe the anteroposterior specificationoftheaxialskeleton. H. expression. synergy andrequirement formaintenancebutnotinitiationofHoxgene Polycomb group genesinmice. and vanLohuizen, M. 499-510. repression of A., Samori,B.andPandolfi,P. P. Mice doublydeficientforthePolycombGroup genes M., Taniguchi, A.andKoseki,H. M.,Alkema,Berns, M.,Vidal, 1522. and Gyurkovics,H. 1358. Transcription factorYY1functionsasaPcGprotein invivo. mouse Polycombtranscriptionalrepressor complex. new murinehomologofthe Satijn, D.P., Otte,A.P., A.andvanLohuizen,M. Berns, 226-240. constituents ofamultimericmammalianPolycombcomplex. and vanLohuizen,M. silencing in renders activedomainsoftargetgenesaccessibleto (1996). Arole for mutant mice,aswellcriticalreading ofthemanuscript.Jacqueline Development HoxD gene expression through chromatin remodeling. mel-18 gene. (2001). The region genes. (1998). Geneticinteractions anddosageeffects of (1997a). IdentificationofBmi1-interactingproteins as 128 Dev. Genet. , aPolycombgroup-related vertebrategene,during Drosophila , 1587-1597. Development Trithorax-mimic (2002). Plzfmediatestranscriptional Dev. Genet. . Genetics (1989). Geneticinteractionsofthe 12 Polycomb protein isamember ofthe RESEARCH ARTICLE , 349-361. 125 159 10 allele of , 3543-3551. , 249-260. , 1135-1150. etween RNAmolecules J. Mol.Biol. Polycomb (1991). Phenotypic Mel18 Development Enhancer ofzeste otsune etal., EMBO J. (1997b). MPc2,a and Genes Dev. -group-dependent os. t, herein 273 Drosophila Bmi1 , 993-1003. 22 122 Dev. Cell reveal , 1347- (2003). (2001). , 1513- 11 nds, 4965 , 3 ,

DEVELOPMENT del MarLorente, M.,Marcos-Gutierrez, C.,Perez, C.,Schoorlemmer, J., B.E.,Kamal,M.,Lindblad-Toh,Bernstein, K.,Bekiranov, S.,Bailey, D.K., 4966 de Napoles,M.,Mermoud,J.E.,Wakao, R.,Tang, Y. A.,Endoh,M., Bernstein, E.,Duncan,E.M.,Masui,O.,Gil,J.,Heard,Bernstein, E.andAllis,C.D. Denisenko, O.N.andBomsztyk,K. Cheng, N.N.,Sinclair, D.A.,Campbell,R.B.andBrock, H.W. Cao, R.,Wang, L.,Wang, H.,Xia,L., Erdjument-Bromage, H.,Tempst, P., Boyer, L.A.,Plath,K.,Zeitlinger, J.,Brambrink,T., Medeiros, L.A.,Lee,T. I., Denisenko, O.,Shnyreva, M.,Suzuki,H.andBomsztyk,K. Colberg-Poley, A.M.,Voss, S.D.,Chowdhury, K.,Stewart,C.L.,Wagner, E. Cao, R.,Tsukada, Y. andZhang,Y. Eid, R.,Koseki,H.andSchughart,K. Dressler, G.R. and Gruss,P. Deschamps, J.,vandenAkker, E.,Forlani,S.,DeGraaff, W., Oosterveen,T., Campbell, R.B.,Sinclair, D.A.,Couling,M.andBrock, H.W. Brown, J.L.,Mucci,D.,Whiteley, M.,Dirksen,M.L.andKassis,J.A. Breiling, A.,Turner, B.M.,Bianchi,M.E.andOrlando,V. Bracken, A.P., Dietrich,N.,Pasini,D.,Hansen,K.H.andHelin, Faust, C.,Lawson,K.A.,Schork,N.J.,Thiel,B.andMagnuson,T. Coré, N.,Bel,S.,Gaunt,S.J.,Aurrand-Lions,M.,Pearce, J.,Fisher, A.and Fritsch, C.,Brown, J.L.,Kassis,A.andMuller, J. Francis, N.J.andKingston,R.E. Czermin, B.,Melfi,R.,McCabe,D.,Seitz,V., Imhof,A.andPirrotta, V. Faust, C.,Schumacher, A.andMagnuson,T. Fujimura, Y., M.,Endoh,Kajita,H.,Mizutani-Koseki, Y., Isono, K.,Vidal, 127 mutations showaPolycombgroup functionforRing1Ainmice. Ramirez, A.,Magin,T. M. andVidal, modifications inhumanandmouse. T. R.etal. Huebert, D.J.,McMahon,S.,Karlsson,E.K.,Kulbokas,J.R.,Gingeras, heritable genesilencingandXinactivation. Polycomb group proteins Ring1A/Blinkubiquitylationof histoneH2Ato Appanah, R.,Nesterova, T. B.,Silva,J.,Otte,A.P., M.etal. Vidal, and RNAare enrichedinfacultativeheterochromatin. (2006). MousePolycombproteins binddifferentially tomethylatedhistoneH3 Mol. Cell.Biol. of the Interactions of ubiquitylation andHoxgenesilencing. Polycomb-group silencing. Jones, R.S.andZhang,Y. . in mesoderm-derivedstructures correlate withthelineargeneorder alongthe Gen. Genet. interactions anddosageeffects of Nature transcription factorsbindpromoters repressed byPolycombgroup proteins. transitions. Genome-wide mappingofPolycombtargetgenesunravelstheirroles incellfate Nature complexes repress developmentalregulators inmurineembryonicstemcells. Levine, S.S.,Wernig,M.,Tajonar, A.,Ray, M.K.etal. 2560-2569. Biol. mutations intheWD40domainofEedblockitsinteractionwithEzh2. during murinedevelopment. F. and Gruss,P. melanogaster 635-650. maintenance of Roelen, B.andRoelfsema,J. protein withhomologytothetranscriptionfactorYY1. The Polycomb methyltransferase activitythatmarkschromosomal Polycombsites. Drosophila Polycomb-M33-deficient mice. Djabali, M. anterior mesodermproduction inmouse. elements inthemurine transgenic embryossuggeststhepresence ofseveral 185-196. homeotic gene. Polycomb group protein pleiohomeoticmediatessilencingofa Nat. Rev.Mol.CellBiol. during gastrulationinthemouseembryo. domains of roles ofPolycomb group geneproducts intranscriptionallyrepressed andactive Takihara, Y., vanLohuizen,M.,Otte,A.,Jenuwein,T. etal. , 5093-5100. Drosophila 18 RESEARCH ARTICLE Drosophila extrasexcombs 412 441 , 5634-5642. -group gene (2005). Genomicmapsandcomparativeanalysisofhistone enhancer ofZeste/ESCcomplexeshaveahistoneH3 , 651-655. , 349-353. Genes Dev. Hoxb8 (1997). Altered cellularproliferationin andmesodermpatterning 246 . Differentiation Genetics polyhomeotic 17 Polycomb group gene Hox Development (1985). Clustered homeoboxesare differentially expressed , 291-300. , 4707-4717. . Development gene expressioninthemouse. patterns eed 20 Hoxb-6 2 138 , 409-421. , 1123-1136. Science is required fornormalmorphogeneticmovements (1989). Anteriorboundariesof , 1151-1162. (2002). RoleofhistoneH3lysine27methylationin with Cell 41 126 Development (1999). Initiation,establishmentand gene. , 193-201. (2001). Mechanismsoftranscriptionalmemory. 43 133 , 3905-3913. Polycomb Polycomb gene displaystranscriptionalrepressor activity. (2005). RoleofBmi-1andRing1AinH2A 298 , 39-45. (1997). Theproduct ofthemurinehomolog Cell (1993). Analysisof , 2371-2381. Dev. Dyn. pleiohomeotic Mol. Cell , 1039-1043. (2000). Loss-andgain-of-function 120 Development Development Dev. Cell group genesof group genesof , 169-181. 124 (1995). The 196 22 , 721-729. , 845-854. (1999). TheDNA-binding , 205-216. 7 cis encodes aDNAbinding , 663-676. Mol. Cell -acting regulatory 121 125 LacZ eed Mol. Cell.Biol. (2006). Polycomb Hox (2001). General , 273-285. , 4495-4506. Drosophila Drosophila Int. J.Dev.Biol. (1998). Point reporter genesin mutation disrupts (1995). Genetic Drosophila gene expression (1994). (2006). Distinct 1 Development , 1057-1064. Cell (2006). (1998). The Mol. Cell. (2004). . (1998). 111 Mol. (2002). 26 , , 43 , Kessel, M.andGruss,P. Kuzmichev, A.,Nishioka,K.,Erdjument-Bromage, H.,Tempst, P. and Katoh-Fukui, Y., Tsuchiya, R.,Shiroishi, T., Nakahara,Y., Hashimoto,N., Kalantry, S.,Mills,K.C.,Yee, D.,Otte,A.P., Panning,B.andMagnuson,T. Kennison, J.A.andTamkun, J.W. Kuzmichev, A.,Jenuwein,T., Tempst, P. andD.R. Kalantry, S.andMagnuson,T. Kuzmichev, A.,Margueron, R.,Vaquero, A.,Preissner, T. S.,Scher, M., García, E.,Marcos-Gutierrez, C.,delMarLorente, M.,Moreno, J.C.and Lee, T. I.,Jenner, R.G.,Boyer, L.A.,Guenther, M.G.,Levine, S.S.,Kumar, R. Gaunt, S.J.,Coletta,P. L.,Pravtcheva,D.andSharpe,P. T. Gaunt, S.J. Gaunt, S.J.,Krumlauf,R.andDuboule,D. Gunster, M.J.,Satijn,D.P., Hamer, K.M.,denBlaauwen,J.L.,deBruijn,D., Gould, A.,Morrison,Sproat, G.,White,R.A.andKrumlauf, Gordon, S.,Akopyan,G.,Garban,H.andBonavida,B. Gilthorpe, J.,Vandromme, M.,Brend, T., Gutman,A.,Summerbell,D., Hernandez-Munoz, I.,Taghavi,Hernandez-Munoz, P., Kuijl,C.,Neefjes,J.andvanLohuizen,M. Hemenway, C.S.,Halligan,B.W. andLevy, L.S. Hashimoto, N.,Brock, H.W., Nomura,M.,Kyba,Hodgson,J.,Fujita,Y., Isono, K.,Fujimura,Y., Shinga,J.,Yamaki, M.,O-Wang, J.,Takihara, Y., Jürgens, G. Jin, Q.,vanEynde,A.,Beullens,M.,Roy, N.,Thiel,G.,Stalmans,W. and 85 and concomitantalterationsof Polycomb andAntennapediamutationsin Dev. human multiprotein complex containingtheEnhancerofZesteprotein. Reinberg, D. 104. M33 Noguchi, K.andHigashinakagawa,T. from differentiation-induced reactivation. (2006). ThePolycombgroup protein Eedprotects theinactiveX-chromosome Genet. dispensable fortheinitiationofrandomX-chromosome inactivation. bithorax complexin H3. containing complexestargetmethylation ofhistoneH1ornucleosomal complexes changeduringcellulardifferentiation. (2005). Compositionandhistonesubstrates ofPolycombrepressive group Kirmizis, A.,Ouyang,X.,Brockdorff, P. N.,Abate-Shen,C.,Farnham, etal. EMBO J. of themammalianPolycombcomplex,andwithtranscriptionfactorYY1. M. Vidal, their regulation. expression intheembryo,butshowstage-andtissue-dependentdifferences in a subfamily, Hox-1.4,-2.6and-5.1,displaysimilaranteroposterior domainsof M., Chevalier, B.,Johnstone,S.E.,Cole,M. F., Isono, K.etal. 1859-1864. overlapping Positive cross-regulation andenhancersharing:twomechanismsforspecifying other membersofthe 3.4 Hox-3.1 overlapping domainsinembryonicgermlayersandorgans:acomparisonof Biol. Polycomb-group protein BMI1andhumanhomologsof polyhomeotic. Identification andcharacterizationofinteractionsbetweenthevertebrate Alkema, M.J.,vanLohuizen,M.,Driel,R.andOtte,A.P. Oncogene factor YY1:structure, function,andtherapeuticimplicationsincancerbiology. 3887-3899. dependent ontheubiquitoustranscriptionfactorNFY. Totty, N.andRigby, P. W. Biol. PRC2/EZH2 andthemaintenanceDNAmethyltransferaseDNMT1. (2005). AssociationofBMI1withPolycombbodiesisdynamicandrequires 2541-2547. interacts withdinGandMPh2:therole ofRINGfingerdomains. group complexes. and M33are membersofheterogeneous multimericmammalian Polycomb Takihara, Y., Shimada,K.andHigashinakagawa,T. Polycomb repression of Mammalian PolyhomeotichomologuesPhc2andPhc1actinsynergytomediate Murahashi, Y., Takada, Y., Mizutani-Koseki,Y. andKoseki,H. 278 Development (EED),andfunctionsasatranscriptionalrepressor. of PP1(NIPP1),interactswiththePolycombgroup protein, EmbryonicEctoderm Bollen, M. , 8136-8140. : Homeoboxsequenceandembryonicexpressioncompared patterns with , 30677-30685. Mol. Cell 17 25 16 mutant mice. 2 , 2326-2335. , 11047-11058. , 2893-2905. and , 656-664. 18 (1985). Agroup ofgenescontrolling thespatialexpression ofthe (1988). Mousehomeoboxgenetranscriptsoccupydifferent but (1999). RYBP, anewrepressor protein thatinteractswithcomponents 25 , 3404-3418. (2003). TheProtein Phosphatase-1(PP1)regulator, NuclearInhibitor Hox Hox-1.5 , 1125-1142. 14 (2002). Histonemethyltransferaseactivityassociatedwitha , 183-193. Development expression patterns. Biochem. Biophys.Res.Commun. Nature Drosophila . Development Hox (1991). Homeotictransformationsofmurinevertebrae Hox 393 gene network. (2002). Spatiallyspecificexpression of genes. 107 , 688-692. (2006). ThePolycombgroup protein EEDis . Hox Nature , 131-141. (1988). Dosage-dependentmodifiersof Mol. Cell.Biol. codes inducedbyretinoic acid. 103 Genes Dev. 316 , 135-144. (1998). Male-to-femalesexreversal in Nat. CellBiol. Development Drosophila , 153-155. (1989). Mousehomeo-geneswithin Proc. Natl.Acad.Sci.USA 11 (1998). TheBmi-1oncoprotein 25 (2004). Different EZH2- Development 133(24) , 900-913. 245 , 6694-6706. . Proc. Natl.Acad.Sci.USA Development (1998). RAE28,BMI1, 8 109 , 356-365. (2006). Transcription , 195-202. , 329-339. (1990). Mouse J. Biol.Chem. Oncogene (2006). (1997). Mol. Cell. (2005). Hoxb4 Cell PLoS (1997). 129 Mol. Cell. Genes 67 , is , 89- 16 102 Hox- , ,

DEVELOPMENT Peytavi, R.,Hong,S.S.,Gay, B.,d’Angeac,A.D.,Selig,L.,Benichou,S., Otte, A.P. andKwaks,T. H.J. Ng, J.,Li,R.,Morgan,K.andSimon,J. Ringrose, L.andParo, R. Lessard, J.,Schumacher, A.,Thorsteinsdottir, U.,vanLohuizen,M., Rinchik, E.M.andCarpenter, D.A. Ramirez-Solis, R.,Zheng,H., Whiting,J.,Krumlauf,R.andBradley, A. Poux, S.,Melfi,R.andPirrotta, V. Pirrotta, V., Poux,S.,Melfi,R. and Pilyugin,M. Paro, R.andHogness,D.S. Polycomb complexesfunctioninparallelpathways Plath, K.,Fang,J.,Mlynarczyk-Evans, S.K.,Cao,R.,Worringer, K.A.,Wang, Neubüser, A.,Koseki,H.andBalling,R. Rietzler, M.,Bittner, M.,Kolanus,W., Schuster, A.andHolzmann,B. Richie, E.R.,Schumacher, A.,Angel,J.M.,Holloway, M.,Rinchik,E.M.and Lund, A.H.andvanLohuizen,M. Mager, J.,Montgomery, F. N.D.,deVillena, P. andMagnuson,T. Levine, S.S.,Weiss, A.,Erdjument-Bromage, H.,Shao,Z.,Tempst, P. and Lorente, M.,Perez, C.,Sanchez,Donohoe,M.,Shi,Y. M. andVidal, Mok, H.,Jelinek,J.,Pai,S.,Cattanach,B.M.,Prchal, J.T., Youssoufian, H. Mihaly, J.,Mishra,R.K.andKarch, F. Mann, R.S. Mak, W., Baxter, J.,Silva,Newall,A.E.,Otte,P. andBrockdorff, N. Montgomery, N.D.,Yee, D.,Chen,A.,Kalantry, S.,Chamberlain,S.J.,Otte, Mok, H.,Mlodnicka,A.E.,Hentze,M.W., Muckenthaler, M.and Müller, J.,Hart,C.M.,Francis,N.Vargas, M.L.,Sengupta,A.,Wild,B., Cell. Biol. predicted structure ofthe of a6-to11-cMsubregion ofthe homolog ofthemurine Benarous, R.andBoulanger, P. Acad. Sci.USA domain withaheterochromatin-associated protein of Dev. protein complexes:adistinctcomplexforeveryoccasion? Pax1 developmental expression of Polycomb andTrithorax group proteins. complementation analysisof31mutations. Completed testingof4557gametesand deletionmappingand Polycomb Magnuson, T. andSauvageau,G. cells. Control ofdevelopmentalregulators byPolycombinhumanembryonicstem tails of The humanWDrepeat protein WAIT-1 specificallyinteracts withthecytoplasmic vertebra anddefectsintheclosurerudiments. ofthesternal 2514. requires atransientinteractionbetweenPCandESC. Chem. Hoxb-4 histone H3lysine27methylationinXinactivation. H., delaCruz,C.C.,Otte,A.P., Panning,B.andZhang,Y. complexes andsilencingmechanisms. mechanisms. Dev. lymphomas. differentiation andsuppresses thedevelopmentofcarcinogen-induced T-cell Magnuson, T. 123 Genet. Genome imprintingregulated bythemousePolycombgroup protein Eed. 22 compositionally andfunctionallyconservedinflieshumans. Kingston, R.E. genetic interactionwiththePolycombgroup gene (2006). Homeotictransformationsoftheaxialskeleton mice. dynamic alterationsiniron homeostasisanderythropoiesis inpolycythaemia and Schumacher, A. Polycomb 1020. with theinactiveXchromosome introphoblast stemcells. (2002). MitoticallystableassociationofPolycombgroup proteins EedandEnx1 required forglobalhistoneH3lysine-27methylation. A. P. andMagnuson,T. iron deficiencyandoverloadinmice. Schumacher, A. complex. Histone methyltransferaseactivityofa Miller, E.L.,O’Connor, M.B.,Kingston,R.E.andSimon,J.A. , 6070-6078. , 312-320. . 13 13 Cell Dev. Biol. Development 274 33 , 448-454. , 2691-2703. ( Hox-2.6 7-integrins. Cell -Group genes -response elements. 125 17 , 502-507. (1997). Whyare , 1635-1645. , 6663-6672. Oncogene Curr. Opin.CellBiol. , 301-313. 111 88 (2002). The 170 (2002). Thecore ofthePolycombrepressive complexis ) mutantmiceshowhomeotictransformationofacervical (2006). Themolecularcircuitry regulating theswitchbetween , 197-208. , 263-267. , 701-716. 131 J. Biol.Chem. (2004). Disruptionofferroportin 1regulation causes 21 , 1859-1868. eed eed (2004). Epigeneticregulation ofcellularmemory bythe (2005). ThemurinePolycombgroup protein Eedis Drosophila , 299-306. Hox Polycomb (1991). ThePolycombprotein shares ahomologous and gene, bindstothematrixprotein ofHIV-1. Pax9 (2003). Generepression byPolycombgroup Mol. Cell genes clustered? Bmi1 (1999). HEED,theproduct ofthehuman , apaired-box-containing generelated to (2001). EstablishmentofPolycombsilencing Fah 16 (2004). Polycombcomplexesandsilencing 273 (1999). (1999). Functionalantagonismofthe , 239-246. -group gene J. Biol.Chem. extra sexcombsrepressor protein. - (1998). Aconservedsequencemotifin Genetica in hemopoieticcellproliferation. Hbb Drosophila , 27459-27466. (1997). Evolutionaryconservationand 1 Annu. Rev.Genet. (1995). Characterizationand , 1065-1066. interval ofmousechromosome 7: N Genetics -ethyl- 117 (2003). AssemblyofPolycomb BioEssays Polycomb group repressor eed Science Ring1 281 N , 191-197. 152 -nitrosourea mutagenesis Curr. Biol. Genes Dev. Drosophila regulates thymocyte , 7946-7951. / , 373-383. Ring1A Curr. Opin.Genet. YY1 Curr. Biol. 300 38 19 Cell , 413-443. , 661-664. , 131-135. (2003). Roleof mutant miceand 15 Mol. Cell.Biol. . . 73 15 Mech. Dev. Proc. Natl. , 942-947. (2002). , 279-294. (2003). , 2509- 12 Genes J. Biol. , 1016- (1998). Mol. (1993). Nat. Satijn, D.P. andOtte,A.P. van derVlag,J.andOtte,A.P. Suzuki, M.,Mizutani-Koseki,Y., Fujimura,Y., Miyagishima,H.,Kaneko,T., Silva, J.,Mak,W., Zvetkova,I.,Appanah,R.,Nesterova, T. B.,Webster, Z., Satijn, D.P., Gunster, M.J.,vanderVlag,Hamer, K.M.,Schul,W., Sanchez-Elsner, T., Gou,D.,Kremmer, E.andSauer, F. van Lohuizen,M., Tijms,M.,Voncken, J.W., Schumacher, A.,Magnuson,T. van derLugt,N.M.T., A.andDeschamps, J. Alkema,M.,Berns, van derLugt,N.M.T., Domen,J.,Linders,K.,vanRoon,M.,Robanus- Tomotsune, D.,Shirai,M.,Takihara, Y. andShimada,K. Takihara, Y., Tomotsune, D.,Shirai,M.,Katoh-Fukui,Y., Nishii,K.,Motaleb, Satijn, D.P., Hamer, K.M.,denBlaauwen,J.andOtte,A.P. Tolhuis, B.,Muijrers, I.,deWit,E.,Teunissen, H.,Talhout, W., vanSteensel, Saurin, A.J.,Shao,Z.,Erdjument-Bromage, H.,Tempst, P. andKingston,R. Schoorlemmer, J.,Marcos-Gutierrez, C.,Were, F., Martinez,R.,Garcia, E., Schwartz, Y. B.,Kahn,T. G.,Nix,D.A.,Li,X.Y., Bourgon,R.,Biggin,M.and Schumacher, A.,Lichtarge,O.,Schwartz,S.andMagnuson,T. Schughart, K.,Utset,M.F., Awgulewitsch, A.andRuddle,F. H. Schumacher, A.,Faust,C.andMagnuson,T. Sewalt, R.G.A.B.,vanderVlag,J.,Gunster, M.J.,Hamer, K.M.,den Shao, Z.,Raible,F., Mollaaghababa,R.,Guyon,J.Wu, C.T., Bender, W. Sham, M.H.,Hunt,P., Nonchev, S.,Papalopulu,N.,Graham,A.,Boncinelli, Shrivastava, A.andCalame,K. Mech. Dev. group proteins anddisplaystumorigenicactivity. 311 Genet. the humanPolycomb-group protein EEDinvolveshistonedeacetylation. Polycomb the anterior-posterior axisinmice. (2002). InvolvementofthePolycomb-group gene Takada, Y., Akasaka,T., Tanzawa, H.,Takihara, Y., Nakano,M.etal. 495. transient recruitment ofEed-Enx1Polycombgroup complexes. Establishment ofhistoneh3methylationontheinactiveXchromosome requires Peters, A.H.,Jenuwein,T., Otte,A.P. andBrockdorff, N. 5151-5155. multi-functional transcriptionalregulator Yin Yang-1. as atranscriptionalrepressor. (1997). RING1isassociatedwiththePolycombgroup protein complexandacts Alkema, M.J.,Saurin,A.Freemont, P. S.,vanDriel,R.andOtte,A.P. of trithoraxresponse elementsrecruit of the abnormalities, andsevere hematopoieticdefectsinmicewithtargeted deletion van Lohuizen,M.etal. Maandag, E.,Riele,H.,vanderValk, M.,Deschamps, J.,Sofroniew, M., of themammalian Hoxb3 699. Development gene leadstoaltered anteroposterior andneuralcrest patterning defects. Targeted disruptionofthemousehomologue M. A.,Nomura,M.,Tsuchiya, R.,Fujita,Y., Shibata,Y. etal. neural tissuein Polycomb group protein EEDinteractswithYY1,andbothproteins induce Polycomb chromatin binding in B. andvanLohuizen,M. Natl. Acad.Sci.USA Structure andexpression ofHox-2.2,amurinehomeobox-containinggene. proteins. E. rhombomere boundariesinthemousehindbrain. repressor thatinteractswiththePolycomb-M33protein andisexpressed at Satijn, D.P., Otte,A.P. M. andVidal, melanogaster Pirrotta, V. implications ofevolutionaryconservation. murine 253. global regulator ofanterior-posterior inmice. patterning mammalian Polycomb-group protein complexes. group proteins Enx1/EZH2andEEDsuggeststheexistence ofdifferent (1998). CharacterizationofinteractionsbetweenthemammalianPolycomb- Blaauwen, J.L.,Satijn,D.P. E.,Hendrix,T., vanDriel,R.andOtte,A.P. Polycomb complex. and Kingston,R.E. the potentialforshared regulatory regions. transcriptswithdifferentialalternative distributionsinthenervoussystemand E. andKrumlauf,R. 3595. (2001). A , 1118-1123. bmi-1 expression inthehindbrainand pharyngealarches by 23 Polycomb Nature -group homologBmi-1isaregulator ofmurine , 474-478. 58 (2006). Genome-wideanalysisofPolycombtargetsin Drosophila proto-oncogene. . 124 , 153-164. Nat. Genet. Xenopus 412 -group gene , 3673-3682. Polycomb Cell , 655-660. 85 (1999). Stabilizationofchromatin structure byPRC1,a (1992). Analysisofthemurine , 5582-5586. Polycomb group complexincludesZesteanddTAFII 98 embryos. (1994). Posteriortransformation,neurological (1999). RING1interactswithmultiplePolycomb- , 37-46. 38 (2006). Genome-wideprofiling ofPRC1andPRC2 group ofgenes. Mol. Cell.Biol. , 700-705. eed Genes Dev. (1999). Transcriptional repression mediatedby (1994). Ananalysisofgenesregulated bythe Drosophila melanogaster Development and itshumanorthologue:functional Mol. Cell.Biol. Drosophila (1997). Ring1Aisatranscriptional Genomics RESEARCH ARTICLE 8 EMBO J. , 757-769. 17 Mech. Dev. (1996). Positionalcloningofa , 4105-4113. 129 Mol. Cell.Biol. Ash1 toUltrabithorax. Mol. Cell.Biol. 21 Ring1B EMBO J. , 4171-4183. , 1360-1369. 11 Hox-2.7 54 Drosophila polyhomeotic Nucleic AcidsRes. , 1825-1836. , 79-88. (2006). NoncodingRNAs . Hox in thespecificationof 98 Nat. Genet. (2000). Regulationof Nature 16 , 165-169. rae28 gene: conserved , 5930-5942. (2003). gene expression. (2001). The Dev. Cell 19 (1997). 18 (1998). The 383 (1988). Drosophila , 57-68. , amember (1996). The , 3586- , 250- 38 22 Nat. Science 4 , 694- , 481- 4967 , Proc.

DEVELOPMENT 4968 Verbout, A.J. Wang, H.,Wang, L.,Erdjument-Bromage, M.,Tempst, H.,Vidal, P., Jones,R. Voncken, J.W., Roelen,B.A.,Roefs,M.,deVries, S.,Verhoeven, E.,Marino, Wang, J.,Mager, J.,Schneider, E.andMagnuson,T. Wang, J.,Mager, J.,Chen,Y., Schneider, E.,Cross, J.C.,Nagy, A.and Cell. Biol. proteins Enx1andEnx2withEed:indicationforseparatePc-Gcomplexes. and Wientjens,E. Embryol. CellBiol. silencing. S. andZhang,Y. 100 causes gastrulationarrest andcellcycleinhibition. S., Deschamps,J.andvanLohuizen,M. Polycomb Magnuson, T. , 2468-2473. RESEARCH ARTICLE 18 Nature group gene. , 3572-3579. (1985). Thedevelopmentofthevertebralcolumn. (2001). ImprintedXinactivationmaintainedbyamouse 431 (2004). RoleofhistoneH2AubiquitinationinPolycomb 90 (1998). InteractionofmousePolycomb-group (Pc-G) , 1-122. , 873-878. Nat. Genet. 28 , 371-375. (2003). Proc. Natl.Acad.Sci.USA Rnf2 (2002). Themouse ( Ring1b Adv. Anat. ) deficiency PcG Mol. Yu, S.E.,Brown, B.D.,Hess,J.L.,Horning, G.A.J.andKorsmeyer, S.J. Wilkinson, D.G.andNieto,M.A. Wang, L.,Brown, J.L.,Cao,R.,Zhang,Y., Kassis,J.A.andJones,R.S. Yu, S.E.andKorsmeyer, B.D.,Hanson,R.Hess,J.L., Horning, S.J. gene Mamm. Genome Nature (1995). Altered and M.L.DePamphilis),pp.361-373.SanDiego:AcademicPress. Mouse Development(MethodsinEnzymology) hybridization totissuesectionsandwholemounts.In 637-646. Hierarchical recruitment ofPolycombgroup silencingcomplexes. 10636. maintenance factorinmorphogenesis. (1998). MLL,amammalian eed 378 is required for , 505-508. Hox 13 expression andsegmentalidentityin , 493-503. Hox trithorax gene repression andextraembryonicdevelopment. (1993). DetectionofmessengerRNAby -group gene,functionsasatranscriptional Proc. Natl.Acad.Sci.USA . Vol. 225(ed.P. M.Wassarman Development 133(24) Guide toTechniquesin Mll -mutant mice. Mol. Cell 95 , 10632- (2004). 14 in situ ,

DEVELOPMENT