Hox Proteins Display a Common and Ancestral Ability to Diversify Their Interaction Mode with the

17 Pages·2012·4.11 MB·English

Hox Proteins Display a Common and Ancestral Ability to
Diversify Their Interaction Mode with the PBC Class
Cofactors
BrunoHudry1,SophieRemacle2,Marie-ClaireDelfini1,Rene´ Rezsohazy2,YacineGraba1,SamirMerabet1*
1InstitutdeBiologieduDe´veloppementdeMarseilleLuminy,IBDML,UMR7288,CNRS,AMU,ParcScientifiquedeLuminy,Case907,Marseille,France,2Molecularand
CellularAnimalEmbryologyGroup,LifeSciencesInstitute,Universite´catholiquedeLouvain,Louvain-la-Neuve,Belgium
Abstract
Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro
analyseshaveestablishedthattheformationofHox/PBCcomplexesreliesonashortconservedHoxproteinmotifcalledthe
hexapeptide(HX).Thisparadigmisatthebasisofthevastmajorityofexperimentalapproachesdedicatedtothestudyof
Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the
BimolecularFluorescenceComplementation(BiFC)assay.ThismethodallowsanalyzingHox-PBCinteractionsinvivoandat
agenome-widescale.WefoundthattheHXisdispensableforPBCrecruitmentinthemajorityofinvestigatedDrosophila
andmouseHoxproteins.WeshowedthatHX-independentinteractionmodesareuncoveredbythepresenceofMeisclass
cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis.
Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins.
Altogether,ourresultshighlightthatflexibilityinHox-PBCinteractionsisanancestralandevolutionaryconservedcharacter,
whichhasstrongimplicationsfortheunderstandingofHoxproteinfunctionsduringnormaldevelopmentandpathologic
processes.
Citation:HudryB,RemacleS,DelfiniM-C,RezsohazyR,GrabaY,etal.(2012)HoxProteinsDisplayaCommonandAncestralAbilitytoDiversifyTheirInteraction
ModewiththePBCClassCofactors.PLoSBiol10(6):e1001351.doi:10.1371/journal.pbio.1001351
AcademicEditor:RobA.H.White,UniversityofCambridge,UnitedKingdom
ReceivedNovember30,2011;AcceptedMay10,2012;PublishedJune26,2012
Copyright: (cid:2) 2012 Hudry et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:AssociationpourlaRecherchecontreleCancer(ARC),AgenceNationalepourlaRecherche(ANR),Fondationpourlarechercheme´dicale(FRM)and
DirectionGe´ne´raledesTechnologies,delaRechercheetdel’EnergieoftheWalloonRegion(WALEOIIgrantnu516054).Thefundershadnoroleinstudydesign,
datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
Abbreviations:AbdA,AbdominalA;AbdB,AbdominalB;Antp,Antennapedia;AP,anterior-posterior; BiFC,BimolecularFluorescenceComplementation;Col,
Collier;Dfd,Deformed;Dll,Distalless;DllR,Dllrepressor;EMSAs,electrophoreticmobilityshiftassays;en,engrailed;Exd,Extradenticle;HD,homeodomain;hth,
homothorax;HX,hexapeptide;Lab,Labial;Scr,Sexcombsreduced;Ubx,Ultrabithorax;VC,C-terminalpartofVenus;VN,N-terminalpartofVenus
*E-mail:[email protected]
Introduction terminal part of both partners [8]. The role of PBC proteins as
directHoxcofactorsiswellestablished[9],allowingtheformation
Hoxgenesencodehomeodomain(HD)-containingtranscription of Hox/PBC/Meis complexes regulating several well-character-
factorsthatspecifycellfatesalongtheanterior-posterior(AP)axis izedtarget genes [10] indifferent developmental contexts[11].
of all bilaterian embryos [1]. Besides early patterning functions, PBC proteins form cooperative DNA-binding complexes with
Hoxgenesareinvolvedinthemorphogenesisofvariousorgans[2] Hoxproteinsfromparaloggroups1to10[12].Theformationof
and inthehomeostasis ofdifferent celltypes inadults [3,4]. Hox Hox/PBC complexes not only improves Hox DNA-binding
functional diversity is accompanied by a high level of transcrip- affinity but also extends the size of cognate DNA sequences.
tionalspecificity,asillustratedbythedistinctdevelopingprograms Moreover, it was shown that the identity of the two central
triggered by each Hox protein during embryogenesis. These nucleotides in the Hox-PBC binding site could discriminate the
specific functions contrast withthepoor DNA-binding stringency HoxproteinengagedintheHox/PBCcomplex[13],althoughthis
of HoxHDs,whichfall onlyintotwospecificity groups [5,6]. observation does not apply to all characterized Hox target
Hox DNA-binding specificity is enhanced by interactions with enhancers [14]. Recent studies further established that the
twofamiliesofcofactors,beingcollectivelyreferredtoasPBCand interaction with the PBC cofactor helps Drosophila Hox proteins
Meis[7].ThesecofactorsbelongtotheTALE(threeaminoacids to recognize distinct DNA-structures [15,16]. This recognition
loop extension) class of HD-containing transcription factors. mode was shown to rely on conserved and paralog-specific
Representatives of PBC are the Drosophila Extradenticle (Exd) or residues[15],anditwassuggestedthatthePBCcofactorcouldbe
vertebratePbx1–4proteins.TheMeisfamilycomprisesMeisand widely used tounlock a ‘‘latent specificity’’ in HoxDNA-binding
Prep subclasses in vertebrates, but Drosophila has only one Meis recognition properties[16].
representativecalledhomothorax(hth)[7].Meisfamilymembersare Biochemical and structural analyses of Hox/PBC complexes
required for the nuclear translocation of PBC proteins, through have identified a generic mode of interaction whereby the
interactions via a highly conserved domain localized in the N- recruitment of PBC cofactors is dependent on a six-residue-long
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Hox-PBCInteractionsAreWidelyHX-Independent
the HX in mouse Hoxb8 leads to dominant-negative phenotypes
Author Summary
thataredifficulttoreconcilewithPBC-dependentfunctions[32].
Hox proteins are key transcriptional regulators of animal Finally,theHXmutationrendersatruncatedLabial(Lab)protein
development, famously helping to determine identity hyperactive in an Exd-dependent context [33], and does not
along the anterior-posterior body axis. Although their compromise the Exd-dependent repression of the Distalless (Dll)
evolution and developmental roles are well established, enhancer by Ubx [34] and AbdominalA (AbdA, [35]) in the
the molecular mechanisms underlying their specific func- Drosophilaembryo.Whethertheseobservationsconstitutepeculiar
tions remain poorly characterized. The current dominant casesorwhetherHoxproteinsfrequentlydisplayHXdispensabil-
viewisthatinteractionwithdifferentmembersofthePBC
ity for mediating interactions with the PBC cofactor remain,
familyoftranscriptionfactorsconfersspecificDNA-binding
however, tobedetermined.
properties on different Hox proteins. However, this idea
In this work, we investigated the requirement of the HX in
conflictswithinvitroevidencethatashort‘‘hexapeptide’’
vertebrateandinvertebrateHoxproteins,focusingonseveralHox
(HX) motif shared by most Hox proteins is solely
paralog groups. Our analysis relied on the Bimolecular Fluores-
responsible for generic PBC recruitment. Here we have
cenceComplementation(BiFC)technology[36]tovisualizeHox-
used the BiFC (bimolecular fluorescence complementa-
tion) method toaddress the globalimportance oftheHX PBC interactions in the Drosophila embryo, mammalian cells, and
motif for Hox-PBC interactions in living cells and living chick embryos. This method allows assessing the requirement of
animalsincludingfruitfliesandchickembryos.Weobserve the HX in vivo. Importantly, BiFC presents the advantage of
that most interactions between Hox and PBC proteins do providing, for the first time, a global measure of protein domain
notdependonHX,andthatalternativeproteinmotifsare requirement for Hox-PBC interactions. Indeed, a loss of fluores-
widelyusedforPBCrecruitmentinvivo.Wealsoshowthat centsignalfollowingproteindomainmutationwillreflectabroad
DNA binding by a second family of cofactors, the Meis use of the domain for the interaction, demonstrating its
proteins,unmasksthesealternativeinteractionmodesand requirement in the regulation of a large set of target genes.
that this property is conserved not only across Bilateria, Results showed a large dispensability of the HX for Hox-PBC
but also in the basal animal phylum Cnidaria. Taken interactionsandidentifywidelyusedalternativeinteractionmodes.
together, our results demonstrate that Hox-PBC partner- We also demonstrated that the HX dispensability is often
ship relies on multiple interaction modes, which can be
unmasked by the additional DNA binding of Meis proteins, and
influenced by additional transcriptional partners. We
that both dispensability and Meis unmasking constitute an
propose that this ancestral feature has been essential for
ancestral character ofHox proteins.
ensuringHoxfunctionalplasticityduringdevelopmentand
evolution.
Results
peptide lying upstream of the Hox HD. This hexapeptide (HX) TheHXIsDispensableinSeveralDrosophilaHoxProteins
motif contains a core Y/FP/DWM sequence in Hox paralog for PBC Recruitment In Vivo
groups1–8,andisconsiderablydivergentinposteriorHoxparalog BiFCreliesonthepropertyofN-andC-terminalfragmentsof
groups 9–10 that retain only a single conserved W residue. This fluorescent proteins to reconstitute fluorescence once they are
residue establishes crucial contacts within a hydrophobic pocket brought in close proximity. This property was used in different
formed in part by the TALE motif of the PBC HD [15,17–19]. model systems to validate the existence of direct interactions
Accordingly, the mutation of the W residue is often sufficient to between two proteins, each fused to a non-fluorescent N- or C-
abolishHoxcooperativeDNAbindingwithPBCproteinsinvitro terminal fragment [36]. Here we have investigated the global
[20,21]. However, in the case of vertebrate proteins, data contribution of the HX of Drosophila Hox proteins for Exd
regardingHox-PBCinteractionsweremostlyobtainedwithnearly recruitment by comparing fluorescent signals resulting from the
identicalHox/PBCbindingsitesinitiallyderivedfromasequence assemblyofExdwithwildtypeorHX-mutatedHoxproteins.Six
(called PRS for Pbx Recognition Sequence) requiring the HX for of the eight Drosophila Hox proteins were fused to the C-terminal
Hox/PBC complex assembly [20–25]. These binding sites thus part (VC) of Venus (a variant of the Green Fluorescent Protein)
presentedastrongbiastowardsHX-dependentassociationmodes. eitheraswildtypeorHX-mutatedversions(Figure1AandTable
Finally, several Hox/PBC structures have been solved with S1).ThecomplementaryN-terminalpartofVenus(VN)wasfused
vertebrate and invertebrate proteins (see [10] for review). These totheExdcofactor.Thischoiceoffusiontopologieswasbasedon
structureswereobtainedusingHoxpeptideslimitedtotheregion previousresultsusingAbdAandExdforestablishingBiFCinthe
encompassing the HD and HX, which excludes protein domains Drosophila embryo [37]. In particular, it was shown that the
that could additionally contribute to the interaction with PBC. combination with VC-AbdA and VN-Exd was best suited for
SuchproteindomainshavebeendescribedintheC-terminalpart BiFCsincethesefusiontopologiesdidnotaffectknownregulatory
of the Drosophila Deformed (Dfd, [26]) and Ultrabithorax (Ubx, functions in vivo [37]. Constructs were cloned downstream of
[27]) proteins, and it was later found that a short motif lying Gal4 UAS sequences for expression through the UAS/Gal4
immediately downstream oftheHD couldconvey Exd-recruiting system. Wild type and HX-mutated forms of Hox proteins were
activitiesinUbx[28].Nevertheless,thegeneralrequirementofthe inserted at the same genomic locus ([38] and Materials and
HX for Hox-PBC interactions in vitro has led to the assumption Methods),allowingsimilarlevelsofproteinexpression(FigureS1).
that thismotifisthemain,ifnotunique,Hoxproteinmotifused For Antennapedia (Antp), Ubx, AbdA, and AbdominalB (AbdB)
for Hox/PBCcomplexassembly in vivo. fusionproteins,weusedGal4driversderivedfromPinsertionsin
TheproposalofasinglegenericHX-mediatedinteractionmode the corresponding genes, which reproduce the endogenous Hox
needs to be reconsidered in light of several in vivo phenotypes gene expression profile [37,39]. These genetic tools allow
associated with the HX mutation. In the mouse Hoxb6 [29], measuring BiFC in cells that normally express the Hox and Exd
Hoxb8 [30], or Hoxa9 [31], the HX mutation does not proteins during embryonic development. In addition, the P(Ubx-
systematically abolish transforming activities expected to be Gal4), P(abdA-Gal4), and P(AbdB-Gal4) correspond to null muta-
PBC-dependent in hematopoietic cells. In addition, the lack of tions, allowing expressing fusion proteins in the absence of the
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Hox-PBCInteractionsAreWidelyHX-Independent
Figure1.BiFCanalysisoftheHXrequirementforHox-ExdinteractionsintheDrosophilaembryo.(A)SchemeofHoxandExdfusion
proteinsusedasUASconstructsforBiFCanalysisintheDrosophilaembryo.TheplaceoftheHXwithregardtotheHomeodomain(HD)istoscalefor
eachHoxprotein.TheHXmutationengineeredineachHoxproteinisindicated,aswellastheHDmutationinExd.VCandVNcorrespondtotheC-
terminalandN-terminalfragmentsoftheVenusfluorescentprotein,respectively.(B–C)Imagesareillustrativeconfocalcapturesofstage10living
embryos.EachlanecorrespondstoadifferentHoxprotein,whosefusionvariantsareexpressedwithaspecificGal4driver,asindicatedontheleft.
FirstandsecondcolumnscorrespondtoBiFCbetweenHoxandthewildtypeorHD-mutatedformofExd,respectively.Thethirdcolumncorresponds
to BiFC between Exd and the HX-mutated Hox proteins. Panels on the right show the statistical quantification, as a boxplot representation, of
fluorescentsignalsresultingfromBiFCineachcondition(seealsoMaterialsandMethods).QuantificationswithmutatedExdandHoxproteinsare
numberedandarerepresentedasapercentageoftheBiFCnormallyobtainedwiththecorrespondingwildtypeproteins.Dotted-whiteboxesin(B)
indicatethezonewhereBiFCsignalshavebeenquantified.SeealsoFiguresS1,S2,S3,S4.
doi:10.1371/journal.pbio.1001351.g001
endogenous Hox product [37,39]. Under our experimental and Figure S1). Since no P insertions are available in close
parameters, fusion proteins were expressed at levels close to proximity of labial (lab) and Sex combs reduced (Scr), we used the
physiologicalconditionsandBiFCwasanalyzedinthecontextofa engrailed(en)-Gal4andAntp-Gal4driverstoexpresstheLabandScr
perfectly viable embryo (as previously described for AbdA [37] fusionproteins,respectively.Inthesecontexts,BiFCwasanalyzed
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Hox-PBCInteractionsAreWidelyHX-Independent
in segments normally specified by Lab and Scr, with levels of HXwasthenanalyzedbyusingHX-mutatedformsofHoxa1and
fusion proteinnot affecting embryonicdevelopment (FigureS1). Hoxb8.Wefoundthatthismutationhasdistinctconsequencesin
We observed that all VC-Hox/VN-Exd complexes produce each protein, with a mean loss of BiFC of 80% for HX-mutated
fluorescent signals in the epidermis of the Drosophila embryo Hoxa1 and 30% for HX-mutated Hoxb8 (Figure 2C–D). Thus,
(Figure 1B–C). By comparison, no BiFC signals were visualized Hoxa1-Pbx1interactionsarestronglydependentontheHX,while
between VC-Hox proteins and the transcription factor Collier most of Hoxb8-Pbx1 interactions do not require this motif in
(Col)fusedtotheVNfragment(FigureS2).ThespecificityofBiFC COS7cells.
in the Drosophila embryo was also previously verified by several WenextinvestigatedthePBC-recruitingfunctionoftheHXof
control experiments [37]. Among these, it was shown that using vertebrate Hox proteins in the chick embryo, extending our
AbdA and Exd fusion proteins mutated in the residue 51 of the analyses to Hoxb6 and Hoxa9. Heterologous gene expression of
HD abolished DNA binding and complex formation in vitro as mouseHoxproteinswasachievedfollowingelectroporationinthe
wellasBiFCinvivo[37].SincetheotherDrosophilaHoxproteins trunk neural tube, which endogenously expresses Hox [42] and
also require the DNA binding of Exd for dimeric complex PBC/Meis proteins [43]. Hox and Pbx1 fusion proteins were
formation in vitro (Figure S3), we performed BiFC between wild electroporated at identical E2 embryonic stages and BiFC was
type VC-Hox fusion proteins and the HD-mutated form of VN- observed 24h later (Materials and Methods). Three of the four
Exd. We observed that abolishing complex formation in vitro investigatedHoxproteinsproduceBiFCwithPbx1(Figure2E–G).
correlates with a strong decrease of BiFC, although to a lesser Hoxa1istheonlyproteinthatdidnotproduceBiFC(unpublished
extent for Lab (Figure 1B–C). This latter result suggests that a
data).Thisnegativeresulthas,however,tobetakencautiously,as
fraction of Lab-Exd interactions could occur outside the DNA in
residentcentralHoxproteinsmay,accordingtothephenomenon
vivo. Altogether, these control experiments highlight that Hox/
ofposteriorprevalence,suppresstheactivityoftheheterologously
Exd complex assembly depends, for a large part, on interactions
expressedHoxa1protein.Inthiscase,posteriorprevalence could
occurring on DNA. They also demonstrate that BiFC properly
partly rely on competition with the PBC cofactor, as recently
reproduces conditions affecting Exd recruitment for all Drosophila
suggested[44].Nonetheless,thisnegativeresultindicatesthatthe
Hox proteinsusedin thisstudy.
trunk neural tube is not appropriate for revealing interactions
We next investigated the contribution of the HX for Hox-Exd
between Pbx1 andHoxproteins ofanterior paralog groups.
interactions,byusingHX-mutatedformsofHoxproteinsforBiFC
The role of the HX was thus analyzed for the central and
analysis. Lab and Sex combs reduced (Scr) show a clear
posteriorHoxb6,Hoxb8,andHoxa9proteins.Wefoundthatthe
dependency for the HX: Lab loses around 70% of the BiFC
HXmutationdoesnotaffectHoxb6-Pbx1(Figure2E)andHoxa9-
signal, whereas for Scr the HX mutation almost completely
Pbx1(Figure2G)interactions,whileitleadstomeanlossof60%of
abolishes BiFC (Figure 1B). In both cases, the loss was similar to
BiFC between Hoxb8 and Pbx1 (Figure 2F). The latter result is
theHDmutationofExd,suggestingthatallLab/ExdorScr/Exd
consistent with BiFC in COS7 cells, which also established a
complexes occurring on DNA were affected. For Antp, Ubx,
significant contribution of the HX for the interaction with Pbx1.
AbdA, and AbdB, results show that BiFC is not significantly
Specificity of BiFC in the chick embryo was confirmed by the
affected upon the HX mutation (Figure 1C). In addition, BiFC
absence of signal when the HD-mutated form of Pbx1, which
with the HD-mutated form of Exd further establishes that Hox-
properly localizes in nuclei of neural cells (see Figure S5 for a
ExdinteractionsarestillmainlyoccurringonDNAinthecontext
comparison with the wild type Pbx1 fusion protein), was used
of theHXmutation (Figure S4).
(Figure 2F–G).
We conclude that all but Lab and Scr Drosophila Hox proteins
These data, together with those in the Drosophila embryo,
marginally requiretheHX forExd interactioninvivo.
demonstratethatHoxproteinsdisplayanunexpectedlevelofHX-
dispensability forinteracting withPBC-class proteinsinvivo.
TheHXIsDispensableinSeveralMouseHoxProteinsfor
PBC Recruitment In Vivo
TheHXIsDispensableinSeveralDrosophilaHoxProteins
The global contribution of the HX of vertebrate Hox proteins
for PBC Recruitment In Vitro
for Pbx1 recruitment in vivo was also assessed by BiFC. Selected
mouse Hox proteins representative of anterior, central, and TofurtherinvestigatethecontributionoftheHXforHox-Exd
posterior classesand Pbx1 proteins were respectively fused to the interactions, we performed electrophoretic mobility shift assays
C-terminal and N-terminal fragments of Venus (Figure 2A). All (EMSAs)ontwotypesofDNAprobes.Thefirsttype,represented
constructs were cloned downstream the pCMV promoter for by Dllcon, is derived from the Dll enhancer [45] and has the
expression (Materials andMethods andTableS1). property to allow the formation of Hox/Exd and/or Hox/Exd/
We first performed BiFC using Pbx1, Hoxa1, and Hoxb8 Hth complexes with all Drosophila Hox proteins. The second type
proteins in mammalian COS7 cells. Of note, these cells corresponds to DNA-binding sites characterized from natural cis-
endogenously express Meis1 [40], and Hoxa1 and Hoxb8 are regulatorysequencesofHoxtargetgenes.Thecontributionofthe
the only vertebrate Hox proteins for which the functional role of HX was evaluated in the context of Hox/Exd (when applicable)
theHX hasbeen examinedin vivo[32,41]. and Hox/Exd/Hth complexes. The latter allows assessing the
ThespecificityofBiFCintheseexperimentswasestablishedby impactof Hth inHox-Exd interactions.
the absence of signals between VC-Hox fusion proteins and the On the Dllcon probe, all tested Hox proteins form dimeric and
Venus VN fragment, and between the VN-Pbx1 fusion protein trimericcomplexeswithExdorExdandHth(Figure3A),withthe
and the Venus VC fragment (Figure 2B and Figure S5 for exception of AbdB, which forms only trimeric AbdB/Exd/Hth
expression level controls). complexes (Figure 3A and unpublished data). In the absence of
WeobservedthatVC-Hoxa1andVC-Hoxb8proteinsproduce Hth, the HX mutation leads to a complete loss of Hox/Exd
BiFCsignals withVN-Pbx1inCOS7cells (Figure2C–D).Asfor complex formation for Lab, Scr, and Antp (Figure 3A). As
Drosophila Hox/Exd complexes, loss of BiFC with a HD-mutated previously described [34,35], the Ubx/Exd and AbdA/Exd
form of Pbx1 demonstrates that Hoxa1/Pbx1 and Hoxb8/Pbx1 dimeric complexes are not affected upon the HX mutation
interactionsmainlyoccuronDNA(Figure2C–D).Theroleofthe (Figure 3A).
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Hox-PBCInteractionsAreWidelyHX-Independent
Figure2.BiFCanalysisoftheHXrequirementforHox-Pbx1interactionsinmammalianCOS7cellsandthetrunkneuraltubeofthe
chickembryo.(A)SchemeofHoxandPbx1fusionproteinsusedforBiFCanalysis.TheHXmutationengineeredineachHoxproteinisindicated,as
wellastheHDmutationinPbx1.(B–D)BiFCbetweenHoxandPbx1fusionproteinsinmammalianCOS7cells.(B)BiFCbetweenHoxa1,Hoxb8,or
Pbx1fusionproteinsandthecomplementaryisolatedVNorVCfragment,asindicated.(C)BiFCbetweenHoxa1andPbx1fusionproteins.(D)BiFC
betweenHoxb8andPbx1fusionproteins.Wildtypeandmutatedfusionconstructsusedineachtransfectionexperimentareindicated.Illustrative
confocalpicturesofBiFCsignals(green)inCOS7cellsareshownwith(firstcolumn)orwithout(secondcolumn)nucleistaining(withDAPI,blue).The
percentage of BiFC levels was deduced from the quantification of the intensity and number of fluorescent signals in approximately 300 cells
(Materials and Methods). Experiments were independently repeated three times. See also Figure S5. (E–G) BiFC between Hox and Pbx1 fusion
proteinsinthetrunkneuraltubeofthechickembryo.(E)BiFCbetweenHoxb6andPbx1fusionproteins.(F)BiFCbetweenHoxb8andPbx1fusion
proteins.(G)BiFCbetweenHoxa9andPbx1fusionproteins.Wildtypeandmutatedfusionconstructsusedineachexperimentareindicated.Fusion
proteinswereco-electroporatedwithamRFP-encodingvector(red)forassessingtheefficiencyoftheelectroporation(MaterialsandMethods).The
quantificationofBiFCsignals(green)inonehemisegmentispresentedasboxplotsingraphsonthebottom.Quantificationswithmutatedfusion
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Hox-PBCInteractionsAreWidelyHX-Independent
proteinsarenumberedandrepresentedasapercentageofBiFCnormallyobtainedwiththecorrespondingwildtypefusionproteins.SeealsoFigure
S5.
doi:10.1371/journal.pbio.1001351.g002
WenextinvestigatedtherequirementoftheHXforHox/Exd in Hox-Exd interactions. In the case of AbdB, we observed the
complexassemblyinthepresenceofHth.Inthatcontext,theHX formationofdimericcomplexeswithHth,butonlywiththeHX-
mutation does not impair the potential of Ubx and AbdA to mutatedformandinabsenceofExd(FigureS6).Thissuggeststhat
interact with Exd (Figure 3A). Interestingly, the presence of Hth the HX mutation uncovers AbdB-Hth contacts that could
eithercompletely(forAntp)orweakly(forLabandScr)rescuesthe potentially be involved in the formation of AbdB/Exd/Hth
loss of Hox/Exd dimeric complex formation induced by the HX complexes.
mutation(Figure3A).Finally,theHXmutationinAbdBalsohas EMSAswithprobesdesignedfromphysiologicalDNA-binding
no effect on the trimeric complex assembly (Figure 3A). For all sitesshowedthatLab(EVIIIprobe),Scr(fkhprobe),andAntp(ap1
except AbdB, no Hox/Hth complexes can be formed on Dllcon, probe) require the HX to recruit Exd in the presence of Hth
either with wild type or HX-mutated forms of Hox proteins (Figure 3B). Of note, the strong monomer binding of the HX-
(FigureS6).WeconcludedthattherescueoftheHXmutationin mutated form of Lab is titrated out in the presence of Exd
thecontextofthetrimericcomplexislikelyindicative ofchanges (Figure 3B), suggesting that the two proteins interact in a DNA-
Figure3.RoleoftheHXofDrosophilaHoxproteinsforExdand/orExd/Hthrecruitmentinvitro.(A)Electomobilityshiftassays(EMSAs)on
theDllconprobe.(B)EMSAsonphysiologicalprobes.Inallpanels,wildtypeandHX-mutatedHoxproteinsareindicatedaboveeachgel(coloredbars,
withasamecolorcodeasinFigure1A).ThepresenceofExd(E)andHth(H)cofactorsisschematizedbydarkandlightgraybars,respectively.Onthe
right of each gel, colored arrows indicate the corresponding Hox monomer binding. Gray and black arrows indicate Hox/Exd and Hox/Exd/Hth
complexes, respectively. On the left, black arrowheads and asterisks depict Exd/Hth complexes and radioactive probes, respectively. Bands
correspondingtodimericandtrimericcomplexeshavebeenquantifiedandaresymbolizedbygradientgrayboxesbeloweachgel.ForeachHox
protein,theeffectoftheHXmutationisnumberedasapercentageofremainingcomplexeswhencomparedtothewildtypeHoxprotein.Nameof
physiologicalprobes(thenucleotidessequencesareprovidedinMaterialsandMethods):fragmentEVIIIisderivedfromtheenhanceroftheLab
targetgeneCG11339[14];fkhisderivedfromtheenhanceroftheScrtargetgeneforkhead[66];ap1isderivedfromtheenhanceroftheAntptarget
geneapterous[67];tshisderivedfromtheenhanceroftheUbxtargetgeneteashirt[68];rhoisderivedfromtheenhanceroftheAbdAtargetgene
rhomboid[58];andcycEisderivedfromtheenhanceroftheAbdAtargetgenecyclinE[46].SeealsoFigureS6.
doi:10.1371/journal.pbio.1001351.g003
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Hox-PBCInteractionsAreWidelyHX-Independent
independentmanner,asobservedinvivobyBiFC(Figure1B).For Hoxa10 (Figure 4B–B9). Surprisingly, the presence of Meis1 did
Ubx,AbdA,andAbdB,onlytrimericcomplexesareobservedon not rescue complex formation for Hoxa7 (Figure 4B) and even
their respective natural DNA-binding sites (Figure 3B). In AbdB providedconstraintsforrestrictingthecomplexassemblytoaHX-
(CycE probe), the HX mutation affects but does not abolish the dependent interactionmodein thecaseofHoxb6 (Figure 4B).
trimericcomplexformation(Figure3B,[46]).Ofnote,noAbdB/ Altogether, these in vitro experiments demonstrate HX
Hthcomplexescanbeobservedinthatcontext(FigureS6).InUbx dispensability for five of the six investigated mouse Hox proteins.
(tshprobe)andAbdA(rhoprobe)theHXmutationhasnoorlittle Results also highlight a role for Meis1 in exerting a control over
effects, respectively (Figure 3B). Hox-PBC interactions, as previously noticed with Drosophila
In conclusion, EMSAs on consensus and physiological binding proteins. This control appears to be influenced by the topology
sites support the view that all except Lab and Scr Drosophila Hox of theHox/PBC/Meis bindingsites insome instances.
proteinshavethepotentialtointeractwithExdintheabsenceof
the HX. In addition, results with the consensus nucleotide probe Meis DNA-Binding Is Important for Promoting HX-
highlight two modes of HX-dispensability: the first one can be
IndependentInteractionsbetweenHoxandPBCProteins
independent of Hth (Ubx and AbdA), while the second requires
In Vitro
thepresence ofthis thirdpartner (otherHox proteins).
The influence of the topology of the Hox/PBC/Meis binding
sitessuggestthatbindingofMeisproteinstoDNAisaprerequisite
TheHXIsDispensableinSeveralMouseHoxProteinsfor
forMeis-mediateduncoveringofalternativeHox-PBCinteraction
PBC Recruitment In Vitro
modes.Toinvestigatethismoredirectly,werepeatedEMSAswith
ThecontributionoftheHXforPbx1recruitmentwasanalyzed
DNA binding deficient Meis proteins. In the case of Drosophila
byEMSAsinthemouseHoxa1,Hoxb6,Hoxb7,Hoxb8,Hoxa9,
proteins,weusedanaturallyHD-lessisoformofHth(Figure5A),
and Hoxa10 proteins. Distinct DNA target sequences were used
which contains only the evolutionary conserved HM domain
for anterior/central and posterior Hox proteins. The first
mediating the direct interaction with Exd [49]. EMSAs were
nucleotide probe (called ant/cent; Figure 4A) contains sequences
performed on Dllcon, which allowed assessing Hth-mediated
previouslydescribedasallowingHoxproteinsfromparaloggroups
uncovering of HX dispensability for Exd recruitment by Lab,
1 to 8 to form cooperative DNA-binding complexes with Pbx1
Scr,Antp,andAbdB.TheroleofHth-HMwasnotanalyzedfor
[12].ForposteriorHoxa9andHoxa10proteins,onenucleotideof
Ubx and AbdA since these two proteins do not require Hth for
the ant/cent Hox/Pbx binding site (underlined in Figure 4A9 and
establishing HX-independent interactions with Exd (Figure 3A).
[5]) was changed for converting the Hox core sequence to a
We observed that the HD-less isoform of Hth is able to form
consensus binding site for posterior Hox proteins (the resulting
trimeric complexes in the context of wild type Hox proteins
probeiscalledpost).Forbothant/centandpostprobes,anidentical
(Figure5B).TheHXmutation,however,abolishestheformation
core binding site for Meis1 (corresponding to the TGACAG
of the trimeric complex in all cases (Figure 5B), highlighting that
consensussequence;[6])wasadded8nucleotidesupstream,andin
Hth is not able to promote HX-independent interaction modes
thesameorientation,oftheHox/Pbxbindingsite.Thistopology
whenitisnot binding to Dllcon sequences.
wasdesignedtomimictheregulatoryelementofHoxa2,forwhich
EMSAs were also performed with mouse Hox proteins of
thecontributionoftheMeisbindingsitehasbeenmolecularlyand
central paralog groups, for which the HX dispensability for PBC
functionallyvalidated[10,47].Ontheseprobes,wefoundthatthe
recruitment was better (Hoxb6 and Hoxb8) or only (Hoxb7)
HX mutation either abolishes (for Hoxa1, Hoxb7, and Hoxa10;
uncovered in the presence of Meis1 on the ant/cent probe
Figure4A–A9)orstronglyaffects(forHoxb6,Hoxb8,andHoxa9;
(Figure 4A). In this case, we used a full-length Meis1 protein
Figure 4A–A9) dimeric complex formation with Pbx1. In all but
mutatedintheAsn54oftheHD.Thismutantproteinwascalled
Hoxa1, the addition of Meis1 either completely (for Hoxb7,
Meis51 (as it was done for Exd and Pbx1) since this residue
Hoxb8, Hoxa9, and Hoxa10) or partially (for Hoxb6) recues
complex formation (Figure 4A–A9). These observations suggest corresponds to the position 51 in classical 60 amino acids long
HDs[50].AlthoughthismutationabolishedtheDNAbindingof
that the Meis partner helps to promote HX-independent
interaction modes, as previously observed with Drosophila Hox Pbx1/Meis1 complexes (red asterisk in Figure 5C), as previously
proteins. Moreover, the role of Meis1 is likely occurring through described [48], trimeric complexes were still observed with wild
the remodeling of Hox-Pbx1 interactions since no Hox-Meis typeHoxproteins(Figure5C).Thesecomplexesare,however,lost
complexesareformedontheant/centorpostprobes,withwildtype in the context of the HX mutation (Figure 5C), highlighting that
or HX-mutated Hoxproteins(Figure S7). DNA binding deficient Meis1 is not able to uncover HX
We next investigated whether the contribution of Meis1 on dispensability forHox-Pbx1 interactions.
Hox-Pbx1 interactions could depend on thenature of its binding The importance of Meis DNA binding for unmasking HX-
site.Thisquestionwasraisedbytheobservationthatthetopology independent interaction modes between Hox and PBC proteins
of the Meis binding site can strongly differ when comparing wasfurthersupportedonPRSsequences.Thesesequencesdonot
different characterized targetenhancers,withstrongvariationsin contain a Meis binding site and were originally described to
sequence, orientation, and distance from the Hox binding site promote HX-dependent interactions between several vertebrate
(Figure S8 and [48]). Here we have tested whether Meis1 could Hox proteins and the Pbx1 cofactor [20,22,24]. Accordingly, we
stillinfluenceHox-Pbx1interactionswhenitsbindingsitemimics observed that the HX-mutated form of Hoxb8 cannot interact
the topology of the Dllcon and Dll repressor (DllR) elements [45]. withPbx1onPRS,eveninthepresenceofMeis1(Figure5D).In
To thisaim, the Meis binding siteof ant/cent and post probes was this last context, the simple addition of a Meis binding site is
changed in one nucleotide position and reversed, leading to the sufficient to partially restore the complex formation (Figure 5E),
ant/centbis and postbis nucleotide probes (Figure 4B–B9). We highlighting theimportance ofMeis DNAbinding forpromoting
observed that the HX mutation led to similar effects in the HX-independent Hox-Pbx1 interaction modes. Consistently, we
context of dimeric Hox-Pbx1 complexes on these modified observed that Meis1 is not able to promote HX-independent
nucleotide probes (Figure 4B–B9). The addition of Meis1 also interaction modes between Hoxa9 or Hoxa10 and Pbx1 on the
led to a rescue of complex formation for Hoxb8, Hoxa9, and post probelackingtheMeis binding site(Figure S9).
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Hox-PBCInteractionsAreWidelyHX-Independent
Figure4.RoleoftheHXofmouseHoxproteinsforPbx1andPbx1/Meis1recruitmentinvitro.(A)EMSAsofwildtypeandHXmutated
formsofHoxa1,Hoxb6,Hoxb7,andHoxb8withthePbx1andMeis1cofactorsontheant/centprobe,asindicated.(A9)EMSAsofwildtypeandHX
mutatedformsofHoxa9andHoxa10withthePbx1andMeis1cofactorsonthepostprobe,asindicated.(B)EMSAsofwildtypeandHXmutated
formsofHoxa1,Hoxb6,Hoxb7,andHoxb8withthePbx1andMeis1cofactorsontheant/centbisprobe,asindicated.(B9)EMSAsofwildtypeandHX
mutated forms of Hoxa9 and Hoxa10 with the Pbx1 and Meis1 cofactors on the postbis probe, as indicated. The sequence of each probe with
orientationsoftheHox,Pbx,andMeisbindingsitesareindicatedabovethegel.Coloredbars,marks,andquantificationsofproteincomplexesare
symbolizedasinFigure3.SeealsoFiguresS7andS8.
doi:10.1371/journal.pbio.1001351.g004
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Hox-PBCInteractionsAreWidelyHX-Independent
Figure5.MeisclassproteinscannotrevealHX-independentinteractionmodesintheabsenceofDNA-binding.(A)Schemeofthe
shortHD-lessisoformofHth,whichcontainsonlytheExdinteractiondomain(HM).ThisformwasfusedtotheHAtag(notschematized).(B)EMSAs
withwildtypeorHX-mutatedformsofDrosophilaHoxproteinsandExdandtheHD-lessisoformofHthontheDllconprobe,asindicated.ForHox
proteinsmakingadimericcomplexwithExd(Lab,ScrandAntp),ananti-HAraisedagainstHth-HMwasalsousedtovalidatethepresenceofthe
trimericcomplex.Inallcases,theHXmutationabolishestrimericcomplexformationwithExdandHth-HM.(C)EMSAswithwildtypeorHX-mutated
formsofcentralmouseHoxproteinsandPbx1andtheHD-mutatedformofMeis1aontheant/centnucleotideprobe,asindicated.TheHDmutation
abolishesDNA-binding[48],asexemplifiedbytheabsenceofDNA-boundPbx/Meiscomplexes(redasterisk).Thismutationalsoabolishes(forHoxb6
andHoxb7)ordrasticallyaffects(forHoxb8)trimericcomplexformationwithHX-mutatedHoxproteins.(D)EMSAswithwildtypeorHX-mutated
formsofHoxb8andPbx1orPbx1/Meis1onthePRSnucleotideprobe,asindicated.Onthisprobe,theHX-mutatedformofHoxb8isnotabletoform
PLoSBiology | www.plosbiology.org 9 June2012 | Volume 10 | Issue 6 | e1001351
Hox-PBCInteractionsAreWidelyHX-Independent
anydimericortrimericcomplexwithPbx1orPbx1/Meis1,respectively.(E)EMSAswithwildtypeorHX-mutatedformsofHoxb8andPbx1orPbx1/
Meis1onthePRSprobecontainingaMeisbindingsite,asindicated.Onthisprobe,theHX-mutatedformofHoxb8isabletoformtrimericcomplexes
withPbx1/Meis1.Ananti-HAagainsttheHAtagofHoxb8wasaddedinthelastreactiontoconfirmthepresenceoftrimericcomplexes.Notethat
Pbx1isabletobindthetwoTGATbindingsitesofPRS(grayarrowheads).Forallgels,coloredbarsandmarksaresymbolizedasinFigure3.Seealso
FigureS9.
doi:10.1371/journal.pbio.1001351.g005
Conserved HX-Dispensability and Role of Meis Class TDWMand,toalesserextent,UbdAmutationsaffecttheAbdA/
Proteins in Controlling Hox-PBC Interactions in Exd complex formation (Figure 7D). Similarly, the UbdA
Cnidarians mutation also strongly affects the Ubx/Exd complex assembly
on Dllcon (Figure7D).
The HX is widely dispensable for PBC recruitment in several
Finally, the TDWM and UbdA motifs were also shown to be
Drosophila and mouse Hox proteins, suggesting that this property
requiredforAbdA/Exd/HthcomplexassemblyonDllRsequences
could be an ancestral character. To test this hypothesis we used
(Figure S12), confirming their importance in the context of
two Hox proteins of the sea anemone Nematostella vectensis, which
belongs toCnidaria, thesister phylum ofBilateria.
Although Hox-like patterning functions remain hypothetical in
this species [51,52], Anthox6a or Anthox1a of Nematostella are,
respectively, considered as anterior and central/posterior Hox
proteins [51,53,54]. The interaction potential of Anthox6a or
Anthox1a (Figure 6A) was analyzed by EMSAs with the mouse
andDrosophilaPBC/Meiscofactors.ResultsshowedthatAnthox6a
and Anthox1a are able to form dimeric and trimeric complexes
with Exd and Exd/Hth on Dllcon, respectively (Figure 6B). The
HXmutationleadstoalossofdimericcomplexes,whiletrimeric
complexes are still present (with a mean loss of 50%; Figure 6B).
Thus,theHXofAnthoxproteinsisnottheuniqueExd-recruiting
domain inthepresence ofHth.
SimilarresultswereobtainedwithPbx1andMeis1ontheant/
centbisprobe(Figure6C),againsuggestingthatMeis-classproteins
are sufficient to promote HX-independent interaction modes in
the assembly of Anthox/PBC/Meis complexes. Of note, no
dimeric Anthox/Meis1 complexes are formed with wild type or
HX-mutated proteins (FigureS10).
AlthoughitremainstobedeterminedwhetherHX-independent
interactions could also exist in the context of PBC and Meis
cofactors of Nematostella vectensis, our observations suggest that the
potentialfordiversifyingthePBCinteractionmodealreadyexisted
in Hox proteins of the last common ancestor of cnidarians and
bilaterians.
Revealing the Existence of Other Widely Used PBC
Interaction Motifs in Two Drosophila Hox Proteins
The existence of HX-independent interaction modes between
Hox and PBC proteins suggest that Hox proteins could contain
other PBC interaction motifs. Besides the HX, only one other
motif has been described to be necessary for PBC recruitment.
This motif, called UbdA, is conserved among Ubx and AbdA
proteins of protostomes [55] and was shown to mediate Ubx/
Exd/Hthcomplexassemblyoncis-regulatorysequencesoftheDll
target gene [28]. In addition, AbdA harbors an HX-like motif,
TDWM, lying in between the canonical HX motif and the HD,
and beingevolutionary conserved ininsect lineages[56].
The global requirement of the TDWM and UbdA motifs for
Exd recruitment in vivo was assessed by BiFC with the
Figure 6. Hox proteins of the anthozoaNematostellavectensis
corresponding mutated VC-AbdA or VC-Ubx fusion proteins
can recruit the PBC/Meis cofactors in absence of the HX in
(Figure7A).WeobservedthattheTDWMorUbdAmutationin
vitro. (A) Scheme of the Anthox6a and Anthox1a proteins. The HX
VC-AbdAleadstoameanlossof60%and50%ofBiFCwithExd, mutationisindicatedbeloweachprotein.(B)EMSAsofwildtypeorHX
respectively (Figure 7B) andthat theUbdAmutation inVC-Ubx mutatedformsofAnthox6aandAnthox1awiththeDrosophilaExdor
leads to a mean loss of 85% of fluorescence (Figure 7C). These Exd/HthcofactorsontheDllconprobe.(C)EMSAswithwildtypeorHX
distincteffectsdonotreflectdifferencesinexpressionlevels(Figure mutated forms of Anthox6a and Anthox1a with the mouse Pbx1 or
Pbx1/Meis1 cofactors on the vertebrate ant/centbis probe. In each
S11).TheroleoftheTDWMandUbdAmotifsaspotentialPBC-
condition,ananti-HAraisedagainsttheHAtagofAnthoxproteinswas
interacting motifs was also tested by EMSAs on Dllcon, which
addedtoverifythepresenceoftrimericcomplexes.Coloredbarsand
allows assessing the PBC-recruiting functions in the presence of marksaresymbolizedasinFigure3.SeealsoFigureS10.
Exdonly.IncontrasttotheHXmutation,whichhasnoeffect,the doi:10.1371/journal.pbio.1001351.g006
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