Selection of distinct Hox–Extradenticle interactionmodes fine-tunes Hox protein activityMehdi Saadaouia, Samir Merabeta,1, Isma Litim-Mecheria, Elise Arbeillea, Nagraj Sambrania, Wim Damenb, Carlo Brenac,Jacques Pradela, and Yacine Grabaa,1

aInstitut de Biologie du Développement de Marseille Luminy, Centre National de la Recherche Scientifique, Université de la Méditerranée, 13288 MarseilleCedex 09, France; bDepartment of Genetics, Friedrich-Schiller University Jena, 07743 Jena, Germany; cDepartment of Zoology, University Museum of Zoology,Cambridge CB2 3EJ, United Kingdom

Edited* by Matthew P. Scott, Stanford University/Howard Hughes Medical Institute, Stanford, CA, and approved December 9, 2010 (received for review May18, 2010)

Hox genes encode transcription factors widely used for diversify-ing animal body plans in development and evolution. To achievefunctional specificity, Hox proteins associate with PBC class pro-teins, Pre-B cell leukemia homeobox (Pbx) in vertebrates, andExtradenticle (Exd) in Drosophila, and were thought to use aunique hexapeptide-dependent generic mode of interaction. Re-cent findings, however, revealed the existence of an alternative,UbdA-dependent paralog-specific interaction mode providing di-versity in Hox–PBC interactions. In this study, we investigated thebasis for the selection of one of these two Hox–PBC interactionmodes. Using naturally occurring variations and mutations in theDrosophila Ultrabithorax protein, we found that the linker region,a short domain separating the hexapeptide from the homeodo-main, promotes an interaction mediated by the UbdA domain in acontext-dependent manner. While using a UbdA-dependent in-teraction for the repression of the limb-promoting gene Distalless,interaction with Exd during segment-identity specification still re-lies on the hexapeptide motif. We further show that distinctlyassembled Hox–PBC complexes display subtle but distinct repres-sive activities. These findings identify Hox–PBC interaction asa template for subtle regulation of Hox protein activity that mayhave played a major role in the diversification of Hox proteinfunction in development and evolution.

transcriptional regulation | AbdominalA | Hox protein specificity anddiversity

Hox genes encode transcription factors widely used for di-versifying animal body plans in development and evolution

(1). Functional diversity likely relies on interaction with proteinpartners (2, 3). Although other proteins presumably are involved,our current knowledge of Hox protein mode of action stemsmainly from the interaction with PBC class of cofactors, Pbx invertebrates andExtradenticle (Exd) inDrosophila (4–7). Extensivedata, including in vitro interaction assays (8, 9), the requirementfor in vivo Exd-dependent processes (10–12), and crystallo-graphic studies (13–16), support the notion that Hox–PBC inter-action relies on a short hexapeptide (HX) motif lying upstreamof the homeodomain (HD) and shared by most Hox proteins.However, some PBC-dependent Hox functions are retained

following mutation of the HXmotif. This retention is illustrated invertebrates by the dominant phenotypes resulting from HX mu-tation of themouseHoxB-8 protein, which are difficult to reconcilewith a lack of Pbx protein interaction (17), and in Drosophila bythe retained capacity of HX-mutated AbdominalA (AbdA) andUltrabithorax (Ubx) proteins to repress the limb-promoting targetgene Distalless (Dll) (11, 18). In Ubx, a short motif located down-stream of the HD conveys Exd interaction potential (18). Thismotif, UbdA, 8 aa long, is shared exclusively by the central paralogproteins Ubx and AbdA and is found only in protostomes (19, 20).Thus, Hox–PBC interactions occur through distinct modes,

suggesting that flexibility in Hox–PBC contacts contributes inspecifying and diversifying Hox protein function and raising

questions about the mechanisms controlling such flexibility. Inthis study, we use Dll regulation and naturally occurring varia-tions in Ubx protein sequences during development and evolu-tion to investigate the molecular control of Hox–PBC interactionand to evaluate its functional impact.

ResultsArthropod Ubx Proteins Display Distinct Exd Interaction Modes for DllRepression. Alignment of available Ubx sequences representativeof the four main arthropod groups, chelicerates (Cupiennius salei;Cs), myriapods (Strigamia maritima; Stm), crustaceans (Artemiafranciscana; Af), and hexapods (Drosophila melanogaster; Dm),shows conserved HX and UbdA motifs, indicating the potentialfor both Hox–Exd interaction modes (Fig. 1A). Significant se-quence divergences are found outside the HD, including fourprotein domains absent in non-DrosophilaUbx proteins (Fig. S1).To investigate whether these sequence divergences could affect

themode ofHox–Exd association, we performed in vitro band shiftexperiments, in which trimeric Ubx/Exd/Homothorax (Hth) com-plex formation on Dll target sequences relies on Ubx–Exd in-teraction (18, 21). Mutations of either the HX or UbdA motifswere introduced in Ubx sequences. Although Dm-Ubx (morespecifically the Dm-UbxIa isoform, see below) requires the UbdAmotif to assemble a Ubx/Exd/Hth complex in vitro (18), Exd in-teraction byCs-Ubx,Af-Ubx (22), and Stm-Ubx (23) implicates theHX motif instead (Fig. 1B). We concluded that sequence diver-gences between Drosophila and other arthropod Ubx proteinsunderlie distinct Exd interactionmodes on theDll target sequence.In the Drosophila embryo, Dll expression is restricted to tho-

racic segments and is repressed in abdominal segments by Ubx(24) and AbdA (25). Accordingly, ectopic expression of Ubx (orAbdA) in thoracic segments represses Dll expression in thoracicsegments (24, 26, 27). Chelicerates, like insects, possess a re-duced number of legs; myriapods and crustaceans bear more legs(28). However, Ubx/AbdA expression domains do not accountfor changes in leg number, and changes in protein activity un-derlie the control of leg suppression in crustaceans (25). If suchchanges also underlie the control of leg suppression in cheli-cerates and myriapods, Cs-Ubx, but not Stm-Ubx, should displayleg-suppressive potential. Because our final aim is to evaluatehow protein domains contribute to leg suppression (not possibleat present in chelicerates and myriapods), we evaluated the leg-repressive potential of arthropod Ubx proteins by quantifying

Author contributions: M.S., S.M., J.P., and Y.G. designed research; M.S., S.M., I.L.-M., E.A.,and N.S. performed research; W.D. and C.B. contributed new reagents/analytic tools; M.S.,S.M., I.L.-M., E.A., N.S., J.P., and Y.G. analyzed data; and Y.G. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence may be addressed. E-mail: samir.merabet@univmed.fr oryacine.graba@univmed.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1006964108/-/DCSupplemental.

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their repressive ability on the limb-promoting gene Dll enhancer[followed by the DME-lacZ reporter (24)] in the Drosophilaembryo. For quantitation, we established experimental conditionsto drive transgene expression of Ubx variants close to physiolog-ical levels of Drosophila Ubx (Experimental Procedures and Fig.S2). Although Stm-Ubx and Af-Ubx are poorly active in this assay(respectively 25% and 30% repression, respectively), Cs-Ubx actsas a more potent repressor (60% repression; Fig. S3).The repressive potential of Cs-Ubx allows us to investigate

whether the distinctive Exd interaction modes seen in vitro applyalso to the Exd-dependent in vivo repression of the Hox targetgene Dll (24, 26, 27). Transgenic lines allowing thoracic expres-sion of HX- or UbdA-mutated forms of Cs-Ubx were generated.Consistent with in vitro observations, HX mutation severelyaffects the repressive activity of Cs-Ubx, but UbdA mutation hasno effect (Fig. 1C). Thus, Cs-Ubx and Dm-Ubx both interact withExd to repressDll but act through distinct protein motifs, HX andUbdA, respectively. This observation indicates that the sequencedivergences between Cs-Ubx and Dm-Ubx are responsible forselecting distinct Ubx/Exd interaction modes.

Linker Region Extension Promotes an UbdA-Dependent InteractionMode for Dll Repression. Among the four predominant sequencedivergences distinguishing Drosophila from other arthropod Ubxproteins, we focused on the domain that separates theHX from theHD, referred to as the “linker region” (LR). Notably, the Dro-sophila Ubx gene encodes several splice variants that are evolu-tionary conserved in theDrosophila lineage (29, 30).These isoformsdiffer only in the size of theLR. Themost abundant isoform, Ia, hasan LR that is 41 aa long and is expressed in the ectodermwhereDllis repressed (31). With the exception of Ib, other isoforms havea smaller LR, intermediate in size between the Drosophila Ia iso-form and other arthropod Ubx proteins, which all have an LR 7 aalong. SuchUbx isoforms,differing in theLR,werenot found innon-Drosophila arthropods, suggesting either that they do not exist orthat proper strategies for recovering them were not employed.To address the role of the LR in the choice of Exd interaction

mode, the HX or UbdA motifs were mutated in the IVa, IVb,and IIb isoforms, displaying LRs of 7, 16, and 33 aa, respectively.In vitro band shift experiments on the Dll regulatory sequenceshow a progressive transition from the HX to the UbdA in-teraction mode that correlates with the extent of LR extension in

Dm-UbxIa FYPWMAIAGECPEDPTKSKIRSDLTQYGGISTDMGKRYSESLAGSLLPDWLGTNGL( )QAIKELNEQDm-UbxIVa FYPWMAIA-------------------------------------------GANGL( )QAIKELNEQStm-Ubx FYPWMAIA-------------------------------------------GANGL( )QAIKELNEQCs-Ubx FYPWMAIA-------------------------------------------GANGV( )QAIKELNEQAf-Ubx FYPWMAIA-------------------------------------------GANGL( )QAIKELNEQ

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Fig. 1. Distinct modes of Exd interaction in arthropod Ubx proteins. (A) Alignment of Ubx peptides (HX to UbdA sequences) from C. salei, S. maritima, A.franciscana, and D. melanogaster UbxIa and IVa isoforms. (B) EMSA of Af-Ubx, Stm-Ubx, Cs-Ubx, and Dm-UbxIa variants, wild type or mutated in the HX orUbdA motifs on the DllR probe. Lanes 1 and 2: DllR probe alone and in the presence of a fixed amount of Exd and Hth; lanes 3–26: fixed amount of Exd, Hth,and a fixed, identical amount of Af-Ubx, Stm-Ubx, or Cs-Ubx variants. Bars above the gel indicate proteins present in the experiment. (C) (Left) Repressivepotentials in Drosophila of Cs-Ubx variants. The arm-Gal4–driven ubiquitous expressions of the variants (red) repress the Dll reporter gene (green) to differentextents. (Right) Quantifications of Dll reporter repression. The error bars represent SDs; the number above the bracket indicates the P value.

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Fig. 2. Drosophila UbxIa and -IVa isoforms repress Dll using different Exd interaction modes. (A) EMSA of Dm-UbxIVa variants, wild type or mutated, in theHX or UbdA motifs on the DllR probe. Lanes 1 and 2: DllR probe alone and in the presence of fixed amount of Exd and Hth. Lanes 3–8: fixed amount of Exd,Hth, and fixed, identical amount of Dm-UbxIVa variants. Bars above the gel indicate proteins present in the experiment. (B) Repressive potentials of Dm-UbxIaand Dm-UbxIVa variants. (Left) armadillo(arm)-Gal4–driven ubiquitous expression of the variants (red) represses the Dll reporter gene (green) to differentextents. (Right) Quantifications of Dll reporter repression. The error bars represent SDs; numbers above the brackets indicate P values.

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the different isoforms (see Fig. 2A for isoforms Ia and IVa andFig. S4 for all isoforms).To gain in vivo evidence for a role of LR extension in selecting

a UbdA-mediated Exd interaction mode, we analyzed the Dllrepressive potential of HX and UbdA variants of the UbxIVaisoform. We observed, as previously reported (24, 26, 27), thatDm-UbxIVa had a weaker Dll repressive potential than Dm-UbxIa (45% vs. 85%) (Fig. 2B). Mutation of Exd-interactingmotifs in Dm-UbxIVa revealed that the HX, and not the UbdAmotif as in UbxIa, is essential for Exd-dependent Dll repression(Fig. 2B). Because the only sequence difference between Dm-UbxIa and Dm-UbxIVa is the LR, we concluded that LR ex-tension promotes a UbdA-mediated Exd interaction mode inDm-Ubx proteins. LR extension may influence the mode of Exdinteraction by modifying the length of the LR (in the foldedprotein) and/or by providing specific sequence motifs promotingthe UbdA interaction mode.

Linker Region Extension During Arthropod Evolution Is Responsiblefor Distinct Mode of Dll Repression. The distinct Exd interactionmodes seen forDm-UbxIa andDm-UbxIVa isoforms suggest that,among the differences accumulated in Ubx protein sequencesduring arthropod evolution (Fig. S1), those regarding the LRmaybe responsible for changing the Exd interaction mode. To addressthis possibility, we generated Drosophila/Cupiennius chimericproteins where the Cupiennius LR was replaced by the extendedDrosophila LR (DmLR) (Fig. 3A). We first analyzed the capacityof such a chimeric protein, intact or mutated in either the HX orUbdA motifs, to interact with Exd on the Dll target sequence invitro. Results showed that grafting the Drosophila LR affects theExd interaction mode, because the chimeric Cs-Ubx(DmLR)requires the UbdA motif and not the HX motif to assemble thetripartite Ubx/Exd/Hth complex (Fig. 3B and Fig. S5). We nextestablished transgenic lines allowing expression of these Dro-sophila/Cupiennius chimeric proteins in the fly embryo. Ouranalysis established that changes in motif requirement for Exdinteraction also are observed in vivo, because Exd-dependent Dllrepression by Cs-Ubx(DmLR) is severely affected by mutation ofthe UbdA motif but not by mutation of the HX motif (Fig. 3C).The role of the LR in the selection of the Exd interaction mode

also was addressed in the context of the AbdA protein. Cs-AbdAhas a shorter LR thanDm-AbdA or Procambarus clarkiiAbdA (Pc-AbdA) (Fig. 4A). We found that interaction with Exd in vitro wasstrongly HX dependent in Cs-AbdA but did not require the UbdAdomain (Fig. 4B and Fig. S6). Extending the LR with Pc, Dm, oreven with a polyalanine stretch imposes a similar requirement forthe UbdA motif, with a variable dependency on the HX motif,which is largely dispensable with Pc-LR but is significantly requiredwith Dm-LR or the polyalanine LR (Fig. 4B and Fig. S6). Trans-genic lines allowing the expression ofCs-AbdA,Cs-AbdA(DmLR),Cs-AbdA(PcLR), and their HX-mutated variants in theDrosophilaembryo indicated that although mutation of the HX resulted ina strong decrease (53%) in Cs-AbdA repressive activity on Dllexpression, it had a moderate effect (39%) in Cs-AbdA(PcLR),and only a weak effect (15%) inCs-AbdA(DmLR) (Fig. 4C). Thesefindings indicate that extending the LRweakens the requirement ofthe HX for Exd-dependentDll repression. It is noteworthy that theeffect of the HX mutation in Cs-AbdA(PcLR) and Cs-AbdA(DmLR) proteins onAbdA/Exd complex assembly in vitro does notcorrelate with the efficiency ofDll repression in vivo (compare Fig.4 B and C). These observations indicate that the LR region affectsnot only DNA binding properties but also activity regulation, aspreviously suggested (24). However, the UbdA dependency for Dllrepression by Cs-AbdA, Cs-AbdA(PcLR), and Cs-AbdA(DmLR)could not be demonstrated, because UbdA-mutated transgenesexpressed transcripts but no detectable proteins (Fig. S7).In summary, the LR in Ubx and AbdA influences the Exd in-

teraction mode. Because the sequence identity of the LR of Dm-

Ubx and Dm-AbdA, and of Dm-AbdA and Pc-AbdA are stronglydivergent, we concluded that divergent sequences can promote theUbdA-mediated Exd interaction mode. The distinct efficiencies inpromoting the UbdA interaction mode by an LR with a distinctsequence identity but a similar number of amino acids suggests theexistence of protein sequence constraints within the LR.

UbdA Motif Is Dispensable for Exd-Dependent Dm-UbxIa–MediatedSpecification of Segment Identity. To address whether the pres-ence of an extended LR generally imposes a UbdA-dependentExd interaction mode, we explored motif requirements for Dm-UbxIa–mediated segment-identity specification. Previous workshowed that although Dm-UbxIa promotes A1 identity, Dm-UbxIa mutated for the HX promotes A2 identity instead, mim-icking the activity of Dm-UbxIa in the absence of zygotic Exdactivity (11, 22). These results suggest that Exd interaction in theprocess of segment-identity specification by Dm-UbxIa occursthrough the HX motif, a conclusion confirmed by the observationthat in the complete absence of exd (maternal and zygotic loss), orin hth mutants that phenocopy complete loss of exd, Dm-UbxIa

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Fig. 3. Evolutionary LR extension promotes UbdA-mediated Exd interactionby Ubx for Dll repression. (A) Schematic representation of Cs-Ubx variants,with Dm-Ubx LR insertion (yellow) and motif mutations indicated by blackcrosses. (B) EMSA of Cs-Ubx variants on the DllR probe. Lane 1: DllR probealone; lanes 2–12: fixed amount of Exd and Hth and increasing identicalamounts of Cs-Ubx variants, as indicated. Bars above the gel indicate theamount of protein present in the experiment. (C) (Left) Repressive potentialsin Drosophila of Cs-Ubx(DmLR) variants. arm-Gal4–driven ubiquitous ex-pression of the variants (red) represses the Dll reporter gene (green) todifferent extents. (Right) Quantifications of Dll reporter repression. The er-ror bars represent SDs; the number above the bracket indicates the P value.

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still promotes A2 identity (Fig. S8). To assess whether the UbdAmotif also contributes to Exd interaction in this context, we in-vestigated the activity of the Dm-UbxIa mutant for UbdA andfound that it promotes T1-like identity, as evidenced by the ap-pearance of the beard, a T1-specific feature under Sex comb re-duced (Scr) control (Fig. 5A). These observations suggest thatDm-UbxIaUbdA has acquired Scr-like activity, as confirmed fur-ther by the absence of ectopic Scr transcripts following Dm-UbxIaUbdA expression (Fig. 5B). Thus, Dm-UbxIaUbdA behavesdistinctly from Dm-UbxIa in the absence of Exd. Together withthe activity of Dm-UbxIaHX, this difference suggests that, in theprocess of segment-identity specification, despite the presence ofan extended LR, the HX, instead of the UbdA motif, promotesExd interaction.

DiscussionInsights into the Selection of Hox–Exd Interaction Modes. The find-ing that the UbdA motif promotes interaction with Exd showedthat Hox–Exd interaction does not occur solely through the HXmotif, introducing diversity in Hox–Exd interaction modes. Inprinciple, interaction with Exd may occur through the exclusiveuse of one or the other of these protein motifs or through a bal-

anced combination of both. In any case, the use of generic (HX)and/or paralog-specific (UbdA) interaction modes needs to becontrolled.We found that LR extension in Ubx, and most likely in AbdA,

promotes the transition from an HX-dependent to a UbdA-de-pendent interaction mode in the process of Dll repression. In ad-dition we found that, despite an extended LR, Dm-UbxIa uses anHX-mediated Exd interaction in the process of segment-identityspecification. These findings indicate that the LR does not pro-mote the UbdA-dependent Exd interaction mode in all situations,suggesting that the identity of the cis regulatory target sequencealso affects the choice of interaction mode. It also emphasizes thatthe function of theUbdAmotif, as already shown for the HXmotif(25, 32), is not dedicated exclusively to Exd interaction.Interestingly, LR size is a paralog-specific feature (3, 33), with

anterior Hox proteins displaying extended LR compared withposterior Hox proteins. Thus, although the UbdAmotif is specificto some central Hox paralogs, the LR generally may fine-tuneHox–PBC interactions by controlling the balance between genericHX-dependent and paralog-specific interaction modes that areyet to be identified in other paralog groups.

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Fig. 5. Motifs’ requirements for segment-identity specification by Dm-UbxIa. (A) Anterior regions of wild-type and of ubiquitously expressing Dm-UbxIa,Dm-UbxIaHX, or Dm-UbxIaUbdA first-instar larvae. Arrows point toward “beards,” small denticles in the naked region of T1 and T1-like transformed segments(T1′). (B) Distribution of Scr transcripts (green) in wild-type and ubiquitously expressing Dm-UbxIa or Dm-UbxIaUbdA embryos (red). No ectopic Scr transcriptsare detected in T2 and T3 segments.

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RYPWMSIA----------------GPNGCP( )RAVKEINEQRYPWMSIENQWRGLTANWNL-—PWSPNGCP( )RAVKEINEQRYPWMTLTDWMGSPFERVVCGDFNGPNGCP( )RAVKEINEQRYPWMSIAAAAAAAAAAAA-----GPNGCP( )RAVKEINEQRYPWMTLTDWMGSPFERVVCGDFNGPNGCP( )RAVKEINEQRYPWMSIENQWRGLTANWNL—-PWSPNGCP( )RAVKEINEQ

arm > Cs-AbdA(DmLR)HXarm > Cs-AbdA(PcLR)HXarm > Cs-AbdAHX

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Fig. 4. Conserved role of the evolutionary LR extension in AbdA. (A) Alignment of AbdA peptides (HX to UbdA sequences) from Cupiennius salei (Cs)Procambarus clarkii (Pc), Drosophila melanogaster (Dm), and the modified Cs-AbdA(DmLR), Cs-AbdA(PcLR), and Cs-AbdA(LRala) proteins. (B) EMSA of Cs-AbdA variants on the DllR probe. Lanes 1 and 2: DllR probe alone and in the presence of fixed amount of Exd and Hth; lanes 3–26: fixed amount of Exd andHth and fixed, identical amount of Cs-AbdA variants. Bars above the gel indicate the amount of protein present in the experiment. (C) (Upper) Repressivepotentials in Drosophila of Cs-AbdA variants. arm-Gal4–driven ubiquitous expression of the variants (red) represses the Dll reporter gene (green) to differentextents. (Lower) Quantifications of Dll reporter repression. The error bars represent SDs, and numbers above the brackets indicate P values.

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Functional Impacts Resulting from Changes in the Hox–Exd InteractionMode.The existence of at least two Exd interaction modes for thecentral Hox paralog proteins Ubx and AbdA raises the questionwhether these qualitatively distinct Hox/Exd complexes havedistinct activities. Our data provide support for such distinct ac-tivities. Modifying the interaction mode does affect the efficiencyof Dll repression: Dm-UbxIa, Cs-Ubx(DmLR), and Cs-AbdA(DmLR) repress Dll with a stronger efficiency than DmUbx-IVa,Cs-Ubx, and Cs-AbdA. These results indicate that repression ofDll is more efficient when the complex is assembled through aUbdA interaction mode.However, in contrast to the effects seen on Dll expression, we

did not found significant differences in binding affinities of Hox/Exd complexes assembled through distinct interaction modes:Changing the balance from an HX- to a UbdA-dependent modeby modifying the LR in Cupiennius Ubx and AbdA proteins doesnot affect binding affinity significantly (Figs. S5 and S6); Dm-UbxIa and Dm-UbxIVa form Ubx/Exd complexes with similaraffinities (21). These observations indicate that, in the case of Dllregulation, modifying the mode of interaction does not affectDNA binding. However, the distinct DNA binding affinities dis-played by Dm-UbxIa and Dm-UbxIVa on a composite Hox/Exdsite (34) suggest that modifying the mode of interaction may havea different effect on other targets.We propose that HX- and UbdA-assembled Hox/Exd com-

plexes have the potential for distinct activities, affecting eitherDNA binding or a later step of transcriptional regulation (effi-ciency of activation or repression). This potential may result fromchanges in the overall or local conformation in the Hox/Exdcomplex that intrinsically affect binding properties and/or modifyinterfaces toward additional protein partners involved in targetgene regulation. Such a scenario also explains the importance ofLR sequence identity (Pc or Dm) in regulating the activity ofAbdA/Exd complexes.

Subtle Changes in Hox Protein Function Through a Co-Option Mech-anism. Hox proteins have served as paradigms for studying howevolutionary changes in protein sequences affect morphologicaldiversity. The few available reports were associated with drasticchanges in Hox protein function (22, 35–38). Our work, instead,highlights protein changes that have distinct features.First, changes in protein activity are subtle rather than drastic

and, in the case studied, do not drive morphological changes:Although the acquisition of the LR changes the Ubx–Exd in-teraction mode, the absence of legs in chelicerates and insectsargues that each of the two interaction modes is sufficiently effi-cient to repress Dll. Such subtle changes in protein activity (de-fined as Dll repressive activity), initially not affecting proteinfunction (defined as the morphological outcome, i.e., suppressionof legs), may contribute to morphological changes when associ-ated with complementary changes in cis regulatory sequences orchanges in levels of protein expression in the context of evolution.Second, although previous work demonstrated that protein

changes rely directly on the functional attribute of the motif thatwas gained or lost, the acquisition of the LR in insect Ubx/AbdAproteins acts through preexisting motifs, the HXmotif (present inall bilaterianUbx/AbdA proteins) and theUbdAmotif (present inall protostome Ubx/AbdA proteins). Co-opting the UbdA motiffor Exd interaction through LR extension may have releasedfunctional constraint on theHXmotif, thereby allowing theHX toacquire novel functions. Such novel functions of the HX have

been demonstrated previously for AbdA (25) and also for Antp(32), suggesting that similar mechanisms may operate in moredistant non-UbdA–containing Hox proteins. We propose that themechanism of protein motif co-option identified here has thepotential to promote functional shuffling of protein domains. Thismode of protein evolution, subtle and regulated rather thandrastic, provides more room for the idea that protein sequence, inaddition to cis-regulatory sequences, is the template for molecularand morphological evolution.

Experimental ProceduresFlies, EggCollections, Immunostaining, inSituHybridization,andCuticlePreparation.Alleles used were exdXP11 and hth P2. Exd maternal deprivation was achievedby generating exd germ-line clones. All transgenes were HA tagged. Embryocollection, immunodetection, and cuticle preparations were performedaccording to standard procedures. Antibodies against HA tag (rat; 1:500;Invitrogen), to Ubx (FP3.38; 1:1000), AbdA (rabbit polyclonal; 1:1,000), andβ-galactosidase (1:500) were used. Digoxigenin RNA-labeled probes weregenerated from Scr or Cs-AbdA full-length cDNAs according to the manu-facturer’s protocol (Boehringer-Mannheim).

Constructs, Transgenic Lines, and in Vivo Reporter Analysis. Ubx cDNAs wereprovided by W. McGinnis (University of California at San Diego, La Jolla, CA) forArtemia and by M. Akam (University of Cambridge, Cambridge, United King-dom) for Strigamia. Cs-Ubx is Ubx2. Accession numbers for sequences used inthis study are Af-Ubx, Q8WRG5; Stm-Ubx, Q1KY84; Dm-UbxIVa, AAF55355;Dm-UbxIVb, AAS65158; Dm-UbxIIb, AAN13719; Dm-UbxIa, AAN13178; andCs-AbdA, AJ007436. The Pc-AbdA sequence is from ref. 39. Hox variantconstructs were generated by PCR from full-length cDNAs. HX and UbdAmutations for Cs-, Af-, and Stm-Ubx and for Cs-AbdA are YPWM to YAAAand KELNEQ to KAAAAQ. Primers used are available upon request. Allconstructs were cloned in the pcDNA3 vector and were sequence verified.Mutations of Dm-Ubx are as previously described (18). Constructs for flytransformation were cloned in the pUAST vector, and transgenic lines wereestablished by P-element–mediated germ-line transformation. Collectedembryos were stained with anti-HA to select the conditions (line and tem-perature) that result in expression levels similar to endogenous Ubx or AbdAlevels in A1 and A2, respectively (Fig. S2) (18, 22). Quantification of Dll re-pression, assessed by measuring the loss of DME-lacZ reporter activity fol-lowing Ubx expression, was achieved using the DME-lacZ insertionpreviously described (18). Error bars represent the deviation from the aver-age value (18 < n < 27). P values were calculated using the nonparametricWilcoxon test.

Protein Expression and Electromobility Gel Shift Assays. Exd, Hth, Ubx, andAbdA proteins were full length. Proteins were produced with the TNT (T7)coupled in vitro transcription/translation system (Promega). Production yieldsof Hox proteins and variants were analyzed after scanning gel containing[35S]methionine-labeled protein and identical amounts of wild-type or mutatedproteins were used in band shift assays, performed as previously described (25)using 4 μL of a doubly programmed Exd/Hth lysate. The probe used was DIIR(24). Relative DNA binding affinity for Ubx or AbdA variants was measured withImageJ software. When several Hox concentrations were used, quantificationswere performed on the intermediate Hox concentration.

ACKNOWLEDGMENTS. We thank W. McGinnis and R. Mann for providingUAS-Af-Ubx and DME-lacZ lines, M. Akam for the Stm-Ubx cDNA, R. Whitefor antibodies, and E. Sanchez-Herrero and C. Alonso for Ubx isoforms,B. Hudry for discussions, and B. Hudry, A. Saurin, J. Deutsch, and M. Akamfor comments on the manuscript. This research was supported by the CentreNational de la Recherche Scientifique, Université de la Méditerranée, and bygrants from the Centre Franco-Indien pour la Promotion de Recherche Avan-cée, the Agence Nationale pour la Recherche, Fondation pour la RechercheMédicale, and Association pour la Recherche sur le Cancer to Y.G., and a fel-lowship fromMinistère de la Recherche et Technologie and Association pourla Recherche sur le Cancer to M.S.

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