Dimer formation via the homeodomain is required for function and specificity of Sex combs reduced in Drosophila

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Genomes and Developmental Control

Dimer formation via the homeodomain is required for function and

speciﬁcity of Sex combs reduced in Drosophila

Dimitrios K. Papadopoulos

1,2

, Kassiani Skouloudaki

1,2

, Yoshitsugu Adachi

3

,

Christos Samakovlis

1

_{, Walter J. Gehring}

n

Department of Cell Biology, Biozentrum, University of Basel, 4056 Basel, Switzerland

a r t i c l e

i n f o

Article history:

Received 29 August 2011 Received in revised form 12 April 2012

Accepted 16 April 2012 Available online 28 April 2012 Keywords:

Sex combs reduced Homeodomain dimerization Antennapedia ELEKEF motif Transcriptional speciﬁcity Homeotic function

a b s t r a c t

Hox transcription factors specify body segments along the anteroposterior axis of the embryo. Despite conservation of the homeodomain (HD), different Hox paralogs instruct remarkably different develop-mental fates. We have unexpectedly found that the Drosophila Sex combs reduced (Scr) protein dimerizes in vivo via the homeodomain, whereas its closest relative, Antennapedia (Antp), does not. Dimerization requires the conserved residue 19 in the ELEKEF motif of the HD and is facilitated by DNA binding. To study Scr dimerization in vivo, we generate a giant transcriptional puff in live salivary gland cells, consisting of a controllable multiple Scr-binding site of the fork head enhancer, and visualize Scr dimer formation upon specific DNA binding. Scr dimerization is required not only for transcriptional activation of the fork head gene but also for Scr homeotic function in the fly (formation of ectopic salivary glands, posterior transformations in the embryo and antenna-to-tarsus transformations). Finally, we attempt to attribute the differential behavior in dimer formation observed between Antp and Scr to diverse amino acid regions between the two proteins that account for dimerization in Scr versus non-dimerization in Antp. By constructing hybrid Antp proteins, we find that the C terminus and linker region between the YPWM motif and the HD of Scr are independently sufficient to confer dimer formation in Antp, whereas the long N terminus of the protein and the HD are largely dispensable. Our results indicate that Scr functions as a homodimer to increase its transcriptional specificity and suggest that the formation of HD homo- or heterodimers might underlie the functional distinction between very similar HD proteins in vivo.

Introduction

Homeotic (Hox) transcription factors specify the identity of body segments along the anteroposterior axis of the embryo. They generally harbor the HD, which is the DNA-binding domain, and the YPWM motif, which binds Hox cofactors, such as Extraden-ticle (Exd), and increases Hox specificity in vivo (Passner et al., 1999). However, few paralog-specific cofactors have been identi-fied to date (Mann et al., 2009). The complication lies in the inability to explain how very similar binding site preferences of different Hox paralogs, resulting from very similar HD sequences, can have very different transcriptional outputs in vivo. These can be as diverse as the development of salivary glands and

prothoracic legs, instructed by Scr, versus the development of middle legs and wings, instructed by Antp. Therefore, other mechanisms might account for the ability of Hox proteins to find their target sequences. Recently, DNA structure in the minor groove has suggested an interesting alternative that accounts for specific binding of Scr on the fork head 250 (fkh250) enhancer (Joshi et al., 2007). Also, using extensive computational analysis, Hox/Exd heterodimers have been found to be able to amplify the differences between DNA sequence recognition properties of Hox paralogs, thus suggesting a novel perception of Hox specificity (Slattery et al., 2011).

We have previously demonstrated that both the Antp protein (Papadopoulos et al., 2011) and the Scr protein (Papadopoulos et al., 2010) N termini are largely dispensable for the in vivo homeotic function of the transcription factors, but are presumably required for the ﬁne-tuning of transcriptional potency in the regulation of target genes. This supports the idea that their speciﬁcity largely lies in the portion of the protein that contains the YPWM motif, linker and HD. For Antp the size of the linker plays an important role in transcrip-tional control (activation versus repression) (Papadopoulos et al., 2011), but no such role has been assigned to Scr yet.

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Developmental Biology

n

Corresponding author.

E-mail address: [email protected] (W.J. Gehring). 1

Present address: Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, 10691, Stockholm, Sweden.

2

These authors contributed equally to this work. 3

Present address: Institute of Psychiatry, King’s College London, SE5 8AF London, UK.

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Using the Bimolecular Fluorescence Complementation system (BiFC), we have unexpectedly observed that Scr forms homodi-mers, whereas Antp does not, as shown by previous in vitro studies using the Antp HD (Affolter et al., 1990). This clear difference between Hox proteins containing very similar HDs prompted us to investigate the contribution of homodimerization to Scr homeotic function and speciﬁcity in vivo.

We found that Scr molecules physically interact via the HD to form homodimers ex vivo. Scr homodimerization requires gluta-mate 19 of the HD, a residue residing in the ELEKEF motif which has been conserved from ﬂies to humans (Fig. S1). Mutation of amino acid 19 completely abolishes dimer formation both in vitro and in vivo.

In order to visualize speciﬁc binding of Scr dimers to their native target sequences in vivo, we have introduced a controllable giant synthetic binding site consisting of 50 repeats of the fkh250 enhancer and examined Scr binding in salivary gland nuclei by BiFC. We have found that Scr molecules bearing the E19G mutation cannot bind the synthetic site in vivo and show that Scr dimer formation is required for Scr-mediated transcriptional regulation of the fkh gene. Therefore, we examine the require-ment of homodimerization for Scr homeotic function in vivo and ﬁnd it necessary for the manifestation of most known Scr phenotypes.

Finally, we attempt to attribute the differential homodimer-ization behavior observed between Scr and Antp to amino acid sequences that differ between the two proteins. Having excluded the long and diverse N termini of the proteins and the N-terminal arm of their HDs, we show that both the Antp linker region and its C terminus – ﬂanking the HD – are required to abolish its homodimerization. Exchange of either of the two protein domains with those of Scr results in Antp hybrids that exhibit a neo-morphic gain of dimerization capacity.

Our results clearly show that the formation of dimers repre-sents a functional requirement for Scr and suggest dimerization as a novel mechanism for increasing Scr speciﬁcity in vivo.

Materials and methods Cell culture and transfections

Human HEK 293 T cells were grown in Dulbecco’s modiﬁed Eagle’s medium containing 10% fetal bovine serum and antibio-tics. A TransPEI transfection method (Eurogentec, Cologne, Ger-many) was used for DNA transfections. The transfected cells were analyzed on the second day.

Immunoprecipitations and western Blot Analysis

Cells were washed with ice-cold PBS and lysed with lysis buffer (20 mM Tris, pH 7.5, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 15 mM Na4P2O7, 0.1 mM EDTA), supplemented with 1 mM NaVO4and a protease inhibitor mixture (Roche). After centrifuga-tion (15,000 g, 15 min, 4 1C) and ultracentrifugacentrifuga-tion (100,000 g, 30 min, 4 1C), the cell lysates containing equal amounts of total protein were incubated for one hour at 4 1C with the anti-Flag M2 resin (Sigma), or with anti-V5 antibody, followed by incubation with 30

mL of protein A-Sepharose beads for two hours. The beads

were then washed extensively with lysis buffer, and bound proteins were analyzed by Western blotting with the following antibodies: M2 antibody to FLAG (Sigma) and mouse monoclonal antibody to V5 (Serotec). For detection of FL-Scr peptides the clean-blot IP detection reagent was used to detect the native antibody (and not the dissociated one) in a dilution of 1:500.

Cloning and ﬂy transgenesis

Plasmids were generated using standard procedures. The sequence containing the 50 repeats of the fkh and UAS sequences were synthesized as individual sequences by Geneart and then assembled into a pH-Pelican vector containing the hsp70 minimal promoter, the lacZ coding sequence and gypsy insulators (Barolo et al., 2000). All UAS-Scr constructs and the UAS-Gal4-mRFP1 line were generated by cloning the corresponding sequences in a PUAST vector. The Venus ﬂuorescent protein Vn or Vc sequences were cloned N-terminally to all Scr constructs and are essentially the ones used in Plaza et al. (2008). Fly transgenesis was then performed as described (Spradling and Rubin, 1982). Additional strains used for this study were sgs3-Gal4 (Bloomington stock center, stock number 6870), dppblink_{-Gal4 (}_{Staehling-Hampton} et al., 1994), fkh-lacZ (Zhou et al., 2001), nullo-Gal4 (Gehring et al., 2009), Dll-Gal4 (MD23) (Calleja et al., 1996) and heat-shock-Gal4 (Bloomington stock center, stock number 1799).

Immunohistochemistry on embryos

Preparation of embryos was performed as inGrieder et al. (2000). For induction of ectopic salivary glands the precise process was followed as inBerry and Gehring (2000). For ectopic expression of fkh, the same process was used as inPanzer et al. (1992). Images were obtained within 24 h using a Leica SP5 Confocal setup. Antibodies were used in the following dilutions:

b

-gal (mouse) 1:500 (Promega) and dCreb-A (rat) 1:2000 (Deborah Andrew). Preparation of embryonic cuticle

Cuticles were prepared according toGibson and Gehring (1988). Preparation of adult cuticle

Escapers or pharate adult flies from the cross of Dll-Gal4 to the various UAS-Scr lines (for the induction of antennal tarsi) or of the cross of dppblink_{-Gal4 to the various UAS-Scr lines (for induction of} eye reduction phenotypes) were collected and fixed in 30% glycerol in ethanol. Then, fly parts were dissected from the flies in the same solution, transferred to microscopic slides into droplets of Faure’s medium, covered with coverslips, flattened and used for imaging.

Electrophoretic mobility shift assays

Gel-shift assays were performed as described by Plaza et al. (2001). Peptides were produced using TNTs

T7 Quick Coupled Transcription/Translation System (Promega).

Bimolecular Fluorescence Complementation in HEK293T cells The same process was followed as inPlaza et al. (2008). Quantiﬁcation of Scr dimer formation using Bimolecular Fluorescence Complementation in live salivary gland cells

For the evaluation of the dimerization capacity of each Scr construct, different transformant lines of the same UAS-Vn-Scr construct were crossed to different transformant lines carrying the corresponding UAS-Vc-Scr construct to generate various lines carrying both UAS-Vn-Scr and UAS-Vc-Scr. Four of these double lines were crossed to sgs3-Gal4 for expression in the salivary glands. Third instar well-fed larvae bearing both UAS-constructs and the Gal4-driver were then dissected in Grace’s medium and the salivary glands were transferred to microscopic chambers in

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PBS. Salivary glands were then imaged immediately using a Leica SP5 confocal microscope. Three nuclei per pair of salivary glands (three pairs in total) were imaged (using the same optical settings), making a total of nine nuclei per Vc/Vn pair and a total of 36 nuclei per investigated Scr construct. The total nuclear ﬂuorescence was then measured using the Leica SP5 software and the background ﬂuorescence intensity was subtracted from the total intensity. Error bars in Fig. 4B represent the standard deviation from the mean value, obtained from 36 measurements. Control of expression of all UAS-constructs generated

25 pairs of salivary glands expressing either Vn-Scr or Vc-Scr constructs were dissected and lysed using a pestle. A standard Western blotting protocol was then followed using different anti-GFP antibodies that recognize either the N-terminal or the C-terminal portion of Venus. The N-terminal portion of Venus was detected using a rabbit monoclonal to GFP (Abcam), whereas for the C-terminal portion a mouse monoclonal to GFP was used (Roche). Both antibodies were used in 1:1000 dilutions.

Results

Scr molecules physically interact to form dimers via a C terminal portion that contains the HD

We have previously observed that heterodimer formation between different HD-containing transcription factors (Antp and Eyeless (Ey)) is mediated by the HD and results in mutual inhibition of their function (Plaza et al., 2008). Therefore, we were interested to know whether HD–HD binding also takes place between molecules of the same transcription factor and whether this could have any functional implication in Hox-mediated gene regulation. Thus, we have analyzed two synthetic Hox genes, Antp and Scr, for their ability to form homodimers in live cells. The synthetic peptides contain the HD, YPWM motif and the small C terminus and their function in vivo has been previously confirmed genetically and biochemically (Papadopoulos et al., 2011;Papadopoulos et al., 2010). To visualize interactions in live cells we used the Bimolecular Fluorescence Complementation (BiFC) system (Hu et al., 2002;Saka et al., 2007) and tagged the synthetic peptides with the N- or C-terminal portion of Venus fluorescent protein. We observed that only Vn-SynthScr/ Vc-SynthScr partners interacted in HEK293T cell nuclei, whereas the fluorescence intensity of the Vn-SynthAntp/Vc-SynthAntp pairs was more than 25 times less (Fig. 1). Because binding studies using the purified Antp HD have revealed that it does not dimerize in vitro (Affolter et al., 1990), dimer formation of SynthScr has been surprising, given the sequence similarity of its HD to the Antp HD (the two HDs differ by 5 out of 60 amino acids only).

In order to understand this result further, we used full-length (FL-Scr) and synthetic Scr (Synth(FL-Scr) peptides to study the physical interaction between Scr molecules in coimmunoprecipitation experi-ments. Each construct was tagged N-terminally with either V5 or Flag epitopes. We have taken into consideration that in this experiment only the differentially tagged transcription factors (i.e. V5-Scr/Flag-Scr dimers) can be detected as interacting partners, whereas the fractions of V5-Scr/V5-Scr and Flag-Scr/Flag-Scr dimers are not detectable, even though they are assumed to form in equal proportions to the V5-Scr/ Flag-Scr fraction. Therefore, to compensate for these technical limita-tions and for the low expression levels and transfection efﬁciency of these constructs in Drosophila S2 cells (data not shown), we have used human HEK293T cells. We have used both the wt and the constitu-tively inactive form of Scr in which threonine 6 and serine 7 of the HD have been substituted by aspartates (ScrDD) (phosphomimicking form), as previously described (Berry and Gehring, 2000;

Papadopoulos et al., 2010). This inactive Scr variant has limited function in vivo, because the introduction of aspartates in the ﬂexible N-terminal arm of the HD prevents its binding to the minor groove of the DNA. Fig. 2A summarizes the full-length and synthetic Scr constructs used in coimmunoprecipitation experiments and the residues mutated in each construct. Coimmunoprecipitations showed that both FL-Scrwtand SynthScrwtpeptides form homodimers (Fig. 3A and B, left columns) and the interaction is comparably strong between them. FL-ScrDDand SynthScrDDpeptides can also interact physically (Fig. 3A and B, right columns), although the synthetic peptides dimerize much weaker than their full-length equivalents. These results show that Scr dimerizes ex vivo and that the long N terminus of Scr is essentially not required for dimer formation. Glutamate 19 of the HD is required for Scr homodimer formation

We have then asked which amino acids could be responsible for dimer formation in Scr. Since homodimerization also occurs in synthetic Scr peptides, the responsible amino acids should be present in the portion of Scr that contains the YPWM motif, the HD and the C terminus (seeFig. 2A for reference). The YPWM motif is the cofactor-interacting motif and therefore not expected to be involved in Scr–Scr binding, and the C terminus has been poorly conserved in evolution (Fig. S1), leaving the HD as the only conserved domain that might account for dimer formation. However, certain sequences in the HD are responsible for its DNA binding activity (Qian et al., 1989), whereas others that are situated away from the protein–DNA inter-face are exposed to possibly participate in protein–protein interac-tions, while still allowing the transcription factor to bind DNA. We have previously shown that glutamate at position 19 of the HD is crucial for binding of the HD to the PAIRED domain (PD) of Ey (Plaza et al., 2008), resulting in partial to complete repression of the eye developmental pathway. This prompted us to test whether the same amino acid, conserved from ﬂies to humans (Fig. S1), would be responsible for Scr dimerization. We therefore mutated glutamate 19 of the Scr HD to glycine (E19G) in both synthetic and full-length Scr

0 20 40 60 80 100 SynthScr dimer SynthAntp dimer Percentage of Venus positive cells Vc-SynthScr/Vn-SynthScr Vc-SynthAntp/Vn-SynthAntp

Fig. 1. Scr, but not Antp, forms homodimers in live cells. Bimolecular Fluorescence Complementation of synthetic Scr and Antp peptides indicates that only Scr forms dimers in HEK293T cell nuclei. Scr dimerizes in about 80% of cotransfected cells, whereas Antp dimerization is negligible (less than 5%). The cytoplasm has been counterstained with Cyan Fluorescent Protein.

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peptides bearing the wt and constitutively inactive N-terminal arm of the HD (ScrDD) (Fig. 2A andFig. 3C and D). Coimmunoprecipitations in HEK293T cells showed that the ability of Scr to form homodimers is completely abolished in the E19G mutants, both in the context of the full-length (Fig. 3D) and the synthetic transcription factor (Fig. 3C). This result shows that glutamate 19 of the HD is critical for Scr homodimerization.

Scr dimer formation is facilitated by DNA binding

Coimmunoprecipitations using total cell lysates provided evi-dence for physical interaction between Scr molecules. However,

how do Scr dimers function in flies? To address this question we generated UAS lines for all Scr constructs fused to the N- or C-terminal portions of Venus (Vc and Vn, respectively) and initially quantified dimer formation in live salivary gland cells, using BiFC. Although individual Vc or Vn UAS constructs were expressed in embryos in approximately equal amounts (Fig. 4C), when coinduced in salivary glands using sgs3-Gal4, only the FL-Scrwtand SynthScrwt lines significantly formed dimers. Dimerization was almost com-pletely abolished in coexpressed Vn- and Vc-ScrE19G constructs and clearly lower in the ones bearing the DD substitution (Fig. 4A). Except for the coexpressed Vn- and Vc-tagged wt full-length and synthetic Scr constructs that strongly bound DNA all along the polytene chromosomes, all other variants examined (those bearing the DD mutation, the E19G mutation, or both; in both the context of the full-length protein and the synthetic peptides) exhibited a small amount of dimerization. The protein dimers appeared excluded from polytene chromosomes and appeared to reside mostly in the nucleoplasm (Fig. 4A). Although we cannot exclude the possibility that part of the observed BiFC fluorescence in E19G mutants might be due to the molecular crowding of the Vn- and Vc-portions of Venus when these are overexpressed in a densely packed polytene nucleus, the efficiency of dimerization of the wt variants and their strong association with the DNA argues in favor of dimerization being facilitated by DNA binding of the transcription factor.Fig. 4B quantitatively summarizes the differences in fluorescence intensity between Vn/Vc partners for different Scr lines.

Scr dimerizes on speciﬁc binding sites in live cells

Our analysis in salivary gland cells provided support that Scr dimers form in vivo and that they depend to a great extent on DNA binding. Therefore, we were interested to know whether dimers can also be observed on native Scr binding sites in live cells. We have previously followed the kinetics of SynthScr binding to polytene chromosomes and found that specific binding of SynthScrwt peptides to polytene chromosomes is best studied at low expression levels, because, under conditions of overexpres-sion, non-specific binding of the transcription factor becomes very pronounced, masking to a large extent the specific-interac-tions (Papadopoulos et al., 2010;Vukojevic et al., 2010). On the other hand, under low expression levels, the identification by live imaging of polytene bands and interbands bound by the tran-scription factor in salivary gland nuclei is difficult, making the Scr coding sequence aa 301 aa417 E19G E19G E19G Flag V5 (cell culture) (flies) UAS Vn UAS Vc (flies) Flag V5 (cell culture) UAS UAS Vn Vc TkrQr TS TS DD TS DD TS TS DD DD

Antp coding sequence

RkrGrQT ce cu ure RkrGrQT RkrGrQTE19G Flag V5 (cell culture) Flag V5 (cell culture) (cell culture) Flag

Exd coding sequence (aa 144-376)

ell cultur la xd codin se uen aa 144-37 diverse N -terminus YPWM motif linker region Scr sequence Antp sequence cr sequ nt Exd sequence homeodomain C terminus PBC-B domain

Fig. 2. Schematic representation of constructs used. (A) Representation of Scr constructs used for coimmunoprecipitation experiments in HEK293T cells and for generation of transgenic flies. The colors represent the different domains of Scr and are explained at the bottom. In the wt full-length Scr construct the seven N-terminal amino acids of the HD (N-terminal arm) are designated (aa 1–7 of the HD), with upper-case letters indicating the four residues that differ between Antp and Scr. A rectangle outlines the sixth and seventh residue of the HD (TS) that has been mutated to aspartates (DD) in the constitutively inactive full-length and synthetic Scr constructs (see text for details). Mutated residues (DD or E19G) are indicated by purple color. The synthetic peptides span the region from aa 301 to aa 417, that corresponds to the YPWM motif, linker, HD and C terminus of the protein. On the left side of full-length and synthetic Scr constructs, the tags used for generating N-terminally tagged proteins for expression in flies or in cell culture are indicated. Vn and Vc designate the N- or C-terminal portion of the Venus fluorescent protein, respectively. Vn- and Vc-tagged constructs have been coexpressed in flies in Bimolecular Fluorescence Complementation experiments. All constructs have been combined with all tags indicated on the left side. (B) Representation of Antp constructs used in coimmunoprecipitation experiments in HEK293T cells. The residues of the N-terminal arm of the HD that are diverse between Antp and Scr are indicated by upper-case letters as in (A). Substitutions of these residues to the corresponding Scr residues in full-length Antp constructs are shown in red. The synthetic Antp constructs span the region between the YPWM motif and the C terminus. Hybrid Antp constructs are drawn to contain either the linker or the C terminus of Scr. Combinations with Flag or V5 epitopes have been generated for all constructs indicated. (C) Representation of the Exd construct used in coimmunopre-cipitations in HEK293T cells and in gel-shift experiments (aa 144–376).

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Scr Scr Scr GFP GFP IP: V5, WB: Flag Lysates, WB: Flag IP: V5, WB: V5 IP: Flag, WB: V5 Lysates, WB: V5

IP: Flag, WB: Flag

Scr Scr Scr GFP GFP SynthScr_DD V5-Scr V5-Scr + + -V5-GFP + -FL-ScrDD V5-Scr V5-Scr + + -V5-GFP + -Flag-Scr Flag-Scr Flag-GFP IP: V5, WB: Flag Lysates, WB: Flag IP: V5, WB: V5 V5-Scr V5-Scr + + -V5-GFP + -SynthScr_wt Scr Scr Scr GFP GFP Flag-Scr Flag-Scr Flag-GFP IP: Flag, WB: V5 Lysates, WB: V5

Scr Scr Scr GFP GFP FL-Scrwt V5-Scr V5-Scr + + -V5-GFP + -V5-Scr V5-Scr V5-GFP + + + -SynthScrDDE19G Scr Scr Scr GFP GFP IP: V5, WB: Flag Lysates, WB: Flag IP: V5, WB: V5 V5-Scr V5-GFP V5-Scr + + + -FL-ScrDDE19G IP: Flag, WB: V5 Lysates, WB: V5

IP: Flag, WB: Flag

Scr Scr Scr GFP GFP Flag-Scr Flag-Scr Flag-GFP IP: V5, WB: Flag Lysates, WB: Flag IP: V5, WB: V5 V5-Scr V5-Scr V5-GFP + + + -SynthScrE19G Scr Scr Scr GFP GFP Flag-Scr Flag-Scr Flag-GFP V5-Scr V5-GFP V5-Scr + + + - -FL-ScrE19G IP: Flag, WB: V5 Lysates, WB: V5

IP: Flag, WB: Flag

Scr

Scr Scr

GFP

Fig. 3. (A and B) Synthetic and full-length Scr molecules physically interact to form dimers. (A) HEK293T cells were transfected with constructs encoding synthetic

Flag-Scrwtor Flag-ScrDD, Flag- or V5-GFP as a control, and synthetic V5-Scrwtor V5-ScrDD, respectively. Immunoprecipitation was conducted using anti-V5 coupled beads. Cell

lysates were analyzed by immunoblotting with anti-Flag antibody. Blots were stripped and reprobed with anti-V5 antibody. Homodimerization of Scr was conﬁrmed by

immunoblotting with anti-Flag antibody. (B) HEK293T cells were transfected as above with FL-Scrwtor FL-ScrDDconstructs, Flag- or V5-tagged. The Flag-tagged Scr

proteins were immunoprecipitated with M2 sepharose beads; precipitation of Flag-Scr immobilized V5-Scr, but not V5-GFP, while precipitation of Flag-GFP did not bind V5-Scr. Protein expression levels were analyzed using an anti-Flag antibody. Red arrowheads show dimer formation. (C and D) Mutation of glutamate 19 of the HD in

synthetic (C) and full-length (D) Scr peptides completely abolishes dimer formation. The same method was used as inFig. 3(A) and (B), respectively, for synthetic and

full-length constructs, bearing the E19G substitution, with or without the DD mutation. Red arrowheads show absence of dimer formation. Non-speciﬁc bands indicate light or heavy chain of the dissociated antibody, wherever present. IP, immunoprecipitation; WB, Westen blot.

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presence of speciﬁc sites along the polytene chromosomes not easily identiﬁable. To bypass these technical limitations, we have introduced a controllable giant synthetic binding site containing

50 repeats of the Scr binding site fork head 250 (fkh250) (Ryoo and Mann, 1999) and 50 repeats of UAS, the Gal4 binding site, upstream of the lacZ coding sequence (Fig. 4D). We observed that

50XUAS 50Xfkh250 lacZ 50 X UAS 50 X fkh250 dppblink_-Gal4 or sgs3-Gal4

UAS-Vc-SynthScr UAS-Vn-SynthScr UAS-Gal4-mRFP1

50Xfkh,50XUAS-lacZ SynthScr wt SynthScrE19G Scr Scr Gal4 Gal4 overlay overlay 0 20 40 60 80 100 120 140 SynthScrE19G SynthScr DD E19G SynthScr DD SynthScr wt FL-Scr wt FL-Scr DD FL-Scr E19G FL-Scr DD E19G

Fluorescence intensity (a. u.)

SynthScrE19G SynthScr_DDE19G SynthScr_DD SynthScr_wt FL-Scrwt FL-ScrDD FL-ScrE19G FL-ScrDDE19G 20 kDa 25 kDa Vc-SynthScr wt Vc-SynthScr DD Vc-SynthScrE19GVc-SynthScr DD E19G 25 kDa 37 kDa Vn-SynthScr wt Vn-SynthScr DD Vn-SynthScrE19GVn-SynthScr DD E19G 50 kDa 75 kDa Vc-FL-Scr wt Vc-FL-Scr DD Vc-FL-ScrE19GVc-FL-Scr DD E19G 50 kDa 75 kDa Vn-FL-Scr wt Vn-FL-Scr DD Vn-FL-ScrE19GVn-FL-Scr DD E19G

Fig. 4. (A–C) Scr dimer formation is facilitated by DNA binding in live salivary gland cells. (A) Bimolecular Fluorescence Complementation images of fly salivary gland cells coexpressing Vc- and Vn-tagged Scr peptides, using sgs3-Gal4. The wt full-length and synthetic transcription factors interact strongly and associate very tightly with polytene chromosomes. Peptides bearing the DD substitution associate weakly, the transcription factors reside predominantly in the nucleoplasm and are excluded from polytene chromosomes. Peptides with an E19G substitution (with or without the DD mutation) fail to dimerize in live cells. (B) Average fluorescence intensity of nuclei expressing combinations of Vn/Vc partners (see Materials and Methods for details). Error-bars indicate the standard deviation obtained from 36 measurements. (C) Control of expression levels of UAS-Vc-Scr and UAS-Vn-Scr levels in salivary glands. All constructs are expressed in comparable levels. Dashes on the sides indicate the closest molecular weight indicators. Different GFP antibodies have been used to detect either Vc- or Vn-tagged peptides (see Materials and Methods for details). (D and E) Specific Scr-binding sites are bound by Scr dimers in vivo. (D) Schematic representation of the generation of a giant synthetic binding site in live salivary gland cells. An array of 50 repeats of the fkh250 enhancer has been cloned upstream of a 50mere of UAS sites in a lacZ vector. In flies bearing this reporter, it is expected that Gal4 expression in

salivary gland cells (driven by sgs3-Gal4 or dppblink_{-Gal4) induces the expression of Vn-/Vc-Scr constructs and Gal4-mRFP1, resulting in neighboring or overlapping giant}

binding sites on polytene chromosomes. (E) Live imaging of salivary gland nuclei expressing the constructs outlined in (D). The SynthScrwtlines present overlapping

accumulation patterns for both Scr (green channel) and Gal4 (red channel) on the synthetic puff. In this case the Scr dimers occupy the polytene chromosomes almost exclusively, whereas the Gal4 peptides reside predominantly in the nucleoplasm. In nuclei expressing the SynthScrE19G peptides, accumulation of only Gal4 is observed on the giant synthetic binding site and Scr is almost completely excluded from polytene chromosomes and resides in the nucleoplasm. Arrows show the sites of Scr-Gal4 accumulation and arrowheads show foci, occupied by Gal4 only.

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salivary gland cells carrying this binding site and expressing Vn-FL-Scrwt, Vc-FL-Scrwtand a red-labeled Gal4 peptide (UAS-Gal4-mRFP1), in the presence of a salivary gland-expressed Gal4 driver build a giant transcriptional puff, in which the red and green peptides colocalize on the giant binding site (Fig. 4E, upper row). The red-labeled Gal4 peptide allowed us to identify that the site of accumulation of Scr dimers is indeed the introduced site. Meanwhile, salivary gland cells expressing the FL-ScrE19G pep-tides showed that only Gal4 peppep-tides accumulate on the binding site (Fig. 4E, lower row). These results indicate that Scr dimers form on speciﬁc binding sites in live cells.

Scr dimers are required for transcription of fkh

Although salivary glands expressing Scr in the presence of the giant binding site also very strongly expressed lacZ (data not shown), we could not exclude the possibility that the tandem repeats of the fkh250 element to some extent artiﬁcially induced dimerization due to the proximity of the binding sites, which are aligned one after the other on the chromosome. At the same time, we wanted to know whether Scr dimers are required for Scr-mediated transcription of the fkh gene in vivo. We therefore expressed all Scr lines in the early embryo, using the nullo-Gal4 driver (Gehring et al., 2009) in the presence of a previously characterized fkh-lacZ reporter (Zhou et al., 2001). The SynthScrwt and the FL-Scrwtwere the only constructs able to induce ectopic expression of fkh in the head region (Fig. 5), lending support that Scr dimer formation is indeed required for expression of the fkh gene.

Dimer formation is required for Scr homeotic function

We have then asked whether Scr dimerization is restricted to certain functions of the transcription factor or whether it represents a generic principle of Scr function in vivo. Due to the unavailability of an E19G mutant line for the endogenous Scr gene, we analyzed the UAS lines in gain-of-function assays in the embryo and adult ﬂies. We tested them for their ability to induce ectopic salivary

glands in the embryo, to transform the second and third thoracic segment of the embryo toward prothoracic identity (posterior Scr gain-of-function phenotype), as well as their transformation capa-city in the antenna. We found that only the SynthScrwt and the FL-Scrwt peptides could exhibit homeotic function in the embryo and adult ﬂies (Fig. 6), whereas as little as substitution of one amino acid in the HD (E19G mutant constructs) completely abolished Scr homeotic function. This result indicates that Scr dimerization is a requirement for a plethora of functions in vivo and is rather the rule in Scr-mediated gene regulation.

We have then evaluated the ability of Scr to cause eye reduction phenotypes in the developing head by overexpressing the various constructs with dppblink-Gal4. This is an appropriate phenotypic indication of heterodimer formation with the Ey transcription factor. We observed that all variants bearing an E19G mutation did not show eye reduction phenotypes (Fig. S2). The SynthScrwt, FL-Scrwt and FL-ScrDD lines showed from com-plete eye absence to strong eye reduction, respectively, whereas SynthScrDDhad almost no effect in eye development. The inability of the SynthScrDDpeptide to interact with the Ey PD might be due to electrostatic interactions introduced by the aspartate residues that have a greater effect in the smaller, synthetic peptide as compared to the full-length protein, as previously hypothesized (Papadopoulos et al., 2010).

Previous genetic and biochemical evidence supports the notion that the E19G mutation does not interfere with the DNA-binding ability of the Antp HD, nor does it alter its homeotic function in the antenna (Plaza et al., 2008). This suggests that the same principle might apply to the Scr HD, which is the HD most closely related to the Antp HD in ﬂies (Fig. S3). Nevertheless, to exclude the possibility that the failure to exhibit gain-of-function phenotypes of the E19G variant is attributed to a defective folding of the protein or a failure to interact with other proteins (e.g. cofactors) that are crucial for Scr function in vivo, we performed additional control experiments. To assess the ability of full-length peptides to bind native enhancers, containing HD-binding sites, we tested them in gel-shift experi-ments using the recently characterized antennal enhancer of the spineless (ss) gene (D4 enhancer) (Duncan et al., 2010) (Fig. S4). The FL-Scrwtand the FL-ScrE19G peptides bound DNA equally as strong, whereas their DD counterparts did not. Like in Antp, this result shows that the DNA-binding activity of the HD is not altered upon introduction of the E19G mutation, suggesting that the transcription factor three-dimensional structure is not gravely impaired.

Second, we have examined whether the E19G substituted protein could physically interact with Exd. Therefore, we coex-pressed V5-tagged FL-Scrwtor FL-ScrE19G proteins with a Flag-tagged Exd protein (amino acids 144–376), that contains the PBC-B domain, HD and C terminus of the protein (Fig. 2C) and sufﬁces to cooperatively bind to Hox/Exd target sites in vitro and in vivo (Papadopoulos et al., 2011). We observed that both the wt and the E19G substituted protein physically interacted with Exd in coimmunoprecipitation experiments (Fig. S5), indicating that the E19G variant retains its capacity to bind Exd.

Finally, we have evaluated the E19G Scr protein for its ability to cooperatively bind HD binding sites in gel-shift assays. In this case we used the BS2 binding site (Muller et al., 1988), the fkh250 enhancer and the D4 enhancer of ss (Fig. S6). In all three cases, we could observe binding of FL-Scrwt and FL-ScrE19G to these binding sites, as well as cobinding with Exd (Fig. S6).

Taken together, these results indicate that the E19G mutation abolishes dimer formation of Scr as well as its homeotic function in vivo, but it does not inﬂuence its capacity to bind HD-binding sites, to physically interact with cofactors like Exd or to coopera-tively bind with cofactors on the DNA.

SynthScrE19G SynthScrDDE19G SynthScrDD SynthScrwt FL-Scrwt FL-ScrDD FL-ScrE19G FL-ScrDDE19G

Fig. 5. Scr dimer formation is required for ectopic expression of fkh in embryos.

Z-projections of embryos carrying a fkh-lacZ reporter, stained forb-galactosidase.

Only embryos expressing the SynthScrwt or FL-Scrwt construct show ectopic

expression of fkh in the region of the embryonic head (cyan arrowheads), in addition to the normal fkh expression. All lines have been induced by a nullo-Gal4 driver.

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SynthScrwt SynthScrDD SynthScrE19G SynthScrDDE19G FL-Scrwt FL-Scr_DD FL-ScrE19G FL-ScrDDE19G

Fig. 6. Scr dimerization is required for its homeotic function in the ﬂy. Homeotic phenotypes induced by ectopic expression of Scr in the embryo (left and middle column), as well as adult ﬂy (right column). The images of lines causing transformations are outlined by red boxes for clarity. (Left column) Induction of additional salivary glands in the

region of the embryonic head is achieved only by synthetic and full-length wt Scr constructs. The constitutively inactive form of Scr (ScrDD) and constructs bearing the E19G

mutation (ScrE19G) fail to cause formation of ectopic salivary glands. Red arrowheads point at the additional salivary glands. Stainings are for dCreb-A, a salivary gland luminal marker. A heat-shock-Gal4 driver has been used throughout. (Middle column) Posterior transformations caused by constitutive expression of Scr constructs during embryogenesis. Only synthetic and full-length Scr lines result in the transformation of mesothoracic and metathoracic segments towards prothoracic segments, identiﬁed by the presence of ectopic beards (cyan arrowheads indicate the normal beards and red arrowheads represent the ectopic beards). A nullo-Gal4 driver has been used throughout.

(Right column) Tarsal transformations in the antenna induced using a Distal-less-Gal4 driver. The SynthScrwtand the FL-Scrwtlines cause a complete transformation, whereas

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The linker and C terminus are independently required to confer homodimerization in Scr, whereas the diverse N terminus and the N-terminal arm of its HD are dispensable for dimer formation

Coimmunoprecipitations, live imaging in HEK293T cells and in Drosophila salivary gland nuclei using BiFC, as well as genetic analyses of Scr gain-of-function phenotypes in the embryo and the antenna, all indicated that homodimerization of Scr is critical for its function. However, we were confronted with an apparent paradox. Antp did not dimerize in vivo and the observed differ-ence in the dimerization capacities between Antp and Scr (where Scr homodimerizes whereas Antp does not) cannot easily be explained by glutamate 19, since this residue is identical in the two paralogs. Therefore, amino acid sequences that are diverse between the two proteins must contribute to their opposite behaviors in the formation of homodimers. Of these, the long N terminus of Scr is not required for dimer formation in vitro or in vivo, because the synthetic peptides, spanning the region between the YPWM motif and the C terminus of the protein (therefore not containing the N terminus) can strongly homo-dimerize, similar to their full-length counterparts. This leaves us with candidate differences in the region between the YPWM motif and the C-terminus of the protein. Of these, the HD of Scr differs in only five positions from the Antp HD, four of which reside in its flexible N-terminal arm (Fig. S3). The terminal histidine of the HD (the fifth diverse residue between the Scr and the Antp HDs) has not been conserved in evolution (Fig. S1) and functional analysis of chimeric Antp-Scr proteins has pre-viously shown that the transcriptional identity of Antp and Scr depend upon the four residues in the N-terminal arm of the HD that differ between the two proteins (Furukubo-Tokunaga et al., 1993).

To this end, we have exchanged these four residues in the full-length Antp protein with the corresponding residues of Scr, creating an Antp quadruple mutant protein (R1T, G4Q, Q6T, T7S), which bears the HD N-terminal ﬂexible arm of Scr

(FL-AntpScr HD N-term. arm), as well as a quintuple mutant construct,

bearing the aforementioned mutations and an additional muta-tion of glutamate 19 to glycine (FL-AntpScr HD N-term. armE19G). Fig. 2B summarizes the Antp constructs used. In this experiment we have expected that, if these diverse residues are responsible for conferring dimerization capacity in Scr, then Antp proteins bearing these substitutions might be able to de novo homodimer-ize. Coimmunoprecipitation experiments in HEK293T cells showed that like the wt Antp protein (FL-Antp), neither of the FL-AntpScr HD N-term. armor FL-AntpScr HD N-term. armE19G peptides could form dimers (Fig. 7), indicating that the N-terminal arm of the Scr HD is not sufﬁcient to confer neomorphic dimerization capacity in Antp.

Therefore, we asked whether other regions that are diverse between the two proteins, but included in the synthetic peptides, are responsible for contributing to dimer formation, leaving us with either the linker between the YPWM motif and the HD or the C terminus of the protein as candidate sequences. To examine these possibilities we constructed Flag- and V5-tagged synthAntp genes, encoding the wt Antp peptide or peptides in which the linker region or the C terminus had been substituted by the corresponding linker or C terminus of Scr (named SynthAntpScr

linkeror SynthAntpScr C term.respectively). In agreement with our

previous analysis in HEK293T cell nuclei by BiFC, we conﬁrmed that the wt SynthAntp construct does not form any homodimer, but both constructs, bearing either the linker or C terminus substitutions to the corresponding regions of Scr, now gained strong dimerization capacity (Fig. 8A). Because it seems not to matter whether the linker or the C terminus of Antp is substituted in order for the protein to de novo gain homodimerization

capacity, this suggests that both the linker and the C terminus of Scr are capable of conferring dimerization capacity indepen-dently from each other.

We have hypothesized that if this would hold true, the two neomorphic variants of SynthAntp should also be able to hetero-dimerize in trans, or with SynthScrwtpeptides, which are capable of forming dimers. Therefore, we tested the combinations of SynthAntpScr linker/SynthAntpScr C term., SynthAntpScr linker/SynthScrwt and SynthAntpScr C term./SynthScrwtin their ability to form hetero-dimers (Fig. 8B). We observed that all variants could interact physically, arguing in favor of the linker and the C terminus of Scr being independently sufﬁcient to confer de novo dimerization in the presence of a wt glutamate residue at position 19 of the HD. Table 1 summarizes our in vitro and in vivo ﬁndings with the synthetic and full-length proteins, as outlined inFig. 2.

Discussion

While Hox transcription factors harbor very conserved and similar HDs, they display very different regulatory properties and instruct different, paralog-speciﬁc fates during segmental differentiation. For

Flag-Antp Flag-Antp Flag-GFP V5-Antp V5-Antp + + -V5-GFP + -FL-Antp V5-Antp V5-Antp + + -V5-GFP + -FL-Antp Scr HD N-term. arm E19G V5-Antp V5-Antp + + -V5-GFP + -FL-Antp Scr HD N-term. arm IP: Flag, WB: V5 Antp Antp GFP Lysates, WB: V5

Antp

GFP

Fig. 7. Scr N-terminal arm of the HD cannot confer neomorphic dimerization capacity to Antp. HEK293T cells were transfected with different combinations of V5- and Flag-tagged Antp constructs. A similar process has been used as in Fig. 3(B) and (D). Neither the wt full-length Antp construct (FL-Antp), nor the Antp

constructs bearing the residues of the N-terminal arm of the HD of Scr (FL-AntpScr

HD N-term. arm, see text for details), with or without the E19G mutation, show any dimerization. V5- and Flag-GFP were used as a negative control. Cell lysates were probed with anti-V5 antibody. Red arrowheads show absence of dimerizing proteins.

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example, the Scr and Antp HDs differ in as little as five amino acids (Fig. S3) and their long and diverse N-terminal sequences are required only for transcriptional fine-tuning and not for their functional specificity in vivo (Papadopoulos et al., 2011; Papadopoulos et al., 2010). Yet, they retain very different roles during development. These lines of evidence have given rise to the question of what determines the mode of action of so closely-related Hox proteins. We have found that, of Antp and Scr, only Scr peptides dimerize in vivo and require

glutamate 19 in the highly conserved ELEKEF motif of the HD. Mutation of this single residue abolished dimerization almost com-pletely. We have also observed that Scr dimerization is greatly facilitated by DNA binding, which implies cooperative binding of Scr transcription factor on the DNA. Moreover, we have shown that Scr dimers bind speciﬁcally to their native target sequences in live salivary gland cells and that dimerization is required for Scr homeotic function in vivo. Finally, we have examined the basis of the IP: Flag, WB: V5

Lysates, WB: V5

IP: Flag, WB: Flag Antp (wt/ hybrids) Antp (wt/ hybrids) GFP Antp (wt/ hybrids) GFP Flag-SynthAntp Flag-SynthAntp Flag-GFP + + -+ -V5-Antp V5-GFP V5-Antp SynthAntp + + -+ -V5-Antp V5-GFP V5-Antp SynthAntp Scr linker + + -+ -V5-Antp V5-GFP V5-Antp SynthAntp Scr C term. Flag-GFP Flag-SynthAntpScr C term. Flag-SynthAntpScr C term. Flag-SynthAntpScr linker Flag-SynthAntpScr linker Flag-GFP Flag-SynthAntpScr C term. Flag-SynthAntpScr C term. Flag-SynthScrwt V5-SynthAn tpScr linker + + -SynthScr V5-SynthAntp Scr linker wt + -IP: Flag, WB: V5 Lysates, WB: V5

IP: Flag, WB: Flag SynthAntpScr linker

SynthScrwt

SynthAntpScr linker SynthScrwt

SynthAntpScr C term. SynthScrwt

Fig. 8. Linker region between the YPWM motif and the HD, and C terminus, are independently sufﬁcient to confer homodimerization capacity in Scr. (A) Synthetic Antp

constructs (SynthAntp) (schematically represented inFig. 2(B)) do not interact physically to form homodimers (lanes 1–3), but if either the Antp linker or the C terminus

are substituted by the corresponding amino acid sequences of Scr (SynthAntpScr linkeror SynthAntpScr C term., respectively) (lanes 4–9 and seeFig. 2(B) for a schematic

representation), dimerization capacity is gained. Precipitation of Flag tagged SynthAntpScr linkeror SynthAntpScr C term.constructs immobilized the corresponding V5-tagged

constructs (red arrowheads), respectively, but not V5-GFP, while precipitated Flag-GFP did not bind any of them. (B) SynthAntpScr linkerand SynthAntpScr C term.interact

physically with each other and with SynthScrwtconstructs to form heterodimers. HEK293T cells transfected with Flag-tagged SynthAntpScr C termand either a V5-tagged

SynthAntpScr linker or a SynthScrwt construct, or alternatively with a Flag-tagged SynthAntpwt and a V5-tagged SynthAntpScr linker construct were lysed and

immunoprecipitation was performed using M2 beads. Precipitates were blotted with the antibodies indicated. Red arrowheads indicate absence or presence of dimer formation.

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distinction in dimerization capacity between Antp and Scr. We have found that, while the long N termini and the N-terminal arms of the HDs of these transcription factors are largely dispensable for instruct-ing/preventing dimer formation, the Antp linker between the YPWM motif and the HD, and its C terminus are necessary for preventing dimer formation. Substitution of either of these domains with the corresponding domains of Scr readily conferred neomorphic dimer-ization capacity in Antp peptides. These results support a novel mechanism for functional specialization of very closely related Hox transcription factors.

Substitution of glutamate 19 by glycine in the Antp HD still allows the transcription factor to bind DNA specifically and instruct Antp-specific phenotypes in flies (Plaza et al., 2008), because AntpE19G mutant proteins are able to transform the antenna to a mesothoracic tarsus. Here, we find that this does not hold true for Scr. While ScrE19G peptides still bind native DNA enhancer sequences in vitro (Fig. S4), physically interact with Exd like the wt protein (Fig. S5) and cooperatively bind with Exd to three Hox/Exd binding sites in vitro (Fig. S6), they still cannot confer any Scr-specific phenotypes in vivo. Our findings are in line with recent functional evidence supporting that the YPWM motif of Scr is required for its interaction with Exd (Lelli et al., 2011).

Structural analysis of the DNA-bound HD has revealed that helix 3 of the HD recognizes AT-rich regions on the major groove and the ﬂexible N-terminal arm of the HD binds via arginine 5 the minor grove of the DNA (Billeter et al., 1993;Qian et al., 1992), whereas helix 1, containing the ELEKEF motif and glutamate 19, is residing at the opposite side of the molecule and does not participate in contacts with the DNA. This suggests that potential interactions of this amino acid region with other peptides might not impair the ability of the transcription factor to remain bound to the DNA.

Previous studies using chimeric Scr and Antp proteins have shown that their specificities rely on the flexible N-terminal arm of the HD and the four amino acids in which the two transcription factors differ therein (Furukubo-Tokunaga et al., 1993). Two of these amino acids in Scr can be phosphorylated, which essentially abolishes DNA binding (Berry and Gehring, 2000). Therefore, dimer or no-dimer formation is by no means the only functional distinction between Antp and Scr. However, in the light of our new findings, we uncover an essential contribution of the homo-dimerization of Scr to its homeotic function in vivo.

Homo- or heterodimerization of HD-containing proteins have been reported to be general mechanisms of gene regulation by transcription factors. For example, the human Pax3 HD binds as a dimer on palindromic DNA sequences (Birrane et al., 2009). HD-mediated dimerization has been observed for Csx/Nkx2.5 during cardiac development (Kasahara et al., 2001) and in differentiating mouse embryonic stem cells the Mixl1 homeoprotein dimerizes on its binding sites (Zhang et al., 2009). Also, stem cell pluripotency has been reported to depend upon the dimerization ability of the HD-containing protein Nanog (Wang et al., 2008). In Drosophila, the specification of the dorsoventral axis in the wing depends on the LIM HD protein Apterous, which binds DNA as a dimer, together with a dimer of its cofactor dLDB/Chip (Milan and Cohen, 1999). Moreover, kinetic studies have shown that also bicoid binds cooperatively as a dimer on hunchback enhancers, containing either one or two HD binding sites (Lopes et al., 2005). This finding suggests that HD-containing transcription factors might form dimers even when only one of the two molecules is bound to the DNA. Also, it is not always clear whether the binding of two molecules of HD-containing proteins on the DNA involves physical interactions between the two peptides, in addition to their interactions with the DNA. In the cases where this holds true, it directly impacts on the stringency of binding site sequence selec-tion, in other words, specificity, because if both peptides are bound

Table 1 Summary of dime r formation capacity and homeotic function of the constructs analyzed in this study. Dimer formation in HEK cell nuclei (BiFC) Dimer formation (Co-IP) Dimer formation in salivary gland nuclei (BiFC) Ectopic expression of fkh-lacZ in the early embryo Induction of ectopic salivary glands in the embryo Posterior transform ations in the cuticle

Antenna-to- tarsus transformation

Eye reduction phe notype Heterodimer formation with Exd (Co-IP) Cooperative binding with Exd (EMSA) FL-Scr wt n/a Yes Yes Yes Yes Yes Yes Yes Yes Yes FL-Scr DD n/a Yes (weak) Yes (weak, excluded from DNA) No No No No Yes n/a n/a FL-ScrE19G n/a No No No No No No No Yes Yes FL-Scr DD E1 9G n/a No No No No No No No n/a n/a SynthScr wt Yes Yes Yes Yes Yes Yes Yes Yes n/a n/a SynthScr DD n/a Yes Yes (weak, excluded from DNA) No No No No No n/a n/a SynthScrE19G n/a No No No No No No No n/a n/a SynthScr DD E19G n/a No No No No No No No n/a n/a FL-Antp n/a No n/a n/a n/a n/a n/a n/a n/a n/a FL-Antp Scr HD N-term. arm n/a No n/a n/a n/a n/a n/a n/a n/a n/a FL-Antp Scr HD N-term. arm E19G n/a No n/a n/a n/a n/a n/a n/a n/a n/a SynthAntp Yes No n/a n/a n/a n/a n/a n/a n/a n/a SynthAntp Scr linker n/a Yes n/a n/a n/a n/a n/a n/a n/a n/a SynthAntp Scr C term. n/a Yes n/a n/a n/a n/a n/a n/a n/a n/a

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to the DNA and also to each other, certain spacing limitations might apply between binding sites and/or DNA conformation.

So far we have studied the genetic and biochemical properties as well as the functional importance of Scr dimer formation in vivo. Structural studies should be helpful to elucidate precise topology properties of Scr dimeric complex formation on the DNA and the interactions that thereby take place among binding partners.

Acknowledgments

This work was supported by the Kantons of Basel-Stadt and Basel-Landschaft, a grant from the Swiss National Science Foun-dation and the European Network of Excellence ‘‘Cells into Organs’’. D.K.P. has been also supported by a European Molecular Biology Organization short term fellowship (ASTF 499-2010), a long term fellowship from the Swiss National Science Foundation (PBBSP-138700) and a long term fellowship from the Federation of European Biochemical Societies. K.S. was supported by a Wenner-Gren stipendium fr ˚an Wenner-Gren Stiftelserna.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version athttp://dx.doi.org/10.1016/j.ydbio.2012.04. 021.

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