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Molecular basis for the inhibition of Drosophila eye development by Antennapedia

2001; Springer Nature; Volume: 20; Issue: 4 Linguagem: Inglês

10.1093/emboj/20.4.802

1460-2075

Serge Plaza,

Invertebrate Immune Response Mechanisms

Article15 February 2001free access Molecular basis for the inhibition of Drosophila eye development by Antennapedia Serge Plaza Serge Plaza Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Frédéric Prince Frédéric Prince Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Johannes Jaeger Johannes Jaeger Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Urs Kloter Urs Kloter Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Susanne Flister Susanne Flister Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Corinne Benassayag Corinne Benassayag Centre de Biologie du Développement–CNRS, 31062 Toulouse, Cedex 04, France Search for more papers by this author David Cribbs David Cribbs Centre de Biologie du Développement–CNRS, 31062 Toulouse, Cedex 04, France Search for more papers by this author W.J. Gehring Corresponding Author W.J. Gehring Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Serge Plaza Serge Plaza Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Frédéric Prince Frédéric Prince Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Johannes Jaeger Johannes Jaeger Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Urs Kloter Urs Kloter Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Susanne Flister Susanne Flister Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Corinne Benassayag Corinne Benassayag Centre de Biologie du Développement–CNRS, 31062 Toulouse, Cedex 04, France Search for more papers by this author David Cribbs David Cribbs Centre de Biologie du Développement–CNRS, 31062 Toulouse, Cedex 04, France Search for more papers by this author W.J. Gehring Corresponding Author W.J. Gehring Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Search for more papers by this author Author Information Serge Plaza1, Frédéric Prince1, Johannes Jaeger1, Urs Kloter1, Susanne Flister1, Corinne Benassayag2, David Cribbs2 and W.J. Gehring 1 1Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland 2Centre de Biologie du Développement–CNRS, 31062 Toulouse, Cedex 04, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:802-811https://doi.org/10.1093/emboj/20.4.802 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia (Antp) in the head induces antenna-to-leg as well as head-to-thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study is to understand the basis of inhibition of eye development. We demonstrate that Antp represses the activity of the eye regulatory cascade. By ectopic expression, we show that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, we demonstrate that this inhibitory mechanism involves direct protein–protein interactions between the DNA-binding domains of EY and ANTP, resulting in mutual inhibition. Introduction The genetic and molecular analysis of the development of different model organisms has yielded a wealth of information about the underlying mechanisms of development. The theme emerging from these studies is that highly conserved genes are involved in the development of animals of strikingly different architecture and embryogenesis. The Hox genes, a subset of the Homeobox gene family, encode transcription factors and are a good example of functional conservation during evolution (Gehring et al., 1994). Hox genes are common to most or all animals, are organized in clusters, and define positional information along the antero–posterior axis. Homeotic mutations in Drosophila have led to the identification of several ‘master control’ genes. This term, initially introduced by Lewis (1992) for the homeotic genes of the bithorax complex, was illustrated by the genetic construction of four-winged and eight-legged flies. Loss- and gain-of-function in these genes lead to opposite homeotic transformations. For example, in Antennapedia (Antp), recessive loss-of-function mutations are lethal at the embryonic or larval stage and lead to a transformation of the second thoracic segment T2 toward the first thoracic segment T1 (Struhl, 1981; Schneuwly and Gehring, 1982; Abbott and Kaufman, 1986). Dominant gain-of-function mutations lead to a transformation in the opposite direction, i.e. from the anterior head and T1 segments toward T2 (Gehring, 1987). By ubiquitous expression of Antp under the control of a heat shock promoter, Schneuwly et al. (1987) changed the body plan of Drosophila by inducing the formation of middle legs in place of the antennae, second thoracic segment structures on the dorsal head capsule and inhibition of eye development. Similar changes in adult pattern have been observed upon ectopically expressing other Hox proteins. These transformations resulting from ectopic selector gene expression can be explained by a combinatorial interaction of two or more homeotic genes in order to specify a given body segment. However, the exact molecular mechanisms remain unknown. Recently, additional Drosophila selector genes have been identified that are capable of inducing organogenesis when expressed ectopically. One of the most striking examples is the transcription factor eyeless (ey), a homolog of Pax-6 in vertebrates (reviewed in Callaerts et al., 1997). In mammals, congenital dominant eye diseases known as aniridia (humans) and small eye (mice and rats) are caused by haploinsufficient loss-of-function mutations of Pax-6. Homozygous embryos lack eyes and nostrils completely, have brain and spinal cord malformations, and die prior to birth. In Drosophila, loss-of-function mutations of ey also show eye defects from subtle restructuring to complete loss (Quiring et al., 1994). In gain-of-function experiments, ectopic eyes are formed on the appendages of the fly (Halder et al., 1995). Ectopic expression of Pax-6 homologs from various species is sufficient to induce ectopic eyes in Drosophila, suggesting remarkably conserved mechanisms for eye differentiation (Callaerts et al., 1997; Gehring and Ikeo, 1999). The Drosophila compound eye develops from the eye–antenna disc, which invaginates from the ectoderm during embryogenesis and grows inside the larva. In the third larval instar, photoreceptor differentiation begins at the posterior margin of the eye disc and spreads anteriorly, led by a depression in the disc known as the morphogenetic furrow. Early determination of the eye primordium requires several nuclear proteins that are likely to act as transcriptional regulators. Like ey, the twin of eyeless (toy) gene encodes a Pax-6 homolog containing a paired and homeo DNA-binding domain (Czerny et al., 1999). Eye gone (eyg) encodes a Pax-like protein (Jun et al., 1998) and sine oculis (so) a homeodomain (HD) protein (Cheyette et al., 1994), while eyes absent (eya) and dachshund (dac) both encode novel nuclear proteins (Bonini et al., 1993; Mardon et al., 1994). Recently, optix, a homeobox gene related to the so gene family, was also shown to play a role in eye development (Toy et al., 1998; Seimiya and Gehring, 2000). Analysis of the expression pattern of these genes combined with a genetic approach in Drosophila has revealed a sequential and hierarchical deployment of these genes during eye development. toy is the first to be expressed and activates ey in the eye primordium (Czerny et al., 1999). eya, so and dac are further downstream and regulated by ey (Halder et al., 1998; Niimi et al., 1999; Zimmerman et al., 2000). eyg and optix are able to induce ectopic eye formation at least in part independently of ey, suggesting that they are involved in a parallel process for eye formation (H.Sun, personal communication; Seimiya and Gehring, 2000). Despite recent advances in understanding the mechanisms involved in the process of organogenesis, it remains unclear how the selector genes' activities are controlled and fine-tuned. For example, ey and toy are also expressed in the central nervous system (CNS) and the peripheral nervous system (Czerny et al., 1999), but only a small number of cells comprising the eye primordium will give rise to an eye. Moreover, when ey is misexpressed ubiquitously, ectopic eye development is restricted to specific regions of the disc (Halder et al., 1998; Chen et al., 1999). These findings show that somehow, resident genetic programs in many cells can inhibit Pax-6 function. The overexpression of various homeobox-containing proteins has been shown to inhibit eye development (Chadwick et al., 1990; Gibson et al., 1990; Zhao et al., 1993; Benassayag et al., 1997; Yao et al., 1999; Curtiss and Mlodzik, 2000). We have investigated the molecular mechanism of dominant eye loss induced by Antp. We find that ectopic ANTP protein induced in the eye is unable to repress ey transcription and translation. Nonetheless, eye development is impaired. Whereas EY is present, the EY target genes so, eya and dac are repressed. These experiments suggest that Antp blocks EY activity. To test this, we ectopically co-expressed ANTP and EY proteins in the same cells of Drosophila imaginal discs, and show that ectopic eye formation (induced by EY) is blocked. Conversely, expression of EY in the antenna disc is able to block the antenna-to-leg transformation induced by ANTP. We show that EY and ANTP interact directly in vitro, via the ANTP HD, and both the paired domain and the HD of EY. In yeast, ANTP inhibits transactivation by EY. Furthermore, in vitro binding of EY to specific DNA target sites is inhibited upon addition of ANTP. These experiments show that homeobox genes can inhibit each other through direct protein–protein interaction. Thus, they support the idea that the relative intracellular level of each protein is crucial for directing the cells into alternative differentiation programs. Results Antp mainly acts in front of the morphogenetic furrow to inhibit eye development by inducing apoptosis in the eye disc The ectopic expression of several homeotic proteins, including ANTP, has previously been shown to inhibit eye development. These results were obtained using different promoters, including the ubiquitous heat shock promoter (Gibson et al., 1990), the dppblink-GAL4 line (Pai et al., 1998) (Figure 1A) or the ey enhancer-GAL4 (EYE-GAL4) driver (Bello et al., 1998) (Figure 1B). The latter two promoters are expressed during early stages of eye differentiation. The eye-specific enhancer of ey induces gene expression in the eye primordia of the embryo, then maintains expression throughout eye morphogenesis. In contrast to endogenous ey expression, enhancer-driven reporter gene expression in the wild-type eye disc is not down-regulated in the differentiating cells posterior to the morphogenetic furrow but extends throughout the disc (Halder et al., 1998). dppblink expression starts in the undifferentiated cells and is maintained thereafter in the developing photoreceptors (Staehling-Hampton et al., 1994). Similar to results with the ey enhancer, ANTP expression in the eye disc directed by the dppblink-GAL4 driver also induces an eyeless phenotype (Figure 1A and B). However, these results do not clarify the question of whether Antp expression in front of (undifferentiated cells) or behind (photoreceptors) the morphogenetic furrow induces eye loss. Therefore, two additional GAL4 driver lines were employed to direct UAS-Antp expression: the GMR-GAL4 line carrying Glass Multimerized Responsive sites (Ellis et al., 1993) expressed in the differentiated ommatidia posterior to the furrow, and OK107-GAL4 (Connolly et al., 1996) expressed in front of the furrow (Figure 1D). Results show that while expression of Antp in the differentiated cells reduces eye pigmentation, it has only a minor effect on the eye size (Figure 1C). In contrast, expression of Antp in front of the furrow results in an eyeless phenotype (Figure 1E) resembling that obtained with EYE-GAL4 or dppblink-GAL4 (Figure 1A and B). Thus, Antp acts as a repressor of eye development in undifferentiated cells. Figure 1.Eye reduction induced by Antp using different drivers. Arrowheads show the lack of eyes (A, B and E), dashed lines delimit the eye size (C). (A) dppblink-Gal4; (B) EYE-GAL4; (C) GMR-GAL4; (E) OK107-GAL4. (D) β-galactosidase expression of OK107-GAL4 in the eye. The morphogenetic furrow is marked by an arrow. (F and G) Acridine orange stainings highlight dead cells (green), (F) wild type and (G) ANTP-expressing disc. Massive cell death is observed in the remaining portion of the disc. Download figure Download PowerPoint Previous work demonstrated that the eye-loss phenotype associated with the regulatory disruption mutation ey2 was a result of cell death in third instar larvae (Halder et al., 1998). This effect is very similar to those of the so1 and eya1 mutants (Bonini and Fortini, 1999). Thus, we tested whether Antp expression in the eye disc also led to increased apoptosis, assessing cell death by staining with the vital dye acridine orange. Massive cell death was observed in eye discs expressing Antp compared with wild type (Figure 1G versus F). These results clearly show a parallel between the Antp-induced gain-of-function phenotype and that for ey (and so and eya) loss-of-function, supporting the idea that Antp inhibits the eye developmental pathway. Antp expression in the eye disc disrupts the ey cascade Ectopic Antp expression induces an eyeless phenotype when expressed in front of the morphogenetic furrow. This observation prompted us to analyze the epistatic relationships among the eye determining genes toy, ey, so, eya, dac, eyg and optix. These genes are normally expressed in front of the furrow following a hierarchical pathway. ey, initially defined as the master control gene for eye morphogenesis, induces the expression of so, eya and dac (reviewed in Gehring and Ikeo, 1999; Treisman, 1999). Additional genes involved in the eye development pathway, like optix and eyg (Seimiya and Gehring, 2000; H.Sun, personal communication), may to some extent act in parallel to ey. In order to analyze the effect of Antp on these genes, we performed in situ hybridizations in the eye–antenna disc after expression of Antp. Whereas ey expression is not affected by ANTP (compare Figure 2A and B), the ey target genes eya, so and dac are repressed (Figure 2, compare C and D, E and F, G and H). Interestingly, the expression of optix (Figure 2I and J), toy (Figure 2K and L) or eyg (Figure 2M and N) is not affected. Altogether, these results indicate that Antp expression in eye precursor cells disrupts the ey regulatory cascade and leads to a phenotype resembling those observed in ey2, so1 and eya1 loss-of-function mutants. Figure 2.ANTP represses the ey regulatory pathway and blocks photoreceptor differentiation despite the presence of EY. In situ hybridization or immunostaining (G, H, O and P) experiments were performed on eye antenna third instar imaginal discs to study gene expression following Antp expression. (A, C, E, G, I, K, M, O and Q) Wild-type discs; (B, D, F, H, J, L, N, P and R) targeted expression of Antp with dppblink-Gal4. The magnification is 2-fold higher for the ANTP-expressing disc as compared with the wild type. (O and P) Immunostaining experiment using an αEY antibody (in green) and the α22C10 neuronal marker (in red). Note, in wild type (O) the expression of EY is restricted anterior to the furrow. (Q and R) Analysis of the EY responsive element so10 enhancer expression in wild-type and Antp-expressing discs. β-galactosidase staining was performed in parallel in wild-type (Q) as well as in Antp-expressing discs (R). Download figure Download PowerPoint Since ey transcription is not affected, we next wanted to know whether the EY protein is normally accumulated in the eye disc when Antp is expressed. Immunohisto chemistry experiments were performed using an anti-EY antibody as well as an antibody raised against the 22C10 neuronal marker. As seen in Figure 2P, following Antp expression the EY protein is easily detected throughout the disc; in contrast, neuronal differentiation is impaired (compare Figure 2O and P). Furthermore, immunostainings performed with an anti-ANTP antibody reveal the presence of ANTP in the region where ey is expressed (Figure 3G), indicating that both proteins co-localize. Figure 3.Ectopic eye induction mediated by ey is inhibited by Antp. Reciprocally, Antp-induced phenotype is inhibited by ey. EY, ANTP and both are expressed using the UAS/GAL4 system with dppblink-Gal4 as driver. (A) Ectopic eyes induced by ey. (B) Antenna-to-leg transformation and eye development inhibition induced by Antp. (C) Lack of ectopic eyes and antenna-to-leg transformation when both proteins are co-expressed. (D) ANTP HD-deleted protein is unable to repress ectopic eye formation. (E–G) EY and ANTP co-localize in the discs, resulting in the inhibition of ectopic eye development. Immunostaining experiments using EY (green) and ANTP (red) antibodies, analyzed by confocal microscopy. Only the merge is presented. (E) Wing disc; (F) leg disc; (G) eye–antenna disc (eye disc at the bottom). (H and I) ANTP-EY co-expression blocks neuronal differentiation and (J and K) so induction. (H) UAS-ey crossed with dppblink-Gal4. Wing disc stained with an EY antibody (green) and the 22C10 neuronal marker (red). (I) Same staining as in (H) but here EY and ANTP are co-expressed. (J and K) β-galactosidase expression of the so enhancer trap line following expression of EY (J) or EY and ANTP (K) in the wing disc. so is not expressed in the wild-type wing disc (not shown). (L–O) Effect on eye development of ANTP-deleted or mutated proteins. Crosses of the UAS constructs indicated in the figure were performed using the driver EYE-GAL4. (L) Wild-type eye; (M) UAS-AntpΔHD construct, HD is deleted; (N) UAS-AntpK50, the Antp DNA-binding specificity is changed to that of Bicoid; (O) UAS-AntpA50,51. Residues involved in DNA contacts have been mutated in order to abolish binding to DNA. Download figure Download PowerPoint Figure 4.ANTP interacts with EY in vitro and inhibits EY transactivation. (A) GST interaction assays were performed with GST or GST–ANTP constructs and 35S-labeled EY or luciferase as control. Input, 20% of the 35S protein involved in the assay. (B) Effect of PAIRED or HOMEO domain deletion on the binding to ANTP. (C) The EY PAIRED and HOMEO domain interact independently with ANTP. (D) Bacterially expressed purified domains are able to interact. One microgram of ANTP HD (pAop2CS; Müller et al., 1988) incubated with 3 μg of GST or GST–PAIRED was subjected to electrophoresis after interaction with GST beads and stained with Coomassie Blue. The arrow shows the ANTP HD. (E and F) Yeast one-and-a-half hybrid experiment. pAS and pACT: empty vectors. pAS-EY: EY encoding vector. pACT-ANTP 287–378: C-terminus of ANTP encompassing the HD is fused to the Gal4 activation domain. (E) Strain carrying the integrated reporter vector pLacZi containing four multimerized HB1 sites upstream of the minimal promoter of the yeast iso-1-cytochrome C gene. (F) Same as in (E) but instead of HB1 one copy of the so10 enhancer is inserted. Arrow: co-expression of ANTP HD and EY inhibit EY transactivation. (G) Bacterially synthesized ANTP HD inhibits PAIRED DNA binding to the 100 bp so probe (Niimi et al., 1999). His-PAIRED (20 ng) and His-ANTP peptides (1 μg) were co-incubated before adding the so probe (10 ng). The arrow shows the retarded complexes. Download figure Download PowerPoint Halder et al. (1998) have demonstrated that so requires ey for its expression in the eye disc. Furthermore, Niimi et al. (1999) recently defined an eye-specific enhancer (so10 fragment) deleted in the so1 mutant, which is directly regulated by ey. This 400 bp element by itself mimics endogenous so expression in the eye disc (compare Figure 2Q with 5A) and constitutes an EY responsive element (Niimi et al., 1999). Therefore, we tested the effect of Antp on so10 fragment expression in order to test whether or not Antp is able to repress this EY responsive element. As seen in Figure 2R, Antp efficiently represses expression directed by the so10 fragment (compare Figure 2Q and R). Taken together, these results strongly support the idea that ANTP blocks EY activity at the protein level. Figure 5.Competition between EY and ANTP in eye morphogenesis. (A–D) β-galactosidase stainings of the so enhancer trap line following expression of the different constructs indicated. dppblink-Gal4 is the driver. (A) so enhancer trap line expression in wild-type eye disc. (B) Antp represses so in the eye. (C) Arrow: ey induces so in the antenna. (D) ey;Antp co-expression results in so repression in the antenna (arrow) and so derepression in the eye. (E–G) Phenotype of adult flies after expression of the different lines indicated using EYE-GAL4. ey/Antp co-expression resulted in the rescue of Antp-induced eye defects. Download figure Download PowerPoint Ey and Antp are mutual inhibitors for directing eye and leg development If the ANTP protein is able to block EY activity, this mechanism should also function in other tissues. Therefore, ectopic eye formation should also be blocked by Antp. To test this prediction, we induced ectopic eyes on wing, antennae and legs using the UAS-GAL4 system (Figure 3A–D). Results show that the ectopic eye formation induced by ey is completely blocked on co-expressing ey and Antp (compare Figure 3A and C). Moreover, the Antp induced antenna-to-leg transformation (Figure 3B) is inhibited by ey (Figure 3C). A series of similar tests employing hs-ey and hs-Antp transgenes, singly or in combination, led to the same conclusions. Furthermore, they revealed a specific requirement for the ANTP HD, since N-terminal deletions of the ANTP protein do not affect its ability to inhibit EY activity, whereas deletion of the HD results in a protein unable to inhibit ey function (data not shown; constructs described in Gibson et al., 1990). Similarly, using the UAS-GAL4 system, the ANTP HD-deleted protein was unable to repress ectopic eye formation (Figure 3D). These results made it necessary to demonstrate that both proteins co-localize in the same cells of the discs. Upon examining protein accumulation by confocal microscopy, we found that both proteins are efficiently co-expressed in these different tissues (Figure 3E, F and G). Furthermore, immunostaining experiments performed using the ey antibody or the neuronal marker 22C10 confirm that, despite the presence of EY in the disc, co-expression of ANTP leads to inhibition of neuronal differentiation (Figure 3H and I). We next tested the effect on subordinate genes taking so as an example, since it is known to be a direct target of EY. Using a so enhancer trap line, we found that expression of Antp blocks so induction, a process normally mediated by EY (compare Figure 3J with K and 5C with D). Taken together, these results parallel those obtained in the eye disc and strongly suggest that the mechanism of EY inhibition is identical under both normal and ectopic conditions. To further confirm this hypothesis, we targeted expression of the HD-deleted ANTP protein using EYE-GAL4 as driver. These results show that the ANTP protein lacking the HD is unable to repress eye development (Figure 3M). Thus, these results indicate that the same mechanism leads to eye inhibition in the ectopic situation as well as in the normal eye. In order to test whether the DNA-binding activity of Antp is not required for the inhibition of eye development, we tested an ANTP mutant in which the DNA-binding specificity was changed (Q50K). Interestingly, this mutated protein is still able to repress eye development (Figure 3N). In addition, we performed mutagenesis experiments to convert Q50 and N51, residues shown to be crucial for DNA contacts (reviewed in Gehring et al., 1994), into alanines. This mutant is unable to bind a DNA PS2 probe containing a Hox/Exd/Hth motif, even in the presence of EXD and HTH in the bandshift assay (not shown). This A50,A51 mutant protein is still capable of inhibiting eye development when expressed in the eye disc using a strong EYE-GAL4 line, although with a lower activity than the wild-type ANTP protein (Figures 3O). EY and ANTP form a protein complex in vitro, mediated by the HD of ANTP and the paired domain and/or HD of EY Based on these in vivo results we asked whether the two proteins ANTP and EY might interact directly and thereby inhibit their respective activities. We first examined potential in vitro interactions between ANTP and EY using glutathione S-transferase (GST)–ANTP fusion proteins immobilized on glutathione–Sepharose beads. These were tested for their ability to retain in vitro synthesized 35S-labeled EY protein. Different portions of the ANTP protein were produced and tested separately for their ability to interact with EY. As seen in Figure 4A (lane 12), only the C-terminal portion of ANTP including the HD is able to interact with EY. This interaction is specific, since [35S]luciferase used as a control did not interact with this ANTP fusion protein (lane 7). The specificity of the interaction is further supported by the inability of EY to interact with GST alone (lane 8), and the other fusion proteins tested (lanes 9–11). To define the regions within EY and ANTP that are required for the interaction of the two proteins, we tested a set of deletion mutants of each protein for their ability to interact in vitro. Structure–function studies of both proteins have delineated specific domains that contribute to their functions as transcription factors as well as their interactions with other proteins. The ANTP HD that mediates DNA binding has also been shown to interact with other HD proteins such as EXD (reviewed in Mann and Affolter, 1998). The EY protein contains two DNA-binding domains, a paired domain and an HD. The paired domain has been shown to interact with different transcription factors (Fitzsimmons et al., 1996; Jun and Desplan, 1996; Bendall et al., 1999). These findings led us to investigate whether these EY paired domains and HDs are involved in the interaction with ANTP. Deleting either of these domains in the EY protein results in a partial loss of the interaction with the ANTP HD (Figure 4B, compare lanes 8 and 9 with 7). Furthermore, either the EY paired domain or the EY HD alone is still able to interact with ANTP (Figure 4C, lanes 10 and 13). These experiments suggest that since each domain is able to interact with ANTP, both domains might cooperate for efficient binding of EY to ANTP (Figure 4B). Moreover, deletion of the C-terminal part of the ANTP protein results in the loss of binding to the paired domain (Figure 4C, lane 12) or HD of EY (Figure 4C, lane 14), confirming that the ANTP HD is essential for the interaction with EY. Because the 35S-labeled proteins used in the binding reactions were synthesized in rabbit reticulocyte lysates, we sought to determine whether the ANTP–EY interaction is direct or dependent on a bridging molecule that might be present in the lysate. Since the complexes were formed using EY paired domain and ANTP HD purified from bacteria (Figure 4D), the two proteins appear to interact directly through their respective DNA-binding regions. ANTP inhibits EY transactivation in yeast and DNA-binding activity in vitro Despite considerable efforts, we were unable to confirm the interaction in a two-hybrid assay in yeast. Thus, we hypothesized that the DNA interface might be important to stabilize the interaction. To address this question, we performed ‘one-and-a-half hybrid’ assays that combine elements of the one- and two-hybrid systems. This allows us to test the effect of ANTP on EY-mediated activation and vice versa in yeast. We generated two reporter constructs cloned upstream of the LacZ gene, one carrying the so10 enhancer as an EY responsive element and the second carrying multimeric ANTP binding sites called HB1 (Haerry and Gehring, 1996; Keegan et al., 1997). Furthermore, we generated an ANTP activator by fusing the GAL4 transactivation domain to the HD (pACT-Antp 287–378). This ANTP protein is able to activate HB1 but not the so10 fragment (Figure 4E and F, lane 4). This further indicates that the so10 enhancer is not directly regulated by ANTP. Moreover, EY activates the so10-LacZ reporter but has no effect on HB1