INTRODUCTION

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In recent years, it has become clear that transcriptional repression is used to regulate many aspects of development and cell differentiation. However, considerably more is understood about the mechanisms and factors that activate gene expression than on how repressors work. This is particularly true for a major class of repressors (so-called active repressors) that bind DNA regulatory sites and are thought to inhibit expression via protein-protein interactions with other factors at the promoter (reviewed in reference 29). Some active repressors can directly target components of the basal transcription machinery (52, 62). However, others are unable to repress transcription by themselves and need to recruit to the promoter accessory proteins (corepressors) that in turn effect repression. Corepressors are likely to act by a variety of mechanisms, including the modulation of chromatin organization mediated by histone deacetylation (reviewed in reference 27).

We have been studying the function and mechanism of action of the Hairy-related family of active repressors in Drosophila (44, 47, 63). These include the Hairy protein, a regulator of embryonic segmentation, and the Deadpan (Dpn) and Enhancer-of-split factors, which act during sex determination and neurogenesis, respectively (8, 16, 17, 37, 38, 50, 54, 68). Like most transcription factors, Hairy-related proteins have a modular structure: they contain a DNA-binding domain of the basic helix-loop-helix class and a separable repressor domain directly involved in mediating repression. This repressor domain includes a C-terminal tetrapeptide (WRPW) which is necessary for repression in vivo and can impose repressor activity on a heterologous DNA-binding domain in cultured cells (15, 23, 65).

The WRPW domain mediates repression via another protein, Groucho (Gro), a maternally contributed factor which contains multiple WD repeats but lacks a known DNA-binding domain (17, 30, 47). The WRPW motif binds specifically to Gro in vitro, and in vivo repression by this motif requires the presence of Gro (23, 33, 47). Thus, Gro behaves as a corepressor that is recruited to target promoters by interactions with the WRPW sequence. Once at the promoter, Gro mediates repression by an as yet unknown mechanism.

Recently, other repressors unrelated to Hairy, such as the homeodomain factor Engrailed (En) and the Rel domain protein Dorsal, have been shown to act via Gro (21, 33). Thus, an En repressor domain (En^R) binds to Gro in vitro, and repression by En^R requires Gro activity in vivo. Interaction with Gro and repressor activity requires a short (7- to 15-amino-acid) conserved sequence in En (En homology region 1 [eh1]; [33, 57]). This sequence does not have similarities with the WRPW motif, suggesting that Gro is a common effector for different classes of repressor domains.

The above studies indicate that the WRPW motif is necessary and sufficient for repression and interaction with Gro (15, 23, 33, 47, 65). The eh1 element is also necessary for these activities (33, 57), but it is not known whether it can act independently of other repressor sequences. The possibility that small protein motifs are sufficient to mediate repression is interesting because most repressor domains examined to date are relatively large, and little is known about their sequence requirements (reviewed in reference 29).

Protein motifs similar to the eh1 sequence are also present in other developmental regulators such as Goosecoid (Gsc), a homeodomain transcription factor. Gsc function has been mainly analyzed in Xenopus embryos, where it is expressed in the organizer region and contributes to specification of the prechordal plate (9, 11; reviewed in reference 18). Drosophila Gsc is implicated in formation of the somatogastric nervous system (26, 28). In this report, we present functional analyses of a Gsc domain (hereafter referred to as Gsc^R) containing an eh1-like element (also known as the Gsc-En homology [GEH] element). We show that Gsc^R mediates effective repression in Drosophila blastoderm embryos and that its activity depends on the eh1/GEH element. In addition, Gsc^R requires Gro for repression in vivo, and the two proteins interact in vitro via the eh1/GEH motif. Evidence is presented that the eh1/GEH element is sufficient for repression and interaction with Gro. We also investigated the oligomeric status of Gro in the nucleus. Our results suggest that Gro is not recruited for repression as a preassembled complex with other proteins. We discuss the implications of these results in terms of the role of protein motifs in transcriptional regulation.

	MATERIALS AND METHODS

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DNA constructs. Plasmid manipulations were carried out according to standard procedures (4, 51). Sequences encoding the Hairy^Gsc derivative were assembled in pBluescript (Stratagene) by cloning a PCR fragment encoding a Drosophila Gsc domain (amino acids 102 to 216) as a BamHI-XbaI fragment downstream of the unique BamHI site in the hairy (h) cDNA. Hairy^GscGEH was made similarly, using as starting material for PCR a plasmid (kind gift of C. Mailhos and C. Desplan) in which the sequence corresponding to amino acids 117 to 118 of Gsc had been mutated to produce a BamHI site. As a result, Hairy^1-268 was fused to a Gsc domain comprising amino acids 119 to 216 and lacking the eh1/GEH motif. To generate Hairy^GEH-17, a synthetic BamHI-XbaI linker encoding the Gsc eh1/GEH core element and its immediate flanking residues (amino acids 106 to 122) was cloned downstream of the BamHI site in h. Hairy^GEH-9, Hairy^GEH-9m, and Hairy^WRPW were made by following the same strategy and using linkers encoding the sequences LFTIDSILG, LETIDSILG, and GGQPWRPW, respectively.

Transgenic flies. Germ line transformation of hb constructs was performed as described previously (58) by DNA injection into y w embryos and selecting for rescue of w eyes. In general, two or more independent lines were analyzed for each construct. Insertions on the X chromosome were maintained in males by using an attached-X chromosome [C(1)M3]; autosomal insertions were kept as unbalanced stocks selecting for transformant males and nontransformant females. To analyze the effects of hb-h^gsc on gro embryos, mosaic gro females (see below) were crossed to males carrying the hb construct on the X chromosome, so that all gro female embryos inherit the transgene. A similar strategy was used to examine the effects of the inactive hb-h^gscGEH transgene in a wild-type background.

Germ line clones and embryo analysis. Embryos deprived of maternal gro function were generated using the strong gro^E48 allele in combination with the ovo^D-FLP-FRT system (12). In this system, all embryos derive from homozygous clones in the female germ line that have lost the dominant sterile mutation ovo^D1. Briefly, males of the genotype hs-FLP1/Y; FRT[82B] ovo^D1/Sb were crossed to FRT[82B] gro^E48/TM3 females, and 1- to 3-day-old progeny were heat shocked daily for 4 h at 37°C for the following 3 to 4 days. Eclosed hs-FLP1/+; FRT[82B] ovo^D1/FRT[82B] gro^E48 virgin females (carrying homozygous gro^E48 clones) were crossed to males carrying the hb constructs. The progeny of these crosses was examined to confirm the expected lethality of all embryos laid due to the lack of maternal gro function.

In vitro binding assays. GST fusions were expressed in Escherichia coli as described previously (47), using the protease-deficient strain SRP84 (gift of C. Higgins). Binding assays were performed by mixing equal amounts of fusion proteins (a total of 30 µl of glutathione-Sepharose beads normalized with beads from a blank bacterial extract), 20 to 30 µl of ³⁵S-labeled Gro protein synthesized by using the TNT coupled rabbit reticulocyte lysate system (Promega), and 180 µl of binding buffer (20 mM HEPES-KOH [pH 7.9], 50 mM KCl, 2.5 mM MgCl₂, 10% glycerol, 1 mM dithiothreitol, 0.2% Nonidet P-40 [NP-40]) supplemented with 3 µl of rabbit serum and 3 µl of a 100 mM phenylmethylsulfonyl fluoride stock. Binding reaction mixtures were rolled overnight at 4°C and rinsed four times with 1 ml of radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.2% NP-40) at room temperature. The beads were boiled for 1 min in sample buffer, and aliquots were examined by electrophoresis, followed by Coomassie staining to confirm the integrity of GST fusions and autoradiography to detect bound Gro protein.

Analysis of embryonic Gro protein. All protein procedures were at 4°C or on ice. Nuclear extracts were prepared from 0- to 12-h embryos as described previously (66). The final extract had a total protein concentration of about 18 mg/ml and was in HEMG.1 buffer (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA, 12.5 mM MgCl₂, 10% glycerol) containing 100 mM KCl, 0.01% NP-40, 1.5 mM dithiothreitol, 1 mM sodium metabisulfide, 0.2 mM aminoethylbenzenesulfonyl fluoride, leupeptin (2 µg/ml), and pepstatin (0.7 µg/ml). For glycerol gradient sedimentation, 100 µl of extract was loaded onto a 14-ml 15 to 35% glycerol gradient in HEMG.1 buffer. Fractions were collected after centrifugation of the gradients in an SW40 rotor for 17 h at 35,000 rpm. The molecular mass markers bovine serum albumin (BSA), aldolase, thyroglobulin, and dextran blue (Pharmacia) were sedimented in parallel gradients. Profiles of the standards were determined by the method of Bradford and by Coomassie staining. The gradient of crude nuclear extract was analyzed by protein immunoblotting with antibodies directed against Gro, Drosophila TAF_II80, and Brahma.

	RESULTS

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The eh1/GEH motif in Gsc mediates Gro-dependent repression. All known Gsc proteins contain a conserved 7- to 15-amino-acid eh1/GEH motif within the N-terminal half of the protein. This motif is present in several classes of homeodomain proteins and contains a highly conserved core of seven amino acids flanked by a variable number of residues that show weak homology within protein families (57). The eh1/GEH element has been shown to bind Gro and to be important for repression by the En^R in Drosophila embryos (33, 57). Figure 1 shows a comparison of the eh1/GEH core sequences present in selected proteins from different organisms.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Alignment of eh1/GEH and octapeptide motifs of different homeodomain proteins from Drosophila melanogaster (D. mel.), Xenopus laevis (X. lae.), mouse (Mus musculus [M. mus.]), and Caenorhabditis elegans (C. ele.). S59 is a protein expressed in muscle precursor cells (20), and Msh is involved in patterning the neuroectoderm and embryonic muscles (14, 32, 40). Note the presence of a strongly conserved seven-amino-acid core which always starts with a Phe residue (position 3 in the alignment). This Phe residue is not conserved in a related motif known as the octapeptide (reviewed in reference 43; see Discussion). Two octapeptide sequences from the Sparkling (Spa [25]) and Pax-5 (2) proteins are shown for comparison. See references 43 and 57 for more detailed sequence alignments.

View larger version (51K): [in this window] [in a new window]   FIG. 2.   GscR directs Gro-dependent repression via the eh1/GEH motif. (A) Diagram of Hairy derivatives expressed under the control of the hb promoter. The Gsc domains are represented by grey boxes; the position of the eh1/GEH (GEH) motif is indicated by a black box. (B to D) Effects on Sxl expression of hb-hgsc (B and C) and hb-hgscGEH (D) in wild-type (B and D) and gro mutant (C) embryos. Repression by HairyGsc requires the eh1/GEH motif and endogenous Gro activity.

GscGEH

Gsc^R interacts with Gro in vitro via the eh1/GEH element. The preceding experiments indicate that Gsc^R is a Gro-dependent repressor domain. To see whether this involves a physical interaction between Gsc^R and Gro, we examined the ability of Hairy^Gsc to bind to Gro in vitro. Hairy^Gsc was expressed in bacteria as a GST fusion, purified with glutathione-Sepharose beads, and incubated with radiolabeled Gro protein. As shown in Fig. 3, strong binding between the two proteins is detected, whereas a Hairy truncation lacking Gsc^R does not bind Gro. Hairy^GscGEH, lacking the eh1/GEH motif, binds much less effectively, showing that this motif plays a direct role in the interaction with Gro.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Binding of Hairy derivatives to Gro in vitro. (A) GST-Hairy fusions were immobilized on glutathione-Sepharose beads and incubated with 35S-labeled Gro protein. After the beads were washed, the bound Gro protein was detected by SDS-PAGE and autoradiography. Hairy and HairyGsc bind Gro with high affinity. In contrast, little or no binding is detected with Hairy1-286 (which lacks the C-terminal 51 amino acids of the protein) and the HairyGscGEH chimera. (B) Coomassie staining of the gel shown in panel A, demonstrating the integrity of the different GST fusions after the binding reaction.

The eh1/GEH and WRPW motifs are sufficient for Gro-mediated repression. Short conserved protein motifs are necessary for Gro binding and Gro-mediated repression. We examined whether they are also sufficient for such binding by fusing them to the Hairy^1-268 truncation. First, we tested if the WRPW tetrapeptide is sufficient for repression in the hb assay, by examining the activity of Hairy^WRPW, a fusion of Hairy^1-268 with the six C-terminal amino acids of Hairy, including the WRPW sequence (Fig. 4A and Materials and Methods). hb-h^WRPW has effects indistinguishable from those of hb-h: it causes high levels of female lethality (>90% in most lines) and clear repression of Sxl (Fig. 4B and data not shown). These results support the work of Fisher et al. (23), who showed that the WRPW tetrapeptide is sufficient to bind Gro in vitro and acts as a portable repressor domain in transfected cells. Our results suggest that this motif is also sufficient for repression in the embryo (see Discussion).

View larger version (60K): [in this window] [in a new window]   FIG. 4.   The WRPW and eh1/GEH motifs are sufficient for repression in vivo and binding to Gro in vitro. (A) Diagram of the HairyWRPW and HairyGEH derivatives. (B and C) Expression of HairyWRPW (B) and HairyGEH-17 (C) under the control of the hb promoter causes efficient repression of Sxl. (D) Binding of GST-Hairy derivatives to Gro in vitro. HairyWRPW, HairyGEH-17, and HairyGEH-9 bind to Gro with similar efficiencies; in contrast, HairyGEH-9m, which carries a mutation in a conserved Phe residue within the eh1/GEH element, does not interact with Gro.

Gro does not form a stable complex with other proteins in the nucleus. As a corepressor, Gro is expected to associate with other proteins. These interactions could be relatively transient, occurring only at target promoters, or may involve stable complexes which are preassembled before Gro is recruited to promoters. The ability to form stable nuclear complexes is a typical feature of proteins of the general transcriptional machinery and other transcriptional cofactors. Recently, several corepressors have been shown to be part of multimeric complexes that survive methods of biochemical purification. One of the best-characterized examples is the yeast Tup1 protein. Tup1 is a WD-containing protein, like Gro, and exerts its function as part of a multimeric complex consisting of several molecules of Tup1 and the Ssn6 protein (35, 49, 64, 67). This complex is thought to be recruited to target promoters by specific repressors such as the 2 regulator (35), presumably through relatively transient interactions. Thus, it is possible that repressors such as Gsc and Hairy do not recruit a single molecule of Gro to target genes but, instead, recruit a Gro-containing complex. Indeed, recent studies have suggested that Gro is assembled into oligomeric complexes of various sizes (e.g., ~170 and 240 kDa [45]).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Gro does not form oligomeric complexes. Embryo nuclear extracts were centrifuged through a glycerol gradient, and the different fractions collected were examined for the presence of Gro and TAF-80 proteins. The migration of markers (BSA and aldolase) and known endogenous complexes (TFIID and the Brahma complex [Brm-C]) is depicted by arrows. Gro migrates with an apparent size similar to its predicted molecular mass (80 kDa). In contrast, TAF-80 (which also has a molecular mass of 80 kDa) migrates with an apparent mass of ~600 to 700 kDa because it forms part of the TFIID complex. (B) Gel filtration analysis of endogenous and purified recombinant Gro by Sephacryl S-300 chromatography. Fractions collected were examined for the presence of Gro by SDS-PAGE and Western blot analysis with an antibody directed against Gro. The elution volume (Ve) is indicated. Only the gels that contained Gro are shown. Positions of the protein standards are indicated as follows: T, thyroglobulin; F, ferritin; C, catalase; A, aldolase, B, BSA. (C) The position of the approximate Gro peak is indicated on the calibration curve.

	DISCUSSION

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An increasing number of studies are currently addressing the mechanism by which active repressors inhibit gene expression. Several repressor domains, distinct from DNA-binding regions, have been identified in different proteins, but their molecular targets are usually not yet known. In this report, we characterize a conserved repressor domain from the Gsc developmental regulator (the eh1/GEH motif) and show that its activity depends on association with the Gro corepressor. We find that the eh1/GEH element is not only necessary but also sufficient to mediate binding to Gro and transcriptional repression. This functional association appears to represent a widespread mechanism of repression, since several other regulators (e.g., the Drosophila En, S59, and Msh proteins [Fig. 1 and reference 57]) contain versions of the eh1/GEH motif. Consistent with this idea, Drosophila Gro is ubiquitously expressed and appears to be involved in many developmental processes. In addition, these interactions may be highly conserved during evolution, since a wide variety of organisms, including nematodes and vertebrates, have both eh1/GEH-containing factors and Gro homologues (42, 48, 53, 57, 59), and the eh1/GEH domain is necessary for Gsc rescue of UV-ventralized Xenopus embryos (41). Furthermore, the Drosophila En^R is functional in vertebrate cell and embryonic systems and on a wide variety of promoters (5, 13).

Association of Gro with small repressor motifs. Previous work showed that the WRPW motif is sufficient to mediate significant (three- to fourfold) repression in cultured cells and to interact with Gro in vitro (23). A WRPW-like (WRPY) motif present in the Drosophila Runt protein, a regulator of segmentation and sex determination, has been shown to be important for repression in vivo and sufficient for binding to Gro in yeast (3). Our experiments indicate that the WRPW motif is able to repress the Sxl promoter completely (Fig. 4B), and we present evidence that the eh1/GEH motif is sufficient for repression in vivo and binding to Gro in vitro (Fig. 4C and D). Thus, the WRP(W/Y) and eh1/GEH elements represent minimal repressor motifs that can recruit Gro independently of other protein domains. The chimeric constructs that we have used contain the N-terminal portion of Hairy (Hairy^1-268) which includes the basic helix-loop-helix region and the so-called Orange domain. Dawson et al. (15) suggested that these domains can mediate a separable mode of repression that could contribute to the ability of the WRPW and eh1/GEH motifs to repress Sxl. However, only the latter motifs appear capable to associate with Gro (references 23, 33, and 47; this report), arguing that this activity is independent of other protein domains. Indeed, recent experiments with the intracellular portion of the Notch receptor argue that this tetrapeptide can act as an autonomous repressor element (1). This autonomous function is also evident in the case of the GEH motif, which can confer repression on protein sequences (Hairy^1-268) to which it is not normally associated.

Gro is not present as a stable complex with other proteins. An important aspect of the function of Gro is whether it forms stable nuclear complexes with other corepressor proteins. The Tup1 protein, which has served as a paradigm for the role of Gro, appears to form a large multimeric complex in the yeast nucleus (49, 64, 67). However, our results suggest that the great majority of Gro protein is not stably associated with other repressor proteins: Gro protein from crude nuclear extracts migrates through glycerol gradients with an apparent size compatible with its monomeric molecular mass (Fig. 5).

Conclusion. Our data suggest that the conserved eh1/GEH element present in Gsc and other developmental regulators acts as an interaction motif that is sufficient to recruit Gro to target promoters. A similar conclusion applies for the WRP(W/Y) motifs present in Hairy-related factors and members of the Runt family (this report; references 3 and 23). Together, these results suggest that interactions between very short peptides and Gro represent ancient functional associations that have been employed repeatedly and in several contexts during evolution. Gro may be the common node in an evolutionarily conserved network of interactions that includes different classes of transcription factors and which serves to repress gene expression in a wide variety of developmental processes.