INTRODUCTION

The Groucho protein of Drosophila binds directly to basic helix-loop-helix proteins (bHLH)¹ of the Hairy family (1). Together, Groucho and Hairy-like proteins repress transcription factors involved in cell determination, such as fushi tarazu, Enhancer of split, and Achaete-scute complex gene products (1). The consequence of Hairy/Groucho regulation is that cells are directed along one of two possible lineages (2). In this way, Groucho takes part in segmentation of the Drosophila embryo, neurogenesis, and sex determination (1).

The repression by Groucho and Hairy-like proteins requires the S/P (Ser-Pro-rich) domain of Groucho and the conserved carboxyl-terminal Trp-Arg-Pro-Trp (WRPW) sequence of the Hairy-like transcription factor for direct interactions between the two proteins (1). In addition to the S/P domain, a WD₄₀ motif was recognized in the carboxyl-terminal half of Groucho. The WD₄₀ sequence is a loosely conserved repeat of 40 amino acids separated by a Trp-Asp dipeptide sequence (3). By comparison with the function of the WD₄₀ repeat of other proteins, it is likely to be another domain in Groucho that is involved in protein interaction and may serve to recruit factors required for transcriptional repression (4, 5). The other sequence recognized in the Drosophila protein was the CcN motif, containing Cdc3 and casein kinase sites and a possible nuclear localization signal (6).

Homologues of the Drosophila Groucho gene have been isolated from mouse (Groucho-related genes, grg) (7, 8, 9), rat (Enhancer of split-related, Esp) (10), and human (transducin-like element, TLE) (11) cDNA libraries. Comparison of the predicted protein sequences revealed that most of the genes encode proteins with a conserved structural organization (11, 12). At the amino terminus, a glutamine-rich region (Q domain) is present, followed by a Gly/Pro-rich sequence (G/P region), and the CcN, S/P, and WD₄₀ domains. Of these, the Q domain and WD₄₀ domain have a high degree of conservation, suggesting that they play a highly conserved function. The S/P domain is less well conserved, implying that each Groucho family member binds preferentially to a particular Hairy-like transcription factor.

The families of Groucho-related genes in mouse, rat, and human also each include one gene that encodes a truncated homologue of Drosophila Groucho, with only the Q domain and part of the G/P sequence (7, 8, 10). In addition, we found that at least one of the mouse genes, grg3, encodes a long (Grg3a) and a short (Grg3b) Groucho-related protein (12). The lack of an S/P domain to interact with Hairy-related transcription factors and a WD₄₀ domain to interact with other proteins suggested to us that the short proteins may act to regulate the activity of the longer proteins. This type of regulation is frequently observed for families of transcription factors, wherein a short protein down-regulates a full-length protein by dimerizing with it and acting as a dominant suppressor (13, 14, 15, 16, 17, 18, 19).

The consideration that the shorter Groucho homologues might act to regulate the longer Groucho homologues lead us to examine the Q domain for possible dimerization motifs. We and others noted that the Q domain contains a putative leucine zipper sequence (8). Alpha-helical projections of this sequence suggested that it might act in homodimerization among Groucho family members, because the charges that confer specificity of Leu zipper binding are arranged with a mirror image symmetry.²

We have tested the ability of the Groucho homologues to dimerize, using Grg5 and Grg3b with other members of the mouse Grg family. Our studies confirm that the short Groucho homologues can dimerize with the long proteins and localize a critical sequence within the Q domain for this activity. Surprisingly, dimerization does not require the leucine zipper.

EXPERIMENTAL PROCEDURES

The plasmids (pGBT9, pGAD424, and pCL1) were obtained in the Matchmaker^TM two-hybrid system (Clontech). To construct the in-frame GAL4(AD)-Grg3b fusion construct, the Grg3b cDNA (12) was digested with SmaI and PstI and cloned into pGAD424, which had been digested with EcoR I, partially filled in with Klenow in the presence of 1 mM dATP, blunt ended with exonuclease VII, and subsequently digested with PstI. To make the in-frame GAL4(BD)-Grg5 fusion construct, the HpaII fragment of pBS Grg5 (12) was cloned into the PstI site of pGBT9 using an adapter (5-CGTGCA-3) (Operon Technologies, Inc.).

All yeast two-hybrid interaction studies were performed using yeast strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112+URA3::GALIlacZ, LYS2::GALHIS3 cyh^r; gift of S. J. Elledge). YPD, 50 ml, was inoculated with a 3-ml overnight culture of Y190 cells and grown for another 3 h at 30 °C. Cells were harvested and washed three times with 1 ml of ice-cold sterile water followed by 1 ml of ice-cold 1 M sorbitol. The final pellet was resuspended in 1 ml of ice-cold 1 M sorbitol. Transformations were performed by electroporation as described by Becker and Guarente (27) and plated on appropriate selective minimal media plates.

Yeast harboring the GAL4(DB) and/or the GAL4(AD) fusion proteins were assayed for -galactosidase activity by the filter method. Yeast transformants were transferred to nylon filters, permeabilized in liquid nitrogen, and placed on Whatman No. 1 filter paper that had been soaked in Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0) containing 2.7 µl/ml -mercaptoethanol and 0.34 mg/ml 5-bromo-4-chloro-3-indolyl -D-galactoside. Positive colonies appeared in 0.5-10 h.

To make the in-frame pGEX2T-Grg3b fusion construct the BamHI-EcoRV fragment of Grg3b was ligated to BamHI linkers (BRL), and cloned into the BamHI site of pGEX2T. To make the in-frame pGEX2T-Grg5 fusion construct pBS/Grg5 was digested with AvaI, filled in with Klenow, and ligated into the filled in EcoRI site of pGEX2T.

Three carboxyl-terminal truncations of Grg5 were created using the unique restriction sites MscI, PpuMI, and StuI located at amino acids 51, 104, and 163, respectively and the unique HindIII site located 95 nucleotides after the translation stop codon. The N50-Grg5 construct was created by digesting pBS/Grg5 with MscI and HindIII, partially filling in with Klenow in the presence of 1 mM dATP and 1 mM dGTP, blunting with exonuclease VII, and re-ligating. The N103-Grg5 construct was created by digesting pBS/Grg5 with PpuMI and HindIII, blunting with exonucleaseVII, and re-ligating. The N162-Grg5 construct was created by digesting pBS/Grg5 with StuI and HindIII, filling in with Klenow, and re-ligating. The three carboxyl-terminal deletion constructs maintained the same reading frame, following ligation at the HindIII site, and terminated at a stop codon located close to the HindIII site. To increase the translation performance of RNA prepared from the N50-Grg5 and N103-Grg5 constructs, the Cap-independent translation Enhancer sequence was isolated from the pCite-1 vector (Novagen) by digestion with EcoRI and MscI and cloned in-frame and amino-terminal to the N50-Grg5 and N103-Grg5 translation start.

Two amino-terminal deletion constructs, C147-Grg5 and C94-Grg5, were created by digesting pBS/Grg5 with MscI and PpuMI, respectively. Both were also cut with EcoRI, which cuts within the Bluescript vector (Stratagene) multiple cloning site eliminating the Grg5 translation start. To regenerate the Grg5 translation start, two oligonucleotides, one coding for the sense strand (5-AATTCGCGACTGACATGATGTTTCTGGCCAAGGTCCTAGG-3) and the other coding for the antisense strand (5-CCTAGGACCTTGGCCAGAAACATCATGTCAGTCGCG-3) (Operon Technologies, Inc.), were annealed creating an EcoRI overhang at one end and an MscI and a PpuMI site close to the other end. The annealed product was digested with MscI and PpuMI and ligated to the C147-Grg5 and C94-Grg5 plasmids, respectively. To increase the translation performance of the RNA prepared from the C147-Grg5 and C94-Grg5 deletion constructs, the 5-untranslated region of Xenopus -globin (28) linked directly to the initiation sequence CGCTAGCCATGT (provided by an oligonucleotide) was cloned in-frame and amino-terminal to the C147-Grg5 and C94-Grg5 translation start.

The 50/103-Grg5 construct containing amino acids 50 to 103 of pBS/Grg5 was created by digesting N103-Grg5 with EcoRI and MscI and regenerating the translation start as in the C147-Grg5 construct. The Cap-independent translation Enhancer sequence was cloned in-frame and amino-terminal to the 50/103-Grg5 translation start as in N103-Grg5.

Site-directed mutagenesis of a putative leucine zipper domain within Grg5 was performed using the Altered Sites II in vitro mutagenesis system (Promega). N103-Grg5 was digested with XhoI, filled in with Klenow, and digested with EcoRI and the fragment was ligated into the EcoRI-SmaI site of the pALTER-1 vector. Site-directed mutagenesis was performed as instructed in the technical manual (Promega) using the mutagenic oligonucleotide CL31 (5-GACCGCATCAAAGATGAGTTCCAG-3) to change the charged residues of lysine to glutamic acid and glutamic acid to lysine at amino acids 31 and 33, respectively. Leucine residues were mutated using oligonucleotides CL32 (5-GAGTTCCAGCTGAAGCAAGCGCAGTA-3) and CL33 (5-TCACAGCCGGAAGCTGG-3) (Operon Technologies, Inc.), changing one leucine to a lysine and a second leucine to an arginine at amino acids 37 (CL32) and 44 (CL33), respectively. Potential positive colonies were identified based on change in antibiotic resistance from tetracycline-resistant to ampicillin-resistant colonies.

All fusion constructs, deletion constructs, and site-directed mutagenesis clones were verified by sequencing using the T7 sequencing kit (Pharmacia Biotech Inc.).

BL-21 (B, F dcm ompT hsdS (r_Bm_B)gal (DE3, Stratagene), a protease deficient strain of E. coli, was transformed with either pGEX2T, pGEX2T-Grg3b, or pGEX2T-Grg5 and grown overnight in YT medium with ampicillin (100 µg/ml). Five ml of the culture was added to 100 ml of medium containing ampicillin and grown at 37 °C to an A₆₀₀ of 0.4-0.5 before adding 1 mM isopropyl-1-thio--D-galactopyranoside to induce expression of fusion proteins. The culture was incubated at 37 °C for a further 3 h. The bacteria was harvested and lysed by sonication in 10 ml of NTEN (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and 1 mM dithiothreitol). One ml of 10% Triton X-100 was added, the suspension was mixed, and insoluble material was removed by centrifugation. The supernatant was mixed with 1 ml of glutathione-Sepharose 4B beads (Pharmacia) and gently rocked for 30 min. The beads were washed twice with NTEN, followed by two washes with PBS (137 mM NaCl, 2.7 mM KCl, 5.4 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) by adding 10 ml of solution, mixing, and centrifuging. The beads were stored in 10 ml of PBS.

Prior to in vitro transciption, plasmids were linearized by digestion with a restriction enzyme that recognized a site outside the coding sequence and produced a 5-protruding-end on the plasmid. Capped RNA transcript was synthesized from the linearized plasmid using T3 RNA polymerase for all the deletion constructs and Mash2, and T7 RNA polymerase for the site-directed mutagenesis clones. Transcripts were subsequently used to synthesize ³⁵S-labeled protein. Transcription and translation reactions were performed as instructed by the manufacturer (Promega) in the rabbit reticulocyte lysate system technical manual .

Equivalent amounts of immobilized fusion proteins were mixed with specific ³⁵S-labeled reticulocyte lysate by gentle rocking at 4 °C for 1 h. The glutathione beads were washed twice with 1 ml of NTEN followed by 1 ml of PBS. Fusion and bound proteins were eluted with 30 µl of 50 mM Tris-Cl (pH 8.0), 15 mM reduced glutathione and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.

RESULTS

We first used the yeast two-hybrid system to detect possible interactions of the short Grg proteins through the Q domain. The yeast two-hybrid assay is a genetic test used to detect protein interactions in vivo (20). It is based on the fact that transcription factors consist of two separable functional domains, the DNA binding domain and the transcription activation domain. Both components are required to activate transcription. In the yeast two-hybrid system used here, the sequences for the two functional domains of GAL4 have been cloned into two different expression vectors. The pGBT9 vector contains the sequence for the GAL4 DNA binding domain (BD) and the pGAD424 vector contains the sequence for the GAL4 activation domain (AD). By fusing sequences of known proteins (X, Y) downstream and in-frame to the GAL4 sequences, co-transformations with these two expression vectors allows for detection of interactions between X and Y combinations of proteins. Interactions result in -galactosidase expression that, upon substrate (X-gal) addition, turns colonies with interacting proteins blue, while colonies with non-interacting proteins remain white.

We placed the Grg3b sequence in-frame to the GAL4(AD) of pGAD424 and the Grg5 sequence into GAL4(BD) of pGBT9. -Galactosidase activity was assayed, and the results are shown in Table I. Yeast transformed with pGBT9, pGAD424, or both resulted in no -galactosidase activity as indicated by white colonies. Yeast transformed with either the pGBT9-Grg5 or pGAD424-Grg3b fusion constructs also resulted in white colonies. However, yeast co-transformed with both fusion constructs resulted in blue colonies, indicating an interaction between Grg3b and Grg5. The positive control plasmid, CL1, encodes the complete GAL4 protein and also produced blue colonies upon -galactosidase staining.

Table I. Results from the yeast two-hybrid assay to test Grg dimerization pGBT9 contains only the DNA binding domain of GAL4 and is unable to induce LacZ expression. pGAD424 has only the activation domain of GAL4 and is therefore also unable to induce LacZ expression. The fusion protein pGBT9-Grg5 or pGAD424-Grg3 cannot independently induce LacZ expression. However, when co-transformed, pGBT9-Grg5 and pGAD424-Grg3 can induce LacZ expression, indicating Grg5 and Grg3 are able to interact. The CL1 plasmid is a positive control, encoding the complete Gal4 protein. Transformation plasmid Colony color 1. pGBT9 (Gal-4 DNA binding domain only) white 2. pGAD424 (Gal-4 activation domain only) white 3. pGBT9 + pGAD424 white 4. pGBT9-mEspN white 5. pGAD424-mEspB white 6. pGBT9-mEspN + pGAD424-mEspB blue 7. CL1 (complete Gal 4 protein) blue

The results of the yeast two-hybrid assay were verified with binding assays using GST fusion proteins. We used pGEX2T for inducible expression of Grg3b and Grg5 as GST fusion proteins in bacteria. Bacterial extracts were mixed with glutathione-Sepharose beads to adsorb the fusion protein. The beads with the adsorbed GST proteins were subsequently incubated with radiolabeled in vitro translated Grg1, Grg3b, and Grg5. Translated products that were retained by the GST-Sepharose beads were visualized by gel fractionation and autoradiography. As a control, Mash2, which is a basic helix-loop-helix protein that does not bind with Groucho, was used. The first four lanes in Fig. 1A show one-tenth of the translation products. The remainder of the gel in Fig. 1A shows that none of the in vitro translated proteins were retained by the GST protein alone. However, translated Grg1, Grg3b, and Grg5 did bind to beads carrying the GST-Grg3b (Fig. 1B) and GST-Grg5 (Fig. 1C) fusion proteins, whereas Mash2 did not. This again demonstrated that the Grg proteins can dimerize and further showed that the short proteins can bind both to long and to short proteins.

To delineate the necessary sequences for dimerization, we constructed a series of deletion constructs of the Grg5 cDNA template used for in vitro transcription (Fig. 2A). These deletions made progressive truncations from the amino terminus and from the carboxyl terminus of the Grg5 protein product. Radiolabeled proteins were synthesized in vitro and incubated with the sepharose beads carrying GST, GST-Grg3b, or GST-Grg5 fusion proteins. Bound proteins were again visualized by gel fractionation and autoradiography (Fig. 2B). One-tenth of the translation products were also visualized (Fig. 2B, lanes 1-7 in the left panel) to determine translation efficiency.

As for the full-length Grg5, no binding was observed when proteins were mixed with GST beads (Fig. 2B, lanes 8-14 in the left gel). However, for the GST-Grg3b and GST-Grg5 beads, binding of the full-length Grg5 and some of the deletion products was observed (Fig. 2B, right panel). We found that removal of 35 amino acids of the carboxyl-terminal region (N162) did not decrease the amount of bound protein. However, further deletion into the Q domain, removing 94 amino acids (N103), significantly reduced the ability of the truncated protein to bind with Grg3b or Grg5. Further deletion from the carboxyl terminus, removing much of the Q domain but leaving the leucine zipper intact (N50), drastically reduced the ability of the truncated protein to dimerize. A deletion of 50 amino acids from the amino terminus, removing the leucine zipper (C147), also significantly reduced the amount of dimerized protein and deletion of 103 amino acids from the amino terminus eliminated dimerization (C94). Deletions of both the amino-terminal 50 amino acids and the carboxyl-terminal 94 amino acids (50/103) resulted in loss of all binding. These results indicate that the critical sequence for dimerization is located between amino acids 50 and 103, but this sequence is insufficient and requires additional amino-terminal or carboxyl-terminal sequence.

To further examine whether the leucine zipper plays any role in dimerization activity, we used the N103 Grg5 cDNA, which contains the leucine zipper and the critical core of sequence required for dimerization. The leucine zipper of N103 was disrupted in two different ways using site-directed mutagenesis (Fig. 3A). In one case, the charged amino acids that flank the aliphatic face of the putative alpha helix were changed (Lys31Glu, Glu33Lys), to possibly disrupt the dimerization specificity of the protein (N103-CHG). In the second mutant (N103-LEU), two of the leucines were changed to charged amino acids (Leu37Lys; Leu44Arg). Mash2 was again used as a negative control. Each of the translation products was run on the gel, as previously, to examine translation efficiency (Fig. 3, lanes 1-4).

The translated proteins were mixed with glutathione-Sepharose beads carrying GST, GST-Grg3b, or GST-Grg5. No binding was observed with GST beads (Fig. 3, lanes 5-8). The N103 product was able to bind to GST-Grg3b and GST-Grg5, as noted above (Fig. 3, lanes 9 and 13), but Mash2 was not (Fig. 3, lanes 12 and 16). Changing either the charged amino acids or the Leu zippers had no effect on the level of protein heterodimerization by N103 to Grg3 (Fig. 3, lanes 10 and 11) but did reduce the ability to form homodimers between N103 (derived from Grg5) and Grg5 (Fig. 3, lanes 14 and 15). Thus, the leucine zipper does not appear to be critical to dimerization of Grg proteins but may strengthen the interactions between homodimers of Grg5.

DISCUSSION

We have demonstrated that the Grg proteins are able to dimerize through the amino-terminal Q domain. This domain is highly conserved in all of the mouse Grg family members (12), as well as in other vertebrate and invertebrate species (11); therefore, we expect that the ability to dimerize is common to all of the Groucho homologues.

Grg dimerization requires a core sequence within the Q domain, plus flanking sequence from either side. The additional sequence may be required for the correct structural conformation of the dimerization domain, or there may be additional dimerization sequences in both flanking regions that are required for maximal binding. Mutagenesis of the leucine zipper indicated that, although it may confer stronger binding of the Grg5 protein, it was not the additional sequence required on the amino-terminal side of the core sequence. Therefore, in contrast to our prediction, the leucine zipper does not appear to play a role in Grg dimerization to other Grg proteins. One possibility is that it instead interacts with the basic helix-loop-helix transcription factors with which it is likely to interact, namely the Hes (Hairy/Enhancer of split homologous) (21, 22, 23) proteins.

The potential interaction between Grg proteins suggests that the short Grg proteins may act to modify activity of the long Grg proteins. At the present time, we have no indication whether this would be a positive or negative regulation. However, the function of the long Grg proteins probably requires binding to Hes and DNA, by analogy to the Drosophila proteins (24); therefore, it will be important to determine if tertiary complexes form between the short and long Grg proteins and Hes. It will also be interesting to examine if both forms of the Grg protein interact with Hes while it is bound to DNA and also the effect on transcriptional regulation by the Hes proteins.

In Drosophila, Groucho is part of a complex network of transcriptional regulatory proteins. This includes basic helix-loop-helix transcription factors that are involved in cell determination, partners that lack a basic domain and act as negative regulators, the Hairy-related proteins, which also act as negative regulators, and Groucho, which is required for repression by Hairy-related proteins (6, 25, 26). The complex regulation of these transcription factors may exist because they are involved in controlling cell proliferation and differentiation, and therefore, inappropriate function would have serious consequences for the host. The existence of two types of Groucho-related proteins and their ability to dimerize suggest that there is yet an additional level of transcriptional regulation in mammalian systems.