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Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095-1569,1 Department of Biochemistry, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel2
Received 19 October 2003/ Returned for modification 22 December 2003/ Accepted 11 February 2004
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ABSTRACT |
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INTRODUCTION |
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Groucho family proteins are characterized by a conserved N-terminal glutamine-rich region (the Q domain) and a conserved C-terminal WD-repeat domain (11). The WD-repeat domain is required for binding to a variety of proteins involved in repression (33, 64, 68). The Q domain has also been implicated in several protein-protein interactions (10, 15, 20, 45, 59), but, perhaps most significantly, this domain is required for the homotetramerization of Groucho (13, 56). The amino-terminal enhancer of split (AES) proteins, which constitute a Groucho subfamily, contain the Q domain but lack the WD-repeat domain, and a number of studies suggest a dominant negative role for AES proteins in Gro-mediated processes (59, 60). This dominant negative function may result from the formation of mixed oligomers between AES proteins and full-length members of the Gro/TLE family, thereby sequestering these factors in an inactive form. Thus, the dominant negative function of AES proteins is consistent with the idea that the Q domain mediates oligomerization in vivo.
Between the highly conserved Q and WD-repeat domains of Groucho family proteins are three less well conserved domains, the GP domain, the CcN domain, and the SP domain. The GP domain may be important for interactions with the histone deacetylase HDAC1 (10, 12, 15), while the entire N-terminal region of the protein, including everything other than the WD-repeat domain, may be important for binding to hypoacetylated forms of the core histone N-terminal tails (23, 53).
Most of the available evidence suggests that Gro mediates long-range repression (16, 22, 43). In other words, this corepressor is able to silence a locus regardless of where it is tethered to the template relative to DNA-bound activators or the core promoter. This contrasts with dCtBP, another Drosophila corepressor, which represses transcription only when bound within about 100 bp of an activator or the core promoter (43, 49, 51). Long-range repression by Gro could relate to its ability to both oligomerize and interact with core histones, thus allowing the corepressor to polymerize along the template and establish a large transcriptionally silent domain. Recruitment of HDAC1 by Gro could serve to reenforce the silent state since histone deacetylation increases Gro binding affinity. It is interesting that other regulatory factors that establish large silent domains, in particular those involved in heterochromatic silencing, have similarly been shown to both oligomerize and bind histones (16, 46).
Although corepressor oligomerization is implicated in many instances of repression, in no case has a definitive link been made between the ability of a factor to oligomerize and its ability to repress transcription in vivo. In the experiments presented here, we show that Gro oligomerization is very probably mediated by two coiled-coil motifs in the Q domain. Furthermore, we show that mutations that disrupt the hydrophobic faces of these coiled coils prevent both tetramerization and the formation of higher-order Gro oligomers. Finally, we show that the same point mutations that disrupt oligomerization prevent Gro-mediated repression in transfected cells and in vivo, strongly suggesting that oligomerization is a prerequisite for repression.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein preparation and gel filtration. FLAG-tagged Gro mutant proteins were expressed in stably transfected S2 cells and purified on anti-FLAG antibody conjugated beads (Sigma). Briefly, a pRM expression construct (9) encoding FLAG-tagged Gro and a plasmid encoding the selectable hygromycin resistance gene (HPH) marker were cotransfected into S2 cells. After selection with hygromycin B, the cells were amplified and Gro expression was induced by the addition of 0.5 mM CuSO4 to the cell culture medium. Gro mutant proteins were purified from nuclear extracts as described previously (14). A Superdex 200 gel filtration column connected to a fast protein liquid chromatography system was used to analyze the native size of Gro mutants. The gel filtration running buffer contained 25 mM HEPES (pH 7.6) and 250 mM NaCl.
Transient-transfection assays and RNAi. Calcium phosphate-mediated cotransfection was performed with S2 cells as described previously (17). The luciferase reporter activity was analyzed by the dual-luciferase reporter assay system (Promega). For the RNA interference (RNAi) experiment, 1 µg of double-stranded RNA (dsRNA) directed against the Gro 3' untranslated region (UTR) (matching the sequence from bp 95 to 683 downstream of the stop codon) or control dsRNA directed against the glutathione S-transferase (GST)-conjugated mRNA was cotransfected with the reporter.
Generation and analysis of flies overexpressing Gro mutants. PCR fragments encoding Gro40,89D and Gro38,87D were inserted into the p131 transformation vector (1) so that the Gro coding region was in frame with the amino-terminal 6xmyc epitope tag. The upstream activation sequence (UAS) constructs were then introduced into the germ line by standard procedures.
To determine lethality rates, Actin-Gal4/CyO flies were crossed with homozygous UAS-myc-Gro40,89D or UAS-myc-Gro38,87D lines. The percent lethality was defined as 100 – [(straight-winged flies/curly-winged flies) x 100]. About 300 curly winged flies were scored per cross.
Clonal overexpression of Gro mutants was achieved as described previously for overexpression of wild-type Gro (31). Briefly, hs-flipase; vgQ-lacZ; Actin>CD2>Gal4/TM6 females were crossed with homozygous UAS-myc-Gro40,89D or UAS-myc-Gro38,87D males. Progeny were heat shocked (40 min at 35°C) as first-instar or early-second-instar larvae. After 2 or 3 days, late-third-instar larvae lacking TM6 were selected for dissection and staining.
Immunocytochemistry and X-Gal staining. Primary antibodies used were mouse anti-Gro monoclonal Ab (kindly provided by Christos Delidakis; 1:2,000), mouse anti-FLAG monoclonal Ab (Sigma; 1:4,000), mouse anti-myc monoclonal Ab (Santa Cruz; 1:1,000), mouse anti-CD2 monoclonal Ab (Serotec; 1:1,000), and rabbit anti-ß-galactosidase polyclonal Ab (ICN/Cappel; 1:1,000). Secondary antibodies were from Sigma: fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (IgG) (1:100), tetramethylrhodamine-5-isothiocyanate-conjugated anti-rabbit IgG (1:200), Cy3-conjugated anti-mouse IgG (1:800), and peroxidase-conjugated anti-mouse IgG (1:5000). For wing disk staining, the anterior halves of late-third-instar larvae were inverted in 1x phosphate-buffered saline fixed, and antibody staining or 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) staining performed as described previously (62).
Coimmunoprecipitation. S2 cells transfected with wild-type or mutant Gal4-Gro were resuspended in lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.8% Triton X-100, 0.5 mM dithiothreitol, 1x complete protease inhibitor cocktail [Roche]) and rocked at 4°C for 15 min. Clarified lysates were incubated with rabbit anti-Gal4 DNA binding-domain polyclonal Ab (Santa Cruz) and protein A-Sepharose beads (Amersham). The beads were then washed with the lysis buffer, eluted with SDS sample buffer, and analyzed by Western blotting with anti-Gro Ab. To detect the interaction between exogenous and endogenous Gro in the Drosophila embryo, Actin-Gal4/CyO flies were crossed with w1118, UAS-myc-Gro40,89D, or UAS-myc-Gro38,87D lines. Overnight embryos were collected and homogenized in lysis buffer. The lysates were subjected to immunoprecipitation with mouse anti-myc monoclonal Ab.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helices as AH1 and AH2, respectively. When helix-breaking proline residues were introduced into AH1 and AH2, tetramerization did not occur (13).
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-helix formation. L38 and L87 are located on the putative hydrophobic faces of AH1 and AH2, respectively, while A40 and T89 are located on the putative hydrophilic faces of AH1 and AH2, respectively (Fig. 1B). A coiled-coil prediction algorithm (42) suggests that the L38 and L87 mutations will disrupt the coiled-coils while the A40 and T89 mutations will have no effect on coiled-coil formation (Fig. 1C). To test the effects of these mutations on Gro oligomerization, we carried out coimmobilization assays. These assays used a histidine-tagged N-terminal Gro fragment containing the Q and GP domains (His-GroN), as well as wild-type full-length untagged Gro. These two forms of Gro were expressed together or separately in an in vitro translation system and then incubated with Ni-NTA beads. When expressed separately, untagged Gro did not bind the beads (Fig. 2A, lane 1). However, untagged Gro was efficiently recruited to the beads when cotranslated with His-GroN, demonstrating the formation of mixed oligomers containing both the full-length and truncated proteins (lane 2). Under the conditions of these experiments, formation of the mixed oligomers required cotranslation of the two proteins. When full-length Gro and His-GroN were mixed posttranslationally, very little untagged Gro was recruited to the beads (data not shown), strongly suggesting that oligomer dissociation and/or reassociation is slow relative to the time of the coimmobilization assay.
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60 to 70%, while the double mutation (His-GroN38,87D) abolished binding (Fig. 2B). To further assess the effects of the mutations on Groucho oligomerization, we analyzed FLAG-tagged Gro40,89D and Gro38,87D expressed in transformed S2 cell lines. Both proteins localized primarily to the nucleus (Fig. 3A) and were purified from nuclear extracts by anti-FLAG immunoaffinity chromatography (Fig. 3B). On analytical gel filtration chromatography (Fig. 3C), Gro40,89D, which oligomerizes as efficiently as the wild-type protein according to the coimmobilization assays, eluted primarily as a tetramer, a finding consistent with our previous analysis of wild-type Gro (13). In addition, some of the Gro40,89D behaved as a higher-molecular-mass aggregate, eluting just after the void volume (the resin has a nominal exclusion limit of 1,300 kDa) but before the 669-kDa marker. The wild-type protein exhibits a similar high-molecular-mass peak on gel filtration chromatography in addition to the tetramer peak (data not shown). In contrast, Gro38,87D, which failed to oligomerize in the coimmobilization assay, behaved exclusively as a monomer during gel filtration chromatography - we observed neither the tetramer nor any sign of the high-molecular-mass aggregate (Fig. 3C). These experiments support the idea that two coiled-coil motifs corresponding to AH1 and AH2 are required for Gro tetramerization, as well as for the formation of higher-order Gro oligomers.
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In the transient-transfection assays, Gal4-Gro fusion proteins containing Q-domain mutations were introduced into S2 cells and tested for their ability to repress a cotransfected luciferase reporter. In addition to Gal4 sites, the reporter contained multiple sites for the activators Dorsal and Twist, and cotransfection of the reporter with vectors encoding Dorsal and Twist resulted in a 30-fold increase in reporter gene expression (Fig. 4A). When a vector encoding Gal4-Growt was also included in the transfection, reporter activity was significantly reduced (1 and 5 µg of a vector encoding Gal4-Growt resulted in 10- and 15-fold repression, respectively). The control mutant Gal4-Gro40,89D also repressed transcription, although with slightly reduced efficiency relative to Gal4-Growt (1 and 5 µg of a vector encoding Gal4-Gro40,89D resulted in 4- and 12-fold repression, respectively). In contrast, Gal4-Gro38,87D exhibited no ability to repress transcription (Fig. 4A). An anti-Gro immunoblot (Fig. 4B) demonstrated that the inability of Gal4-Gro38,87D to repress is not due to poor expression, since Gal4-Gro38,87D was expressed better than Gal4-Growt and Gal4-Gro40,89D.
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In these experiments, we looked at the role of endogenous Gro in repression by the wild-type Gal4-Gro fusion protein and by Gal4-Gro fusion proteins containing mutations disrupting single coiled-coil motifs. The mutant fusion proteins contained single proline substitutions at either L38 (disrupting AH1) or L87 (disrupting AH2). Transient-transfections assays demonstrated that the single proline substitutions reduced but did not abolish repression (Fig. 4C, top). While the wild-type fusion protein repressed transcription by 13-fold, the single mutants repressed transcription by 2- to 3-fold. Although the RNAi treatment significantly reduced the levels of endogenous Gro (Fig. 4C, bottom), there was little or no effect on repression of the luciferase reporter by the wild-type Gal4-Gro fusion protein (Fig. 4C, top). On the other hand, repression by the single-mutant forms of Gal4-Gro was dependent on endogenous Gro, since it was largely abolished on treatment of cells with Gro dsRNA. This finding is consistent with the idea that repression by the single mutants requires oligomerization with endogenous wild-type Gro. Coimmunoprecipitation assays confirmed that both single-mutant forms of Gal4-Gro can oligomerize with endogenous Gro (Fig. 4D). Consistent with the results of the Ni-NTA pulldown assays (Fig. 2), the single-mutant form of Gal4-Gro bound endogenous Gro less avidly than did wild-type Gal4-Gro, while double-mutant Gal4-Gro completely failed to bind endogenous Gro.
In conclusion, the robust repression directed by wild-type Gal4-Gro is largely independent of endogenous Gro. However, when oligomerization is weakened by the disruption of a single coiled-coil motif, repression is enhanced by endogenous wild-type Gro. This further supports the idea that oligomerization contributes to repression, since the endogenous Gro lacks a Gal4 DNA binding domain and can presumably be recruited to the reporter only by oligomerization with Gal4-Gro.
Oligomerization is required for the lethality and developmental defects that result from Gro overexpression. To determine the effects of mutations in the putative coiled-coil motifs on repression in the fly, we overexpressed mutant forms of Gro by using the binary Gal4-UAS overexpression system. For these experiments, we generated lines containing transgenes encoding myc-tagged Gro38,87D and myc-tagged Gro40,89D under control of the UAS. To determine the levels of myc-tagged Gro expression in various transgenic lines, the UAS-myc-Gro lines were crossed with a line containing an Actin-Gal4 driver, which directs ubiquitous expression, and overnight embryos were analyzed by anti-myc immunoblotting. We observed that while there was some line-to-line variability, the range of expression levels of the two forms of Gro was comparable (Fig. 5A). Consistent with the results of the Ni-NTA pulldown assay (Fig. 2), myc-tagged Gro40,89D but not myc-tagged Gro38,87D could oligomerize with endogenous Gro (Fig. 5B).
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Survivors containing the UAS-myc-Gro40,89D transgene and the Actin-Gal4 driver always exhibited visible defects. These included wing blisters, abundant extra wing vein tissue, and missing pigment bands on the 6th and 7th abdominal tergites (data not shown). In contrast, flies expressing the UAS-myc-Gro38,87D transgene never exhibited visible defects, consistent with the dramatically reduced ability of the protein to oligomerize.
Oligomerization is required for Gro-mediated repression of Brk targets in the wing disk. To further assess the requirement for oligomerization in Gro function, we examined the ability of various mutant forms of Gro to mediate repression by Brinker in the wing disk. In the wild-type wing disk, a Dpp signal emanating from the center of the disk results in the graded activation of the transcription factor heterodimer Mad/Med (2, 47). The Mad/Med heterodimer then cooperates with the transcriptional repressor Schnurri to repress Brinker expression. As a result, Brinker concentrations are highest at the edges of the disk and gradually decrease toward the center. A number of promoters, including the optomotor blind (omb) and vestigal (vg) promoters, are activated by Mad/Med and repressed by Brinker. Antagonism between the opposing Dpp and Brinker gradients is critical in determining the anteroposterior boundaries of the expression domains of these genes. omb is expressed in a narrower anteroposterior domain than is vg, apparently reflecting the somewhat greater sensitivity of this promoter to repression by Brinker. A previous study showed that overexpression of Gro in the wing disk changed the expression pattern of reporters under the control of omb and vg regulatory modules by strengthening repression by Brinker (31), and, as described below, we have taken advantage of this observation to examine the activity of Gro mutations in the wing disk.
To examine the effects of mutant Gro overexpression on the activity of an omb-lacZ reporter, we employed the C765-Gal4 driver, which directs expression throughout the wing disk (Fig. 5C, left panel). In the absence of Gro overexpression, the reporter is expressed in a broad stripe at the center of the third-instar larval wing disk (right panel). Overexpression of wild-type Gro or Gro40,89D, but not of Gro38,87D, resulted in a narrowing of the stripe as well as a reduction in its intensity (Fig. 5D and data not shown). To quantitate this change, we measured the width of the stripe relative to the width of the disk in 40 disks of each genotype (Fig. 5E). The measurements reveal that overexpression of wild-type Gro or Gro40,89D reduced the average width of the stripe by about one-third while overexpression of Gro38,87D had no effect on the width or intensity of the stripe.
Using a strategy previously employed for wild-type Gro (31), we similarly overexpressed the Gro Q-domain mutants in marked clones in the wing disk. We then assessed the resulting effects on the expression patterns of vgQ-lacZ, a reporter containing the dpp-responsive vgQ enhancer from the vg gene. Both clones overexpressing wild-type Gro (Fig. 5F) and clones overexpressing myc-Gro40,89D (Fig. 5G) exhibited cell-autonomous repression of vgQ-lacZ. In contrast, clones overexpressing myc-Gro38,87D exhibited no repression of the vgQ-lacZ reporter (Fig. 5H).
Previous analysis showed that the Q domain is not required for binding to Brinker (31). However, this result was obtained with a Q-domain deletion mutant. To show that the Q-domain point mutations do not alter Gro conformation in a way that prevents binding to Brinker, we carried out coimmbolization assays using GST-Brk fusion proteins. As expected, full-length Gro bound to full-length GST-Brk (Fig. 6A, lane 3) as well as to GST-Brk(441-589) (lane 4), a GST fusion protein containing the central region of Brk, which includes an FKPY Gro-interaction motif (31, 67). In contrast, Gro(2-194), containing the Gro Q and GP domains, bound very poorly to GST-Brk (lane 7) and not at all to GST-Brk(441-589) (lane 8). When we introduced mutations to disrupt AH1 (Fig. 6B, lanes 4 to 6), AH2 (lanes 7 to 9), or both (lanes 10 to 12), we observed a modest reduction in the binding to both GST-Brk (Fig. 6B) and GST-Brk(441-589) (data not shown). This reduced binding can be accounted for by the diminished ability of the mutants to oligomerize, thus reducing the number of Gro protomers immobilized by each GST-Brk/Gro binding interaction. Similar results were observed for the binding of wild-type and mutant Gro to GST-Hairy and GST-Huckebein (data not shown). Thus, the Q-domain point mutants retain the ability to bind Brinker, Hairy, and Huckebein. This reinforces the conclusion that the inability of these mutants to repress transcription is probably the result of their inability to oligomerize.
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DISCUSSION |
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The ability of the same domain to mediate both tetramerization and the formation of higher-order oligomers could easily be explained by the idea of "domain swapping" (8, 40). This is a process whereby a relatively small closed oligomer can be converted into a larger open oligomer by an exchange of a structural element between subunits. In the case of Gro, the tetramer may represent a closed oligomer while the high-molecular-mass form may represent an open oligomer (Fig. 7).
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Long- versus short-range repression. Repressors that employ Gro as a corepressor, such as Hairy and Dorsal, generally mediate long-range repression (7, 66). That is, they are often able to silence a locus completely, regardless of where they are bound relative to the core promoter or relative to the binding sites for activators. In contrast, a number of Gro-independent repressors, including Krüppel, Knirps, and Snail, function as short-range repressors (4, 26). Specifically, they block activation only by activators bound to the template within about 100 bp of the repressor. Short-range repressors are able to interfere with the action of one enhancer while still allowing another enhancer to function. This so-called enhancer autonomy is important at complex loci such as the pair rule loci, in which a single promoter is under the control of multiple enhancers that need to function independently of one another. Interestingly, the short-range repressors mentioned above all rely on the corepressor dCtBP (50). Thus, Gro and CtBP may function by distinct mechanisms that ensure long and short-range repression, respectively.
Studies of other types of long-range repression, particularly heterochromatic silencing, suggest that long-range repression involves the establishment of large, transcriptionally silent chromosomal domains with altered chromatin structure (28, 41). The establishment of a large silent domain often involves the recruitment of enzymes such as histone deacetylases and histone methyltransferases that change the posttranslational modification state of histones. For example, Gro recruits the histone deacetylase HDAC1 via its GP domain, a region just C-terminal to the Q domain (10, 12, 15). Paradoxically, however, the short-range corepressor dCtBP also appears to function, at least in part, by recruiting histone deacetylases to the template (18, 63). Therefore, the difference between long- and short-range repression is not determined by whether a corepressor serves to recruit histone-modifying enzymes. Indeed, studies with yeast have shown that histone-modifying enzymes such as histone deacetylases may normally act in a very local manner to modify only about a 100- to 200-bp region surrounding the site of recruitment (34, 61).
Our finding that Gro can form high-order oligomers and that oligomerization is essential for Gro-mediated repression suggests another way in which long-range repression might be distinguished from short-range repression. In particular, long-range repressors may generally possess the ability to polymerize, perhaps allowing them to spread along the chromatin template and establish a transcriptionally silent domain. In addition to Gro, other long-range corepressors that may have the ability to polymerize include Sir3/Sir4 (29), which is involved in heterochromatic silencing in budding yeast, and HP1/Swi6, which mediates heterochromatic silencing in fission yeast, metazoans, and plants (52). In addition to having the ability to oligomerize, these long-range corepressors bind to specifically modified forms of histones—both Sir3/Sir4 and Gro bind hypoacetylated histone tails (23, 29, 53), while HP1 binds very specifically to the lysine 9-methylated histone H3 tail (6, 36). This interaction between the template and the corepressor may facilitate spreading along the template from a nucleation site. In each case, the corepressor has the additional ability to recruit a histone-modifying enzyme that changes the modification state of the template in such as way as to increase the affinity of the corepressor for the template. Specifically, Gro recruits HDAC1 (a class I histone deacetylase) (10, 12, 15), Sir3/Sir4 recruits Sir2 (an NAD+-dependent histone deacetylase) (32, 37), and HP1 recruits Su(var)3-9 (a histone H3 lysine 9 methyltransferase) (30, 58). This may result in the establishment of a self-reinforcing transcriptionally silent state and, in the case of heterochromatic silencing, may be important for the epigenetic stability of the silenced state.
Overexpression of Gro makes Brinker a more efficient repressor. Dpp is expressed at the anteroposterior midline of the wing disk, from which it is thought to diffuse, resulting in the formation of a Dpp concentration gradient (38, 48). Since the Dpp signal results in the activation of Mad/Med (a transcriptional activator) and the transcriptional silencing of brk (which encodes a Gro-dependent repressor) (44, 65), the gradient of Dpp activity results in a parallel gradient of Mad/Med activity but an opposing gradient of Brk activity. Competition between Mad/Med and Brk is then thought to determine the domains of transcription of dpp target genes such as omb (2, 47). Based on this model, one might expect the on/off state of Dpp target genes such as omb to depend only on the balance between Mad/Med and Brk. However, the findings presented here and elsewhere (31) suggest that the concentrations of coregulators can also be critical. Specifically, when functional Gro was overexpressed in the wing disk, the domain of omb expression was narrowed, indicating that limiting concentrations of Brk can be compensated for by increased levels of Gro. This finding is compatible with the idea that Gro-dependent repression depends on the cooperative assembly of a nucleoprotein complex termed a repressosome. In addition to DNA-bound repressors, this repressosome probably includes corepressors, nucleosomes, and architectural factors and may be analogous to the enhanceosomes that mediate transcriptional activation (16).
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant GM44522 to A.J.C. and by Israel Science Foundation (116/00-1) and Król Charitable Foundation grants to Z.P. P.H. was supported by a Clore Foundation PhD Scholarship. Z.P. is a Braun Lecturer in Medicine.
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FOOTNOTES |
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(lanes 4 and 8). (B) Full-length GroWT (lanes 1 to 3), Gro38P (lanes 4 to 6), Gro87P (lanes 7 to 9), or Gro38,87P (lanes 10 to 12) were incubated with glutathione-agarose beads bearing GST (lanes 2, 5, 8, and 11) or GST-Brk (lanes 3, 6, 9, and 12). The percentage of input protein that bound GST-Brk beads is indicated at the bottom of the GST-Brk lanes.