INTRODUCTION

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Transcriptional repressors that can function at a distance, analogously to transcriptional activators, with separable DNA binding and effector domains, have been termed active repressors (18). Many higher eukaryotic transcription factors have been found to possess such activities (reviewed in references 13 and 23). One such protein that has been well-characterized both in cultured cells and in vivo is the product of the engrailed locus of Drosophila. The Engrailed protein (EN) contains a homeodomain (HD) related in DNA binding specificity to that of members of the Antennapedia class (3) but representing a separate, conserved class with two known members in both insects and mammals. Several members of the Antennapedia class have been shown to be transcriptional activators, including the fushi tarazu protein FTZ. FTZ is a strong, context-independent activator in cultured cells (16, 37) and participates in a direct positive feedback on its own gene in Drosophila embryos (10, 31, 39). By swapping HDs between FTZ and EN, it was shown that EN domains can confer a dominant negative activity on the FTZ HD, counteracting endogenous FTZ protein to generate a ftz mutant phenotype in embryos (21). Indications that this repression is active, rather than simply a disruption of binding by factors that normally interact with ftz, include the dominant repression of the endogenous en gene, another FTZ target in vivo, even in regions in which FTZ is not expressed, and the loss of repression of the endogenous ftz gene upon deletion of a portion of EN from the chimeric repressor that is also required for active repression in culture. This deleted protein, even though it is unable to repress endogenous ftz, still interacts with FTZ target sites in the ftz upstream enhancer, since it is still capable of repressing a transgene driven by this enhancer by competing for binding sites with the endogenous FTZ protein (21). Using a novel assay, we have confirmed this active repression by EN in vivo and have compared the domains required for repression in vivo with those required for active repression in culture. We find that the EN repression function is contributed by multiple domains in both assays but that different domains have different potencies in the two systems. One conserved region (eh1 [25]) is particularly important in vivo (32) but shows very little activity in standard active repression assays involving transient transfection of cultured cells (see reference 11; confirmed in this report). This region mediates interaction with the Groucho (GRO) corepressor. GRO is related to the yeast corepressor TUP1, which mediates active repression by the HD protein 2 (22), as well as to mammalian homologs of the transducin-like Enhancer of Split (TLE) family (35). GRO has been shown to be recruited to DNA by members of other DNA binding protein families, including the Hairy-related basic-helix-loop-helix (HLH) proteins (29) and Runt domain proteins (1). Two other repression domains (one immediately flanking the EN HD) are more potent in transient transfections of cultured cells than in vivo. The differences between their functional characteristics and those of eh1, which mediates the interaction with GRO, suggest that they utilize a distinct mechanism. This distinction appears to hinge on the integrated state of the target gene in vivo, since on integrated target genes in the same cultured cells, the relative potencies of different repression domains closely parallel those seen in vivo.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Embryo preparation and staining. P-element transformations (33), cuticle preparations (36), and in situ hybridization to fixed embryos (7) were performed essentially as described previously. Antibody staining was performed essentially as described elsewhere (28) with a polyclonal -EN antiserum (a kind gift of Charles Girdham and Patrick O'Farrell) that had been prepared against full-length, partially purified, glutathione S-transferase (GST)-tagged EN and affinity purified against a His-tagged peptide with the N-terminal 150 amino acids of EN. Either alkaline phosphatase (AP) or peroxidase-coupled secondary antibodies (Vector Laboratories) were used both for microscopic examination of fixed embryos, for which either 5-bromo-4-chloro-3-indolylphosphate toluidinium (BCIP) and nitroblue tetrazolium (for AP) or 3,3'-diamino-benzidine (DAB) substrates were used for staining (Boehringer Mannheim), and for quantitation of antibody signals, for which the AP substrate p-nitrophenyl phosphate (Sigma) was used as described before (28). Incubation times were determined to be in the linear range of the assay by incubating sets of embryos with different signal intensities for various times.

Transfections and Western blots. Cell culture assays for passive and active repression were performed with Drosophila S2 cells as described before (18), with 2 µg of one of two target genes (T₃N₆D-33Cat_A and N₆T₃D-33Cat_B [18]) per 60-mm culture dish. Active repression assays with each of these target genes gave qualitatively similar results. The values shown in Fig. 2, 3, 5, and 6 were from transfections with the former plasmid. For active repression assays, 0.04 µg of pP_Ac-GR (38) was used to express the rat glucocorticoid receptor (GR). For passive repression assays, 0.3 µg of FTZ expression plasmid pP_Ac-ftz (16, 37) was used. Chloramphenicol acetyltransferase (CAT) assays, as well as -galactosidase assays for expression of the cotransfected reference gene pLac82SU (5), were performed as described elsewhere (18). Cotransfected plasmids used to express EFE and its derivatives were the same as those used for P-element transformation (see below). See figure legends for additional details.

Plasmid constructions and Drosophila strains. Expression plasmids for EFE derivatives were modifications of a P-element transformation vector capable of providing inducible expression of EFE in transformed Drosophila from a heat shock promoter, as described by John et al. (21). Modifications were made using either PCR-based methods (for 5 and 6), synthetic DNA adaptors to create deletions adjacent to unique restriction sites (for 234, 23, 34, 3, and 4), or a combination of the two (eh1, FE, and Meh1). Resulting deletion end points and amino acid substitutions are described in figure legends and the text. All regions containing synthetic or PCR-synthesized DNA were subsequently sequenced (automated) to confirm the expected structure. Appropriate restriction fragments were combined to generate the combined deletion plasmids 46 and 456. Details are available on request. These plasmids were introduced into flies using standard methodologies (33). Homozygous viable insertions on either the second or third chromosome were used in all analyses of repression activity. Additional details are either contained in figure legends or text or are available on request.

Yeast two-hybrid system and in vitro interaction assays. A Drosophila embryonic library (39) in pACT (6) was screened with an EN clone in pAS2 (14) encoding amino acids (aa) 1 to 349 in frame with the Gal4 DNA binding domain as bait. After transformation of Saccharomyces cerevisiae Y190 (14) with bait and library plasmids, 2 × 10⁶ cells viable on synthetic medium lacking Leu and Trp (DOBA -Leu -Trp; Clontech [with both plasmids]) were plated at a density of 300/cm² onto DOBA medium (-Leu -Trp -His) with 30 mM 3-aminotriazole, grown at 30°C until single colonies were visible, replica plated onto DOBA medium (-Leu -Trp), and grown overnight, and replicas were transferred to filters. Cells on the filters were permeabilized by freeze-thaw and were stained for -galactosidase activity. Positive colonies were restreaked and tested for expression of -galactosidase. Plasmids were isolated from positive colonies and tested by cotransformation against several negative control bait plasmids, and the original interaction was verified. Clones surviving all tests were grouped by partial sequencing and restriction mapping.

	RESULTS

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An in vivo assay for active repression. We sought to determine whether the requirements for active repression by EN, as defined in transient transfection assays with cultured cells, were substantially the same as or different from the activities required for repression of endogenous genes in vivo. Previous studies suggested that repression of the endogenous ftz gene by a chimeric repressor termed EFE (EN with its HD replaced by that of FTZ) was analogous to active repression in culture, principally because the activity required a region of the protein well separated from the HD in the primary sequence (32). To validate the use of this assay in a detailed comparison of repression activities, we wanted to test definitively whether this in vivo assay involved active repression, rather than simply a competition for binding sites with an activator. The assay that we developed does not depend on the modularity of repression and targeting domains. Previous studies showed that the EFE derivative FE (which carries a single amino acid substitution in the conserved eh1 repression domain) had lost most of its ability to repress the endogenous ftz gene. With the new assay, we asked whether FE can act as an activator when it is competing with the fully active EFE for sites in vivo. This assay can distinguish whether FE has lost active repression function per se or simply the ability to compete for binding sites. If it had lost only DNA binding ability, then producing FE in combination with EFE, even if it were still able to partially displace EFE, would not prevent repression but instead would have no effect or might augment repression, if the total occupancy of the site increased.

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Passive activation by FE in vivo. FE is a derivative of the EN-FTZ chimera EFE, which carries a single amino acid change in the conserved eh1 repression domain (see text). Passive activation refers to the relief of repression by FE when it competes with the active repressor EFE for target sites. Transgenic lines were heat pulsed for 6 min at 37°C, between 2 h and 40 min and 2 h and 46 min after the end of a 15-min collection. (a) Recipient strain showing the normal pattern of endogenous ftz gene expression; (b) transgenic embryo carrying a heat-inducible EFE transgene; (c) transgenic embryo carrying both the same EFE transgene and, on a separate chromosome, an inducible transgene encoding the point-mutated derivative EFE-FE. (a to c) Embryos from each line were heat shocked and stained in parallel for endogenous ftz RNA by in situ hybridization as previously described (32). The probe does not detect the ftz HD sequence contained in the EFE transgenes. Representative embryos from each strain are shown (see text). (d) Hatching rates were determined, cuticles were prepared 28 h later, and the severity of pattern defects was assessed for lines carrying an EFE transgene insert on chromosome III (EFE3), either without or with an EFE-FE insert on chromosome II (FE2), or carrying an EFE transgene on the second chromosome (EFE2), either without or with EFE-FE on the third chromosome (FE3). Embryos showing a pair-rule pattern of defects in the ventral denticle bands, those showing more severe defects than the pair-rule pattern, and those showing less severe defects were each counted. Very few embryos showed ambiguities between different regions, consistent with pre- vious studies (21) which showed that ftz-dependent pattern elements are deleted preferentially in response to EFE induction, resulting in mostly pair-rule deletions. The percentage of cuticles showing severe (pair-rule or more) defects was multiplied by the fraction that had failed to hatch, and the results are shown as percentages of severe pattern defects. This assumes that all hatched embryos had less severe defects, as previously determined by analyzing hatched larval cuticles (data not shown). Values shown are the averages and ranges from at least two separate experiments with at least 120 embryos per experiment.

Repression activity in cultured cells is determined by multiple EN domains. Previous results showed that EFE, like EN, was capable of repressing transcription in cultured cells independent of the context of its binding sites. Specifically, both basal-level transcription of various promoters and transcription activated by a variety of activators are effectively repressed, even when binding sites for the activator and repressor are separated by more than 400 bp (11, 17, 18). Repression occurring over a distance, which depends on specific binding sites in the target gene, as well as on an activity of the repressor functionally separable from DNA binding activity, was termed active repression. In contrast, passive repression, wherein the repressor directly competes with activators for binding sites, requires only a DNA binding domain, such as the HD (18).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   (A) Features of the EFE chimeric protein. The diagram indicates which portions of the coding sequence derive from EN and which derive from FTZ, our numerical designations of regions of EN (1 to 6 [not including the FTZ HD]), and the locations of known features within those regions (eh1, eh2, eh5, and R). eh1, eh2, and eh5 are peptide sequences found in all known EN homologs (25) from widely divergent species, including insects and mammals; eh1 is also similar to regions of other classes of HD proteins (32), and R is an autonomous active RD identified in cell culture studies (11). Homologies eh2 and eh5 are part of the conserved regions flanking the EN HD, which also include a sequence termed eh3 (immediately flanking the N terminus of the EN HD) that has been implicated in nuclear localization (16a) and thus was left intact in our analyses. Locations of region boundaries in the amino acid sequence are indicated at the bottom. Deletions and other alterations of these regions are described in detail in subsequent figures or in the text. (B) Repression by EFE and derivatives in cultured cells. Drosophila S2 cells were cotransfected with a CAT reporter plasmid, which contains binding sites for both the GR and the FTZ HD, separated by 40 bp, upstream of a basal promoter, and a plasmid that expresses either FTZ or GR (see Materials and Methods for details). Each of the latter two activate reporter expression by 50- to 100-fold above the basal level (shown as 100%). The ability of either EFE or the indicated derivatives to repress this activated transcription was determined by cotransfection of an appropriate expression plasmid. The same amount of a given expression plasmid was used in both repression assays, but the amounts were adjusted among the derivatives to give approximately equal levels of passive repression to allow a more accurate assessment of the potency for active repression. Thus, 4 µg of expression plasmid was used for 234, 3, and 6; 3 µg was used for EFE, 23, and 34; 1 µg was used for 46, 456, and 5; and 0.5 µg was used for 4. The nonrepressed level was determined by cotransfection of 3 µg of empty parental expression plasmid, which is a P-element transformation vector (see Materials and Methods). CAT activities were determined and normalized to the activities of a cotransfected reference gene (see Materials and Methods for details). The graph represents the averages and ranges for at least two independent transfections in at least two separate experiments. (C) Comparison between active repression in culture and hatching rates in vivo in response to EFE derivatives. Active repression was determined as described above, except that the amounts of expression plasmid for 4 and 5 were the same as that for EFE (3 µg). Hatching rates were determined for the wild-type recipient strain (none) and for transgenic lines expressing the indicated EFE derivatives following induction of expression by a 15-min heat pulse at 37°C between 2.5 and 3 h after egg deposition. Both hatched and unhatched egg casings were counted 28 h after egg deposition (hatching normally occurs at 24 h). Error bars indicate the ranges of values obtained with at least four collection plates (with at least 100 eggs per plate) in at least two separate experiments. Similar results were obtained with at least two independent homozygous insert lines for each derivative. Hatching rates in the absence of induction were higher than 95% for each line. (D) Western blot analysis of proteins from transfected cultures. Nuclear extracts of S2 cell cultures were transiently transfected with expression plasmid for the indicated EFE derivatives followed by PAGE, electroblotting, and immunodetection with polyclonal antiserum to the N-terminal region of EN (antiserum affinity-purified by using regions 1 and 2, which were contained within each of these derivatives). Cultures in 60-mm dishes that were 20% confluent were each transfected with 20 µg of expression plasmid and harvested 60 h later, and nuclear extracts were prepared as previously described (11). See Table 1 footnotes for a description of 6'.

Multiple domains also contribute to active repression in vivo but have different relative potencies. We assessed the activity of EFE derivatives in vivo by multiple criteria. A set of transgenic flies were utilized (32), each expressing a deletion derivative of EFE from a heat-inducible promoter. A brief heat pulse induces ubiquitous expression from the transgene. Such expression of EFE causes rapid and persistent loss of ftz expression in the trunk region (Fig. 1) (32). Repression of ftz and other FTZ target genes results in the generation of pair-rule deletions in the cuticle pattern at the end of embryogenesis that mimic those seen in ftz mutants (21). Such heat treatment had no effect on endogenous ftz expression in wild-type embryos (Fig. 1A) (21). In testing derivatives from which EN-derived portions of EFE were deleted, we discovered that multiple regions contribute to activity. In addition to examining their ability to generate a ftz mutant phenotype at the end of embryogenesis (Table 1), we looked at their general ability to disrupt development of embryos when ectopically expressed. For this, we tested the ability of transgenic embryos to hatch following induction. For each derivative, we found a close correlation between its ability to repress the endogenous ftz gene and its ability to prevent hatching (Table 1; Fig. 2C). We also found that each derivative that prevented hatching caused preferential deletion of ftz-dependent pattern elements (Table 1) (see reference 21 for details of the developmental effects of EFE). However, 4 and 5, which repress ftz more strongly than EFE, appear to have lost some specificity in vivo, since they also caused a higher incidence of other defects (results summarized in Table 1).

The eh1 homology mediates repression in vivo but not in transient transfections of cultured cells. The clear difference in potency of 3 between the in vivo and cell culture assays is striking. The ftz repression activity of region 3 was previously attributed (32) to the engrailed homology region eh1 (25). In order to test whether eh1 is required for repression by EFE in cultured cells, we tested both a small deletion within eh1, and a single point mutant at the most conserved position (see reference 32 for a description of the conservation). Both a 15-aa deletion removing the most conserved portion of eh1 and a change of the invariant Phe to Glu (FE [used in the experiments depicted in Fig. 1]) resulted in derivatives of EFE with strongly reduced abilities to generate the ftz mutant phenotype (Table 1) and to prevent hatching (Fig. 3). The levels of ftz RNA are reduced only slightly relative to that of the wild type following induction of each of these derivatives (32). Thus, each of these changes in eh1 had an effect on EFE activity indistinguishable from that of removing region 3 entirely. To determine whether the conservation of this region from flies to mammals had preserved function, we tested a substitution of the 15-aa region of the Drosophila protein with the corresponding region from the mouse EN1 protein. This resulted in four nonconservative and three neutral substitutions and one conservative substitution within the region. This replacement fully restored the ability of EFE to prevent hatching (Fig. 3 [Meh1]) and to produce an ftz mutant cuticle pattern in Drosophila embryos (data not shown), indicating that the function required for this activity, presumably active repression, is conserved. In contrast to the drastic effect of mutating region 3, combining two deletions that each reduce active repression in culture, 4 and 6, resulted in a protein (46) with strong repression activity in vivo (Fig. 3 and data not shown) (see also reference 32). Previously, examination of protein levels produced in embryos showed that for those mutated in region 3, the less active proteins were produced at slightly higher levels than were the more active ones, while all were about equally stable (32). For 46, the levels were slightly higher initially, and the protein was considerably more stable than EFE, perhaps contributing significantly to its activity. However, 46 is less stable than 34 (32), but nonetheless has much greater activity in vivo (compare Fig. 2C and Fig. 3), consistent with the strong in vivo activity of region 3. Western blot analysis of extracts from transfected cultures (Fig. 2D) showed that, as found for 46 and other EFE derivatives (described above), passive repression activities parallel the levels of protein for the derivatives shown in Fig. 3. Thus, eh1, FE, and Meh1 showed levels of protein indistinguishable from that of EFE, while 46 and 456 showed increased levels (two- to threefold) and 6 showed slightly decreased levels (about twofold [data not shown]). Hatching rate was found to accurately parallel the ability of each derivative to generate an ftz mutant phenotype. In addition, all derivatives were localized to nuclei (data not shown). Thus, eh1 mediates the in vivo activity of EFE, while removal of other regions that effect active repression in culture have a less dramatic impact on activity in vivo. Strikingly, as with deletion of region 3, these mutations fail to strongly affect the repression activity of EFE in transient transfections of cultured cells. This series of comparisons clearly shows that while the active repression function of EFE is required for its function in both assays, different domains of EN are responsible for the predominant repression activity in each case. Thus, these different domains, exemplified by eh1 on the one hand and regions 4 and 6 on the other, are likely to function by distinct mechanisms.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Mutations in eh1 more strongly affect activity in vivo, while mutating eh5 (in 6) has a stronger effect in culture. Passive and active repression by EFE and derivatives and hatching rates of transgenic lines were determined as described in the legend to Fig. 2. In each case, hatching rate is an accurate reflection of the ability to repress endogenous ftz and generate ftz mutant cuticle patterns (see text). 6 is a 9-aa deletion, aa 523 to 531, within the conserved region flanking the EN HD (eh5 [Fig. 2A]). 4 removes the RD identified by Han and Manley (11), while 5 removes the conserved region N terminal to the HD. Note that mutating either regions 4 and 6 together or the three regions that contribute strongly to repression in cultured cells (456) abolishes activity in culture, but not in vivo, whereas mutating eh1 has the converse effect.

eh1 is required for interaction with the corepressor GRO. Using as bait an N-terminal fragment of EN (aa 1 to 350) that contains both eh1 and the cell culture RD of region 4, we screened a yeast expression library for interacting proteins using a two-hybrid system (6). After carrying out several tests for specificity and grouping the clones by partial sequencing and restriction mapping, we obtained (from 2 × 10⁶ initial colonies) clones representing 38 distinct cDNAs. We specifically looked for candidate eh1 region interactors by rescreening each group of clones with the same N-terminal EN region but containing the FE mutation. Only one showed a significant reduction in interaction intensity with the point mutant, and, in this case, the interaction was essentially abolished (Fig. 4A). This group, represented by four identical isolates, encoded the C-terminal conserved (WD40 repeat) region of GRO. To further test the specificity of interaction between EN and GRO, we removed the region 4 RD from the N-terminal clone of EN. This resulted in no apparent reduction in the interaction (Fig. 4A). Full-length EN also interacted strongly with the C terminus of GRO, and, conversely, full-length GRO interacted strongly with both the N-terminal region of EN and full-length EN. In each case, the interaction was virtually abolished by the point mutant FE (Fig. 4A). Strong interaction was restored (Fig. 4A) by substituting the eh1 region from the mouse EN1 protein (25). Thus, the requirements for interaction with GRO in this system are the same as the requirements in vivo for the repression activity of the eh1 region.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   (A) Interaction of EN and GRO in yeast. Using a two-hybrid system, we tested the abilities of several EN regions to interact with either full-length GRO (aa 1 to 719) or the GRO WD40 repeat region (aa 399 to 719). The EN derivatives used as bait (fused to the GAL4 DBD) are indicated between the panels. F.L., full length (aa 1 to 552); N.T., N-terminal region (aa 1 to 348); FE, point-mutated derivatives in which the invariant Phe (aa 175) in eh1 was changed to Glu; Meh1, the 15-aa core of the eh1 homology (aa 172 to 186) in the Drosophila protein replaced by the homologous region from the mouse EN1 protein; w/o TCRD (aa 1 to 227), the cell culture RD removed from the N-terminal region; C.T., the C-terminal region of EN (aa 348 to 552); + ctrl, positive control, i.e., mouse p53 as bait interacting with simian virus 40 large T antigen (both sides);  ctrl, negative control, i.e., mouse p53 and GRO (aa 1 to 719 on the left or 399 to 719 on the right). (B) GST-EN interacts with the GRO WD40 repeats in vitro. The EN N-terminal region (aa 1 to 348, without and with the FE point mutation) fused in frame with GST was produced in E. coli, purified via the GST tag, and mixed with in vitro-translated GRO (aa 399 to 719). Following incubation with glutathione-agarose beads, centrifugation, washing, elution (see Materials and Methods), and SDS-PAGE, interacting proteins were visualized by autoradiography. The lower band present also in the GST alone lane is seen even without programming the system with GRO-encoding DNA (not shown).

Stably integrated target genes respond to eh1 in cultured cells. A number of possibilities are suggested by the differences in relative potencies of different RDs when the in vivo and transient transfection assays are compared. For example, a corepressor required for eh1 function in vivo might be missing in S2 cells. However, GRO is present in abundance in these cells (8). Alternatively, if the function of eh1 in vivo requires a normal chromatin environment, the chromatin state of the target gene might be sufficiently different in transient transfections to preclude its function. To attempt to distinguish between these possibilities, we tested whether we could see a more stringent requirement for the eh1 region if the target gene in the cell culture assay were integrated stably into the genome. To this end, we established stably transformed populations of S2 cells containing the same target gene used in the previous transient assays (Fig. 5). We then transfected these cells with activator plasmid encoding GR, along with each of the EFE derivatives shown in Fig. 5A. In sharp contrast to the results with transiently transfected target genes, the repression activity now showed a strong dependence on the eh1 homology region. Rather than causing a reduction of 10% or less in active repression activity (Fig. 3), the point mutation Phe-to-Glu in this region (FE in Fig. 5A) caused a 70% loss of activity. In addition, replacing Drosophila eh1 with the corresponding mouse EN1 region clearly restored activity (Meh1 in Fig. 5A), rather than having an unmeasurable effect, as it did in transient transfection assays in the cells (Fig. 3). As in the transient transfections, 34 had little or no activity in this active repression assay. To confirm and extend these results to the normal EN protein (with its native HD), we transfected the stably transformed cells with the EN derivatives shown in Fig. 5B. Here again, removing eh1 caused a precipitous loss of activity, in contrast to the standard transient transfection assay, in which its removal had no discernible effect (data not shown). Consistently, replacing the Drosophila sequence with the mouse homologous region again restored much of the activity (Fig. 5B [Meh1]). Direct comparison with the 4 and 6 EN derivatives showed that removing eh1 caused more of a loss of activity than removing these other RDs, in sharp contrast to the results with the transiently transfected target gene (compare Fig. 3 and 2). Thus, when an integrated target gene is assayed, the relative potency of EN domains closely parallels that seen in vivo. The eh1 region apparently has an activity that is invisible in the normal transient transfection analysis; this is due not to a difference in cellular environment relative to the in vivo situation but rather to some difference in the assay itself. Perhaps the activity of eh1 requires a more normal chromatin environment than that occurring on transiently transfected DNA.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Repression of integrated target genes in cultured Drosophila cells. A pool of S2 cells stably transfected with the same CAT-expressing reporter used in Fig. 2 and 3 (selected on 200 µg of hygromycin B per ml after cotransfection of reporter with the hygromycin-resistant gene expression plasmid pCop-hygro) were transiently transfected with the activator expression plasmid encoding GR (see Materials and Methods), either alone or with the indicated repressor expression plasmids. Parallel transfections with empty expression vector were used to determine the background of expression without activation, which was subtracted from the results shown. This background (B.G.) amounted to 50 to 80% of the maximum activity, which is the activity with GR alone. (A) EFE and derivatives (with the FTZ HD) were transfected in parallel cultures. Each received 0.1 µg of GR plasmid, 5 µg of the indicated repressor expression plasmid, and 0.5 ng of the reference gene. Values given were normalized to the amount of CAT activity (divided by reference gene activity) with activator, but with empty repressor expression vector (pCaSpeR-hs), which is shown as 100%. The averages and ranges of two independent transfections are shown. Similar results were obtained for four additional independent transfections in two separate experiments. (B) EN and derivatives (with the EN HD) were transfected in parallel cultures as described for panel A, and expression levels were normalized to the level with activator alone, as in panel A. Similar results were obtained in four additional independent transfections in two separate experiments, each with a different pool of stably transfected S2 cells.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Transient transfections with low amounts of reporter and repressor plasmids. S2 cells were transfected as described in the legend to Fig. 2, except that reporter plasmid was reduced by 4-fold to 0.5 µg per 60-mm culture dish, activator plasmid was reduced by 5-fold to 8 ng, and repressor expression plasmids (for EN and EN derivatives) were 12-fold lower (0.4 µg). Total DNA was reduced by 2-fold to 5 µg per dish. The averages and ranges of two independent transfections for each plasmid, normalized to the activity of a cotransfected reference gene and to the activated level without repressor (shown as 100%, corresponding to 18-fold activation above the nonactivated level), are shown. Similar results were obtained in four independent transfections in two additional experiments, one using 0.2 µg of each repressor expression plasmid.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Multiple EN domains contribute to active repression. Analysis of EN repression function in two assays has shown that multiple domains contribute to activity. In the first assay, EN was retargeted in vivo to the endogenous ftz gene (by replacing the EN HD with that of FTZ), resulting in repression of the ftz gene. In the second assay, this chimeric repressor, EFE, actively repressed artificial target genes in cultured cells. Strikingly, one region predominantly affects repression activity in vivo. This region (region 3 [Fig. 2A]) contains the single conserved domain (eh1) not closely associated with the HD in the primary sequence. Deleting the core of this homology region, which is found in all EN class homeoproteins, or mutating the most conserved amino acid, Phe 175 (FE), strongly reduces repression activity in vivo, to a degree equivalent to deleting all of region 3 (Fig. 2 and 3). In contrast, none of these mutations strongly affects repression in cultured cells (Fig. 3), although the effect of deleting regions 3 and 4 appears to be significantly greater than that of deleting region 4 alone (Fig. 2B and C). We established that this region contributes to active repression per se and is not simply defective in binding to endogenous sites by showing that the point-mutated protein FE can actually reduce repression when it is coexpressed with the unmutated EFE (Fig. 1). We interpret this to mean that FE displaces EFE from sites in the ftz gene but is defective in active repression.

The eh1 region interacts with the GRO corepressor. We identified an EN corepressor in a yeast two-hybrid screen, using as bait an N-terminal region of EN that contained both eh1 and the region 4 RD. The interacting clone that we obtained encodes the C-terminal region of GRO, which consists of a tandem array of WD40 repeats highly homologous to the C terminus of both the yeast corepressor TUP1 and mammalian TLE proteins (35). This region of TUP1 mediates its interaction with the 2 protein (22), which, like EN, is a homeodomain-containing repressor. GRO is also recruited to DNA by both Hairy-related bHLH repressors (29) and Runt domain proteins (1). We found that the FE mutation in eh1, which abolishes the repression activity of eh1 in embryos, virtually eliminates interaction with GRO (both full length and the WD40 repeat region) in the yeast assay, in the context of both full-length EN and the N-terminal region (Fig. 4A). This was confirmed in vitro by GST pulldown assays with the N-terminal region of EN (Fig. 4B). Furthermore, just as substituting the mouse eh1 region for that of Drosophila restores repression activity in Drosophila embryos, the same substitution restores interaction with GRO in the yeast assay (Fig. 4A). Similarly, Jiménez et al. (20) recently showed that the eh1 region of EN is required for GRO-dependent repression by a Hairy-EN fusion protein in Drosophila embryos. The GRO-EN interaction appears to be completely independent of the cell culture RD of region 4, since removing it entirely has no apparent effect on the strength of the interaction in yeast (Fig. 4A). Thus, the requirements for EN-GRO interaction correlate well with the requirements for repression by the eh1 region, while the apparent lack of involvement of the region 4 RD in interaction with GRO is consistent with its distinct functional characteristics, as discussed below.

Distinct mechanisms of active repression in vivo and in culture. A detailed comparison of the relative potencies of different RDs in the in vivo and cell culture assays leads to the conclusion that multiple mechanisms of active repression are likely to be encoded by EN. The most striking example is highlighted by the comparisons of Fig. 3, in which it is shown that alterations of region 3 and the eh1 homology that it contains clearly have distinct effects from alterations in regions 4 and 6. Region 3, which interacts with GRO, primarily affects activity in vivo, while regions 4 and 6 have much stronger effects in transient transfection assays in culture. This difference is not due simply to one assay being more stringent than the other; rather, the eh1 domain is dispensable for repression in transient transfections, but not in vivo, while the R domain and region 6 are dispensable in vivo, but not in transient assays. This distinction suggests that these two types of RD confer mechanistically different activities on EFE that are each preferentially active in different contexts. Three possibilities for the critical difference in context are (i) the cell type in which the assay is done (cultured cells versus embryonic tissues), (ii) the target gene assayed (reporter genes in culture versus the endogenous ftz gene), and (iii) the integration state of the target gene (transiently transfected DNA versus a normal chromatin environment). The first two of these possibilities are ruled out by our assays of stably integrated target genes in cultured cells (discussed below).

Stable integration of target genes reveals a GRO-dependent repression activity invisible in transient transfections. One difference between the in vivo assay for active repression by EFE and the standard transient assay in cultured cells is the state of the target gene. We tested whether the activity of the eh1 region might be sensitive to this difference by testing stable transformants. Cultured cells stably transformed with the same target gene that showed very little sensitivity to mutation of eh1 in transient assays were transfected either with EFE or with derivatives mutated in the eh1 region that no longer interact strongly with GRO. This transient-on-top-of-stable assay allowed us to directly compare the activities of different repressors in the same population of cells containing the stably integrated target gene. When eh1 was mutated in the context of either EFE or normal EN, the ability to repress the integrated target gene was severely compromised. This is in striking contrast to the effect in the standard transient transfection assay, in which removing eh1 had very little effect. In addition, replacing Drosophila eh1 with mouse eh1 restored activity. Thus, when the target gene is integrated into a chromosome, its repression by the different EN domains closely parallels that of a natural target gene in vivo. This suggests that the state of the target gene is important for repression by the conserved eh1 domain and, by inference, GRO, but not by the other class of EN domains that are more active in transient transfections. One plausible explanation for this difference is that one RD class, exemplified by eh1, represses by stabilizing or inducing a repressive chromatin structure, while the other, exemplified by regions 4 and 6, acts on another target, perhaps the basal transcriptional machinery.