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Originally published In Press as doi:10.1074/jbc.M313200200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 11, 9725-9732, March 12, 2004
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Repression by TTK69 of GAGA-mediated Activation Occurs in the Absence of TTK69 Binding to DNA and Solely Requires the Contribution of the POZ/BTB Domain of TTK69*

Sara Pagans{ddagger}§, David Piñeyro, Ana Kosoy§||, Jordi Bernués, and Fernando Azorín**

From the Department de Biologia Molecular i Cellular; Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi Girona Salgado, 18-26, 08034 Barcelona, Spain

Received for publication, December 3, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
tramtrack 69 (TTK69) is known to repress GAGA-mediated activation of the eve promoter in S2 cells. Here, we show that repression by TTK69 occurs in the absence of bona fide TTK69-binding sites on the template, indicating that it does not require the binding of TTK69 to DNA. Consistent with this interpretation, the POZ/BTB domain of TTK69, which does not bind DNA, is sufficient for repression. Moreover, a fusion protein in which the POZ/BTB domain of GAGA is replaced by that of TTK69 is not capable of activating the eve promoter but efficiently represses GAGA-dependent activation. Repression involves GAGA-TTK69 interaction because TTK69 is not capable of repressing basal transcription. Most probably, GAGA-TTK69 interaction occurs at the promoter because GAGA·TTK69 complexes are fully competent in binding DNA in vitro. Our results also show that repression by TTK69 of GAGA-dependent activation of the eve promoter is not mediated by any of the co-repressors known to interact with TTK69 (dMi2 or C-terminal binding protein) or by trichostatin A-sensitive histone deacetylases. Altogether, these observations strongly suggest that the binding of TTK69 prevents the interaction of GAGA with the transcription machinery and, therefore, compromises its activation potential.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Trithorax-like (GAGA) and tramtrack (TTK)1 are zinc finger transcription factors of Drosophila that play essential roles during development. Many developmentally regulated genes in Drosophila are known to be activated by GAGA (1), and it has been shown that GAGA acts as a transcription activator in vitro or upon transient transfection in cultured S2 cells (2–4). On the other hand, at the preblastoderm stage, TTK acts as a maternally provided repressor of several pair-rule genes that was proposed to contribute to the establishment of the precise timing of their zygotic expression (5–7). TTK is also known to play a critical role in the development of the nervous system by repressing neuronal identity and stabilizing non-neuronal fates (8–11). GAGA exists as two different splice forms that, differing in the C-terminal glutamine-rich domain, appear to play highly redundant functions in vivo (2, 12) and have indistinguishable DNA binding, chromatin remodeling, and transcription activities in vitro (2). The tramtrack gene also encodes two splicing forms, TTK69 and TTK88, which differ in the C-terminal zinc finger region and thus have different DNA binding specificities (13). The repressing activity of TTK69 is well established. Ectopic expression of TTK69 early in embryo development was shown to cause repression of various pair-rule genes including even-skipped (eve) (6, 7), and overexpression of TTK69 in cultured S2 cells repressed GAGA-dependent activation of the eve promoter (14). Moreover, overexpression of TTK69 during early eye development blocks neuronal photoreceptor differentiation and promotes non-neuronal cone cell specification (15).

Many genes in Drosophila, such as eve, contain at their regulatory regions binding sites for GAGA and TTK, suggesting a coordinated contribution to the regulation of their expression pattern. Actually, as mentioned above, TTK69 represses GAGA-dependent activation of the eve promoter but not basal transcription (14). In this paper, the mechanisms of repression by TTK69 of the eve promoter were analyzed in S2 cells. It was shown earlier that repression by TTK69 of the eve promoter involves direct GAGA-TTK69 interaction (14). Here, we show that repression does not require the binding of TTK69 to DNA because it occurs in the absence of TTK69-binding sites on the template and the POZ/BTB domain of TTK69, which does not bind DNA, is sufficient for repression. Several mechanisms have been proposed to account for the repression activity of TTK69. TTK69 was found to interact both biochemically and genetically with the transcriptional co-repressor dCtBP (15) as well as with the dMi2 subunit of the nucleosome remodeling and histone deacetylase or NuRD co-repressor complex (16). dMi2 is not likely to mediate repression by TTK69 of the eve promoter because it binds TTK69 through a 100 aa region lying immediately upstream of the zinc fingers of TTK69 outside of the POZ/BTB domain (16). On the other hand, the POZ/BTB domain of TTK69 is known to contribute to the interaction with dCtBP (15). Our results, however, indicate that repression by TTK69 of GAGA-dependent activation of the eve promoter is neither affected by changing dCtBP levels nor is sensitive to trichostatin A (TSA) treatment. These observations, together with the known requirement for the presence of GAGA at the promoter (14), argue in favor of a quenching or masking mechanism in which the binding of TTK69 compromises the activation potential of GAGA by preventing its interaction with the transcription machinery. Most likely, GAGA-TTK69 interaction occurs at the promoter because GAGA·TTK69 complexes bind DNA with an apparent higher affinity than GAGA itself.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins—The proteins used in these experiments correspond to the GAGA519 (2, 3) and TTK69 isoforms (8, 13). POZTTK corresponds to a truncated TTK69 form that carries the first 114 residues of TTK69. Proteins were expressed in BL21LysS or BL21 Escherichia coli cells as His6-tagged proteins using pET14 expression vectors (Novagen) and/or as GST fusions proteins using pGEX-KG vectors (Amersham Biosciences). Rabbit polyclonal antibodies raised against bacterial expressed GAGA519 and TTK69 proteins were obtained and purified by conventional methods.

Transcription Assays in S2 Cells—Repression by TTK69 of GAGA-dependent activation of the eve promoter was analyzed by transient expression experiments in cultured S2 cells using a pGL3 reporter plasmid (Promega) carrying a luciferase reporter gene fused to a 1.8-kb fragment of the eve promoter (from position +102 to position –1759) that was found to drive transcription within the limits of stripe 2 (17) or similar plasmids carrying truncated promoters containing only the proximal promoter region. Reporter plasmids were co-transfected with pAct5CPPA vectors expressing the proteins indicated in each case. POZTTK corresponds to a truncated TTK69 form carrying the first 114 aa of TTK69. {Delta}TTK580 is a C-terminal deletion to aa 580 of TTK69. {Delta}POZGAGA is a N-terminal deletion to aa 122 of GAGA519 that was expressed as a N-terminal His6-tagged polypeptide, which contrary to untagged {Delta}POZGAGA (14) was found to be efficiently expressed in S2 cells (data not shown). POZTTK{Delta}POZGAGA corresponds to a fusion protein in which the first 122 aa of GAGA519 were replaced by the first 114 aa of TTK69. dCtBP (18) was expressed as a C-terminal FLAG fusion protein.

For each assay, 7 µg of the corresponding reporter plasmid were co-transfected to S2 cells together with increasing amounts of pAct5CPPA vectors expressing the proteins indicated in each case. 3 µg of pCMV-{beta}-galactosidase plasmid (Stratagene) were added to the transfection mixture to allow correction for variations in transfection efficiencies. Total DNA was adjusted to 20 µg by the addition of pAct5CPPA. Transfection was carried out by the calcium phosphate method. Cells were harvested 48 h after transfection and lysed according to the {beta}-galactosidase gene reporter kit (Roche Applied Science). Luciferase activity was determined according to the luciferase activity assay kit (Promega) and normalized with respect to {beta}-galactosidase activity. When the effect of TSA was determined, cells were treated with 300 nM TSA 20 h before harvesting.

For RNAi experiments, a EcoRI/HindIII fragment (~500 bp) of the dCtBP-coding sequence was inserted in pBluescript (Stratagene) and transcribed in vitro using T3/T7 RNA polymerases. Increasing amounts of double-stranded RNA were co-transfected to S2 cells as indicated.

EMSA Experiments—For EMSA experiments, increasing amounts of bacterial expressed GAGA519 and/or TTK69 were incubated with 1 ng of 32P-labeled –173eve DNA fragment obtained from the corresponding pGL3 plasmid in 100 mM KCl, 15 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.5 mM dithiothreitol, and 8% glycerol in the presence of 1 µg of bovine serum albumin and a 100-fold excess (w/w) of E. coli DNA for 20 min at 20 °C in a final volume of 25 µl. Protein-DNA complexes were analyzed on 0.8% agarose, 0.5x TBE gels. Electrophoresis was performed at 100 V and 4 °C. After electrophoresis, gels were dried and autoradiographs were recorded on Hyperfilm (Amersham Biosciences) at –80 °C.

DNase I Footprinting—For DNase I footprinting, protein-DNA complexes were prepared as described above for EMSA experiments and treated with DNase I in 8 mM MgCl2, 2 mM CaCl2. Digestion was allowed to proceed at 20 °C for 1 min and stopped by the addition of 4 volumes of 50 mM Tris-HCl (pH 8.0), 0.1 mM NaCl, and 0.5% SDS. Samples were then analyzed electrophoretically in 6% polyacrylamide, 8 M urea, and TBE-denaturing gels. After electrophoresis, gels were dried and autoradiographs were recorded on Hyperfilm at –80 °C.

GST Pull-down Assays—GST and GST-POZTTK proteins were expressed in E. coli and bound to glutathione-Sepharose 4B beads. Protein concentrations, determined by gel electrophoresis, were adjusted by dilution with unbound beads. Agarose-bound GST fusion proteins were equilibrated in buffer D (20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, 5 mg/ml bovine serum albumin), and 25 µl of beads were incubated for 30 min at room temperature with 0.2 ng of 32P-labeled –173eve fragment that was preincubated or not with 1 µg of recombinant GAGA519 for 30 min at room temperature in a final volume of 100 µl. Beads were washed three times with 450 µl of buffer D and once with 450 µl of 0.2 M KCl-buffer D. DNA was recovered by incubation with 100 µl of 1% SDS, 1% NaHCO3 twice, phenol/chloroform extraction, and ethanol precipitation. Samples were analyzed on 8% PAGE-0.5x TBE, dried, and exposed to x-ray-sensitive film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Most Proximal eve Promoter Region, Which Contains Binding Sites for GAGA but Not for TTK69, Is Sufficient for TTK69 Repression of GAGA-dependent Activation—We showed previously (14) that, in S2 cells, overexpression of TTK69 represses transcription from a truncated eve promoter spanning 1.7 kb of the 5' region flanking the transcription start site to position –1759. This truncated promoter contains the core promoter elements and all cis-regulatory sequences necessary to drive transcription within the limits of stripe 2 (17). Our previous results also indicate that repression by TTK69 of the eve stripe 2 promoter occurs only when it is activated by GAGA (14). In the eve stripe 2 promoter, most of the GAGA and TTK69-binding sites cluster at the proximal promoter region between positions +1 and –400 (Fig. 1A). As determined by DNase I footprinting, this region contains at least eight binding sites for GAGA and four for TTK69 (19). A deleted eve promoter that contains only the proximal promoter elements up to position –413 recapitulates the results reported earlier with the full eve stripe 2 promoter (14). The profile of activation by GAGA of the –413eve promoter construct (Fig. 1B, left panel) is not significantly different from that obtained with the full eve stripe 2 promoter, and TTK69 represses very efficiently GAGA-dependent activation of the deleted –413eve promoter (Fig. 1B, right panel).



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  		FIG. 1.
The most proximal eve promoter region is sufficient for repression by TTK69 of GAGA-dependent activation. Constructs used in these experiments are shown in panel A (see under "Results" for details). The distribution of binding sites for GAGA (open ellipses) and TTK69 (full ellipses) is indicated. S2 cells were co-transfected with reporter plasmids carrying a luciferase gene fused to the –413eve promoter (panel B) or to the –173eve promoter (panel C) and increasing amounts of the plasmid overexpressing GAGA (left) or with the same reporter plasmid, 4 µg of the plasmid overexpressing GAGA, and increasing amounts of the plasmid overexpressing TTK69 (right). These data correspond to the average of six independent experiments.

 
Within the proximal eve promoter, the four TTK69-binding sites map between positions –200 and –400 (19) so that the most proximal promoter region from position +1 to –173 contains no binding sites for TTK69, although it still carries several GAGA-binding sites of high affinity (Fig. 1A). The absence of TTK69-binding sites within the deleted –173eve promoter was corroborated in vitro by EMSA (Fig. 2) and DNase I footprinting experiments (Fig. 3). No significant retardation of the –173eve promoter fragment was observed upon the addition of up to 720 ng of bacterially expressed TTK69 (Fig. 2B). Similarly, no detectable DNase I footprints were observed even in the presence of up to 1200 ng of TTK69 (Fig. 3B). On the other hand, TTK69 binds the –413eve promoter fragment with significant affinity, showing 50–60% binding in the presence of 100 ng of protein (data not shown). The binding of GAGA to the –173eve promoter fragment was also confirmed by EMSA and DNase I footprinting (Figs. 2 and 3). Significant binding was observed in the presence of 400 ng of GAGA, and full retardation was obtained upon the addition of 600 ng of GAGA (Fig. 2A). Under these conditions, four strong DNase I footprints were detected (Fig. 3A) that corresponded to the four central GAGA-binding sites contained within the –173eve promoter fragment (Fig. 3D), the two furthermost 5'- and 3'-GAGA-binding sites not being resolved under the experimental conditions of Fig. 3A.



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  		FIG. 2.
The binding of GAGA and TTK69 to the –173eve promoter is analyzed by EMSA (panels A–C) and GST pull-down (panel D). The binding of GAGA (A) or TTK69 (B) to the –173eve promoter DNA fragment is presented as a function of increasing amounts of protein: 0 ng (lanes 0); 100 ng (lanes 1); 200 ng (lanes 2); 400 ng (lanes 3); and 600 ng (lanes 4). In C, the binding of TTK69 was determined in the presence of 280 ng of GAGA and is shown as a function of increasing amounts of TTK69: 0 ng (lane 0); 240 ng (lane 1); 480 ng (lane 2); and 720 ng (lane 3). In D, 0.2 ng of radioactively labeled –173eve fragment DNA was incubated either in the absence (lanes 1 and 3) or in the presence of 1 µg of GAGA (lanes 2 and 4) and then incubated with either GST (lanes 1 and 2) or GST-POZTTK (lanes 3 and 4) Sepharose beads. The amount of radioactively labeled DNA retained was then analyzed electrophoretically in 8% PAGE-0.5x TBE gels. Lane 0 shows 50% of the input DNA.

 



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  		FIG. 3.
The binding of GAGA and TTK69 to the –173eve promoter is analyzed by DNase I footprinting. The binding of GAGA (A) or TTK69 (B) to the –173eve promoter DNA fragment is presented as a function of increasing amounts of protein: 0 ng (lanes 0); 200 ng (lanes 1); 400 ng (lanes 2); 800 ng (lanes 3); and 1200 ng (lanes 4). In C, DNase I footprinting was carried out in the absence (lane –) or in the presence of 100 ng of GAGA (lanes 0–4) and increasing amounts of TTK69: 0 ng (lane 0); 200 ng (lane 1); 400 ng (lane 2); 800 ng (lane 3); and 1200 ng (lane 4). Lanes C, correspond to G+A sequencing ladders. Regions showing footprints are indicated. 5' to 3' direction are also indicated. Summary of the results is shown in D. Observed footprints are indicated by boxes, and previously reported GAGA-binding sites (19) are underlined.

 
GAGA activated transcription from the deleted –173eve promoter (Fig. 1C, left panel). This activation was strictly dependent on the ability of GAGA to bind DNA because a H361P mutation in the DNA-binding domain of GAGA, which impairs DNA binding (20), fully abolished activation (data not shown). Interestingly, TTK69 can still repress very efficiently GAGA-dependent activation of the –173eve promoter (Fig. 1C, right panel). These results indicate that repression by TTK69 of GAGA-dependent activation occurs in the absence of bona fide TTK69-binding sites on the template, suggesting that it does not require direct interaction of TTK69 with DNA.

The POZ/BTB Domain of TTK69 Is Sufficient for Repression of GAGA-dependent Activation—As reported previously (14), repression by TTK69 of GAGA-dependent activation of the eve promoter requires the contribution of the POZ/BTB domain of TTK69 because a truncated {Delta}POZTTK form is not competent in repressing GAGA-mediated activation. Actually, the POZ/BTB domain of TTK69 was sufficient to repress GAGA-mediated activation because overexpression of POZTTK, a truncated form that carries only the POZ/BTB domain of TTK69, was capable of repressing the eve stripe 2 promoter (Fig. 4), although less efficiently than full-length TTK69. POZTTK lacked the two zinc fingers that mediated DNA binding. Therefore, these results are in good agreement with the strong repression observed in the absence of bona fide TTK69-binding sites on the template and provide additional evidence in favor of a model in which repression by TTK69 of GAGA-mediated activation does not require the binding of TTK69 to DNA.



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  		FIG. 4.
The POZ/BTB domain of TTK69 is sufficient for repression of GAGA-dependent activation. S2 cells were co-transfected with the luciferase-reporter plasmid carrying the full eve stripe 2 promoter, 4 µg of the plasmid overexpressing GAGA, and increasing amounts of plasmids overexpressing POZTTK (panel A) or {Delta}TTK580 (panel B). Results were normalized with respect to the activation obtained when only the plasmid overexpressing GAGA was cotransfected (bars 0). Data corresponds to the average of six independent experiments.

 
The essential contribution to repression of the POZ/BTB domain of TTK69 was also revealed when the POZ/BTB domain of GAGA was replaced by that of TTK69. Such a fusion protein, POZTTK{Delta}POZGAGA, was not capable by itself of activating the eve stripe 2 promoter (Fig. 5A) but, on the other hand, was still capable to efficiently repress GAGA-mediated activation (Fig. 5B). It might be argued that, in this case, repression is the consequence of a direct competition for binding to the template because POZTTK{Delta}POZGAGA carries the DNA-binding domain of GAGA; therefore, it can bind the same DNA sequences as GAGA. To test for this hypothesis, we analyzed the effects on GAGA-mediated activation of the eve promoter of a truncated form of GAGA, {Delta}POZGAGA, which was missing the POZ/BTB domain. {Delta}POZGAGA binds DNA very efficiently (21), but it cannot by itself activate transcription (Fig. 5C). Moreover, this truncated form repressed GAGA-mediated activation very inefficiently since only a modest repression was observed at very high protein concentration (Fig. 5D). In these experiments, POZTTK{Delta}POZGAGA and {Delta}POZGAGA were expressed to equivalent levels as judged by Western blotting and accumulated in the nucleus (data not shown). These results indicate that repression by POZTTK{Delta}POZGAGA of GAGA-dependent activation was not likely the result of competition for the GAGA-binding sites.



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  		FIG. 5.
POZTTK{Delta}POZGAGA efficiently represses GAGA-dependent activation. A, S2 cells were co-transfected with the luciferase-reporter plasmid carrying the full eve stripe 2 promoter and increasing amounts of the plasmid overexpressing POZTTK{Delta}POZGAGA (full bars). For a comparison, the activation profile obtained in the presence of increasing amounts of the plasmid overexpressing GAGA is included (empty bars). B, as in A but co-transfection was carried out in the presence of 4 µgofthe plasmid overexpressing GAGA and increasing amounts of the plasmid overexpressing POZTTK{Delta}POZGAGA. Results were normalized with respect to the activation obtained when only the plasmid overexpressing GAGA was co-transfected (bar 0). C, as in A but co-transfection was carried out in the presence of increasing amounts of the plasmid overexpressing {Delta}POZGAGA (full bars). The activation profile obtained in the presence of increasing amounts of the plasmid over-expressing GAGA is also included (empty bars). D, as in A but co-transfection was carried out in the presence of 4 µgofthe plasmid overexpressing GAGA and increasing amounts of the plasmid overexpressing {Delta}POZGAGA. Results were normalized with respect to the activation obtained when only the plasmid overexpressing GAGA was cotransfected (bar 0). These data correspond to the average of six independent experiments.

 
GAGA·TTK69 Complexes Bind DNA in Vitro—The results reported above show that repression by TTK69 of GAGA-mediated activation of the eve promoter does not involve the binding of TTK69 to DNA. Repression, however, involves direct GAGA-TTK69 interaction because it requires the contribution of the POZ/BTB domain of GAGA (14) as well as that of TTK69, which is necessary (14) and, as shown above, sufficient for repression. Therefore, it might be possible that GAGA-TTK69 interaction would prevent GAGA from binding DNA, thus impairing activation. Inconsistent with this hypothesis, in vitro, the binding of GAGA to the –173eve promoter was facilitated in the presence of TTK69 (Fig. 2C) so that when binding is assayed by EMSA at a GAGA concentration showing ~50% retardation (Fig. 2C, lane 0), full retardation is observed upon the addition of TTK69 at concentrations that by themselves show no binding to the –173eve promoter fragment (Fig. 2, compare lanes 1–3 in panel C with lanes 2–4 in panel B). Similar results were obtained by DNase I footprinting (Fig. 3C). The addition of increasing amounts of TTK69 at a GAGA concentration that by itself does not give rise to any detectable footprint resulted in the formation of clear footprints (Fig. 3C, compare lane 0 with lanes 1–4). These footprints were restricted to the GAGA-binding sites contained within the –173eve promoter because no detectable protection to DNase I cleavage was observed outside of them. Sites 2, 3, and 4 were significantly protected, but under these conditions, site 1 was only weakly protected.

The binding of GAGA to the –173eve promoter fragment resulted in the formation of complexes of very high molecular mass that can only be resolved electrophoretically in low percentage (0.8%) agarose gels (Fig. 2A). The high molecular mass of GAGA·DNA complexes reflect the oligomeric character of GAGA and the presence of multiple GAGA-binding sites on the template (21). Under these experimental conditions, ternary TTK69·GAGA·DNA complexes were likely to behave as a heterogeneous population of very high molecular mass rather than as a single electrophoretic species. Actually, in the presence of GAGA and TTK69, a smear of very high molecular mass was observed (Fig. 2C, lanes 2 and 3), probably reflecting the formation of such ternary complex. Consistent with this interpretation, in GST pull-down assays, the complex formed by GAGA with the –173eve promoter fragment bound very efficiently a GST-POZTTK fusion protein (Fig. 2D). In these experiments, radioactively labeled –173eve DNA fragment was first incubated with GAGA to form the protein-DNA complex and then assayed for binding to either GST-POZTTK or GST. As shown in Fig. 2D, the GAGA·DNA complex was efficiently bound by GST-POZTTK (Fig. 2D, lane 4) but not by GST (Fig. 2D, lane 2). Moreover, the binding of the complex to GST-POZTTK was strictly dependent on the presence of GAGA, because no retention was observed when preincubation with GAGA was omitted (Fig. 2D, lane 3). Altogether, these results indicate that binding of TTK69 does not prevent binding of GAGA to DNA. On the contrary, DNA binding increases in the presence of TTK69, suggesting that GAGA·TTK69 complexes bind DNA with a higher affinity than GAGA.

Repression by TTK69 of GAGA-dependent Activation Is Not Affected by Changing dCtBP Levels Nor Is It Sensitive to Treatment with TSA—It is known that TTK69 interacts physically and genetically with dCtBP (15). Actually, a P-DLS consensus dCtBP binding motif was located at the C-terminal region of TTK69 (aa 591–595). Mutations at this motif modified the functional properties of TTK69 (15). This motif, however, did not appear to contribute to repression because a truncated {Delta}TTK580 form carrying a C-terminal deletion up to aa 580 repressed GAGA-mediated activation of the eve promoter as efficiently as full-length TTK69 (Fig. 4B). Nevertheless, in vitro, the binding of dCtBP to TTK69 appeared to be largely mediated by the POZ/BTB domain (15) that, as shown here, was sufficient for repression of GAGA-dependent activation (Fig. 4A). Therefore, in this case, dCtBP could mediate repression through its binding to the POZ/BTB domain rather than by binding to the P-DLS motif. Inconsistent with this hypothesis, no synergism was observed when dCtBP and TTK69 were co-expressed. As shown in Fig. 6A, the efficiency of repression by TTK69 did not increase when dCtBP was overexpressed. In these experiments, dCtBP was efficiently expressed and showed a nuclear localization (data not shown). Moreover, the dCtBP form used here was found to be functional in S2 cells because in a different promoter context, its overexpression was shown to interfere with activation (22). In good agreement with these results, co-transfection of increasing amounts of RNAi directed against dCtBP showed no significant effect on the ability of TTK69 to repress GAGA-mediated activation of the eve promoter (Fig. 6B). In parallel experiments, a significant decrease in dCtBP levels was detected when overexpressed in the presence of increasing amounts of RNAi directed against dCtBP (data not shown).



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  		FIG. 6.
Repression by TTK69 of GAGA-dependent activation is insensitive to changing levels of dCtBP or treatment with TSA. A, S2 cells were co-transfected with the luciferase-reporter plasmid carrying the full eve stripe 2 promoter, 4 µgofthe plasmid overexpressing GAGA, 0.5 µgofthe plasmid overexpressing TTK69, and increasing amounts of the plasmid overexpressing dCtBP. Results were normalized with respect to the activation obtained with 4 µg of the plasmid overexpressing GAGA in the absence of TTK69 and dCtBP (bar –). These data correspond to the average of six independent experiments. B, as in A but cells were co-transfected with 4 µg of the plasmid overexpressing GAGA, 0.5 µg of the plasmid overexpressing TTK69, and the amounts indicated of RNAi directed against dCtBP. These data correspond to the average of two independent experiments. C, as in A but cells were co-transfected with 4 µg of the plasmid overexpressing GAGA and the amounts indicated of the plasmid overexpressing TTK69 and then either treated with 300 nM TSA 20 h before harvesting () or not ({square}). Results were normalized with respect to the activation obtained with 4 µg of the plasmid overexpressing GAGA in the absence of TTK69 and, in this case, are presented as percent repression as a function of increasing TTK69 overexpression. These data correspond to the average of six independent experiments.

 
POZ/BTB is an evolutionary conserved domain present in many transcriptional repressors. In several cases, its contribution to repression was found to be mediated by TSA-sensitive histone deacetylases (23–30). However, repression by TTK69 of GAGA-dependent activation of the eve promoter was insensitive to treatment with TSA. As shown in Fig. 6C, the ability of TTK69 to repress GAGA-mediated activation was not significantly affected when cells were treated in the presence of 300 nM TSA, although under these conditions, significant changes in histone acetylation levels were detected (data not shown). Altogether, these results strongly suggest that repression by TTK69 of GAGA-dependent activation of the eve promoter was not likely mediated by dCtBP or TSA-sensitive histone deacetylases.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this paper, we have shown that repression by TTK69 of GAGA-dependent activation of the eve promoter occurred in the absence of TTK69 binding to DNA. Several models could account for these results (Fig. 7). It would be possible that GAGA-TTK69 hetero-oligomers would not be capable of binding DNA (Fig. 7B) or that, by interacting with the transcription activation machinery, TTK69 would prevent its recruitment by GAGA to the promoter (Fig. 7C). However, earlier results (14) show that repression by TTK69 of the eve stripe 2 promoter depends on its activation by GAGA and requires the contribution of the POZ/BTB domains of GAGA and TTK69, strongly suggesting that it actually involves direct GAGA-TTK69 interaction. Moreover, in vitro, GAGA-TTK69 hetero-oligomers appeared as fully competent in binding DNA (Fig. 2). These results argue in favor of models in which GAGA contributed to the recruitment of TTK69 to the promoter (Fig. 7, D and E). In agreement with this hypothesis, repression occurred in the absence of TTK69-binding sites on the template and the POZ/BTB domain of TTK69 showed by itself a significant repressive effect. Nevertheless, TTK69 forms capable of binding DNA were more efficient than POZTTK in repressing GAGA-mediated activation, indicating that TTK69-DNA interactions further stabilize the ternary protein-DNA complex and therefore increase the efficiency of repression. This possibility becomes particularly evident in promoter constructs containing binding sites for TTK69. However, also in the absence of TTK69-binding sites as in the –173eve promoter fragment, GAGA-TTK69 hetero-oligomers appeared to bind DNA with higher affinity than GAGA homo-oligomers themselves (Figs. 2 and 3). The binding of GAGA-TTK69 hetero-oligomers to the –173eve promoter was constrained to the GAGA-binding sites because no additional DNase I footprints were detected in the presence of TTK69 (Fig. 3), suggesting that, in this case, direct protein-protein interactions or, more likely, non sequence-specific TTK69-DNA interactions help to stabilize the binding of GAGA to DNA.



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  		FIG. 7.
Possible models for repression by TTK69 of GAGA-dependent activation. GAGA activates transcription from promoters carrying GAGA-binding sites (A). TTK69 might repress GAGA-dependent activation by rendering GAGA unable to bind to the promoter (B) or by interacting with the transcription activation machinery and preventing its recruitment to the promoter (C). Alternatively, GAGA-TTK69 hetero-oligomers could bind to the promoter but would either be inefficient in recruiting the activation complex (D) or actively participate in the recruitment of co-repressor complexes (E).

 
Altogether, the observations discussed above strongly suggest that repression involves direct GAGA-TTK69 interaction at the promoter. GAGA·TTK69 complexes could actively participate in the recruitment of co-repressor complexes (Fig. 7E). Actually, TTK69 is known to interact with several co-repressors such as dMi2 and dCtBP (15, 16). However, dMi2 did not appear to mediate repression by TTK69 of GAGA-dependent activation because dMi2-TTK69 interaction occurs through a region lying outside of the POZ/BTB domain of TTK69, which as shown here is sufficient for repression (Figs. 4 and 5). Similarly, in this context, dCtBP did not seem to have a contribution to repression because no significant effects were observed upon dCtBP overexpression or RNAi-mediated inhibition (Fig. 6). However, our results point out the essential contribution to repression of the POZ/BTB domain of TTK69. In several cases, the contribution of POZ/BTB domain to repression was shown to be mediated by TSA-sensitive histone deacetylases (23–30). However, repression by TTK69 of GAGA-mediated activation of the eve promoter was insensitive to treatment with TSA. These results indicate that, if mediated by co-repressors, repression by TTK69 of GAGA-dependent activation of the eve promoter did not likely involve any of the co-repressors known to interact with TTK69 or TSA-sensitive histone deacetylases. Alternatively, the binding of TTK69 might have simply interfered with the ability of GAGA to load and/or assemble the activation complex at the promoter (Fig. 7D). Several repressors have been found to act through such a quenching or masking mechanism (reviewed in Refs. 31–33). In favor of this hypothesis, TTK69 efficiently repressed GAGA-mediated activation of the eve promoter but not basal transcription (14). Similarly, POZTTK{Delta}POZGAGA did not affect basal transcription (Fig. 5A). These results, together with the reported interactions of TTK69 with dCtBP and dMi2, suggest that depending on the promoter and the developmental stage, TTK69 might repress transcription through different mechanisms.

How could TTK69 interfere with the interaction of GAGA with the transcription machinery? It was shown earlier that the C-terminal glutamine-rich domain of GAGA is essential for GAGA-dependent activation both in vitro and in vivo (4). However, the results reported here show that, in addition, the POZ/BTB domain also appeared to have a direct contribution to activation because {Delta}POZGAGA did not efficiently activate the eve promoter. It might be argued that the inability of {Delta}POZGAGA to activate transcription reflects the contribution of the POZ/BTB domain to oligomerization. However, POZTTK{Delta}POZGAGA did not activate transcription (Fig. 5), although it is capable of oligomerization because the POZ/BTB domain of TTK69 also mediates homo-oligomerization (14). The binding of TTK69 occurs through the POZ/BTB domain of GAGA (14). Therefore, it is feasible that the binding of TTK69 would quench the contribution to activation of the POZ/BTB domain of GAGA. In this context, it is interesting to note that the POZ/BTB domain of bric-à-brac was reported to interact with dTAFII155 (34). If conserved, this interaction might account for the contribution of the POZ/BTB domain of GAGA to activation, providing at the same time a rationale for repression by TTK69 of GAGA-dependent activation. TTK69 might disrupt GAGA-dTAFII155 interaction by displacing dTAFII155 from the complex and/or competing with GAGA for dTAFII155 binding.

The inability of POZTTK{Delta}POZGAGA to activate transcription suggests that, although highly homologous (40% identity), the POZ/BTB domains of GAGA and TTK69 are not functionally exchangeable. The crystal structure of the POZ/BTB domain of the human promyelocytic leukemia zinc finger protein was solved (35, 36). The structure revealed a tightly interwound dimer with a surface-exposed groove that only existed in the dimer and was proposed to be involved in ligand binding (36). Interestingly, as shown in Fig. 8, the POZ/BTB domains of GAGA and TTK69 differ significantly at regions {alpha}1/{beta}2 (aa 22–37) and {alpha}3/{beta}4 (aa 58–68) that formed the floor and walls of the groove mentioned above, suggesting that they might sustain binding of different ligands. At this region, bric-à-brac shows a higher homology to TTK69 than to GAGA (Fig. 8).



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  		FIG. 8.
Sequence comparison of regions {alpha}1/{beta}2 and {alpha}3/{beta}4 of the POZ/BTB domains of GAGA, TTK69, and bric-à-brac. Identical residues are shaded in black, and similar residues are shaded in gray. Similarity groups: Ile-Val-Leu, Phe-Tyr, Asn-Gln, Asp-Glu, Arg-Lys, and Ser-Thr. Numbers indicate the amino acid position corresponding to the GAGA sequence.

 
Repression by TTK69 of GAGA-mediated activation of the eve promoter solely requires the contribution of the POZ/BTB domain, which is shared by TTK88, strongly suggesting that the results reported here can also be extrapolated to TTK88. GAGA and TTK are maternally provided transcription factors that in the early Drosophila embryo regulate the expression of several pair-rules genes (5–7). In this context, GAGA-TTK interaction might contribute to the establishment of the program of their zygotic expression by preventing the activator from activating. Additional elements facilitating or resolving GAGA-TTK interaction would also contribute to properly regulate their expression program.


   FOOTNOTES
 
* This work was financed by grants from the Ministero de Ciencia y Tecnologie (BMC2000-878 and BMC2000-898) and the Commissió Interdepartamental de Recerca i Innovacio Tecnologica (CIRIT) (2001SGR-00344). This work was carried out within the framework of the Centre de Referència en Biotecnologia de la Generalitat de Catalunya. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141. Back

§ Supported by a doctoral fellowship from CIRIT. Back

Supported by a Formación Profesora de Universitario doctoral fellowship from Ministero de Educación y Ciencia. Back

|| Present address: Derald H. Ruttenberg Cancer Center, 1425 Madison Ave., New York, NY 10029. Back

** To whom correspondence should be addressed: Dept. de Biologia Molecular i Cellular, Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi Girona Salgado, 18-26, 08034 Barcelona, Spain. Tel.: 3493-4006137; Fax: 3493-2045904; E-mail: fambmc{at}cid.csic.es.

1 The abbreviations used are: TTK, tramtrack; GAGA, trithorax-like; POZ/BTB, poxvirus zinc finger protein/bric-à-brac, tramtrack, broad complex; dCtBP, C-terminal binding protein; TSA, trichostatin A; eve, even-skipped; aa, amino acid; POZTTK, a truncated TTK69 form that carries the first 114 residues of TTK69; GST, glutathione S-transferase; RNAi, RNA interference; EMSA, electrophoretic mobility shift assay; TBE, Tris borate-EDTA. Back


   ACKNOWLEDGMENTS
 
We thank Dr. D. Arnosti and Dr. M. Sutrias for providing us with dCtBP cDNA. We also thank Dr. M. L. Espinás for helpful discussions and advice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wilkins, R. C., and Lis, J. T. (1997) Nucleic Acids Res. 25, 3963–3968[Abstract/Free Full Text]
  2. Benyajati, C., Mueller, L., Xu, N., Pappano, M., Gao, J., Mosammaparast, M., Conklin, D., Granok, H., Craig, C., and Elgin, S. (1997) Nucleic Acids Res. 25, 3345–3353[Abstract/Free Full Text]
  3. Soeller, W. C., Oh, C. E., and Kornberg, T. B. (1993) Mol. Cell Biol. 13, 7961–7970[Abstract/Free Full Text]
  4. Vaquero, A., Espinás, M. L., Azorín, F., and Bernués, J. (2000) J. Biol. Chem. 275, 19461–19468[Abstract/Free Full Text]
  5. Brown, J. L., Sonoda, S., Ueda, H., Scott, M. P., and Wu, C. (1991) EMBO J. 10, 665–674[Medline] [Order article via Infotrieve]
  6. Brown, J. L., and Wu, C. (1993) Development 117, 45–58[Abstract/Free Full Text]
  7. Read, D., Levine, M., and Manley, J. L. (1992) Mech. Dev. 38, 183–196[CrossRef][Medline] [Order article via Infotrieve]
  8. Xiong, W. C., and Montell, C. (1993) Genes Dev. 7, 1085–1096[Abstract/Free Full Text]
  9. Guo, M., Bier, E., Jan, L. Y., and Jan, Y. N. (1995) Neuron 14, 913–925[CrossRef][Medline] [Order article via Infotrieve]
  10. Giesen, K., Hummel, T., Stollewerk, A., Harrison, S., Travers, A., and Klämbt, C. (1997) Development 124, 2307–2316[Abstract]
  11. Lai, Z.-C., and Li, Y. (1999) Genetics 152, 299–305[Abstract/Free Full Text]
  12. Greenberg, A. J., and Schedl, P. (2001) Mol. Cell Biol. 21, 8565–8574[Abstract/Free Full Text]
  13. Read, D., and Manley, J. L. (1992) EMBO J. 11, 1035–1044[Medline] [Order article via Infotrieve]
  14. Pagans, S., Ortiz-Lombardía, M., Espinás, M. L., Bernués, J., and Azorín, F. (2002) Nucleic Acids Res. 30, 4406–4413[Abstract/Free Full Text]
  15. Wen, Y., Nguyen, D., Li, Y., and Lai, Z.-C. (2000) Genetics 156, 195–203[Abstract/Free Full Text]
  16. Murawsky, C. M., Brehm, A., Badenhorst, P., Lowe, N., Becker, P. B., and Travers, A. A. (2001) EMBO Reports 2, 1089–1094[CrossRef][Medline] [Order article via Infotrieve]
  17. Small, S., Blair, A., and Levine, M. (1992) EMBO J. 11, 4047–4057[Medline] [Order article via Infotrieve]
  18. Poortinga, G., Watanabe, M., and Parkhurst, S. M. (1998) EMBO J. 17, 2067–2078[CrossRef][Medline] [Order article via Infotrieve]
  19. Read, D., Nishigaki, T., and Manley, J. L. (1990) Mol. Cel. Biol. 10, 4334–4344[Abstract/Free Full Text]
  20. Kosoy, A., Pagans, S., Espinás, M. L., Azorín, F., and Bernués, J. (2002) J. Biol. Chem. 277, 42280–42288[Abstract/Free Full Text]
  21. Espinás, M. L., Jiménez-García, E., Vaquero, A., Canudas, S., Bernués, J., and Azorín, F. (1999) J. Biol. Chem. 274, 16461–16469[Abstract/Free Full Text]
  22. Ryu, J.-R., Olson, L. K., and Arnosti, D. N. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12960–12965[Abstract/Free Full Text]
  23. Dhordain, P., Albagli, O., Lin, R. J., Ansieau, S., Quief, S., Leutz, A., Kerckaert, J. P., Evans, R. M., and Leprince, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10762–10767[Abstract/Free Full Text]
  24. Hong, S. H., David, G., Wong, C. W., Dejean, A., and Privalski, M. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9028–9033[Abstract/Free Full Text]
  25. Huynh, K. D., and Bardwell, V. J. (1998) Oncogene 17, 2473–2484[CrossRef][Medline] [Order article via Infotrieve]
  26. Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H. J., and Evans, R. M. (1998) Nature 391, 811–814[CrossRef][Medline] [Order article via Infotrieve]
  27. Grignani, F., DeMatteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M., Fanelli, M., Ruthardt, M., Ferrara, F. F., Zamir, I., Seiser, C., Lazar, M. A., Minucci, S., and Pelicci, P. G. (1998) Nature 391, 815–818[CrossRef][Medline] [Order article via Infotrieve]
  28. He, L. Z., Guidez, F., Tribiolo, C., Peruzzi, D., Ruthardt, M., Zelent, A., and Pandolfi, P. P. (1998) Nat. Genet. 18, 126–135[CrossRef][Medline] [Order article via Infotrieve]
  29. Wong, C.-W., and Privalsky, M. L. (1998) J. Biol. Chem. 273, 27695–27702[Abstract/Free Full Text]
  30. David, G., Alland, L., Hong, S. H., Wong, C. W., DePinho, R. A., and Dejean, A. (1998) Oncogene 16, 2549–2556[CrossRef][Medline] [Order article via Infotrieve]
  31. Johnson, A. D. (1995) Cell 81, 655–658[CrossRef][Medline] [Order article via Infotrieve]
  32. Courey, A. J., and Jia, S. (2001) Genes Dev. 15, 2786–2796[Free Full Text]
  33. Hanna-Rose, W., and Hansen, U. (1996) Trends Genet. 12, 229–234[CrossRef][Medline] [Order article via Infotrieve]
  34. Pointud, J. C., Larsson, J., Dastugue, B., and Couderc, J. L. (2001) Dev. Biol. 237, 368–380[CrossRef][Medline] [Order article via Infotrieve]
  35. Li, X., Peng, H., Schultz, D. C., Lopez-Guisa, J. M., Rauscher, F. J., III, and Marmonstein, R. (1999) Cancer Res. 59, 5275–5282[Abstract/Free Full Text]
  36. Ahmad, K. F., Engel, C. K., and Privé, G. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12123–12128[Abstract/Free Full Text]

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