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Brinker is a sequence-specific transcriptional repressor in the Drosophila embryo.

A Dpp activity gradient specifies multiple thresholds of gene expression in the dorsal ectoderm of the early embryo. Some of these thresholds depend on a putative repressor, Brinker, which is expressed in the neurogenic ectoderm in response to the maternal Dorsal gradient and Dpp signaling. Here we show that Brinker is a sequence-specific transcriptional repressor. It binds the consensus sequence, TGGCGc/tc/t, and interacts with the Groucho corepressor through a conserved sequence motif, FKPY. An optimal Brinker binding site is contained within an 800-bp enhancer from the tolloid gene, which has been identified as a genetic target of the Brinker repressor. A tolloid-lacZ transgene containing point mutations in this site exhibits an expanded pattern of expression, suggesting that Brinker directly represses tolloid transcription. We discuss other examples of transcriptional repressors constraining the activities of signaling pathways.

Dpp signaling gradients pattern the dorsal ectoderm of gastrulating embryos and the wing imaginal disks of third-instar larvae (for review, see Podos and Ferguson 1999). In disks, the gradient is formed by the localized transcription of dpp at the anterior-posterior compartment boundary (e.g., Sanicola et al. 1995). The secreted Dpp protein is thought to diffuse away from this localized source to form a broad gradient across the presumptive wing blade. In embryos, dpp is uniformly transcribed throughout the dorsal ectoderm, but its activity is inhibited by an extracellular inhibitor, Sog, which is expressed in the neurogenic ectoderm (St. Johnston and Gelbart 1987;Francois et al. 1994;Holley et al. 1995;Marques et al. 1997). Sog-Dpp interactions create a Dpp activity gradient, with peak signaling in the dorsal-most regions (far from the localized source of Sog inhibitor) and lower levels in dorsolateral and lateral regions (Fer-guson and Anderson 1992;Holley et al. 1995;Ashe and Levine 1999).
Dpp directs several thresholds of gene activity. Peak levels activate target genes such as Race and hindsight within the presumptive amnioserosa (Rusch and Levine 1997;Yip et al. 1997;Ashe et al. 2000). Intermediate levels activate tail-up and u-shaped in the amnioserosa and dorsal epidermis (Frank and Rushlow 1996;Cubadda et al. 1997;Jazwinska et al. 1999b), whereas low levels directly or indirectly activate pannier and tolloid throughout the dorsal ectoderm (Ramain et al. 1993;Winick et al. 1993;Kirov et al. 1994;Jazwinska et al. 1999b;Ashe et al. 2000). Some of these thresholds depend on a putative repressor, Brinker, which is expressed in lateral stripes within the neurogenic ectoderm just outside the limits of the dorsal ectoderm (Jazwinska et al. 1999bAshe et al. 2000. Here we present evidence that Brinker functions as a sequence-specific transcriptional repressor.

Groucho is required for Brinker-mediated repression
A GST-Brinker fusion protein was mixed with 35 S-labeled dCtBP and Groucho proteins that were synthesized with a rabbit reticulocyte lysate. The fusion protein contains the entire Brinker sequence, amino acid residues 1-704. It weakly binds dCtBP (Fig. 1A, asterisk), but strongly interacts with Groucho (Fig. 1A, arrow). At least 20% of the total input Groucho protein specifically binds to the fusion protein. The conversion of the FKPY motif into AAGA results in a ∼10-fold reduction in binding (Fig. 1B).
Brinker was misexpressed in transgenic embryos using the eve stripe 2 enhancer (Kosman and Small 1997;Fig. 2, cf. B with A). Normal and mutant versions of the gene (containing AAGA in place of FKPY) were examined. The anterior portion of the pannier expression pattern is repressed in embryos carrying the wild-type stripe2-Brinker transgene (Fig. 2C, arrowhead;Ashe et al. 2000). In contrast, the mutant transgene lacking the FKPY motif fails to repress pannier (Fig. 2D). This transgene me-diates weak repression of dpp (Fig. 2F, arrowhead), although the wild-type transgene produces a more substantial gap in the dpp pattern (Ashe et al. 2000;Fig. 2E, arrowhead).

Identification of Brinker binding sites
Brinker contains a potential helix-turn-helix motif, suggesting that it might bind specific DNA sequences. SELEX assays were done with random oligonucleotides and a GST-Brinker fusion protein that contains the putative helix-turn-helix motif (amino acid residues 44-99). After four rounds of selection, a total of 12 random clones were selected and sequenced (Fig. 3A). Alignment of these sequences reveals a tightly conserved core motif, TGGCG. Eleven of the 12 sequences contain a T at position 1, whereas all 12 contain G, G, C, and G at positions 2, 3, 4, and 5, respectively. In addition, all 12 sequences contain either C or T at positions 6 and 7. Thus, the SELEX assays identify the following consensus sequence: T-G-G-C-G-t/c-t/c.
Previous studies have identified several potential target genes that are regulated by the Brinker repressor, including tolloid, pannier, and tail-up (Jazwinska et al. 1999b;Ashe et al. 2000). Among these genes, tolloid has been studied in the most detail (Kirov et al. 1994; see below). The tolloid 5Ј regulatory region contains an 800bp enhancer that has the potential to direct gene expression throughout precellular embryos (Kirov et al. 1994). It is kept off in ventral and lateral regions by the maternal Dorsal gradient. After cellularization, the enhancer directs restricted expression within the dorsal ectoderm in direct or indirect response to Dpp signaling. This pattern expands into lateral regions in Brinker − mutant embryos (Jazwinska et al. 1999b;see below).
Inspection of the 800-bp enhancer sequence identifies three potential Brinker binding sites (Fig. 3B). Gel shift assays suggest that only one of these, site 2, binds Brinker (Fig. 3C, lanes 2,3). This site contains a perfect match to the consensus sequence. In contrast, sites 1 and 3 exhibit little or no binding (Fig. 3C, lanes 7,8,10,11) and contain 6 of 7 matches to the consensus recognition sequence. Additional binding assays were done with a larger DNA fragment containing both sites 1 and 2 (Fig. 3C,lanes 13,14). There is no evidence for cooperative DNA-binding interactions between sites 1 and 2 because a single protein-DNA complex is observed, similar to the Brinker-site 2 complex. Point mutations were introduced into the core TGGCG motif within the site 2 recognition sequence (Fig. 3B). The mutagenized binding site essentially fails to bind the GST-Brinker fusion protein ( Fig. 3C, lanes 16,17).

Brinker functions as a sequence-specific repressor in vivo
By the onset of gastrulation, tolloid expression is restricted to the dorsal ectoderm and excluded from the neurogenic ectoderm in lateral regions (e.g., Fig. 4A, arrowheads). There is extensive modulation of the tolloid staining pattern along the anterior-posterior axis. Male embryos hemizygous for a null mutation in the Brinker gene, brk M68 , exhibit altered patterns of tolloid expression, including lateral expansion into the neurogenic ectoderm (Fig. 4B, arrowheads;Jazwinska et al. 1999b).
To determine whether Brinker-tolloid interactions are direct, site 2 was mutagenized in the context of an otherwise normal tolloid/lacZ transgene (see diagrams beneath Fig. 4C,D). The wild-type enhancer directs lacZ expression throughout the dorsal ectoderm; the pattern is quite similar to that observed for the endogenous gene ( Fig. 4, cf. C with A). At this stage in development (midnuclear cleavage 14), expression is no longer under the control of the maternal Dorsal gradient but, instead, depends on Dpp signaling. Staining is excluded from the lateral neurogenic ectoderm where Brinker is expressed.
The mutagenized tolloid enhancer directs a consistently expanded staining pattern (arrowheads, Fig. 4, cf. D with C). This altered pattern is similar to, but perhaps not quite as severe as, the expansion observed for the endogenous tolloid gene in Brinker − embryos (Fig. 4, cf. D with B). These results suggest that Brinker directly represses tolloid transcription in the neurogenic ectoderm.

Discussion
Brinker is the fourth sequence-specific repressor that has been shown to interact with Groucho through the tetrapeptide motif, aromatic-basic-pro-aromatic. The first version of this motif that was identified is WRPW, located at the carboxyl terminus of the Hairy repressor (Paroush et al. 1994;Fisher et al. 1996). The related WRPY motif was subsequently shown to mediate Runt-Groucho interactions (Aronson et al. 1997;Levanon et al. 1998), and FRPW permits Huckebein to bind Groucho (Goldstein et al. 1999). The Brinker repression domain identified in this study, FKPY, conforms to the other three Groucho motifs except for the lysine residue at position 2.
Genetic studies are consistent with the occurrence of Brinker-Groucho interactions in the early embryo. The tail-up and pannier expression patterns appear to expand into lateral regions of embryos derived from groucho germ-line clones (H. Ashe, unpubl.). It is conceivable that Brinker mediates both Groucho-dependent and Groucho-independent transcriptional repression because the removal of the FKPY motif does not abolish the ability of an otherwise normal stripe2--Brinker transgene to repress dpp expression (see Fig. 2). The residual activity of the mutagenized transgene might be mediated by cryptic Groucho interaction motifs in Brinker (see Fig.   1B). Alternatively, Brinker might repress certain target enhancers via competition between Smad activators and the Brinker repressor to overlapping DNA-binding sites. A similar situation has been described for the Kruppel and Knirps repressors. They require the dCtBP corepressor to regulate some, but perhaps not all, target genes (Nibu et al. 1998a;La Rosee-Borggreve et al. 1999;Keller et al. 2000). The Groucho and dCtBP corepressors might be required only when activators and repressors bind to distinct, nonoverlapping sites within a target enhancer.
Dpp-Brinker interactions represent a particularly vivid example of how sequence-specific transcriptional repressors can limit inductive interactions by extracellular signaling molecules. Brinker helps promote neurogenesis by blocking Dpp signaling in the neurogenic ectoderm (Jazwinska et al. 1999b;Ashe et al. 2000). It might also work as a gradient repressor to subdivide the dorsal ectoderm into dorsal epidermis and amnioserosa. There are other examples of repressors limiting signaling pathways. High levels of the Spaetzle ligand lead to optimal activation of the Toll-Dorsal signaling pathway and the induction of the Snail repressor in the presumptive mesoderm of early embryos (for reviews, see Belvin and Anderson 1996;Rusch and Levine 1996). Snail prevents high levels of Spaetzle from activating neurogenic genes (e.g., Brinker, acaete-scute, and rhomboid) in the mesoderm.

Figure 2.
Brinker represses pannier and dpp. Embryos were collected from transgenic strains containing either wild-type (C,E) or mutant (D,F) stripe2-Brinker transgenes. The embryos in A and B were hybridized with a Brinker antisense RNA probe, those in C and D were hybridized with a pannier probe, and the embryos in E and F were hybridized with a dpp probe. All embryos are undergoing cellularization and are oriented with anterior to the left. (A) Wild-type embryo. Brinker is expressed in two broad lateral stripes within the presumptive neurogenic ectoderm. (B) Same as A except that the embryo contains a copy of the wild-type stripe2-Brinker transgene (see diagram). The FRT-STOP-FRT cassette was removed by introducing the transgene into a parental male that expresses the FLP recombinase under the control of a sperm-specific tubulin promoter. The transgenic embryo exhibits both lateral neurogenic stripes and an ectopic stripe2-Brinker pattern. A similar staining pattern is observed for a mutant form of the stripe2-Brinker transgene containing AAGA in place of FKPY (data not shown). (C) The anterior portion of the pannier expression pattern ("stripe 1") is repressed by the wild-type stripe2-Brinker transgene (arrowhead). (D) Same as C except that the embryo contains the mutant stripe2-Brinker transgene lacking the FKPY Groucho interaction motif. A normal pannier expression pattern is observed. In particular, stripe 1 is restored in anterior regions (arrowhead; cf. with C). (E) The wild-type stripe2-Brinker transgene produces a broad gap in the dpp expression pattern (arrowhead). (F) The mutant transgene retains some repression activity, although the gap in the pattern is not as pronounced as compared with that obtained with the wild-type transgene.
The interplay between extracellular signaling molecules and localized transcriptional repressors is reminiscent of the segmentation pathway in the early Drosophila embryo. Pair-rule stripes of gene expression are established by broadly distributed transcriptional activators, such as Bicoid and dStat. The stripe borders are formed by localized gap repressors, including Hunchback, Kruppel, and Knirps (e.g., Small et al. 1991;Yan et al. 1996). Similarly, the activation of tolloid and pannier might depend on broadly distributed Smad proteins, whereas the lateral limits of the expression patterns are established by the localized Brinker repressor. It is likely that vertebrates also employ one or more transcriptional repressors to restrict TGF-␤ signaling interactions.

GST pull-down assays
Brinker coding sequences (codons 1-704 and 23-103) were cloned into the pGEX-5X-1 expression vector (Pharmacia). GST fusion proteins were induced in Escherichia coli strain BL21::De3pLysS as described by Nibu et al. (1998b). The fusion proteins were immobilized on glutathioneagarose beads (Sigma), and aliquots containing 5 µg of GST or GST-Brinker (1-704) were mixed with 5 µL of 35 S-labeled dCtBP or Groucho proteins. These proteins were synthesized in the TNT transcription/ translation system (Promega) using pBluescript plasmids. Labeled proteins were eluted and fractionated by SDS-PAGE as described by Zhang et al. (1996).

SELEX assays
A mixture of 76mer oligonucleotides containing 26 random residues was labeled with [␣-32 P]dCTP (800 Ci/mmol; purchased from NEN) as described by Pollock (1996). Aliquots containing 1 µL of the labeled oligonucleotides (∼0.4 ng) were mixed with 0.65-1.3 µg of GST-Brinker (23-103), and protein-DNA complexes were fractionated on polyacrylamide gels as described by Catron et al. (1993). The shifted complexes were eluted from the gel using a buffer containing 0.5 M ammonium acetate and 1 mM EDTA (pH 8). The eluted oligonucleotides were amplified by PCR (Pollock 1996) and mixed with the GST-Brinker fusion protein, as described above. After four rounds of selection, the DNA was amplified by PCR using 5Ј and 3Ј primers containing BamH1 and EcoRI restriction sites, respectively, and cloned into the pBluescript SK (+) vector (Stratagene). Twelve randomly selected recombinant plasmids were sequenced.

Transgenic assays
Wild-type and mutant Brinker coding sequences were cloned into the P-element transformation vector 22FPE containing two tandem copies of the eve stripe 2 enhancer (MSE) and an FRT-Stop-FRT cassette (Kosman and Small 1997). The FKPY motif (ttt aag ccc tat) at codons 461-464 was mutated to AAGA (gca gcc ggc gca) using PCR and mutant oligos. The Figure 3. DNA binding assays. (A) A GST-Brinker fusion protein containing amino acid residues 23-103 from Brinker was incubated with a mixture of 76mer oligonucleotides containing a variable central region of 26 nt. Twelve of the oligonucleotides were sequenced after four rounds of binding and gel fractionation (see Materials and Methods). All 12 sequences contain a common core motif that is indicated in boldface. (B) Summary of putative binding sites in the 800-bp tolloid enhancer. There are three sequences in the enhancer that contain at least a 6 of 7 match to the consensus (T-G-G-C-G-c/t-c/t). The sequences of each site are indicated beneath the diagram. Only site 2 contains a perfect match to the consensus. It was mutagenized (site 2M) by replacing the five core nucleotides (TGGCG into GTTTT). (C) Gel shift assays were done with double-stranded oligonucleotides containing each of the three potential Brinker binding sites from the tolloid enhancer. The oligonucleotides were labeled with 32 P, mixed with the GST-Brinker (23-103) fusion protein, and fractionated on a polyacrylamide gel. A strong protein-DNA shifted complex is observed for site 2 (lanes 2,3).  (lanes 13,14). The shifted complex is similar to the one obtained with the site 2 oligonucleotide (lanes 2,3), suggesting that the binding of the GST-Brinker protein to site 2 does not facilitate binding of site 1 via cooperative protein-protein interactions. There is a marked reduction in the binding of the GST-Brinker fusion protein to a mutant version of the site 1 + site 2 oligonucleotide containing the five substitutions in site 2 (lanes 16,17; see panel B) mutation introduces a NgoMIV site (gccggc). SpeI-NgoMIV and NgoMIV-Eag1 PCR fragments were ligated into SpeI-EagI digested SKAsc2brk (Ashe et al. 2000), and the mutant Brinker gene was transferred as an AscI fragment into the 22FPE vector. Both the wild-type and mutant (⌬FKPY) stripe2-Brinker transgenes were crossed into males that express the yeast Flp recombinase in sperm (Kosman and Small 1997). The recombinase removes the Stop cassette, thereby permitting expression of the transgenes in F 1 embryos.
Previous studies have identified an 800-bp enhancer from the tolloid 5Ј flanking region that is sufficient to direct the expression of a lacZ reporter gene within the dorsal ectoderm of transgenic embryos (Kirov et al. 1994). The enhancer was cloned into a modified version of the −42-bp eve-lacZ P-element transformation vector, which contains the eve promoter and lacZ reporter gene (Small et al. 1992). This modified vector, eve2gypsy, contains a unique NotI cloning site and two copies of the 340-bp gypsy insulator DNA (e.g., Cai and Levine 1995) inserted into the EcoRI and PstI sites. The insulators are located 5Ј of the tolloid enhancer and 3Ј of the lacZ gene to limit the effects of neighboring cis-regulatory elements (position effects). Both the wild-type enhancer and a mutant version containing point mutations in site 2 within the core TGGCG motif (Fig. 4) were cloned into the P-element vector.
Transformation vectors were introduced into the yw 67 strain as de-scribed by Small et al. (1992). The expression patterns of the lacZ reporter gene and various endogenous genes (e.g., Brinker, pannier, dpp) were visualized by in situ hybridization using digoxigenin-labeled antisense RNA probes (Jiang et al. 1991). The pattern has been resolved into a series of stripes and bands along the anterior-posterior axis (e.g., arrowheads). (B) Mutant embryo derived from brk M68 heterozygous female. The tolloid staining pattern is expanded and extends into the lateral neurogenic ectoderm (e.g., arrowheads). (C) Transgenic embryo that expresses the wild-type tolloid/lacZ transgene. The lacZ staining pattern is restricted to the dorsal ectoderm and includes various stripes and bands along the anterior-posterior axis. Two of the stripes are indicated by arrowheads. The overall lacZ pattern is very similar to the expression pattern of the endogenous tolloid gene (A). (D) Same as C except that the embryo contains a mutant version of the tolloid enhancer with nucleotide substi-tutions in the core TGGCG motif. The lacZ staining pattern is somewhat broader than the normal pattern. In particular, the two stripes indicated by arrowheads expand into lateral regions. This expansion is similar to the one observed for the endogenous tolloid gene in brk M68 mutants (B).