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First published online May 23, 2008
doi: 10.1242/10.1242/dev.015826

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Multiple modular promoter elements drive graded brinker expression in response to the Dpp morphogen gradient

¹ Department of Developmental and Cell Biology and the Developmental Biology Center, University of California Irvine, Irvine, CA 92612, USA.
² Department of Biology, New York University, New York, NY 10003, USA.

View larger version (62K): [in this window] [in a new window]   Fig. 1. The brk regulatory region contains multiple elements that mediate activation. (A) The brk promoter with arrows indicating transcription start sites for brk and unc-119. Scale is in kb and EcoRI sites (R1) are marked. Filled fragments drive expression in wing discs and embryos after stage 11; open fragments are transcriptionally inactive at these stages. The B14 construct of Muller et al. (Muller et al., 2003) is in gray, with `r' signifying repressor elements and E the activator. (B-I) Reporter expression in wing discs oriented anterior upwards, ventral leftwards. (B) The brkX47 enhancer trap reproduces wild-type brk pattern. (C,D) L1 and L2 are non-overlapping promoter fragments that direct patterns resembling endogenous brk. L1 drives low-level expression and the image was enhanced by increased exposure time. (E) L4 does not display detectable expression. (F) L5 is expressed strongly and excluded from only a narrow central domain. The central stripe of expression corresponds to the AP compartment boundary, where pMad levels are reduced (Tanimoto et al., 2000). (G,H) L6 and L7 also drive laterally restricted expression. L6 directs expression at lower levels than L7, and was enhanced by increasing exposure time. (I) Expression of L8 cannot be detected. (J-N) Reporter expression in late stage 12/13 embryos, oriented laterally. (J,K) Both L1 and L2 reporters mimic brk expression in ventral and lateral stripes in the ectoderm. (L) No embryonic expression is detected with L4. (M,N) L6 and L7 reporters can be detected in a brk-like pattern in the ectoderm and midgut.

View larger version (80K): [in this window] [in a new window]   Fig. 2. A compact promoter fragment is sufficient to generate a brk-like pattern. (A,B) A 580 bp fragment (L12) located 3 kb upstream of the transcription start drives expression in (A) wing discs and (B) embryos. (C) Upregulation of Dpp signaling by expression of TkvA using en-Gal4, results in repression of the L12 reporter in the posterior compartment. (D) L12 is derepressed in shn- embryos.

View larger version (51K): [in this window] [in a new window]   Fig. 3. A modular promoter element contains closely linked sites that mediate activation and Dpp-dependent repression. (A) Schematic showing L12 and derivatives. The red oval marks the SMM site containing C rather than A/G at position 2. The triangle in L12+Spacer marks the location of the insert. (B) Lysates from S2 cells transfected as indicated were used to gel-shift an oligo containing the SMM site. Lane 1 contains probe alone. The presence of Mad/Med results in a slower mobility complex (lane 2) that is further retarded by anti-Flag (lane 3) or Myc-ShnCT (lane 4). The Shn/Mad/Med complex is supershifted by incubation with anti-Myc (lane 5). (C) Wild-type (WT) or mutant (M) SMM oligos were incubated with GST-Mad or Med. Both proteins bind wild-type (lanes 3, 7) but not the mutant site (lanes 4, 8). Excess wild-type (lanes 5, 9), but not mutant, oligos (lanes 6, 10) block Mad/Med binding. L12M containing the mutant SMM site is ubiquitously expressed in (D) wing discs and (E) embryos. (F-I) Expression patterns of L12 derivatives in wing discs. (F) L12-a drives laterally restricted expression. (G) L12-b, which lacks the SMM site, is derepressed medially. (H) A 187 bp L12-d fragment drives brk-like expression, indicating the presence of closely linked SMM and activator sites. (I) Insertion of a 380 bp spacer between the SMM site and activator sequences (L12+Spacer) results in broad expression.

View larger version (28K): [in this window] [in a new window]   Fig. 4. The brk promoter contains multiple modular regulatory elements. (A-E) Transgenic reporters containing 780 bp fragments (green bars), centered on SMM sites drive brk-like expression patterns in the wing disc. Red ovals mark the location of 11 predicted SMM sites (#1-11) within the brk promoter at -271, -2165, -3097, -5175, -6627, -7653, -8023, -8174, -8206, -13,833 and -15,983 bp. (A-D) Expression patterns derived from fragments L17, L16, L15 and L14 containing modules 10, 7/8/9, 5 and 4, respectively. Module 4 drives the highest levels of expression laterally. Module 5 drives weak expression (the image was enhanced by increasing exposure time). (E) Module 3 (L12) is shown for comparison.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Shn interacts with the short-range co-repressor dCtBP. (A) Full-length Shn and ShnCT, a polypeptide sufficient for Dpp-dependent repression of brk, are shown. Zinc-finger domains are marked in blue. A 100 residue domain required for repression (red bar) includes a CtBP interaction motif PMDLT, which was mutated in ShnCTM as shown. (B) ShnCT interacts with dCtBP but not with Gro. Extracts from S2 cells transfected as indicated, were immunoprecipitated with anti-Myc and probed with anti-Flag. Expression levels were monitored by probing separate blots with anti-Flag or anti-Myc. Wild-type ShnCT bound dCtBP but not Gro, while ShnCTM failed to interact with dCtBP. (C-E) The CtBP interaction motif contributes to repression in vivo. (C) The brkX47 reporter is expressed ubiquitously in shn- embryos. (D) In shn- embryo, Hsp70-Gal4-driven expression of ShnCT restores brk-lacZ repression in the dorsolateral ectoderm and rescues dorsal closure defects. (E) ShnCTM is unable either to repress brk-lacZ or to rescue the shn- morphology.

View larger version (86K): [in this window] [in a new window]   Fig. 6. The brk promoter integrates inputs from multiple modules. Confocal images of wing discs (dorsal upwards, anterior leftwards) showing reporter-lacZ (red) and omb-Gal4>UAS-eGFP expression (green). The module number and organization (green oval represents activator, red the SMM complex) is depicted schematically. Discs in A-C were stained in parallel and visualized using identical settings. Graphs show signal intensity in the red and green channels, measured at the white bar. (A) A 1x/+ reporter containing a single copy of module-3 (L12) was expressed in the wing pouch region lateral to omb>GFP. (B) The 2x/+ and (C) 3x/+ reporter drive increasingly higher levels of expression, but showed no significant overlap with omb>GFP.

View larger version (61K): [in this window] [in a new window]   Fig. 7. The impact of Dpp-insensitive mutant modules is ameliorated in multimodular brk reporters. Wing discs stained for lacZ to visualize reporter expression. The module number and organization are depicted schematically. (A) Wild-type L13 reporter. (B,D,F) In fragments containing individual modules (see Fig. 4 for nomenclature; mutant modules indicated in superscript), disruption of SMM sites results in expression throughout the wing pouch. (C,E,G) By contrast, multimodular L13 reporters containing the same lesions do not show derepression in the medial region (compare with B,D,F, respectively). (H) L13M3+M7/8/9, which contains two mutant modules shows significant expansion of lateral expression but continues to be repressed medially, where Dpp activity is highest. The narrow central stripe of expression corresponds to the AP boundary where pMad levels are lower (Tanimoto et al., 2000).

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