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A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer

Abteilung Zellbiologie, Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

View larger version (59K): [in a new window]   Fig. 1. The HOMRE does not activate transcription in the same domain as the entire lab550 enhancer. The sequence of the lab550 enhancer is shown in A. The HTH-, LAB- and EXD-binding sites are shown in green, red and blue boxes, respectively (Grieder et al., 1997; Ryoo et al., 1999). The extent of the 48/95 HOMRE is indicated by the red arrows and the extent of the 50 bp element (see text) by the green arrows. Four MAD/MEDEA-binding sites are underlined and four CREs are boxed in blue. The repressor element shaded in orange. A GATA site is boxed in red and a HMG-binding site in green. Nucleotides conserved between the D. melanogaster lab550 enhancer and the corresponding element from D. hydei are printed in bold; non-conserved residues are not bold. The sequence stretch from 185 to 215 is not conserved between the two species. (B-D) The expression of endogenous lab (brown, B) is compared with the expression driven by lab550 (C; lab in blue, lab550 driven ß-GAL expression in brown) and the expression driven by the HOMRE 48/95 (D; lab in blue, HOMRE in brown) in stage 13 embryos. Clearly, lab550 mimics the expression of lab, whereas HOMRE-driven expression is mostly posterior to lab. (E-J) Using confocal microscopy, the expression of lab550 (green; E,G) and the HOMRE (green; H,J) is compared with endogenous lab (red; F,G,I,J) in stage 14 embryos. Expression levels were higher in later stages and it was not possible to use confocal microscopy in stage 13 embryos.

View larger version (78K): [in a new window]   Fig. 2. Neither the HOMRE nor the DPPRE shows the same genetic requirement for expression as lab550. Expression of lab550 (A-E) is compared with the expression of 48/95 (HOMRE; F-J), 92/546 (DPPRE; K-O) and 92/546m300(DPPREm300; P-T). Expression is analyzed in wild-type embryos (A,F,K,P) or in embryos mutant for abd-A (B,G,L,Q), wg (C,H,M,R) or dpps4 (E,J,O,T). Expression was also analyzed in embryos expressing a constitutive active version of the DPP receptor TKV (tkvQD) in the endoderm (D,I,N,S). Note that in contrast to lab550, 92/546m300 is strongly expressed in the gastric caeca endoderm (arrows, P,Q).

View larger version (58K): [in a new window]   Fig. 3. The mutated DPPRE is independent of the activity of the HOX complex. Expression of lab550 (A,B) is compared with the expression of lab550m300 (C,D) both in wild-type (A,C) and lab mutant embryos (B,D). Embryos were double stained for ß-gal activity driven by the enhancers (brown) and LAB protein (blue). The mutated element is strongly active in lab mutants both in ps 7 and in the gastric caeca (arrow). Note that the diagnostic LAB staining in the CNS (arrowheads) is missing in the mutants.

View larger version (12K): [in a new window]   Fig. 4. 92/546 acts as a strong DPP response element in COS cells. The expression of luciferase under the control of the lab550 enhancer or derivatives thereof was analyzed in COS cells after co-transfection with combinations of MAD-, MEDEA- and TKVQD-expressing plasmids. Cells were transfected with 4 µg of the reporter plasmids lab1-550luc (A) or its derivatives, schematically represented at the bottom of the graph, and (B) co-transfected with 2 µg of each indicated expression plasmids. enhancerless: parental luciferase plasmid pt81-luc. The amount of transfected DNA was kept constant (10 µg) by addition of psG5 plasmid. Bars represent the luciferase activity of transfected cell extracts (mean±s.e.m. of three to ten independent experiments, each carried out in duplicate), expressed as -fold activation over the basal activity of the reporter construct. Values were normalized by co-transfection of 0.1 µg of a pCMV-ß-gal plasmid as an internal standard. lab550 activity is increased 70-fold after the co-transfection of all four plasmids. (A) Although MAD and MEDEA together also stimulated expression of lab550 (14-fold), the addition of TKVQD further increased expression fivefold; TKV did not stimulate lab550 expression in the absence of co-transfected MAD and MEDEA. In contrast to lab550, the activity of HOMRE (B) was increased only threefold by DPP signaling. DPPRE activity was increased 84-fold and the activity of DPPRE carrying mutation 300, which allowed for a strong DPP response in the endoderm, was increased 32-fold. Thus, mutation 300 did not show a stimulatory effect in COS cells but rather somewhat reduced the DPP response. The reduced induction of 92/546m300 when compared with 92/546 was due to a higher basal level of 92/546; the measured activity levels after induction were similar (data not shown).

View larger version (74K): [in a new window]   Fig. 5. lab550 can be induced by DPP signaling in the absence of the visceral mesoderm. In a twi mutant embryo, expression of the lab550 construct is absent in the endoderm (A). The endodermal expression driven by lab550 can be restored in twi mutants upon ectopic expression of an activated form of the TKV receptor, tkvQD, under UAS control, using the endoderm-specific GAL4 line 48Y (B, arrow). When ectopic WG or MAPK signaling were induced in a similar, cell-autonomous, way in twi mutants, using 48Y and an activated form of arm, armS10 (C), and of Ras, rasV12 (D) under UAS control, endodermal expression of lab550 was not restored.

View larger version (27K): [in a new window]   Fig. 6. MAD and CRE sites are important for the DPP response, both in the embryo and in COS cells. (A) Luciferase activity of the lab550 enhancer and two mutant derivatives upon stimulation of DPP signaling in transfected COS-1 cells. The DPP response is expressed as -fold activation over the basal activity of each reporter plasmid alone, which is indicated at the bottom of the graph (see the legend of Fig. 4 for details). The introduction of the four MAD mutations and the introduction of the four CRE mutations lead to a reduction in the response to DPP signaling from 70-fold to 14-fold and 11-fold, respectively. In the embryo, lab550-driven expression (B) was reduced by the introduction of mutations in either the MAD sites (C) or the CRE sites (D). 92/546m300-driven expression (E) was also reduced by mutations in the MAD sites (F) and was hardly detectable after mutating the CRE sites (G).

View larger version (29K): [in a new window]   Fig. 7. Extensive deletion analysis defines an essential 50 bp element A large number of deletion constructs were analyzed for expression in the embryo. M stands for the presence of the mutation at site 300, which leads to a dramatic increase in the sensitivity of the enhancers to DPP. Clearly, deletion of sequences between 444 (1/444m300) and 394 (1/394m300) (deletion of the 50 bp element) leads to a loss of expression in endodermal cells adjacent to ps 3 and ps 7. The equivalent deletion was also analyzed in the context of lab550.

View larger version (113K): [in a new window]   Fig. 8. The 50 bp element is required for lab550 activity. 92/394m300 was not active in wild-type embryos (D), while the same enhancer containing the 50 bp element (92/444m300) was active both in the endoderm of the gastric caeca and adjacent to ps7 (A). Mutation of either the GATA- (G) or the HMG- (H) binding site in 92/546m300 resulted in strongly reduced expression. In the absence of the mutation in the putative repressor binding site 300 and in the presence of the HOMRE, expression of the construct that lacked 50 bp (1/394) was weak in wild-type embryos (E) and inactive in abd-A mutants (F). Expression driven by 1/444 was strong in wild-type embryos (B) and expanded in abd-A mutants (C). Expression of a minimal enhancer construct (1/140;243/444) was also strong in wild-type embryos (I) and strong and posteriorly expanded in abd-A mutants (data not shown).

View larger version (10K): [in a new window]   Fig. 9. The 50 bp element is required for full DPP responsiveness in COS cells. Luciferase activity of the lab550 enhancer and two 3' deleted derivatives upon stimulation of DPP signaling in transfected COS-1 cells. The DPP response is expressed as -fold activation over the basal activity of each reporter plasmid alone, which is schematically represented as a bar with the nucleotide position of the deletion at the bottom of the graph (see the legend of Fig. 3 for details). Deletion of the 50 bp element reduced the induction of 1/394 (22-fold) compared to 1/444 (37-fold) in COS cells. The 50 bp element (a slightly extended version was put into the expression vector for cloning purposes) is induced 10-fold by the activated TKV receptor and the co-transfected Smad-expressing plasmids.

View larger version (37K): [in a new window]   Fig. 10. The lab550 enhancer contains multiple elements that are all required for its HOX- and DPP-dependent regulation in the developing midgut endoderm. (A) Schematic representation of the developing midgut, depicting the visceral mesoderm (VM) and the endoderm. Cells in which DPP is transcribed are shown in dark blue; apparent distribution of the DPP ligand is indicated by the light blue squares. The expression of the endogenous lab gene in the endoderm is indicated by the stripes. The expression domains driven by lab550, HOMRE, DPPRE and DPPREm300 are shown below with brown bars. Embryos depicting the situation in stage 14 embryos are shown in B-D; DPP mRNA is in blue, and ß-gal expression driven by lab550 (B), HOMRE (C) and DPPREm300 (D) in brown. A schematic summary of the factors that have been demonstrated or proposed to regulate lab550 expression is shown in E.

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