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First published online March 7, 2005
doi: 10.1242/10.1242/dev.01721

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Recognition of distinct target sites by a unique Labial/Extradenticle/Homothorax complex

Andreas Ebner^*, Clemens Cabernard, Markus Affolter and Samir Merabet^*,,

Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

View larger version (78K): [in a new window]   Fig. 4. The site of crucial importance for in vivo activity of enhancer EVIII is specifically recognized by Lab and its two co-factors Exd and Hth. (A) Band-shift experiments showing that Lab (lane 3), Lab/Exd (lane 4) and Lab/Exd/Hth (lane 5) specifically bind to the oligonucleotide EVIII. Specificity of the binding was verified by adding a polyclonal anti-Lab antibody to the reactions (lanes 6-8), or by mutating in a G stretch the putative core binding sites of Lab (lanes 11-13), Exd (lanes 16-18) and Hth (not shown). We noted that the Exd-binding site mutation affected Lab monomere binding in presence of the co-factor (lanes 17-18). No different DNA-binding activities were observed between the truncated (158-629) and the full-length form of Lab on the consensus Lab/Exd/Hth-binding site of the lab48/95 enhancer (lanes 19-26). Lanes 1, 9, 14 and 19 are probes alone. Lanes 2, 10, 15 and 20 are probes in presence of the rabbit reticulocyte lysate. For each gel, the following lanes correspond to the Lab protein alone (3, 6, 11, 16, 21 and 24), with Exd (4, 7, 12, 17, 22 and 25), or with Exd and Hth proteins (5, 8, 13, 18, 23 and 26). Lab proteins were full length, except in lanes 21-23, where the 158-629 form (see Materials and methods) described in previous studies was used. (B) Gel mobility experiments with oligonucleotide EVIII, in presence of the Hox proteins Lab (lanes 3-5), Proboscipedia (Pb; lanes 6-8), Sex combs reduced (Scr; lanes 9-11), Deformed (Dfd; lanes 12-14), Antennapedia (Antp; lanes 15-17), Ultrabithorax (Ubx; lanes 18-20), AbdominalA (AbdA; lanes 21-23) and AbdominalB (AbdB; lanes 24-26). Hox proteins are alone (lanes 3, 6, 9, 12, 15, 18, 21, 24), with Exd (4, 7, 10, 13, 16, 19, 22, 25) or with Exd and Hth (5, 8, 11, 14, 17, 20, 23, 26). Exd and Hth proteins correspond to the truncated isoforms described in previous studies (see Materials and methods). Lanes 1 and 2 correspond to probe alone or with rabbit reticulocyte lysate, respectively. Hox/Exd or Hox/Exd/Hth complexes are only observed with Lab (lanes 4 and 5). Lab proteins were full length, except in lanes 3-5. Lab binding, alone or in combination with the Exd and Hth co-factors, is indicated by arrowheads.

View larger version (73K): [in a new window]   Fig. 5. Sequence requirements for Lab, Lab/Exd and Lab/exd/Hth binding to EVIII. (A) Sequences of the mutated EVIII oligonucleotides tested for band-shift experiments with Lab and the co-factors Exd and Hth. Positions of each nucleotide within the Lab/Exd sequence are numbered 1 to 10, and orientations of each binding site are indicated (arrowheads). The two central nucleotides of the Lab/Exd-binding site are highlighted in yellow, while mutated nucleotides are in red. (B) Band shift experiments with the control wild-type and the mutated versions of the EVIII oligonucleotide listed in A. The identity of the oligonucleotide used is indicated below the gel. For each gel, the first two lanes correspond to the probe, alone (first lane) or with the rabbit reticulocyte lysate (second lane). The next three lanes are in the same order: Lab alone (lanes 3, 8, 13, 18, 23, 28, 33, 38, 43 and 48), Lab with Exd (4, 9, 14, 19, 24, 29, 34, 39, 44 and 49) and Lab with Exd and Hth (lanes 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50).

View larger version (60K): [in a new window]   Fig. 2. Enhancer characterisation of the genomic region responsible for CG11339 expression in endoderm. (A) Scheme of the 100B4-B5 genomic region of CG11339. The two isoforms are indicated, as well as the sequence of the consensus Lab/Exd/Hth-binding site found by in silico approach. Genomic fragments tested for enhancer activities are shown: fragments testing positive are shown in blue. (B) ß-Gal staining in lines carrying the lacZ transgene under the control of the different genomic regions, corresponding to fragments A-E and sub-fragments EI-EVIII derived from fragment E. All embryos are shown for stage 14 of embryogenesis.

View larger version (49K): [in a new window]   Fig. 1. Expression of CG11339 in Drosophila midgut endoderm is regulated by lab, exd and hth. (A-L) All embryos are shown at stages 12 (A,C,E,G,I,K) and 14 (B,D,F,H,J,L). Anterior towards the left and posterior towards the right. (A,B) Expression of lab RNA. (C,D) Expression of CG11339 RNA. (E,F) Confocal analysis of CG11339 (in situ; green) and Lab (immunostaining; red). (G,H) Loss of CG11339 RNA expression in labvd1 homozygous mutant embryos. (I,J) Loss of CG11339 RNA expression in hthP2 homozygous mutant embryos. (K,L) Loss of CG11339 RNA expression in maternal and zygotic exdY012 mutant embryos.

View larger version (60K): [in a new window]   Fig. 3. The divergent Lab/Exd/Hth-binding site is crucial for in vivo activity of EVIII enhancer of CG11339. (A) Sequence alignment of the Drosophila melanogaster (Dm) 150 bp fragment EVIII with the corresponding genomic region of Drosophila pseudoobscura (Dp). Identical nucleotides are outlined in red. The putative Lab/Exd- and Hth-binding sites are indicated (broken boxes), as well as the oligonucleotide used for gel mobility experiments in Fig. 4 (broken blue lines). (B-I) All embryos are shown at stages 12 (B,D,F,H) and 14 (C,E,G,I). (B,C) Expression of ß-Gal protein under the control of wild-type enhancer EVIII. This expression was lost when fragment EVIII carried mutations in the two putative Lab/Exd-binding sites (D,E) or in the divergent Lab/Exd/Hth-binding site (F,G); and was diminished with mutations in the atypically oriented Hox/Exd-like binding site (H,I).

View larger version (40K): [in a new window]   Fig. 6. Midgut expression comparisons between several enhancers regulated by Lab. (A) Sequences of the Lab/Exd-binding sites of enhancers thought to be regulated by Lab (red) and its co-factors Exd (blue) and Hth (bold). The two central nucleotides of the Lab/Exd-binding site are highlighted in yellow. These enhancers are derived from the mouse Hoxb1 (repeat3), or the Drosophila Lab (lab550 and lab48/95) genes. The sequence of the divergent Lab/Exd/Hth-binding site of the EVIII enhancer of CG11339 is also shown for comparison. (B-I) All embryos are shown at stages 12 and 14. Confocal analysis of Lab (immunostaining; green) and ß-gal (immunostaining; red) expressions in lines carrying the lacZ transgene under the control of enhancers lab550 (B,C), lab48/95 (D,E), repeat3 (F,G) and EVIII of CG11339 (H,I).

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