Vallin J et al. (2001), Cloning and characterization of three Xenopus s...

XB-ART-8903

J Biol Chem 2001 Aug 10;27632:30350-8. doi: 10.1074/jbc.M103167200.

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Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling.

Vallin J , Thuret R , Giacomello E , Faraldo MM , Thiery JP , Broders F .

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In amphibians and birds, one of the first steps of neural crest cell (NCC) determination is expression of the transcription factor Slug. This marker has been used to demonstrate that BMP and Wnt molecules play a major role in NCC induction. However, it is unknown whether Slug expression is directly or indirectly regulated by these signals. We report here the cloning and characterization of three Xenopus Slug promoters: that of the Xenopus tropicalis slug gene and those of two Xenopus laevis Slug pseudoalleles. Although the three genes encode proteins with almost identical amino acid sequences and are expressed with similar spatiotemporal patterns, their 5'-flanking regions are quite different. A striking difference is a deletion in the X. tropicalis gene located precisely at the transcription initiation site that results in the X. tropicalis promoter being inefficient in X. laevis. Additionally, we identified two regions common to the three promoters that are necessary and sufficient to drive specific expression in NCCs. Interestingly, one of the common regulatory regions presents a functional Lef/beta-catenin-binding site necessary for specific expression. As the Lef.beta-catenin complex is a downstream effector of Wnt signaling, these results suggest that Xenopus Slug is a direct target of NCC determination signals.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: slc12a3 snai2 wnt1

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	FIG. 1. The X. tropicalis slug promoter is inefficient in X. laevis embryos. A, constructs containing different fragments of the X. tropicalis slug promoter (Prom Xt Slug) were injected into X. laevis embryos (upper panel). GFP expression was detected by RT-PCR performed on stage 19 NF embryos (middle panel). Histone H4 was used as an internal control for semiquantitative analysis (lower panel). The bars indicate the means S.D. of three independent experiments. Negative control lanes of the gel correspond to samples where no reverse transcriptase was added. The p200-GFP and p3900-GFP constructs gave the strongest expression. B, to compare the efficiency of the X. tropicalis slug promoter with that of the endogenous promoter, a modified X. laevis Slug cDNA was inserted downstream from the p200 and p3900 promoters (upper panel). With a single set of primers (U and D), both endogenous (endo) and exogenous (exo) Slug cDNAs were amplified (middle panel). This made it possible to compare mRNA levels (lower panel). The relative amount of exogenous product was much lower than expected with the Sleeping Beauty expression system, indicating that the X. tropicalis slug promoter is inefficient in X. laevis embryos. a.u., arbitrary units; u, uninjected; St, stage.
	FIG. 2. Comparison of the three Xenopus Slug genes. A, all three genes displayed the same organization, with three exons (dark-gray boxes) and two introns (i1 and i2; white boxes) of similar length. Asterisks indicate the positions of the stop codons in the coding regions. The 5 -flanking regions are divergent except for two conserved regions, each 300 bp long (regions A and B). Region A is located just upstream from the CAP site, whereas the position of region B is variable. The repeat boxes indicate repeats of a 79-nucleotide motif in X. laevis (X.l.) sequences. The GT box indicates a region of GT repeats in the X. tropicalis (X.t.) sequence. B, comparison of the sequences of X. tropicalis and X. laevis promoters around the transcription start site (indicated by arrows). Asterisks indicate differences between the two species. We predicted CAP sites and determined score as described by Bucher (21). In the X. laevis promoter, a 9-bp insertion creates an extra CAP site, with a better predicted score than the initial CAP site and that of X. tropicalis.
	FIG. 3. The two X. laevis Slug genes have identical expression patterns. A, RT-PCR experiments were performed on X. laevis embryos at various stages of development. A specific primer was designed for each gene based on the 5 -UTR. The graph shows quantification using histone H4 as an internal control. The two Slug mRNAs displayed similar temporal expression patterns and were produced at similar levels. B, the distribution of Slug and Slug mRNA was determined in stage 14 NF embryos by in situ hybridization using 5 -UTR sequences as short specific probes. The two genes were expressed at the same site in the presumptive cephalic NCCs (arrowheads) as previously described for Slug mRNA. a.u., arbitrary units.
	FIG. 4. Both X. laevis Slug promoters drive specific expression in NCCs. Constructs containing the GFP coding region under the control of the slug or slug promoter were injected into one blastomere of a two-cell stage embryo, and GFP levels were monitored throughout early development. The dashed lines indicate the midline of the embryo. e, eye vesicle; c, cement gland. With both constructs, a pattern similar to that for endogenous Slug was obtained. Note that at stage (St.) 25 NF, migrating cells colonizing the branchial arches were still GFPpositive, but the signal was weaker because the acquisition time required to detect it was three times longer (inset in the upper right panel).
	FIG. 5. The second CAP site is necessary for X. laevis Slug promoter function, whereas the first intron is not. A, the second CAP site was deleted from the slug promoter ( 1700- CAP construct) (left panel). This construct was injected into embryos, and GFP expression was quantified by RT-PCR analysis and compared with that driven by the full-length promoter (right panel). No fluorescence was detected with this construct in living embryos, and the mRNA level at stage 19 NF was about one-fifth that obtained with the full-length promoter. A construct containing the full-length promoter and the first intron gave results similar to those obtained with the promoter alone. Int1, intron 1; n, number of embryos analyzed. B, shown is the comparison of the sequences surrounding the transcription initiation site in the 1700- CAP construct and in the X. laevis and X. tropicalis Slug promoters. The transcription start sites are indicated by an uppercase G. Note that the X. laevis construct lacking the second CAP site was very similar to the X. tropicalis promoter in this region. a.u., arbitrary units.
	FIG. 6. Combination of the two conserved regions is necessary and sufficient to drive specific expression in NCCs. Various constructs (left panel) were injected into one blastomere of a two-cell stage embryo at two different concentrations. In 1 injections, 2 1017 mol of plasmid was injected in 15 nl. This corresponded to 100 pg of the 3000- GFP construct. In 8 injections, eight times as many plasmids were injected in the same volume. GFP expression was analyzed both by fluorescence monitoring in living embryos and by semiquantitative RT-PCR with stage 19 NF total RNA. n, number of embryos analyzed; fluo, localization and intensity of the observed fluorescence (major phenotype); nc, neural crest; ep, presumptive epidermis. The bars indicate the amount of GFP mRNA relative to histone H4 mRNA. The images show the GFP expression pattern obtained with the 70TATA-GFP (TATA), 200A -GFP (A ), 270A-GFP (A), and 700BA-GFP (BA) constructs. Charact
	FIG. 7. Region B contains a functional Lef/ -catenin site necessary for positive regulation. A, shown are the results from a mobility shift assay with a fragment of region B including the predicted Lef/ -catenin-binding site as a probe. Purified Lef-1 bound to this fragment (arrow). This binding was inhibited in a dose-dependent manner by adding various amounts (2 to 50) of a DNA fragment including the published Lef-1 consensus binding site. No competition effect was detected with a mutated Lef-1 site (mut. 10 lane). The arrowhead indicates free probe. comp., competitor. B, if Lef-1 was incubated with a GST/ -catenin fusion protein before adding the probe, a DNALef-1 -catenin complex was detected by electrophoretic mobility shift assay (middle arrow). This complex was not very stable under the conditions used, but its existence was confirmed by adding an anti- -catenin antibody (Ab; upper arrow). The lane is a negative control in which the probe was incubated with GST/ -catenin alone. The GST lane is a negative control in which Lef-1 was incubated with GST alone before adding the probe. The arrowhead indicates free probe. C, GFP expression in stage 19 NF embryos injected with the indicated constructs was assessed by RT-PCR. The bars indicate the means S.D. of three independent experiments. The 3000 Lef construct, which contained a promoter lacking the Lef-binding site, gave levels of GFP much lower than those obtained with the full-length 3000 promoter, but similar to those obtained with the negative 70TATA control. a.u., arbitrary units. 30356