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All-trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis-element of Snail2 (previously known as Slug) gene plays a role in repression by all-trans retinoic acid. We analyzed the cis-acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all-trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one-hybrid screen, we found that a polypyrimidine tract-binding protein (PTB), which is known to be a regulator of the alternative splicing of pre-messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein-PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.
Fig. 1. Analysis of effects of all-trans retinoic acid on the expression of the Xenopus Snail2 gene. (A) In situ hybridization of Xenopus
embryos at stage 20. (a) Snail2 gene was expressed in the neural crest area of the control embryo, which is indicated by an arrowhead.
(b) Snail2 gene was repressed when the Xenopus embryo was incubated in 2 · 10)6 mol â L all-trans retinoic acid from stage 10 to
stage 12. (B) Schematic illustration of the subcloning strategy. In the upper trace, the boxes indicate the coding sequence for the
Xenopus Snail2. The 5¢ flanking sequence is subcloned into a site immediately upstream of the firefly-luciferase-coding sequence in the
reporter plasmid pGL3-Basic, while the intron 2 sequence is subcloned into a site immediately downstream of the luciferase-coding
sequence (the lower trace). (C) Schematic illustration of the 5¢ flanking sequence and intron 2 sequence of Xenopus Snail2. The
TGTGT â ACACA sequence is shown as the black boxes. Two neighboring binding sites for Sox9 and Snail2 are shown as arrowheads.
(D) The sensitivity of the genomic Xenopus Snail2 fragments to all-trans retinoic acid. While the 5¢ flanking sequences of the reporter
constructs are shown schematically on the left side of the histogram, the intron 2 sequences are shown on the right. The firefly
luciferase reporter activity normalized to the pRL-CMV internal standard activity is represented in the histogram as the percentages of
the value of the cells without retinoic acid. Data are presented as the mean ± SD of at least three independent experiments.
Fig. 2. Analysis of the sensitivity of the human Snail2 gene and
(TG â CA)26 to all-trans retinoic acid. (A) Schematic illustration of
the 5¢ flanking sequence and intron 1 sequence of the human
Snail2 gene. The TGTGT â ACACA sequence is shown as the
black boxes. (B) Schematic illustration of the reporter construct
containing (TG â CA)26. (TG â CA)26 is subcloned into a site
immediately upstream of the SV40 promoter sequence of the
plasmid pGL3-Promoter as well as into a site immediately
downstream of the luciferase-coding sequence. (C) The
sensitivity of the human Snail2 gene as well as (TG â CA)26 to alltrans
retinoic acid. The remaining luciferase activities are
represented as described for Fig. 1C. (D) Dose-dependence of
the repression of (TG â CA)26-(TG â CA)26 construct by all-trans
retinoic acid. Twenty-four hours after transfection with the
(TG â CA)26-(TG â CA)26 construct, HEK293 cells were incubated
with all-trans retinoic acid (10)11 to 10)7 mol â L) for 24 h. Data
are presented as the mean ± SD of at least three independent
experiments.
Fig. 3. Involvement of PTB2 in the retinoic acid-induced
repression through TG â CA repeats. (A) Gel mobility shift assay of
the TG repeat oligonucleotide. The indicated amount of PTB2
protein was incubated with the (TG)10 oligonucleotide.
Competitor was added at a 125-fold molar excess of the labeled
probe. An arrow indicates the PTB2-DNA complex. (B) The
effects of knockdown and overexpression of polypyrimidine tractbinding
protein (PTB) on the retinoic acid-induced repression of
gene expression. siRNAs designed to reduce the expression of
PTB1 and PTB2 are coexpressed with the reporter constructs.
PTB2 overexpression was induced by cotransfection of PTB2-
pCI with the reporter constructs. Data are presented as the
mean ± SD of at least three independent experiments.
Fig. 4. Suppression of gene expression by overexpression of
PTB1. Xenopus PTB1 was overexpressed in the left anterior
domain of the embryo and in situ hybridization was carried out at
stage 20. (A) Snail2. (B) Pax2. (C) En2. (D) Xrx1. (E) Xotx2.
Suppressed expression of each gene is indicated by a white
arrowhead. The expression of LacZ as a tracer is verified in
Snail2, En2, and Rx1.
Fig. 5. PTB1 morpholino oligonucleotide (MO)-mediated
interference of suppression of Snail2 expression by retinoic acid
and the rescue by overexpression of PTB1. Antisense
morpholino oligonucleotide directed against Xenopus PTB1 was
injected into left side of the embryo and these embryos were
reared until stage 20. Higher expression of Snail2 gene in the
injected side is indicated by a white arrowhead. When Xenopus
PTB1 mRNA was co-injected with PTB1 MO, the expression of
Snail2 was rescued, showing lower than the normal level of
expression of Snail2. The reduced expression of the Snail2 gene
is indicated by a white arrowhead. When control MO was
injected in the left side of the embryo, the embryo showed
almost the same expression of Snail2 as the right side of the
embryo. LacZ, which was used as a tracer, was stained red.
Fig. 6. Increased expression of Otx2 and En2 by PTB1 morpholino oligonucleotide (MO). PTB1 MO was injected into animal pole region
of the left side of the embryo and these embryos were reared until stage 20. Higher expression of Otx2 and En2 is indicated by a white
arrowhead. When Xenopus PTB1 mRNA was co-injected with PTB1 MO, expression of Otx2 and En2 was rescued, showing lower than
normal level of expression of Otx2 and En2. The reduced expression of Otx2 and En2 is indicated by a white arrowhead. When control
MO was injected into animal pole region of the left side of the embryo, almost the same expression as the right side was observed as to
Otx2 and En2, showing the specificity of PTB1 MO. The expression of Otx2 or En2 in the injected side of the embryo is indicated by a
white arrowhead. LacZ, which was used as a tracer, was stained red.
Fig. 7. Expression pattern of
Xenopus PTB1. Whole-mount in situ
hybridization for Xenopus PTB1 was
carried out on developing Xenopus
embryos. (A) Polypyrimidine tractbinding
protein (PTB) expression of
stage 14 embryos. (B) Sox2
expression of stage 14 embryos. (C)
PTB expression of stage 16 embryos.
(D) Snail2 expression of stage 14
embryos.
ª 2010
Fig. 8. Intracellular localization of human PTB2 in 293T cells.
293T cells were transiently transfected with human PTB2-
pEGFP-C2 plasmid DNA and the fluorescence was observed.
(A) Fluorescence image. (B) 4´ 6´ -diamidino-2-phenylindole
dihydrochloride (DAPI). (C) Merged image.
Supplementary Fig. S-1. No toxic effects of PTB1 on Xenopus embryos. Xenopus embryos are injected with mRNA encoding for GFP (A), GFP and PTB1 (B), and GFP and Bax (C). These embryos are reared until stage 30, and fluorescence of GFP was observed.
Supplementary Fig. S-2. Sites of TG/CA repeats in four genes. Sites of TG/CA repeats in Otx2 (A), En2 (B), RPE65 (C), and Tbx1 (D) of human genome are depicted by arrows.
