December 15, 1997;
LEF-1/TCF proteins mediate wnt-inducible transcription from the Xenopus nodal-related 3 promoter.
The Xenopus nodal
-related 3 gene (Xnr3
) is expressed in the Spemann organizer
of the embryo
and encodes a member of the transforming growth factor beta family that mediates some activities of the organizer
is transcriptionally activated by wnt signaling during gastrulation in the Xenopus embryo
. Here we show that a small region of the Xnr3
promoter is sufficient to confer wnt-inducible transcription. By mutational analysis of the promoter, we have identified two distinct sequence elements required for the response to wnt signals. One regulatory sequence interacts with a factor which accumulates in Xenopus gastrulae independent of wnt signaling. The other functionally important site can bind mammalian LEF-1
protein, a member of the LEF-1
/TCF family of transcription factors. In addition, misexpression of LEF-1
explants induces transcription of the endogenous Xnr3
gene. Taken together, these data provide further evidence for a role of LEF-1
/TCF proteins in wnt signaling and identify the Spemann organizer
-specific gene Xnr3
as a direct target of these transcription factors in vertebrates.
[+] show captions
FIG. 1. (a) Sequence of the Xnr3 promoter from the end of the cDNA to an upstream PstI site located 257 bp upstream. Numbering is
relative to the end of the previously isolated Xnr3 cDNA (Smith et al., 1995). End points of a series of exonuclease III deletions are
indicated with an asterisk (*) above the last base pair of the sequence. Wnt responsive elements, WRE1 and WRE2, are underlined. (b)
Deletion analysis of the Xnr3 promoter. The results from one representative assay are shown in which a selection of the truncated
promoters were tested. The wnt inducibility of plasmids was calculated as the amount of luciferase activity present in extracts from
gastrulae injected with the plasmid along with Xwnt8 mRNA, divided by the amount of luciferase activity present in extracts from
gastrulae which were injected with the plasmid alone. The results from this and other experiments show that the 5* end of a wntresponsive
element lies between position -160 and position -142.
FIG. 2. (a) Sequence of linker-scanning mutations of the Xnr3 promoter. Eight- to 10-bp stretches (between 0141 and 063) of the
Xnr3 promoter in p160Xnr3 were replaced with a NotI site as shown. The replacement sequences are in lowercase letters and
surrounding sequences are shown for orientation. These new plasmids, p160LS3 through p160LS11, were tested for their wnt inducibility.
(b) Wnt responsiveness of linker-scanning mutants of the Xnr3 promoter. Two representative assays which show that mutations
in either p160LS3 or p160LS8 reduce the wnt inducibility of the promoter about 10-fold are shown. Two independent isolates of each
mutant construct were tested at least twice and in separate experiments. (c) Mutational analysis of WRE1. Mutation of 5*-ATTA to
5*-AGGA at position 0143/4, significantly reduces the wnt inducibility of the Xnr3 promoter. (d) The b-catenin response of the Xnr3
promoter is dependent on WRE2. The plasmids p160Xnr3 and p160LS8 were tested for their ability to respond to Xwnt8 and bcatenin.
The response of the promoter to b-catenin also requires the sequences removed by the LS8 replacement and so is dependent
FIG. 3. LEF-1 can interact with the Xnr3 promoter. DNase I foot-
print analysis of the Xnr3 promoter fragment using purified LEF-1
HMG polypeptide. Xnr3 fragments were 3* end labeled on the top strand
(left) or bottom strand (right) and incubated with no protein
(lanes 1 and 5) or with 10 ng (lanes 2 and 6), 30 ng (lanes 3 and 7),and
100 ng (lanes 4 and 8) of LEF-1 HMG polypeptide. The brackets indicate
the region protected by LEF-1.
FIG. 4. Mammalian LEF-1 can bind to the sequence from -125
to -63. Embryos were injected with 1 ng LEF-1 mRNA, 1 ng LEF-
1DN mRNA, or 1 ng Xwnt mRNA at the single-cell stage. They
were harvested at gastrulation and extracts were incubated with a
radiolabeled copy of the sequences between -125 and -63 of the
Xnr3 promoter. A novel shifted complex is detected in extracts
from embryos injected with LEF-1 mRNA (lane 2). A complex of
greater mobility is detected in extracts from embryos injected with
LEF-1D56 mRNA (lane 3), indicating that mammalian protein can bind
these sequences directly.
FIG. 5. (a) Embryo extracts contain proteins which bind WRE1
specifically. Extracts were made from midgastrula embryos (stage
11) and mixed with a radiolabeled probe containing sequences from
0160 to 0129 of the Xnr3 promoter and complexes separated by
electrophoresis. Lane 1 contains no competitor, whereas 100-fold
excess of unlabeled competitor DNA was added to the binding
reactions loaded in lanes 2, 3, and 4. Competitor DNAs are wildtype
sequences from the Xnr3 promoter between 0160 and 0129
(lane 2), or 0160 to 0129 with a 2-bp point mutation 5*-ATTA to
5*-AGGA (lane 3), or carrying the same sequence replacement as
is present in p160LS3 (lane 4). (b) A developmentally regulated
complex binds sequences between 0160 and 0104 specifically. Embryos
were injected at the single-cell stage with a-amanitin (an
inhibitor of RNA polymerase II transcription) or left uninjected.
Extracts were then prepared from embryos at different stages of
development as indicated, and a gel retardation assay was performed
using the sequences between 0160 and 0104 as probe.
FIG. 6. (a) Misexpression of either LEF-1 or b-catenin in Xenopus ectodermal explants leads to accumulation of Xnr3 transcripts. The animal pole of Xenopus embryos was injected with 100 pg LEF-1 (lanes 9 and 10), 100 pg b-catenin (lanes 3 and 4), or 1 ng Xwnt8 mRNA (lanes 11 and 12) or left uninjected. Animal pole tissue was explanted at the blastula stage and allowed to develop in isolation until gastrula stages, and then the amount of Xnr3 transcript was assayed by RT-PCR. Explanted animal pole tissue does not normally express Xnr3 (lanes 1 and 2), but injection of any of these three components of the wnt signaling pathway, LEF-1, b-catenin, or Xwnt8, induced Xnr3 expression. Induction of Xnr3 expression by b-catenin was decreased by coinjection of 333 pg of the HMG box of LEF-1 (lanes 5 and 6) and completely abrogated by 1 ng of HMG LEF-1 (lanes 7 and 8). (b) The differential activation of the Xnr3 promoter on the dorsal side of Xenopus gastrulae is dependent on the LEF-1 binding sites. pXnr3lux was injected into the dorsal or ventral side of a four-cell stage embryo. Expression of the reporter from this construct
was between 6- and 15-fold higher after dorsal injection com-pared to ventral injection. This differential activation on the dorsal side of the embryo was not observed when the LEF-1 binding sites in the Xnr3 promoterwere mutated as in the plasmid pXnr3luxmLEF.