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Fig. 7. Smad3 induces expression of Xlim1 in a direct manner; induction of
Chordin by Xlim1-3m requires Smicl. (A) Smad3 but not Smad2 is a
direct inducer of Xlim1. The experimental regime was identical to
that described in Fig. 6A,
except that expression of Xlim1 and not Chordin was analysed
by quantitative RT-PCR. (B) Xlim1/3m is a direct inducer of Chordin.
RNA encoding Xlim1/3m (400 pg) was injected into embryos of Xenopus
laevis at the one-cell stage. Animal caps were dissected from such
embryos and cultured in control medium or medium containing cycloheximide, as
described in Fig. 6A. Induction
of Chordin is not inhibited by cycloheximide. (C) Smicl is required
for Xlim1/3-mediated induction of Chordin. RNA encoding Xlim1/3m (400
pg) was injected into embryos at the one-cell stage in the presence of 80 ng
morpholino oligonucleotide coMO or XlMO. Animal pole regions were dissected
from such embryos at mid blastula stage 8 and cultured until control embryos
reached stage 11. (D) Smicl is not required for expression of Xlim1
in Xenopus laevis. Morpholino oligonucleotides XlMO or coMO (80 ng)
were injected into Xenopus laevis embryos at the one-cell stage and
RNA was extracted at early gastrula stage 10.5. Expression of Xlim1
is not inhibited by XlMO. (E) Smicl and Xlim1/3 can interact. The indicated
combinations of expression constructs encoding HA tagged XtSmicl, Flag tagged
Xlim1/3m, Myc tagged Smad3 and caALK4 were co-transfected into HEK 293T cells.
Extracts were subjected to immunoprecipitation using an anti-Flag antibody and
the presence of HA XtSmicl was analysed by western blotting by using an
anti-HA antibody. Smad3 can bind Xlim1/3 in a Smic1-dependent fashion as shown
by western analysis of Xlim1/3 immunoprecipitates using an anti-Myc antibody.
(F) Binding of Flag-tagged Xlim and HA-tagged Smicl to Chordin
promoter sequences containing Xlim-binding sites. Extracts of HEK293T cells
transfected with Xlim and/or Smicl expression constructs were incubated with
streptavidin-agarose beads and biotinylated double-stranded oligonucleotides.
Upper panel: precipitated complexes were subjected to western blotting using
anti-Flag and anti-HA antibodies. Xlim1, but not Smicl, interacts with this
region of the Chordin promoter, but Smicl can form part of a ternary
complex with the Chordin promoter region and Xlim1. Xlim does not
bind to Chordin promoter oligonucleotides with mutations in the
Xlim-binding sites (see Materials and methods). Lower panel: western blotting
of whole extracts shows similar expression levels of Flag-tagged Xlim and
HA-tagged Smicl.
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