Figure 1. Activin-responsive transcription via the goosecoid
DE. (A) Activin-responsive transcription through the DE is
partially dependent on new protein synthesis. One-cell embryos
were injected with REF–globin internal control together
with globin reporters driven by the minimal g-actin
promoter (gA), or by multiple copies of the DE or ARE upstream
of the mimimal promoter. Animal caps, cut at stage 8
were cultured for 6 hr with or without activin in the absence
or presence of 5 μg/ml cycloheximide. Globin transcripts
from reporter genes (Test-globin) or the internal control (REFglobin)
were detected by RNase protection (Howell and Hill
1997). Transcriptional activation was calculated as a ratio of
the levels of Test-globin to REF-globin. Activin-induced transcription
is expressed as fold inductions. (B) An activin-inducible
protein (DEBP) binds the goosecoid DE. Whole-cell
extracts prepared from stage 8 or stage 11 embryos or stage
11 embryos overexpressing activin, were analyzed by bandshift
assay using the single DE probe. Activin-inducible protein
DEBP is indicated. Competitor oligonucleotides were
used at a 50-fold molar excess over probe where indicated.
(Bottom) Sequences of wild-type DE and mutant oligonucleotides
indicating only the altered nucleotides. The pairedlike
homeodomain-binding site comprising two inverted
TAAT motifs (Wilson et al. 1993) is denoted by arrows;
a third homeodomain core-binding site at the 38 end is
also indicated by an arrow. (Thick dotted line) Sequence
reminiscent of a half-site for T-box protein (AGGTGTGAAATT)
(Kispert et al. 1995); (underline) almost perfect
binding site for the ZFH-1 family of zinc finger homeodomain
proteins (AGGTGAGCAA) (Funahashi et al. 1993). (C) Formation
of DEBP requires new protein synthesis. Extracts
were made from uninjected stage 8 embryos (lane 1), stage 10.5 embryos (lanes 2,3), or stage 10.5 embryos overexpressing activin (lanes
4,5) and analyzed by bandshift using the DE probe. Where indicated, embryos had been preincubated in cycloheximide before stage 8.
Figure 2. DEBP specifically interacts with the effector domain of Smad2. (A) Whole-cell extracts prepared from either stage 8 embryos
(lanes 1–4), stage 10.5 embryos (lanes 5–8), or stage 10.5 embryos overexpressing activin (lanes 9–12) were analyzed by bandshift using
the DE probe. Extracts were mixed with either purified GST protein (100 ng) (lanes 4,8,12) or two concentrations (20 and 100 ng) of
purified GSTSmad2C (lanes 2, 3, 6, 7, 10, 11) before addition of probe. (Open arrow) DEBP; (black arrow) GSTSmad2C bound to DEBP.
(B) The interaction of DEBP with Smad2C is specific. The assay was as in A using GST fusions of Smad1C, Smad2C, or Smad4C as
Figure 3. Homeodomain proteins, Mixer and Milk, but not Mix.1, interact with Smad2C. (A) Mixer and Milk are good candidates for the endogenous protein DEBP. Whole-cell extracts were prepared from stage 10.5 embryos or embryos injected at the 1-cell stage with mRNA encoding myc-tagged Mixer, Milk, Mix.1, or activin, and DE-binding activity was assayed by bandshift. Anti-myc antibody or purified GSTSmad2C were added where indicated. (Open arrow) DEBP; (gray arrow) supershifted complexes. (B) Interaction of GSTSmad2C with members of the Mix family and Fast-1. In vitro-translated Mixer, Milk, Mix.1, and Fast.1 were assayed by bandshift for their interaction with purified GSTSmad2C or GST using the appropriate radiolabeled DE or ARE probes. Transcription factors complexed with probe are indicated; (black arrow) ternary complex with GSTSmad2C.
Figure 4. Characterization of the SIM. (A) Schematics of Mix.1, Mixer, and Milk, with the conserved homeodomains and a carboxy-terminal acidic domain indicated. (Black box) A region conserved in Milk and Mixer, also present in Xenopus Fast-1 and mouse Fast-2 (expanded below where the black line denotes the boundaries of the conserved sequences). (Black shading) Identical amino acids; (gray shading) similar amino acids. The numbers indicate the positions of these amino acids in the full-length sequences of the individual proteins. (B) Carboxy-terminal deletion mutants of Mixer, Milk, and Fast-1 (schematized below) were produced in vitro and their interaction with GSTSmad2C assayed by bandshift using the DE or ARE probe as appropriate. Complexes of transcription factors and probe are indicated; (black arrow) ternary complex with GSTSmad2C. Smad interaction motif (SIM) and DNA-binding domains are indicated. Note that Milk gives rise to two complexes, both of which shift with GSTSmad2C, which correspond to a dimer of Milk and a higher order complex. (C) Mutation of the prolines in the PPNK core motif abolishes the interaction with Smad2C. Full-length Mixer or a mutant derivative (Mixer PP mut), in which the two prolines in the PPNK-containing motif are mutated to alanines, were produced in vitro and assayed for interaction with GSTSmad2C by bandshift using the DE probe. (D) Interaction of Mixer, Milk, and Fast-1 with Smad2C in solution. 35S-labeled transcription factors as indicated were incubated with Sepharose-bound GST (lanes2,5,8,11,14) or GSTSmad2C (lanes 3,6,9,12,15) and bound protein was visualized by SDS-PAGE and autoradiography. A fraction of input protein was analyzed for comparison (lanes1,4,7,10,13).
Figure 5. The Smad interaction motif is sufficient to interact with Smad2. (A) A peptide containing the SIM of Mixer competes specifically for interaction of Mixer with Smad2C. In vitro-translated Mixer was incubated with DE probe alone (lane 1) or in the presence of 1 or 10 pmoles of wild-type peptide (lanes2,3) or mutant peptide (lanes 4,5). GSTSmad2C (20 ng) was included in the reactions in lanes6–14, with the addition of 0.3, 1, 3, or 10 pmoles of wild-type peptide (lanes 7–10) or mutant peptide (lanes11–14). Mixer complexed with probe is indicated; (black arrow), ternary complex with GSTSmad2C. (B) A peptide containing the Mixer SIM specifically disrupts the interaction of DEBP with Smad2C. Whole-cell extracts made from activin-injected stage 10.5 embryos were analyzed by bandshift assay with the DE probe in the absence (lane 1) or presence of GSTSmad2C (20 ng; lanes2–10) with the addition of 5, 15, 30, and 100 pmoles wild-type peptide (lanes 3–6) or mutant peptide (lanes7–10). DEBP is indicated; (black arrow) ternary complex with GSTSmad2C. (C) A peptide containing the Mixer SIM specifically disrupts the formation of ARF. Whole-cell extracts made from activin-injected stage 8 embryos were analyzed by bandshift assay with the ARE probe in the absence (lane 1) or presence of 10, 30, 60, 100, or 200 pmoles of wild-type peptide (lanes 2–6) or mutant peptide (lanes 7–11). The endogenous ARF complex is indicated.
Figure 6. Mixer and Milk interact with activated Smads in vivo. (A) Mixer forms a ligand-dependent complex with Smad2 and Smad4 in solution. Extracts were prepared from NIH-3T3 cells transfected with myc–Smad2, myc–Smad4, and either Flag–Fast-1, Flag–Mixer, or a Flag-tagged mutant derivative (Mixer PP mut), which had been incubated with or without TGF-β1 (2 ng/ml) for 1 hr. Extracts were assayed either by immunoprecipitation of complexes with anti-Flag antibody followed by Western blotting with anti-Myc antibody (top), or Western blotting the whole extract with anti-Flag antibody (middle), or with anti-Myc antibody (bottom). (B,C) Fast-1 and Mixer form ligand-dependent complexes on DNA with endogenous Smad2 and Smad4. Extracts were prepared from NIH-3T3 cells transfected with Flag-tagged Fast-1, Mixer, or Mixer (PP mut), which had been incubated with or without TGF-β1 (2 ng/ml) for 1 hr. Extracts were analyzed by bandshift assay on the ARE (B) or DE (C) probe. Anti-flag, anti-Smad2, or anti-Smad4 antibodies were included in the binding reactions where indicated. In B ARF and antibody-supershifted ARF are indicated. In C, Mixer or Mixer (PP mut) bound to probe, the Mixer–Smad complex and antibody-supershifted Mixer–Smad complex are indicated.
Figure 7. Mixer and Milk mediate TGF-β-dependent transcriptional activation through the DE. (A) NIH-3T3 cells were transfected with the CAT reporters, and plasmids expressing transcription factors, Smad2 and Smad4, as indicated. Cells were cultured with or without TGF-β1 (2 ng/ml) for 8 hr. Cells were harvested and CAT activity measured relative tolacZ activity from the internal control. The data are from a representative experiment, and similar results were obtained in other independent experiments. (B) Mixer mediates TGF-β-dependent transcriptional activation through the DE in the absence of protein synthesis. NIH-3T3 cells were transfected with the (DE)4-globin reporter and REF-globin internal control with or without Mixer expression plasmid. Cells were cultured with or without TGF-β1 (2 ng/ml) for 4 hr in the absence or presence of 50 μg/ml cycloheximide. Globin transcripts from the reporter genes (Test-globin) or the internal control (REF-globin) were detected by RNase protection and quantitated as in Fig. 1.
Figure 8. The temporal and spatial expression patterns of Mixer and Milk in Xenopus embryos makes them good candidates for
mediating transcription of goosecoid in response to an endogenous activin-like signal. (A) Coexpression of goosecoid with Mixer and
Milk at early gastrula stages. Xenopus embryos were fixed at stage 10.25, cut in half through the middle of the dorsal lip, and processed
for in situ hybridization with probes against goosecoid, Mixer, Milk, or Cerberus (as a control to visualize the prospective mesoendodermal
boundary). (Arrowhead) Blastopore lip. The mRNAs are visualized with a dark blue/purple stain. It is clear that goosecoid
is expressed on the dorsal side in prospective mesodermal and endodermal cells expressing Mixer and/or Milk. (Right) Schematic of
stage 10.25 embryo cut in the same way as the embryo halves. (Red) Prospective notochord; (orange) prospective head mesoderm; (pink)
somitic mesoderm; (light orange) ventral migrating mesoderm; (yellow) prospective endoderm (adapted from Keller 1991). (B) Time
course of expression of goosecoid (Gsc), Mixer, Milk, and the FGF receptor (FGFR) assayed by RNase protection. The Milk probe also
detects Bix3 (Tada et al. 1998). Embryos were sampled at stage 8 and subsequent times indicated. In lanes 9–16 the embryos had been
preincubated with cycloheximide from 30 min before stage 8. (C) A model showing that TGF-b/activin-activated Smads translocate
to the nucleus, where they interact with homeodomain transcription factors, Mixer and Milk through the SIM to activate transcription.
(D) A model indicating the proposed role of the Mixer/Milk/Bix3-Smad complexes in the formation of mesoderm and endoderm
in early Xenopus embryos. (Black arrows) Induction of gene expression; (gray arrows) activation of protein complexes. Milk/Bix3–Smad
complexes are involved in the initiation of transcription of mesoendodermal genes andMixer/Milk/Bix–Smad complexes are involved
in the maintenance of gene expression. For discussion, see text.