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Supplementary methods
Embryo manipulations
Eggs were fertilized in vitro, dejellied and resultant embryos cultivated as previously
(28). Synthetic mRNAs were injected into blastomeres at 2 or 4 nl each unless indicated
otherwise. Synthetic mRNA were prepared using Message Machine Kit (Ambion).
Luciferase assays were described previously (7).
Plasmid construction
The -357(4)Xtwn/Luc reporter was generated using-357(3)Xtwn/Luc (8) as template
DNA. The “downstream” Xtwn promoter primer (8) was used with the primer 5’GTAAGcgaccttttgcaAGGTGTCATGTaccgag-3’to produce a 3’ fragment containing a
mutation in Lef1 site 4 (Figure 1). Lowercase letters represent nucleotides changes that
are different from the wild-type promoter. In a second reaction, the “upstream” Xtwn
promoter primer was used with the primer 5’CACCTtgcaaaaggtcgCTTACTCAGACTTCAGTGG-3’ to produce a 5’ fragment. 5’ and
3’ fragments were mixed together and used as template for a third PCR reaction using the
“upstream” and “downstream” primers. The amplified fragment was cloned between the
BamHI and HindIII sites of pOLuc. The -143Xtwn/Luc was constructed by subcloning a
PCR product containing sequences from -143 to +24 of the Xtwn gene into pOLuc. The
(Gal4)5 Luc reporter construct consists of a pentamer of Gal4UAS sequences inserted
upstream of a minimal thymidine kinase promoter (tk5’-46) driving luciferase (15).
3X(TCF)/Luc, a luciferase reporter containing three tandemly repeated Lef-1/TCF sites
(5'-TCGACAAGATCAAAGGGGGTAAGATCAAAGGGGGTAAGATCAAAGGGG3') upstream of a minimal IFN promoter, was generated by replacing the Ig repeats of
p-55IgLuc (16) with synthetic Lef-1/TCF sites.
DNase I footprint assay
The 3’ end of the promoter fragment was 32P end-labeled by filling-in HindIII linearized
–357Xtwn/Luc using 32P-dATP and Klenow enzyme. After labeling, the 392bp promoter
fragment was released by digestion with BamHI, followed by gel purification. For
footprinting reactions, GST, GST-purified MH1-Smad and purified Lef1 were preincubated in binding buffer (20mM Hepes-KOH (pH 7.9), 20% glycerol, 2mM DTT,
7.5mM MgCl2, 55g/ml poly(dI.dC) for 10 minutes on ice. 100K cpm of asymmetrically
32
P-labeled probe was added to the mixture, followed by DNaseI treated on ice for 1
minute. The reaction was stopped by adding Stop solution (0.8% SDS, 25mM EDTA).
DNA was precipitated and electrophoresed. GST-fusion construct of Smad4-MH1
(amino acids 1-151) and full-length GST-Smad1-4 were obtained from L. Attisano and P.
ten Dijke, and purified Lef1 protein was a gift from M. Waterman.
GST-pulldown assays
Glutathione-sepharose slurry (600l of 50% in TBST [10mM Tris, pH8.0, 100mM NaCl,
0.2% Tween]) was incubated with GST-Smad4 protein at 4oC for one hour. Beads were
precipitated and resuspended in TBST+0.2% BSA, then incubated at 4oC for 10 minutes.
This step was repeated and beads were then resuspended in 280l of TBST+0.2% BSA.
20l of this slurry (containing ~1g of GST-Smad4 protein) was mixed with 2-20l of
35
S-labeled in vitro translated Lef1 and TBST+0.2%BSA to a final volume of 200l and
incubated at room temperature for one hour. Beads were washed three times in TBST.
Precipitates were analyzed by SDS-PAGE.
Supplementary figure legend for Fig 4
Fig 4f. Control reporter lacking Gal4 binding sites was not activated (data not shown).
For all transcriptional activation assays shown, a single representative experiment is
shown; error bars represent the values of duplicate transfections done for each data point.
Fig 4g. The decrease in reporter activity at high levels of Smad4 is consistent with our
unpublished observation (with several promoters) that a high Smad4/Lef-1 ratio lowers
reporter activation.