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Fig. 1. (A) Cos-7 cells were transfected with increasing amounts of an expression
vector containing the Gal4 DNA-binding domain (DBD) fused to full-length Dach1
(Gal4-WT), along with the 4R-UAS-Luc reporter plasmid, which contains four
Gal4-DBD-binding motifs. Transcription was repressed by Dal4-WT in a
dose-dependent manner (12.5- to 23-fold repression). (B) Using the same
reporter, a low amount of Gal4-Dach1 was expressed in Cos-7 cells, resulting
in weak repression. However, co-expression of mouse Sin3a strongly enhanced
the repressor activity. (C) Co-expression of Gal4 DBD-Smad1 and BMP4 activated
the 4R-UAS-Luc reporter. Expression of Dach1 weakened this BMP4-mediated
activation. Addition of mouse Sin3a further repressed this activation. When
Dach1 expression was removed, mouse Sin3a-mediated repression was no longer
observed. (D) An immunoprecipitation assay was carried out using HA-tagged
Dach1 and Flag-tagged mouse Sin3a expressed in Cos-7 cells. An irrelevant IgG
did not precipitate either Dach1 or Sin3a (IP IgG). When the anti-HA antibody
was used, both Dach1 and Sin3a were detected (IP Dach1=HA) by the anti-Flag
antibody (IB Flag=mSin3a) and the anti-HA antibody (IB HA), respectively.
Input, 5% of the extract used in this immunoprecipitation assay. (E)
Flag-tagged Smad1 and HA-tagged Dach1 were co-expressed in Cos-7 cells, and
cell lysates were immunoprecipitated with the anti-HA antibody or an
irrelevant IgG (as a negative control). In the precipitant of the irrelevant
IgG, neither Smad1 nor Dach1 was detected (IP IgG). By contrast, both Smad1
and Dach1 were present in the precipitant when the anti-HA antibody was used
for IP (IP Dach1=HA). Input, 5% of the extract used in this
immunoprecipitation assay. (F) Addition of Dach1 repressed the
BMP-4/Smad1/Smad4-mediated transcription of the Xvent2-Luc reporter.
Co-expression of VP=Dach1 activated this reporter even in the presence of
Dach1. (G) By contrast, co-expression of DD2 did not activate this promoter,
but instead cancelled the Dach1-mediated repression.
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