Abstract
The Ski protein has been proposed to serve as a corepressor for Smad4 to maintain a transforming growth factor-beta (TGF-beta)-responsive promoter at a repressed, basal level. However, there have been no reports so far that it indeed acts on a natural promoter. We have previously cloned the human Smad7 promoter and shown that it contains the 8-base pair palindromic Smad-binding element (SBE) necessary for TGF-beta induction. In this report, we have characterized the negative regulation of Smad7 promoter basal activity by Ski. We show that Ski inhibits the Smad7 promoter basal activity in a SBE-dependent manner. Mutation of the SBE abrogates the inhibitory effect of Ski on the Smad7 promoter. Moreover, mutation of the SBE increases the Smad7 promoter basal activity. Using the chromatin immunoprecipitation assay, we further show that Ski together with Smad4 binds to the endogenous Smad7 promoter. Finally, we show that RNAi knockdown of Ski increases Smad7 reporter gene activity in transient transfection assays as well as elevating the endogenous level of Smad7 mRNA. Taken together, our results provide the first evidence that Ski is indeed a corepressor for Smad4, which can inhibit a natural TGF-beta responsive gene at the basal state.
Highlights
The 8-bp palindromic sequence (GTCTAGAC) was initially identified to be a high affinity binding site for Ski, screened by using nuclear extracts from c-Ski-transformed cells [11]
SnoN mRNA is induced by TGF- treatment [16, 19, 20], which may play a role in turning off the TGF- signal
We show that Ski, recruited to the Smad-binding element (SBE) by interacting with Smad4, inhibits the basal level of the endogenous Smad7 gene
Summary
The 8-bp palindromic sequence (GTCTAGAC) was initially identified to be a high affinity binding site for Ski, screened by using nuclear extracts from c-Ski-transformed cells [11]. We show that Ski, recruited to the SBE by interacting with Smad4, inhibits the basal level of the endogenous Smad7 gene. Mutation of the SBE leads to a significantly higher basal transcriptional activity of the Smad7 reporter gene in HepG2 cells (Fig. 1A).
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