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The Ski protein has been proposed to serve as a corepressor for Smad4 to maintain a transforming growth factor-β (TGF-β)-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-β 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-de- pendent 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-β responsive gene at the basal state. [ABSTRACT FROM AUTHOR]