Transcriptional regulation of the human Smad7 promoter

Transforming growth factor beta (TGF-β) signaling is central to the regulation of fundamental cellular processes. Abnormal TGF-β signaling can lead to the development of a number of diseases, such as cancer and fibrosis. Smad7 is a component of the TGF-β pathway. It inhibits TGF-β signal propagation by inhibiting R-Smad phosphorylation and by targeting receptor complexes for degradation. Interestingly, Smad7 mRNA level is rapidly induced by TGF-β, which makes Smad7 part of the negative feedback loop for TGF-β signaling.; The aim of this dissertation is to clone the human Smad7 (hSmad7) promoter and to characterize its regulation at the basal state as well as in the presence of TGF-β. We have isolated approximately 5.5 kb of the hSmad7 promoter upon screening of a human genomic placenta library with a Smad7 probe. Primer extension assays revealed the presence of two promoters, hSmad7P1 and hSmad7P2 separated by 48 by within the hSmad7 regulatory region. Sequence analysis of the Smad7 promoter indicated the presence of the consensus Smad binding element (SBE) 5′GTCTAGAC3′ 210 by upstream of the transcription start site. We have demonstrated that the SBE is important for TGF-β induction of the hSmad7 promoter. Our DNA binding studies indicated that TGF-β treatment rapidly induces the Smad3-Smad4 complex binding to the SBE and that the Smad3-Smad4 complex can bind to the SBE, a high affinity binding site, on its own.; In addition, we have discovered that the SBE also plays an important role in maintaining the Smad7 promoter in a repressed state in the absence of TGF-β signaling. Using chromatin immunoprecipitation assays, we demonstrated that at basal state, the Ski protein is recruited to the SBE of the Smad7 promoter through interaction with Smad4. Depletion of Ski from cells by siRNA leads to transcriptional activation of Smad7 reporter constructs and the upregulation of endogenous Smad7 mRNA. Our study is the first data on negative regulation of the hSmad7 promoter. Moreover, our study provides the first evidence that Ski is indeed a corepressor for smad4 and inhibits a natural TGF-β responsive gene at basal state.