Abstract

Abstract Transforming growth factor‐β (TGF‐β) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor‐activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF‐β regulates the transcription of target genes in a cell context‐dependent manner by screening a double‐stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti‐Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF‐β. The preference of the activated Smad complexes for conventional Smad‐binding motifs such as Smad‐binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor‐binding motifs other than SBE or CAGA, including the Fos/Jun‐binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor‐binding motifs together with Smad‐binding motifs indicated that some of the motifs may be involved in cell type‐dependent transcriptional activation by TGF‐β. The results suggest that the CASTing method is useful for elucidating the molecular basis of context‐dependent Smad signaling.