title: Promoter-wide analysis of Smad4 binding sites in human epithelial cells.
abstract: Smad4, the common partner Smad, is a key molecule in transforming growth factor-beta (TGF-beta) family signaling. Loss of Smad4 expression is found in several types of cancer, including pancreatic cancer and colon cancer, and is related to carcinogenesis. Here we identified Smad4 binding sites in the promoter regions of over 25 500 known genes by chromatin immunoprecipitation on a microarray (ChIP-chip) in HaCaT human keratinocytes. We identified 925 significant Smad4 binding sites. Approximately half of the identified sites overlapped the binding regions of Smad2 and Smad3 (Smad2/3, receptor-regulated Smads in TGF-beta signaling), while the rest of the regions appeared dominantly occupied by Smad4 even when a different identification threshold for Smad2/3 binding regions was used. Distribution analysis showed that Smad4 was found in the regions relatively distant from the transcription start sites, while Smad2/3 binding regions were more often present near the transcription start sites. Motif analysis also revealed that activator protein 1 (AP-1) sites were especially enriched in the sites common to Smad2/3 and Smad4 binding regions. In contrast, GC-rich motifs were enriched in Smad4-dominant binding regions. We further determined putative target genes of Smad4 whose expression was regulated by TGF-beta. Our findings revealed some general characteristics of Smad4 binding regions, and provide resources for examining the role of Smad4 in epithelial cells and cancer pathogenesis.
authors.affiliation: Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo.
authors.forename: Daizo
authors.initials: D
authors.lastname: Koinuma
authors.forename: Shuichi
authors.initials: S
authors.lastname: Tsutsumi
authors.forename: Naoko
authors.initials: N
authors.lastname: Kamimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Aburatani
authors.forename: Kohei
authors.initials: K
authors.lastname: Miyazono
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D051901:Smad4 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta
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