title: Repression of Smad2 and Smad3 transactivating activity by association with a novel splice variant of CCAAT-binding factor C subunit.
abstract: Activation by transforming growth factor-beta (TGF-beta)/activin receptors leads to phosphorylation of Smad2 (Sma- and Mad-related protein 2) and Smad3, which function as transcription factors to regulate gene expression. Using the MH2 domain (Mad homologue domain of Smad proteins 2) of Smad3 in a yeast two-hybrid screening, we isolated a novel splice variant of CAATT-binding factor subunit C (CBF-C), designated CBF-Cb, that associated with Smad3. CBF-C is one of the subunits that form a heterotrimeric CBF complex capable of binding and activating the CAATT motif found in the promoters of many eukaryotic genes. CBF-Cb is 62 amino acids shorter than the wild-type CBF-C in the N-terminal region. In addition, CBF-Cb is expressed ubiquitously in various mouse tissues. By an immunoprecipitation assay, we detected an in vivo association of CBF-Cb with Smad2 and Smad3, independent of signalling by activated TGF-beta type I receptors. In transient transfection experiments, overexpression of CBF-Cb was able to repress the transactivating activity of Smad2 and Smad3, mediated either by direct binding to the Smad-responsive element or through their association with the Smad-interacting transcription factor FAST-2 (forkhead activin signal transducer-2). The Smad-mediated transcriptional response after TGF-beta receptor activation was also inhibited by overexpression of unspliced CBF-C. In addition, the repressive activity of CBF-Cb on Smad2- and Smad3-mediated transcriptional regulation was abrogated by co-expression of the general transcription activator p300. The association of CBF-Cb with Smad2 was competitively inhibited by overexpression of p300. These data indicate a novel mechanism for modulation of the transcriptional activity of Smad proteins, whereby the interaction of CBF-Cb, as well as canonical CBF-C, with the MH2 domain of Smads may prevent the association of Smads with transcriptional co-activators.
authors.affiliation: Department of Medical and Molecular Genetics and the Walther Oncology Center, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis, IN 46202, USA.
authors.forename: Feifei
authors.initials: F
authors.lastname: Chen
authors.forename: Kenji
authors.initials: K
authors.lastname: Ogawa
authors.forename: Xubao
authors.initials: X
authors.lastname: Liu
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Stringfield
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D023081:CCAAT-Binding Factor; D017931:DNA Primers; D004268:DNA-Binding Proteins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators