Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene

• Published in: The Journal of biological chemistry 2002-02, Vol.277 (5), p.3176-3185
• Main Authors: López-Rovira, Teresa, Chalaux, Elisabet, Massagué, Joan, Rosa, Jose Luis, Ventura, Francesc
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins - metabolism; Bone Morphogenetic Proteins - pharmacology; Cell Line; Cloning, Molecular; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Gene Expression Regulation - drug effects; Genes, Reporter; Helix-Loop-Helix Motifs; Id1 gene; Inhibitor of Differentiation Protein 1; Kinetics; Luciferases - genetics; Promoter Regions, Genetic; Recombinant Proteins - metabolism; Repressor Proteins; Signal Transduction; Smad Proteins; Smad1 protein; Smad4 protein; Trans-Activators - chemistry; Trans-Activators - metabolism; Transcription Factors - genetics; Transcriptional Activation; Transfection; Transforming Growth Factor beta
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M106826200

Bone morphogenetic proteins (BMPs) are potent inhibitors of myoblast differentiation and inducers of bone formation both in vivo and in vitro. Expression of Id1, a negative regulator of basic helix-loop-helix transcription factors, is up-regulated by BMPs and contributes to the antimyogenic effects of this family of cytokines. In this report, we have identified a specific BMP-2 immediate early response enhancer in the human Id1 gene. Transcriptional activation of the enhancer was increased by overexpression of BMP-responsive Smads, and Smad4 and was completely abrogated in Smad4-deficient cells. Deletion analysis demonstrates that the responsive region is composed of two separate DNA binding elements, a set of overlapping GC boxes, which bind BMP-regulated Smads upon BMP stimulation, and three repeats of CAGAC boxes. Gel shift and oligonucleotide pull-down assays demonstrated that these two types of motifs were capable of binding their corresponding Smads. However, deletion or mutation of either DNA binding element was nonadditive, since disruption of either GC or CAGAC boxes resulted in complete or severe loss of BMP-2 responsiveness. These data suggest the simultaneous requirement of two independent DNA binding elements to allow functional cooperativity of BMP-regulated Smads and Smad4 in BMP-activated gene promoters.