Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene.
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.
López-Rovira
Teresa
T
Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, 08907 L'Hospitalet de Llobregat, Spain.
Chalaux
Elisabet
E
Massagué
Joan
J
Rosa
Jose Luis
JL
Ventura
Francesc
F
D000818:Animals; D001665:Binding Sites; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism* / Q000494:pharmacology*; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D018257:Helix-Loop-Helix Motifs; D051798:Inhibitor of Differentiation Protein 1; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D015398:Signal Transduction; D051785:Smad Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D051798:Inhibitor of Differentiation Protein 1; D011994:Recombinant Proteins; D012097:Repressor Proteins; D051785:Smad Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D008156:Luciferases