SMAD 8 binding to mice Msx1 basal promoter is required for transcriptional activation.
The Msx1 gene in mice has been proven to be induced by BMP (bone morphogenetic protein) proteins, and three binding sites for SMAD, an intracellular BMP signalling transducer, have already been identified in its promoter. Gel shift analyses were performed and they demonstrated that the consensus found very near the transcription start site, a region designed BP (basal promoter), is functional for binding nuclear proteins from 10.5, 11.5 and 13.5 dpc (days post-coitum) embryos. Notably, this binding occurs only when the SMAD-binding consensus sequence is maintained, suggesting that it is required for the formation of a protein complex over BP. Binding of purified SMAD 1 and SMAD 4 as well as supershift assay with SMAD 1/SMAD 5/SMAD 8 antibody proved that a SMAD protein is present in this complex. Transfection assays in cell cultures with fragments from BP driving the expression of luciferase confirmed that only in the presence of the SMAD consensus site is Msx1 expression activated. A proteomic analysis of the complex components after immunoprecipitation identified several proteins necessary to activate transcription including SMAD 8. Our results suggest that BMP2/BMP4 signalling through SMAD 8 is required for transcriptional activation of the mouse Msx1 gene.
Binato
Renata
R
Laboratório de Biologia Molecular Maury Miranda, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Alvarez Martinez
Cristina E
CE
Pizzatti
Luciana
L
Robert
Benoit
B
Abdelhay
Eliana
E
D000818:Animals; D001665:Binding Sites; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002457:Cell Extracts; D016384:Consensus Sequence / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D051957:MSX1 Transcription Factor / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D015398:Signal Transduction; D051904:Smad8 Protein / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000378:metabolism
C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D002457:Cell Extracts; D051957:MSX1 Transcription Factor; C484084:Msx1 protein, mouse; D046912:Multiprotein Complexes; D051904:Smad8 Protein; C491621:Smad9 protein, mouse; D016212:Transforming Growth Factor beta