Dystrophin Dp71 expression is down-regulated during myogenesis: role of Sp1 and Sp3 on the Dp71 promoter activity.
Dp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5'-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3.
de León
Mario Bermúdez
MB
Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del I.P.N., 07360 México.
Montañez
Cecilia
C
Gómez
Pablo
P
Morales-Lázaro
Sara Luz
SL
Tapia-Ramírez
Victor
V
Valadez-Graham
Viviana
V
Recillas-Targa
Félix
F
Yaffe
David
D
Nudel
Uri
U
Cisneros
Bulmaro
B
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics*; D016189:Dystrophin / Q000031:analogs & derivatives* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
D004268:DNA-Binding Proteins; D016189:Dystrophin; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; C085339:apo-dystrophin 1; D051705:Sp3 Transcription Factor