Dystrophin Dp71 expression is down-regulated during myogenesis: Role of Sp1 and Sp3 on the Dp71 promoter activity

Mario Bermúdez De León, Cecilia Montañez, Pablo Gómez, Sara Luz Morales-Lázaro, Victor Tapia-Ramírez, Viviana Valadez-Graham, Félix Recillas-Targa, David Yaffe, Uri Nudel, Bulmaro Cisneros

Research output: Contribution to journalArticle

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Abstract

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, cotransfecfion 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.
Original languageEnglish
Pages (from-to)5290-5299
Number of pages10
JournalJournal of Biological Chemistry
DOIs
Publication statusPublished - 18 Feb 2005
Externally publishedYes

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Dystrophin
Muscle Development
Myoblasts
Binding Sites
Muscle
Assays
Skeletal Muscle Fibers
Sp3 Transcription Factor
Sp1 Transcription Factor
Electrophoretic mobility
Mutagenesis
Proteins
Down-Regulation
5' Flanking Region
Genetic Promoter Regions
Chromatin
Duchenne Muscular Dystrophy
Chromatin Immunoprecipitation
Site-Directed Mutagenesis
Genes

Cite this

De León, M. B., Montañez, C., Gómez, P., Morales-Lázaro, S. L., Tapia-Ramírez, V., Valadez-Graham, V., ... Cisneros, B. (2005). Dystrophin Dp71 expression is down-regulated during myogenesis: Role of Sp1 and Sp3 on the Dp71 promoter activity. Journal of Biological Chemistry, 5290-5299. https://doi.org/10.1074/jbc.M411571200
De León, Mario Bermúdez ; Montañez, Cecilia ; Gómez, Pablo ; Morales-Lázaro, Sara Luz ; Tapia-Ramírez, Victor ; Valadez-Graham, Viviana ; Recillas-Targa, Félix ; Yaffe, David ; Nudel, Uri ; Cisneros, Bulmaro. / Dystrophin Dp71 expression is down-regulated during myogenesis: Role of Sp1 and Sp3 on the Dp71 promoter activity. In: Journal of Biological Chemistry. 2005 ; pp. 5290-5299.
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abstract = "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, cotransfecfion 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.",
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De León, MB, Montañez, C, Gómez, P, Morales-Lázaro, SL, Tapia-Ramírez, V, Valadez-Graham, V, Recillas-Targa, F, Yaffe, D, Nudel, U & Cisneros, B 2005, 'Dystrophin Dp71 expression is down-regulated during myogenesis: Role of Sp1 and Sp3 on the Dp71 promoter activity', Journal of Biological Chemistry, pp. 5290-5299. https://doi.org/10.1074/jbc.M411571200

Dystrophin Dp71 expression is down-regulated during myogenesis: Role of Sp1 and Sp3 on the Dp71 promoter activity. / De León, Mario Bermúdez; Montañez, Cecilia; Gómez, Pablo; Morales-Lázaro, Sara Luz; Tapia-Ramírez, Victor; Valadez-Graham, Viviana; Recillas-Targa, Félix; Yaffe, David; Nudel, Uri; Cisneros, Bulmaro.

In: Journal of Biological Chemistry, 18.02.2005, p. 5290-5299.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Dystrophin Dp71 expression is down-regulated during myogenesis: Role of Sp1 and Sp3 on the Dp71 promoter activity

AU - De León, Mario Bermúdez

AU - Montañez, Cecilia

AU - Gómez, Pablo

AU - Morales-Lázaro, Sara Luz

AU - Tapia-Ramírez, Victor

AU - Valadez-Graham, Viviana

AU - Recillas-Targa, Félix

AU - Yaffe, David

AU - Nudel, Uri

AU - Cisneros, Bulmaro

PY - 2005/2/18

Y1 - 2005/2/18

N2 - 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, cotransfecfion 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.

AB - 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, cotransfecfion 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.

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DO - 10.1074/jbc.M411571200

M3 - Article

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EP - 5299

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

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