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Am J Physiol Cell Physiol 297: C1113-C1123, 2009. First published August 5, 2009; doi:10.1152/ajpcell.00189.2009
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Muscle Cell Biology and Cell Motility

Identification of Sp1 and GC-boxes as transcriptional regulators of mouse Dag1 gene promoter

Alessandro Rettino,1 Francesca Rafanelli,1 Giannicola Genovese,1 Martina Goracci,1 Rosa Anna Cifarelli,3 Achille Cittadini,1,2 and Alessandro Sgambato1,2

1Centro di Ricerche Oncologiche "Giovanni XXIII"-Istituto di Patologia Generale, Università Cattolica del Sacro Cuore, Rome; 2Laboratorio di Oncologia Molecolare, Centro di Riferimento Oncologico di Basilicata-Istituto Di Ricovero e Cura a Carattere Scientifico, Rionero in Vulture (PZ); and 3X-life Lab, Ospedale Madonna delle Grazie, Matera, Italy

Submitted 30 April 2009 ; accepted in final form 4 August 2009

Dystroglycan is a widely expressed adhesion complex that anchors cells to the basement membrane and is involved in embryonic development and differentiation. Dystroglycan expression is frequently reduced in human dystrophies and malignancies, and its molecular functions are not completely understood. Several posttranslational mechanisms have been identified that regulate dystroglycan expression and/or function, while little is known about how expression of the corresponding Dag1 gene is regulated. This study aimed to clone the Dag1 gene promoter and to characterize its regulatory elements. Analysis of the mouse Dag1 gene 5'-flanking region revealed a TATA and CAAT box-lacking promoter including a GC-rich region. Transfection studies with serially deleted promoter constructs allowed us to identify a minimal promoter region containing three Specificity protein 1 (Sp1) sites and an E-box. Sp1 binding was confirmed by chromatin immunoprecipitation assay, and Sp1 downregulation reduced dystroglycan expression in muscle cells. Treatment with 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A increased Dag1 mRNA expression levels in myoblasts, and methylation decreased promoter activity in vitro. Furthermore, Dag1 gene promoter methylation was reduced while its expression increased during differentiation of C2C12 myoblast cells in myotubes. In conclusion, for the first time we have characterized the activity of the mouse Dag1 gene promoter, confirming a complex regulation by Sp1 transcription factor, DNA methylation, and histone acetylation, which might be relevant for a better understanding of the physiopathology of the dystroglycan complex.

dystroglycan; promoter; epigenetic modification; muscle differentiation; gene expression


Address for reprint requests and other correspondence: A. Sgambato, Istituto di Patologia Generale-Centro di Ricerche Oncologiche "Giovanni XXIII," Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy (e-mail: asgambato{at}rm.unicatt.it).






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