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Originally published In Press as doi:10.1074/jbc.M102100200 on March 19, 2001

J. Biol. Chem., Vol. 276, Issue 23, 20719-20726, June 8, 2001
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The Mouse Dystrophin Enhancer Is Regulated by MyoD, E-box-binding Factors, and by the Serum Response Factor*

Philip Marshall, Nathalie Chartrand, and Ronald G. WortonDagger

From the Ottawa Hospital Research Institute, Ottawa, Ontario K1H 8L6, Canada

In vivo studies in the mouse have revealed that the muscle promoter of the mouse dystrophin gene can target the right ventricle of the heart only, suggesting the need for other regulatory elements to target the skeletal muscle as well as other compartments of the heart. In this study we report the identification of the mouse dystrophin gene enhancer that is located ~8.5 kilobases downstream from the mouse dystrophin gene muscle promoter. The enhancer was tested in myogenic G8, H9-C2, and nonmyogenic 3T3 cell lines and is mostly active in G8 myotubes. Sequence analysis of the mouse dystrophin gene enhancer revealed the presence of four E-boxes numbered E1-E4, a putative mef-2 binding site, and a serum response element. Site-directed mutagenesis studies have shown that E-boxes 1, 2, and 3 as well as the serum response element are required for enhancer activity. Gel shift analysis revealed two binding activities at binding sites E1 and E3 which were specific to myotubes, and supershift assays confirmed that myoD binds at both these sites. Our study also shows that werum response factor binds the serum response element but in myoblasts and fibroblasts only, suggesting that serum response factor may repress enhancer function.

* This work was supported by the Canadian Genetic Diseases Network and the Heart and Stroke Foundation of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Ottawa Hospital Research Institute, 501 Smyth, Ottawa, Ontario K1H 8L6, Canada. Tel.: 613-737-8802; Fax: 613-737-8803; E-mail: rworton@ohri.ca.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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