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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 ϳ8.5 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 Eboxes 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.The 2.5-megabase gene encoding the cytoskeletal protein, dystrophin (1, 2) is the largest gene yet identified and is complex with a minimum of seven promoters. Three promoters, muscle (3), brain (4), and Purkinje (5), express full-length dystrophin isoforms that localize in the skeletal/cardiac muscle, the cerebral cortex, and in cerebellar Purkinje cells, respectively. Four other promoters located in introns 29, 55, 59, and 68 encode shorter isoforms Dp 260 (6), Dp 140 (7), Dp 116 (8), and Dp 71 (9, 10), which are expressed in retina, the central nervous system, the peripheral nervous system, and non-muscle tissues, respectively. The exact role of dystrophin has yet to be determined, but many studies suggest that in muscle it maintains the cytoarchitecture of the cell by bridging intracellular F-actin filaments to the extracellular matrix via contacts with the dystroglycan complex (11,12). What is known is that mutation in the gene resulting in loss of dystrophin is the major cause of Duchenne muscular dystrophy, whereas mutations that alter or reduce the amount of the protein generally cause the milder Becker muscular dystrophy.Although many mutations in the dystrophin gene affect coding sequences, a few of the known mutations affect non-coding sequences, and often these impact on transcription of the gene. This type of mutation is not frequently observed in Duchenne muscular dystrophy or Becker muscular dystrophy but is more common in a group of X-linked dilated cardiomyopathy patients (13), in which promoter deletions (14) or splice site mutations (15) abolish expression of the muscle isoform of dystrophin in skeletal and cardiac muscle. These individuals continue to express full-length dystrophin in their skeletal muscle from the brain and/or Purkinje promoters (15, 16), normally silent in th...

Duchenne muscular dystrophy is a muscle wasting disease that results from a dystrophin deficiency in skeletal and cardiac muscle. Studies concerning the regulatory elements that govern dystrophin gene expression in skeletal and/or cardiac muscle in both mouse and human have identified a promoter and an enhancer located in intron 1. In transgenic mice, the muscle promoter alone targets the expression of a lacZ reporter gene only to the right ventricle of the heart, suggesting the need for other regulatory elements to target skeletal muscle and the rest of the heart. Here we report that the mouse dystrophin enhancer from intron 1 can target the expression of a lacZ reporter gene in skeletal muscle as well as in other heart compartments of transgenic mice. Our results also suggest that sequences surrounding the mouse dystrophin enhancer may affect its function throughout mouse development.

The Dlx homeobox genes of vertebrates are transcribed in multiple cells of the embryo with overlapping patterns but often with different onsets of expression. Here we describe the interaction between two dlx genes, dlx3 and dlx4, during zebrafish development. The observation that dlx3 expression precedes that of dlx4 in the otic vesicle led us to investigate whether dlx3 had the ability to control expression of dlx4. Truncated versions of dlx3 were overexpressed in zebrafish embryos and the expression patterns of dlx4 were examined later in development. Overexpression of truncated forms of Dlx3 or of a Dlx3-Dlx2 chimera was found to result in perturbations in dlx4 expression. In addition, cotransfection experiments indicated the ability of Dlx3 to activate transcription through a 1.7-kb fragment of the 5' flanking region of dlx4. These results suggest that dlx4 is one of the target genes of dlx3 in embryos and that cross-regulatory interactions between Dlx genes may be one of the mechanisms responsible for their overlapping expression.

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