Molecular and Functional Analysis of the Muscle-Specific Promoter Region of the Duchenne Muscular Dystrophy Gene

Abstract

Duchenne muscular dystrophy (DMD) gene transcripts are most abundant in normal skeletal and cardiac muscle and accumulate as normal myoblasts differentiate into multinucleated myotubes. In this report we describe our initial studies aimed at defining the cis-acting sequences and trans-acting factors involved in the myogenic regulation of DMD gene transcription. A cosmid clone containing the first exon of the DMD gene has been isolated, and sequences lying upstream of exon 1 were analyzed for homologies to other muscle-specific gene promoters and for their ability to direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs. The results indicate that the transcriptional start site for this gene lies 37 base pairs (bp) upstream of the 5' end of the published cDNA sequence and that 850 bp of upstream sequence can direct CAT gene expression in a muscle-specific manner. Sequence analysis indicates that in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. Our observation that only 149 bp of upstream sequence is required for muscle-specific expression of a chimeric CAT gene construct further implicates the CArG and myocyte-specific enhancer-binding nuclear factor 1 binding homologies as important domains in the regulation of this gene. On the other hand, the unique profile of myogenic cell line-specific induction displayed by our DMD promoter-CAT gene constructs suggests that other as yet undefined cis-acting sequences and/or trans-acting factors may also be involved.

Multiple CArG Boxes in the Human Cardiac Actin Gene Promoter Required for Expression in Embryonic Cardiac Muscle Cells Developing In Vitro from Embryonal Carcinoma Cells

“…In particular, CArG boxes 1, 3, and 4 appear to play important roles. A number of other genes encoding muscle-specific proteins have CArG boxes in their promoter regions (8,12,24,25,27,31,35,36,39,44), and these sequences have often been shown to play important roles in the activities of these promoters (15,37,39). C-rich regions are also present around a number of muscle genes (24,25), and the cardiac actin promoter contains three (35), at positions -276, -214, and -83 bp relative to the transcription start site (Fig.…”

“…Many of these muscle-specific genes have been cloned. The regulatory sequences involved in their activation are being delineated by transient expression experiments of chimeric gene constructs following their transfection into myogenic cell lines (8,12,15,24,25,27,31,35,36,43,44) and by the creation of transgenic mice (51).…”

Dp71 Point Mutations Induce Protein Aggregation, Loss of Nuclear Lamina Integrity and Impaired Braf35 and Ibraf Function in Neuronal Cells

“…DMD is caused by mutations in the DMD gene that lead to the absence or dysfunction of dystrophin, the major DMD product in muscle [ 2 ]. The DMD gene consists of 79 exons and seven alternative internal promoters, which direct the expression of different tissue-specific dystrophin isoforms [ 3 , 4 , 5 , 6 ]. Dystrophins form a mechanical link between the actin cytoskeleton and the extracellular matrix through the conformation of the dystrophin-associated protein complex (DAPC), which includes α- and β-dystroglycans (α-DG and β-DG, respectively); α and β dystrobrevins; α, β and δ sarcoglycans; and syntrophins [ 7 , 8 ].…”