Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse.

Spontaneous and engineered mouse mutants have facilitated our understanding of the pathogenesis of muscular dystrophy and they provide models for the development of therapeutic approaches. The mouse myodystrophy (myd) mutation produces an autosomal recessive, neuromuscular phenotype. Homozygotes have an abnormal gait, show abnormal posturing when suspended by the tail and are smaller than littermate controls. Serum creatine kinase is elevated and muscle histology is typical of a progressive myopathy with focal areas of acute necrosis and clusters of regenerating fibers. Additional aspects of the phenotype include sensorineural deafness, reduced lifespan and decreased reproductive fitness. The myd mutation maps to mouse chromosome 8 at approximately 33 centimorgans (cM) (refs. 2, 4-7). Here we show that the gene mutated in myd encodes a glycosyltransferase, Large. The human homolog of this gene (LARGE) maps to chromosome 22q. In myd, an intragenic deletion of exons 4-7 causes a frameshift in the resultant mRNA and a premature termination codon before the first of the two catalytic domains. On immunoblots, a monoclonal antibody to alpha-dystroglycan (a component of the dystrophin-associated glycoprotein complex) shows reduced binding in myd, which we attribute to altered glycosylation of this protein. We speculate that abnormal post-translational modification of alpha-dystroglycan may contribute to the myd phenotype.

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5' end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3' to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Tissue-selective expression of alpha-dystrobrevin is determined by multiple promoters.

alpha-Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a dystrophin-associated and dystrophin-related protein. Knockout of the gene in the mouse results in muscular dystrophy. The control of the alpha-dystrobrevin gene in the various tissues is therefore of interest. Multiple dystrobrevin isoforms differing in their domain content are generated by alternative splicing of a single gene. The data presented here demonstrate that expression of alpha-dystrobrevin from three promoters, that are active in a tissue-selective manner, also plays a role in the function of the protein in different tissues. The most proximal promoter A is active in brain and to a lesser extent in lung, whereas the most distal promoter B, which possesses several Sp1 binding sites, is restricted to brain. Promoter C, which contains multiple consensus myogenic binding sites, is up-regulated during in vitro myoblast differentiation. Interestingly, the organization and the activity of the alpha-dystrobrevin promoters is reminiscent of those in the dystrophin gene. Taken together we suggest that the multipromoter system, distributed over a region of 270 kilobases at the 5'-end of the alpha-dystrobrevin gene, has been developed to allow the regulation of this gene in different cell types and/or different developmental stages.