CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus.

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.

Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy.

Expansion of a CTG trinucleotide repeat in the 3' UTR of the gene DMPK at the DM1 locus on chromosome 19 causes myotonic dystrophy, a dominantly inherited disease characterized by skeletal muscle dystrophy and myotonia, cataracts and cardiac conduction defects. Targeted deletion of Dm15, the mouse orthologue of human DMPK, produced mice with a mild myopathy and cardiac conduction abnormalities, but without other features of myotonic dystrophy, such as myotonia and cataracts. We, and others, have demonstrated that repeat expansion decreases expression of the adjacent gene SIX5 (refs 7,8), which encodes a homeodomain transcription factor. To determine whether SIX5 deficiency contributes to the myotonic dystrophy phenotype, we disrupted mouse Six5 by replacing the first exon with a beta-galactosidase reporter. Six5-mutant mice showed reporter expression in multiple tissues, including the developing lens. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Our results suggest that SIX5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.

Fragile-X syndrome and myotonic dystrophy: parallels and paradoxes.

Fragile-X syndrome and myotonic dystrophy are caused by triplet repeat expansions embedded in CpG islands in the transcribed non-coding regions of the FMR1 and the DMPK genes, respectively. Although initial reports emphasized differences in the mechanisms by which the expanded triplet repeats caused these diseases, results published in the past year highlight remarkable parallels in the likely molecular etiologies. At both loci, expansion is associated with altered chromatin, aberrant methylation, and suppressed expression of the adjacent FMR1 and DMAHP genes, implicating epigenetic mediation of these genetic diseases.

Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP.

Myotonic dystrophy, or dystrophia myotonica (DM), is an autosomal dominant multisystem disorder caused by the expansion of a CTG trinucleotide repeat in the 3' untranslated region of the DMPK protein kinase gene on chromosome 19q13.3 (refs 1-3). Although the DM mutation was identified more than five years ago, the pathogenic mechanisms underlying this most prevalent form of hereditary adult neuromuscular disease remain elusive. Previous work from our laboratory demonstrated that a DNase l-hypersensitive site located adjacent to the repeats on the wild-type allele is eliminated by repeat expansion, indicating that large CTG-repeat arrays may be associated with a local chromatin environment that represses gene expression. Here we report that the hypersensitive site contains an enhancer element that regulates transcription of the adjacent DMAHP homeobox gene. Analysis of DMAHP expression in the cells of DM patients with loss of the hypersensitive site revealed a two- to fourfold reduction in steady-state DMAHP transcript levels relative to wild-type controls. Allele-specific analysis of DMAHP expression showed that steady-state transcript levels from the expanded allele were greatly reduced in comparison to those from the wild-type allele. Together, these results demonstrate that CTG-repeat expansions can suppress local gene expression and implicate DMAHP in DM pathogenesis.

Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis.

Genetic studies have demonstrated that MyoD and Myf5 establish the skeletal muscle lineage, whereas myogenin mediates terminal differentiation, yet the molecular basis for this distinction is not understood. We show that MyoD can remodel chromatin at binding sites in muscle gene enhancers and activate transcription at previously silent loci. TGF-beta, basic-FGF, and sodium butyrate blocked MyoD-mediated chromatin reorganization and the initiation of transcription. In contrast, TGF-beta and sodium butyrate did not block transcription when added after chromatin remodeling had occurred. MyoD and Myf-5 were 10-fold more efficient than myogenin at activating genes in regions of transcriptionally silent chromatin. Deletion mutagenesis of the MyoD protein demonstrated that the ability to activate endogenous genes depended on two regions: a region rich in cysteine and histidine residues between the acidic activation domain and the bHLH domain, and a second region in the carboxyl terminus of the protein. Neither region has been shown previously to regulate gene transcription and both have domains that are conserved in the Myf5 protein. Our results establish a mechanism for chromatin modeling in the skeletal muscle lineage and define domains of MyoD, independent of the activation domain, that participate in chromatin reorganization.