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Query: UNIPROT:P06889 (Mol)
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The mechanism of expansion of the (CTG)n repeat in myotonic dystrophy (DM1) patients and the cause of its pathobiological effects are still largely unknown. Most likely, long repeats exert toxicity at the level of nuclear RNA transport or splicing. Here, we analyse cis- and trans-acting parameters that determine repeat behaviour in novel mouse models for DM1. Our mice carry 'humanized' myotonic dystrophy protein kinase (Dmpk) allele(s) with either a (CTG)84 or a (CTG)11 repeat, inserted at the correct position into the endogenous DM locus. Unlike in the human situation, the (CTG)84 repeat in the syntenic mouse environment was relatively stable during intergenerational segregation. However, somatic tissues showed substantial repeat expansions which were progressive upon aging and prominent in kidney, and in stomach and small intestine, where it was cell-type restricted. Other tissues examined showed only marginal size changes. The (CTG)11 allele was completely stable, as anticipated. Introducing the (CTG)84 allele into an Msh3-deficient background completely blocked the somatic repeat instability. In contrast, Msh6 deficiency resulted in a significant increase in the frequency of somatic expansions. Competition of Msh3 and Msh6 for binding to Msh2 in functional complexes with different DNA mismatch-recognition specificity may explain why the somatic (CTG)n expansion rate is differentially affected by ablation of Msh3 and Msh6.
Hum Mol Genet 2002 Jan 15
PMID:Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch-repair proteins. 1180 28

Myotonic dystrophy is a complex neuromuscular disorder associated with DNA expansion mutations in two different genes. In DM1 a CTG repeat in the 3'-untranslated region of DMPK is expanded, whereas in DM2 an intronic CCTG expansion occurs in the gene ZNF9. Transcripts containing expanded repeats form foci in the nuclei of DM1 and DM2 cells. Recent work using antibodies has shown that proteins related to Drosophila muscleblind co-localize with repeat foci in DM1 and DM2 cells. We show that rather than there being a single human muscleblind gene producing multiple proteins through alternative splicing, there are in fact three different muscleblind genes, MBNL, MBLL and MBXL, which map to chromosomes 3, 13 and X, respectively, and which show extensive alternative splicing. Two of the genes, MBNL and MBLL, are expressed in many adult tissues whereas MBXL is expressed predominantly in the placenta. Green fluorescent protein-tagged versions of MBNL, MBLL and MBXL co-localize with nuclear foci in DM1 and DM2 cells, suggesting that all three proteins may play a role in DM pathophysiology.
Hum Mol Genet 2002 Apr 01
PMID:Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. 1192 53

Myotonic dystrophy 1 (DM1) is the most common inherited neuromuscular disease in adults. The disorder, characterized by myotonia, muscle wasting and weakness, cataract, insulin resistance, and mental impairment, is caused by the expansion of an unstable CTG repeat located in the 3' untranslated region of DMPK. The repeat expansion suppresses the expression of the homeobox gene SIX5. We describe here an experimental system to identify downstream transcriptional targets of mouse Six5 in order to elucidate the role of SIX5 in the pathogenesis of DM1 and development. By overexpressing a constitutively active Six5 (VP16-Six5wt) using adenovirus-mediated gene transfer in P19 cells and subsequent expression profiling using cDNA arrays, 21 genes, whose expression level increased by the treatment, were identified as potential target genes. Genes expressed in the somites, skeletal muscles, brain and meninges comprised the majority, suggesting the role of Six5 in the development and function of mesodermal tissues and brain. We provide evidence that Igfbp5 encoding a component of IGF signaling is a direct Six5-target. Moreover, the overall expression level of Igfbp5 was decreased in Six5-deficient mouse fibroblasts, and the response of human IGFBP5 to MyoD-induced muscle conversion was altered in cells of DM1 patients. Our results not only identify Six5 as an activator that directs Igfbp5 expression but also suggest that reduced SIX5 expression in DM1 might contribute to specific aspects of the DM1 phenotype.
Hum Mol Genet 2002 May 01
PMID:Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1. 1197 64

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.
Mol Cell 2002 Jul
PMID:Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. 1215 Sep 6

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG expansion in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase gene (DMPK). Transcripts from this altered gene harbor large CUG expansions that are retained in the nucleus of DM1 cells and form foci. It is believed that the formation of these foci is closely linked to DM1 muscle pathogenesis. Here we investigated the possibility of using a nuclear-retained hammerhead ribozyme expressed from a modified tRNAmeti promoter to target and cleave mutant transcripts of DMPK. Accessible ribozyme target sites were identified in the 3'UTR of the DMPK mRNA and a hammerhead ribozyme was designed to cut the most accessible site. Utilizing this system, we have achieved 50 and 63% reductions, respectively, of the normal and CUG expanded repeat-containing transcripts. We also observed a significant reduction in the number of DMPK mRNA-containing nuclear foci in human DM1 myoblasts. Reduction of mutant DMPK mRNA and nuclear foci also corroborates with partial restoration of insulin receptor isoform B expression in DM1 myoblasts. These studies demonstrate for the first time intracellular ribozyme-mediated cleavage of nuclear-retained mutant DMPK mRNAs, providing a potential gene therapy agent for the treatment of myotonic dystrophy.
Mol Ther 2003 May
PMID:Hammerhead ribozyme-mediated destruction of nuclear foci in myotonic dystrophy myoblasts. 1271 10

Transcripts of the myotonic dystrophy protein kinase (DMPK) gene, a member of the Rho kinase family, are subject to cell-type specific alternative splicing. An imbalance in the splice isoform profile of DMPK may play a role in the pathogenesis of DM1, a severe multisystemic disorder. Here, we report how structural subdomains determine biochemical properties and subcellular distribution of DMPK isoforms. A newly developed kinase assay revealed that DMPK is a Lys/Arg-directed kinase. Individual DMPK isoforms displayed comparable transphosphorylation activity and sequence preference for peptide substrates. However, DMPK autophosphorylation and phosphorylation of MYPT1 (as putative in vivo target of DMPK), were dependent on presence of an alternatively spliced VSGGG motif and the nature of the C terminus. In-gel effects of the VSGGG motif on the migration behavior of full-length kinase provide evidence for a model in which this motif mediates 3-D-conformational changes in DMPK isoforms. Finally, different C termini endow DMPK with the ability to bind to either endoplasmic reticulum or mitochondria or to adopt a cytosolic location. Our results suggest that DMPK isoforms have cell-type and location dependent substrate specificities with a role in organellar and cytoarchitectural dynamics.
Mol Cell Biol 2003 Aug
PMID:Alternative splicing controls myotonic dystrophy protein kinase structure, enzymatic activity, and subcellular localization. 1289 25

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x<y. S-DNAs formed many different species of slipped isomers, as indicated by its multiple electrophoretic species. In contrast, SI-DNAs formed distinct structures, as indicated by the limited electrophoretic species for all possible repeat length pairings. Sister SI-DNAs with an excess of CAG repeats always migrated slower than their sister SI-DNAs with an excess of CTG repeats. Strikingly, both the propensity to form slipped structures and the pattern of S-DNAs, but not SI-DNAs, varied for similar lengths of CTG/CAG repeats between the DM1 and SCA1 loci, highlighting a role for flanking cis-elements in S-DNA but not SI-DNA formation. Slipped structures bound structure and nucleotide-specific anti-DNA antibodies. Binding of anti-B-DNA antibodies was reduced for both S-DNAs and SI-DNAs relative to their linear forms. SI-DNAs bound anti-Z-DNA antibodies, while both S and SI-DNAs bound anti-cruciform antibodies, revealing shared characteristics between the corresponding DNA structures and slipped DNAs. Such features of the repeats may be recognized by cellular proteins known to bind such structures.
J Mol Biol 2003 Sep 19
PMID:Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies. 1296 69

Myotonic dystrophy (DM1) is a multisystemic disorder caused by a CTG repeat expansion within the 3'-UTR of the DMPK gene. DM1 is characterized by delayed muscle development, muscle weakness and wasting, cardiac conduction abnormalities, cognitive defects and cataracts. Recent studies have demonstrated that the disease mechanism involves a dominant gain-of-function conferred upon mutant transcripts by expanded repeats. However, further attempts to model aspects of DM muscle pathology in cultured myoblasts suggest that 3'-UTR sequences flanking the CTG repeat tract are also required for full expression of the disease phenotype. Here, we report that overexpression of the DMPK 3'-UTR including either wild-type (11) or expanded (91) CTG repeats results in aberrant and delayed muscle development in fetal transgenic mice. In addition, transgenic animals with both expanded and wild-type CTG repeats display muscle atrophy at 3 months of age. Primary myoblast cultures from both 11 and 91 repeat mice display reduced fusion potential, but a greater reduction is observed in the 91 repeat cultures. Taken together, these data indicate that overexpression of the DMPK 3'-UTR interferes with normal muscle development in mice and that this is exacerbated by inclusion of a mutant repeat. This suggests that the delayed muscle development in DM1 involves an interplay between the expanded CTG repeat and adjacent 3'-UTR sequences.
Hum Mol Genet 2004 Mar 15
PMID:Inhibition of myogenesis in transgenic mice expressing the human DMPK 3'-UTR. 1473 27

Myotonic dystrophy 1 (DM1) is a multi-system disorder characterized by endocrine defects that include testicular and tubular atrophy, oligospermia, Leydig cell hyperproliferation and increased follicle stimulating hormone (FSH) levels. DM1 results from a CTG expansion that causes transcriptional silencing of the flanking SIX5 allele. Loss of Six5 results in male sterility and a progressive decrease in testicular mass with age. We demonstrate a strict requirement of Six5 for both spermatogenic cell survival and spermiogenesis. Leydig cell hyperproliferation and increased intra-testicular testosterone levels are observed in the Six5-/- mice. Although increased FSH levels are observed in the Six5+/- and Six5-/- mice, serum testosterone levels and intra-testicular inhibin alpha and inhibin beta B levels are not altered in the Six5 mutant animals when compared with controls. Significantly, steady-state c-Kit levels are reduced in the Six5-/- testis. Thus, decreased c-Kit levels could contribute to the elevated spermatogenic cell apoptosis and Leydig cell hyperproliferation in the Six5-/- mice. The results support the hypothesis that the reduced SIX5 levels contribute to the male reproductive defects in DM1.
Hum Mol Genet 2004 Jul 15
PMID:Six5 is required for spermatogenic cell survival and spermiogenesis. 1516 33

Myotonic dystrophy (DM1) is a neuromuscular disorder caused by a CTGn expansion in the 3'-untranslated region (UTR) of myotonic dystrophy protein kinase (DMPK). SIX5 is a homeodomain gene located just downstream of the repeat, and myotonic dystrophy WD protein (DMWD) is located close upstream of DMPK. It has been hypothesized that the expansion might influence the expression of the three myotonic dystrophy locus genes (DM1-locus), contributing to the complex and varied phenotype in this disorder. Real-time quantitative reverse transcription-polymerase chain reaction, or TaqMan, is a very sensitive method that enables quantification of expression levels of genes from small amounts of tissue and lowly expressed genes. Because data are collected during the assay, the quantification is possible over a wide range of expression levels. By the use of a standard curve and an endogenous control, we have applied the TaqMan system for absolute quantification of the expression levels of the three genes (DMPK, DMWD, and SIX5) in the same tissue sample.
Methods Mol Biol 2004
PMID:Real-time RT-PCR for CTG repeat-containing genes. 1520 50


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