Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Prior studies of the DM1 locus have shown that the CTG repeats are a component of a CTCF-dependent insulator element and that repeat expansion results in conversion of the region to heterochromatin. We now show that the DM1 insulator is maintained in a local heterochromatin context: an antisense transcript emanating from the adjacent SIX5 regulatory region extends into the insulator element and is converted into 21 nucleotide (nt) fragments with associated regional histone H3 lysine 9 (H3-K9) methylation and HP1gamma recruitment that is embedded within a region of euchromatin-associated H3 lysine 4 (H3-K4) methylation. CTCF restricts the extent of the antisense RNA at the wild-type (wt) DM1 locus and constrains the H3-K9 methylation to the nucleosome associated with the CTG repeat, whereas the expanded allele in congenital DM1 is associated with loss of CTCF binding, spread of heterochromatin, and regional CpG methylation.
Mol Cell 2005 Nov 11
PMID:Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF. 1628 29

Myotonic dystrophy type 2 (DM2) is caused by a CCTG expansion mutation in intron 1 of the zinc finger protein 9 (ZNF9) gene. The mean expansion size in patients is larger than for DM1 or any previously reported disorder (mean=5000 CCTGs; range=75-11 000), and similar to DM1, repeats containing ribonuclear inclusions accumulate in affected DM2 tissue. Although an RNA gain-of-function mechanism involving DM1 CUG or DM2 CCUG expansion transcripts is now well established, still debated are the potential role that flanking sequences within the DMPK 3'-UTR may have on disease pathogenesis and whether or not decreased expression of DMPK, ZNF9 or neighboring genes at these loci contribute to disease. To address these questions in DM2, we have examined the nucleic acid content of the ribonuclear inclusions and the effects of these large expansions on ZNF9 expression. Using cell lines either haploid or homozygous for the expansion, as well as skeletal muscle biopsy tissue, we demonstrate that pre-mRNAs containing large CCUG expansions are normally spliced and exported from the nucleus, that the expansions do not decrease ZNF9 expression at the mRNA or protein level, and that the ribonuclear inclusions are enriched for the CCUG expansion, but not intronic flanking sequences. These data suggest that the downstream molecular effects of the DM2 mutation are triggered by the accumulation of CCUG repeat tract alone.
Hum Mol Genet 2006 Jun 01
PMID:DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression. 1662 43

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG expansion in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. It has been hypothesized that the pathogenesis in DM1 is triggered by a toxic gain of function of the expanded DMPK RNA. This expanded RNA is retained in nuclear foci where it sequesters and induces alterations in the levels of RNA-binding proteins (RNA-BP). To model DM1 and study the implication of RNA-BP in CUG-induced toxicity, we have generated a Drosophila DM1 model expressing a non-coding mRNA containing 480 interrupted CUG repeats; i.e. [(CUG)20CUCGA]24. This (iCUG)480 transcript accumulates in nuclear foci and its expression leads to muscle wasting and degeneration in Drosophila. We also report that altering the levels of two RNA-BP known to be involved in DM1 pathogenesis, MBNL1 and CUGBP1, modify the (iCUG)480 degenerative phenotypes. Expanded CUG-induced toxicity in Drosophila is suppressed when MBNL1 expression levels are increased, and enhanced when MBNL1 levels are reduced. In addition, (iCUG)480 also causes a decrease in the levels of soluble MBNL1 that is sequestered in the CUG-containing nuclear foci. In contrast, increasing the levels of CUGBP1 worsens (iCUG)480-induced degeneration even though CUGBP1 distribution is not altered by the expression of the expanded triplet repeat. Our data supports a mechanism for DM1 pathogenesis in which decreased levels of MBNL and increased levels of CUGBP mediate the RNA-induced toxicity observed in DM1. Perhaps more importantly, they also provide proof of the principle that CUG-induced muscle toxicity can be suppressed.
Hum Mol Genet 2006 Jul 01
PMID:MBNL1 and CUGBP1 modify expanded CUG-induced toxicity in a Drosophila model of myotonic dystrophy type 1. 1672 74

Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion of the repeating tetranucleotide CCTG*CAGG sequence from <30 repeats in normal individuals to approximately 11,000 for the full mutation in certain patients. This repeat is in intron 1 of the zinc finger protein 9 gene on chromosome 3q21. Since prior work demonstrated that CTG*CAG and GAA*TTC triplet repeats (responsible for DM1 and Friedreich's ataxia, respectively) can expand by genetic recombination, we investigated the capacity of the DM2 tetranucleotide repeats to also expand during this process. Both gene conversion and unequal crossing over are attractive mechanisms to effect these very large expansions. (CCTG*CAGG)n (where n=30, 75, 114 or 160) repeats showed high recombination crossover frequencies (up to 27-fold higher than the non-repeating control) in an intramolecular plasmid system in Escherichia coli. Furthermore, a distinct orientation effect was observed where orientation II (CAGG on the leading strand template) was more prone to recombine. Expansions of up to double the length of the tetranucleotide repeats were found. Also, the repeating tetranucleotide sequence was more prone to expansions (to give lengths longer than a single repeating tract) than deletions as observed for the CTG*CAG and GAA*TTC repeats. We determined that the DM2 tetranucleotide repeats showed a lower thermodynamic stability when compared to the DM1 trinucleotide repeats, which could make them better targets for DNA repair events, thus explaining their expansion-prone behavior. Genetic studies in SOS-repair mutants revealed high frequencies of recombination crossovers although the SOS-response itself was not induced. Thus, the genetic instabilities of the CCTG*CAGG repeats may be mediated by a recombination-repair mechanism that is influenced by DNA structure.
J Mol Biol 2006 Jun 30
PMID:DM2 CCTG*CAGG repeats are crossover hotspots that are more prone to expansions than the DM1 CTG*CAG repeats in Escherichia coli. 1675 77

Myotonic dystrophy (DM), the most common form of adult onset muscular dystrophy, affects skeletal muscle, heart, and the central nervous system (CNS). Mortality results primarily from muscle wasting and cardiac arrhythmias. There are two forms of the disease: DM1 and DM2. DM1, which constitutes 98% of cases, is caused by a CTG expansion in the 3' untranslated region (UTR) of the DMPK gene. DM2 is caused by a CCTG expansion in the first intron of the ZNF9 gene. RNA containing CUG- or CCUG-expanded repeats are transcribed but are retained in the nucleus in foci. Disease pathogenesis results primarily from a gain of function of the expanded RNAs, which alter developmentally regulated alternative splicing as well as pathways of muscle differentiation. The toxic RNA has been implicated in sequestration of splicing regulators and transcription factors thereby causing specific symptoms of the disease. Here we review the proposed mechanisms for the toxic effects of the expanded repeats and discuss the molecular mechanisms of splicing misregulation and disease pathogenesis.
Prog Mol Subcell Biol 2006
PMID:Misregulation of alternative splicing causes pathogenesis in myotonic dystrophy. 1707 68

Although cataract is a characteristic feature of myotonic dystrophy type 1 (DM1), little is known of the underlying mechanisms. We generated four lens epithelial cell lines derived from DM1 cataracts and two from age-matched, non-DM cataracts. Small-pool PCR revealed typical large triplet repeat expansions in the DM1 cells. Furthermore, real-time PCR analysis showed reduced SIX5 expression and increased expression of the Ca(2+)-activated K(+) channel SK3 in the DM1 cells. These cells also exhibited longer population doubling times which did not arise through reduced proliferation, but rather increased cell death as shown by increased release of lactate dehydrogenase (LDH). Using (86)Rb(+) as a tracer for K(+), we found no difference in the resting K(+) influx or efflux kinetics. In all cases, the ouabain sensitive component of the influx contributed approximately 50% of the total. However, stimulating internal Ca(2+) by exposure to ionomycin not only caused greater stimulation of K(+) ((86)Rb) efflux in the DM1 cells but also induced a higher rate of cell death (LDH assay). Since both the hyper-stimulation of K(+) efflux and cell death were reduced by the highly specific SK inhibitor apamin, we suggest that increased expression of SK3 has a critical role in the increased Ca(2+)-induced fragility in DM1 cells. The present data, therefore, both help explain the lower epithelial cell density previously observed in DM1 cataracts and provide general insights into mechanisms underlying the fragility of other DM1-affected tissues.
Hum Mol Genet 2006 Dec 15
PMID:Increased SK3 expression in DM1 lens cells leads to impaired growth through a greater calcium-induced fragility. 1710 31

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder associated with an expansion of CTG trinucleotide repeats in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The RNA gain-of-function hypothesis proposes that mutant DMPK mRNA alters the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Previously, we found alternative splicing variants of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1), which excluded exon 22, in skeletal muscle of DM1 patients. In the present study, we analyzed the molecular mechanisms responsible for the splicing dysregulation of SERCA1. Five 'YGCU(U/G)Y' motifs that could potentially serve as Muscleblind-like 1, (MBNL1)-binding motifs, are included downstream from the SERCA1 exon 22. Exon trapping experiments showed that MBNL1 acts on the 'YGCU(U/G)Y' motif, and positively regulates exon 22 splicing. Of the five MBNL1 motifs in intron 22, the second and third sites were important for regulation of exon 22 splicing, but the other three binding sites were not required. Overexpression of the CUG repeat expansion of DMPK mRNA resulted in exclusion of exon 22 of SERCA1. These results suggest that sequestration of MBNL1 into the CUG repeat expansion of DMPK mRNA could cause the exclusion of SERCA1 exon 22, and the expression of this aberrant splicing form of SERCA1 could affect the regulation of Ca(2+) concentration of sarcoplasmic reticulum in DM patients.
Hum Mol Genet 2007 Dec 01
PMID:Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1. 1772 22

The genetic basis of myotonic dystrophy type 1 (DM1) is a CTG expansion in the 3' untranslated region (UTR) of DMPK. The pathogenic mechanism involves an RNA gain of function in which the repeat-containing transcripts accumulate in nuclei and alter the functions of RNA-binding proteins such as CUG-binding protein 1 (CUGBP1). CUGBP1 levels are increased in DM1 myoblasts, heart, and skeletal muscle tissues and in some DM1 mouse models. However, the molecular mechanisms for increased CUGBP1 in DM1 are unclear. Here, we demonstrate that expression of DMPK-CUG-repeat RNA results in hyperphosphorylation and stabilization of CUGBP1. CUGBP1 is hyperphosphorylated in DM1 tissues, cells, and a DM1 mouse model. Activation of PKC is required for CUGBP1 hyperphosphorylation in DM1 cells, and PKCalpha and betaII directly phosphorylate CUGBP1 in vitro. These results indicate that inappropriate activation of the PKC pathway contributes to the pathogenic effects of a noncoding RNA.
Mol Cell 2007 Oct 12
PMID:Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. 1793 5

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.
Mol Hum Reprod 2008 Jul
PMID:CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation. 1857 25

The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K(+) (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K(+) channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.
Cell Mol Biol Lett 2009
PMID:Potassium currents in human myogenic cells from healthy and congenital myotonic dystrophy foetuses. 1919 65


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