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Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model
- Source :
- Human Molecular Genetics. 8:1975-1984
- Publication Year :
- 1999
- Publisher :
- Oxford University Press (OUP), 1999.
-
Abstract
- The mutation causing myotonic dystrophy (DM) has been identified as a CTG expansion in the 3'-untranslated region (3'-UTR) of the DM protein kinase gene ( DMPK ), but the mechanism(s) of pathogenesis remain unknown. Studies using DM patient materials have often produced confusing results. Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts. By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3'-UTR, we identified both cis and trans effects that are mediated by the DM mutation. Our data show that a mutant DMPK 3'-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci. We determined by deletion analysis that an expanded (CTG) (n) tract alone was sufficient to mediate these cis effects. Furthermore, in contrast to the normal DMPK 3'-UTR mRNA, a mutant DMPK 3'-UTR mRNA with (CUG)(200)selectively inhibited myogenic differentiation of C2C12 myoblasts. Genetic analysis and the Cre- loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3'-UTR transcript. Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) (n) expansion and/or the 3' end of the DMPK 3'-UTR. These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a 'riboregulator' to inhibit myogenesis.
- Subjects :
- Recombinant Fusion Proteins
Mutant
Cis effect
Protein Serine-Threonine Kinases
Biology
Transfection
medicine.disease_cause
Myotonin-Protein Kinase
Cell Fusion
Mice
Myoblast fusion
Trinucleotide Repeats
Genes, Reporter
Genetics
medicine
Animals
Humans
Myotonic Dystrophy
RNA, Messenger
Muscle, Skeletal
3' Untranslated Regions
Molecular Biology
Cells, Cultured
Genetics (clinical)
Sequence Deletion
Reporter gene
Mutation
Reverse Transcriptase Polymerase Chain Reaction
Myotonin-protein kinase
Genetic Complementation Test
Cell Differentiation
General Medicine
Molecular biology
Gene Expression Regulation
Haploinsufficiency
C2C12
Subjects
Details
- ISSN :
- 14602083
- Volume :
- 8
- Database :
- OpenAIRE
- Journal :
- Human Molecular Genetics
- Accession number :
- edsair.doi.dedup.....c1bb717a33dc1adbd1485b1c1b1dae4e