Myotonic dystrophy: A study of the expression of the myotonic dystrophy gene in affected tissues and cells.
Creators: name: Sabourin, Luc.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1995
Subjects: subject: Biology, Molecular.

description: Recently, the molecular basis of myotonic dystrophy(DM) has been characterized as an unstable trinucleotide CTG repeat amplification in the 3' untranslated region of a gene encoding a protien with serine/threonine kinase activity. As a first step towards understanding the molecular mechanisms underlying DM, we have analyzed the amplification of the CTG repeat and the DM kinase (DMK) mRNA steady state levels in tissues and cell lines obtained from normal and congenital DM individuals. We have raised polyclonal antibodies against human DMK fusion protein and undertook DMK protein expression analysis in freshly sampled muscle tissues from normal and DM individuals. Our antibody detected DMK protein isoforms of 72 and 84 kDa, for which the levels and distribution were not significantly altered in tissues from adult and congenital DM patients. In addition, we have demonstrated that the previously reported decrease in DMK mRNA expression in affected tissues may be the result of a significant loss of type I myofibers, which preferentially express DMK. In contrast to previous reports, our results also showed that the mutant DMK allele was clearly transcribed as a high molecular weight mRNA in muscle tissue of a severely affected patient. We have examined DMK expression during muscle differentiation in vitro and subsequently investigated the effect of DMK over-expression on the terminal differentiation of the murine myoblast cell line C2C12. We demonstrated that DMK is up-regulated 2 to 3-fold during skeletal myogenesis and that constitutive over-expression of DMK mRNA in myoblasts caused a marked inhibition of myoblast terminal differentiation. Surprisingly, this activity mapped to the 3'UTR of the DMK transcript.When the DMK 3'UTR was placed downstream of a hygromycin resistance gene, the same inhibition of myogenesis was observed. Over-expression of the DMK 3' UTR in NIH 3T3 fibroblasts did not have any effect on their proliferation, suggesting that the 3' UTR does not prevent cell cycle withdrawal and differentiation by promoting growth. Further characterization of the 3' UTR sequences mediating the observed inhibition of terminal differentiation mapped these elements to a 239 bp conserved segment of the 3' UTR located upsteam of the CTG repeat. Furthermore, the DMK 3' UTR did not have any significant effect on the activity of the myogenic regulator MyoD when co-transfected into 10T1/2 cells along with a reporter construct bearing a muscle specific enhancer element. This suggested that the 3' UTR did not interfere with the ability of MyoD to transactivate muscle-specific genes. However, when the mRNA levels for two early myogenic regulators were analysed, myoblast clones over-expressing the 3' UTR expressed normal levels of MEF-2C, but showed reduced myogenin mRNA levels compared to controls, following the induction of differentiation. In addition, in contrast to controls, myogenin protien levels were found to be unchanged during myogenesis in these clones. These data suggested that over-expression of the DMK 3' UTR may alter the expression of specific mRNAs leading to a delay in terminal differentiation. (Abstract shortened by UMI.)