Premature termination codons in the <i>DMD</i> gene cause reduced local mRNA synthesis.

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the <i>DMD</i> gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in <i>DMD</i> expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize <i>DMD</i> gene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore <i>DMD</i> transcription dynamics revealed a lower rate of <i>DMD</i> transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in <i>mdx</i> mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in <i>mdx</i> mice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression in <i>mdx</i> mice. Overall, our findings show that transcription dynamics across the <i>DMD</i> locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.