Premature termination codons in the DMD gene cause reduced local mRNA synthesis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 28; pp. 16456 - 16464
• Main Authors: García-Rodríguez, Raquel, Hiller, Monika, Jiménez-Gracia, Laura, van der Pal, Zarah, Balog, Judit, Adamzek, Kevin, Aartsma-Rus, Annemieke, Spitali, Pietro
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 14.07.2020
• Subjects: Animal models; Animals; Biological Sciences; Chromatin; Codon, Nonsense - genetics; Codon, Nonsense - metabolism; Codons; Conformation; Disease Models, Animal; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - genetics; Dystrophin - metabolism; Dystrophy; Gene expression; Gene sequencing; Histone deacetylase; Histones; Humans; Hybridization; Immunoprecipitation; Mice; Mice, Inbred mdx; Muscles; Muscular dystrophy; Muscular Dystrophy, Duchenne - genetics; Muscular Dystrophy, Duchenne - metabolism; Mutation; Myotubes; Nonsense Mediated mRNA Decay; Reduction; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; Rodents; Transcription termination; RNA degradation; nascent RNA; premature termination codons; skeletal muscle; Duchenne muscular dystrophy
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1910456117

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD 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 DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD 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 DMD transcription dynamics revealed a lower rate of DMD 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 mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcription dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.