The current view of gene expression suggests the existence of chromosome territories where an intimate relationship between three-dimensional (3D) nuclear organization and gene activity exists. Although, until recently, DNA and associated proteins were thought to mainly dictate nuclear structures, the discovery of long non-coding RNAs (lncRNA) has uncovered new levels of gene expression regulation, based on their pivotal role in the control of genome architecture.
By RNA profiling via next-generation sequencing performed on murine C2C12 myoblasts and in vitro differentiated myotubes, we recently identified the lncRNA Charme, a highly conserved transcript required for the maintenance of long distance interactions between its transcriptional site and skeletal muscle expressed gene loci. Intriguingly, the genetic ablation of Charme in mouse produces an overgrown heart in which the overall architecture of the cardiac muscle is remodeled. This is particularly promising since, in spite of the clear role of protein-coding RNAs in heart development and disease, the involvement of lncRNAs is poorly known and animal models are demanded.
Critically, no molecular analysis has yet been performed to dissect the contribution of Charme in myocardial physiology. Therefore, this proposal aims to extend the knowledge generated by the ablation of Charme in vivo by characterizing its contribution to the regulatory networks controlling cardiomyogenesis. By taking advantage of the CRISPR-Cas9 technology these studies will be further extended to the human transcript, whose primary sequence is evolutionary conserved (~45% identity to mouse homologue). For this reason, the iPSC-derived cardiomyocyte system will offer an attractive experimental platform to model cardiac differentiation and will advance our understanding of the role of the human Charme in myocardial physiology.
