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.
Well-established master protein regulators have been deeply characterized and integrated in dynamic regulatory circuitries controlling cardiac muscle development and differentiation. Whereas a large body of evidence unveiled the function of many short ncRNAs (i.e. miRNA, piRNA, snoRNA and snRNA) our knowledge on the functions of long non-coding RNAs needs to be expanded. This proposal aims to re-evaluate the established molecular networks controlling cardiac differentiation in light of the epigenetic role of nuclear lncRNAs. Although a general consensus on their tissue- and developmental-specific expression, it is still debated whether lncRNAs universally have a function, or they are rather transcriptional by-products of conventional coding gene biogenesis. Suggestive of functionality is the observation that lncRNAs show more phylogenetic conservation than introns or untranscribed intergenic sequences (Ponjavic J et al, Genome Res 2007; Guttman M et al, Nature 2009). Second, waves of lncRNA expression are often associated with a variety of cellular processes, such as pluripotency, immune response and cell cycle regulation, suggesting how their appearance is intentional and reasonably functional. Finally, their expression levels, generally lower than those of most protein-coding genes, suggest a role as fine-tuners. In line with this idea, about a third of lncRNAs turned to be associated to chromatin-modifying complexes (Khalil AM et al, PNAS USA 2009) establishing new paradigms for epigenetic regulation (Rinn JL & Chang HY Annu Rev Biochem 2012). These studies, besides disclosing a role for lncRNAs interfacing the chromatin in modulating gene expression, opened the intriguing possibility that lncRNA-based epigenetic mechanisms might influence different sets of genes during the execution of crucial metabolic pathways, as cellular differentiation programs (Bertani S et al, Mol Cell 2011).
The proposed project is based on a solid biological background: over the years I gained solid expertise and knowledge in studying different aspects of non-coding RNA biogenesis and function. The synergistic match among my specific know-how, the proposed state-of-art technologies and the longstanding expertise in myogenesis of the hosting lab, lay the basis for a successful implementation of this project. Further value comes from the recent discovery of novel lncRNAs, implicated in the control of in vitro skeletal muscle differentiation (Ballarino et al, Mol Cell Biol. 2015); among them, Charme (Ballarino et al, submitted). Here we aim to extend the contribution of Charme to the regulatory networks controlling cardiac differentiation. This proposal presents several challenges involving the set up of essential tools. However, using genetic approaches similar to those here described, our recent work proves its full feasibility. Since we are extremely motivated in addressing these biological questions, we have already developed part of the critical tools and genetic models required for this project, including 1) Charme-/- mouse model, to study the involvement of Charme in vivo; 2) ESCs in vitro differentiation into cardiomyocytes; 3) KO iPSCs for Charme, to study iPSC differentiation into cardiac fate. Overall, these models and tools are an invaluable asset that greatly increases the feasibility of this project. Along this direction, we plan to:
i) perform high-throughput RNA sequencing in mouse and human in vitro cardiac differentiation systems;
ii) evaluate whether Charme controls the transcriptional state of specific targets and how this activity impinges on cell differentiation;
iii) employ and develop state-of-art technologies to unveil chromatin contacts in specific phases of cell differentiation.
Part of the project will be dedicated to studying the role of the human hs_Charme. Patient-specific iPSC-derived cardiomyocytes (iPSC-CMs) offer an attractive experimental platform to model cardiovascular diseases, to study the earliest stages of human development, to accelerate predictive drug toxicology tests and to advance potential regenerative therapies. This approach overcomes the obstacle to work on myocardial tissue biopsies, which is an invasive procedure and requires a special surgical interference (e.g., abdominal operation), which is difficult to carry out. In addition, the biopsy size is small and differentiated cardiac cells have low survival and proliferation potentials.
The overall strategy will allow us to get mechanistic insights into the largely unclear activity of lncRNAs in cardiogenesys and to elucidate the function played in nuclear phenomena, as a prerequisite toward understanding novel molecular aspects of cardiac diseases.