The discovery and characterization of functional long noncoding RNAs (lncRNAs) updated the notion that proteins are the unique determinants for cellular phenotypes, revealing the requirement of these transcripts in cell growth, differentiation, apoptosis, organ development and function. Our lab contributed to advance the field through the identification of Charme (Chromatin architect of muscle expression), a muscle-restricted and evolutionary conserved lncRNA contributing to myogenesis through the regulation of myoblasts fusion and contraction genes. The expression of many of these Charme targets was found altered in human cardiomyopathies. In line with this, Charme null mice (CharmeKO) showed reduced lifespan as a consequence of muscle hyperplasia and a pronounced phenotype of cardiac remodeling at developmental onset. The mechanistic understanding of Charme mode-of-action required extensive efforts and still call for high-level competences, technological applications and model systems to be deepened further. We know that in muscle, the functional isoform (pCharme) retains an evolutionary conserved and long-sized intron (intron-1) of 11 kb in length. pCharme functions in myotubes as a chromatin architect lncRNA, which controls the expression of its direct targets by influencing their 3-dimensional genomic proximity. This epigenetic control is in line with emerging studies indicating that lncRNAs can act as modular scaffolds for chromatin regulators to shape the formation of chromosome territories where co-regulated gene expression occurs. The project proposed here aims to clarify the contribution of RNA-protein interactions to Charme-mediated epigenetic regulation. Although intron-1 mutations produce in vivo phenotypes similar to Charme ablation, evidences supporting the contribution of this region to the lncRNA chromatin performance are still preliminary and requires more investigation.
The field of interest applies to the study of muscle differentiation and related disorders. In this context, the main novelty resides on the study of the epigenetic networks controlled by lncRNAs, a layer of regulation that has been only recently identified and represents an area of intense investigation. Accumulating studies indicate that lncRNA-based epigenetic mechanisms can influence a large range of functions such as gene expression, genetic imprinting, histone modification and chromatin dynamics, which are temporally and spatially regulated though the interaction between the RNA moiety and proteins or nucleic acids. These interactions can be stable, leading to the formation of ribonucleoprotein (RNP) complexes in which RNA serves as a protein scaffold bringing in proximity factors that otherwise would not be able to interact and functionally cooperate. The use of RNA scaffolds presents several advantages in respect to protein ones, as i) a single lncRNA can capture simultaneously more proteins and ii) lncRNAs can act immediately after transcription, while proteins would require at least the step of translation before being functional (Chujo T, et al Biochim. Biophys. Acta. 2015). This structural versatility is unique and further amplified by differential splicing, which can lead to a variety of RNA structures by joining alternative combinations of sequences, thus enlarging the number of tethering¿module combinations. Furthermore, intron retention, which is normally deleterious for proteinogenic transcripts, in the case of lncRNAs, which intrinsically lack any coding constraint, can favour their structural and functional potential. In this view, introns, considered for long time as junk material, must be reinterpreted as functional modules, which generate transcriptome diversity and contribute to shape cell identities.
By exploiting cutting edge technologies and multiple in vitro and in vivo systems, our proposal aims to apply an original large-scale approach to identify functional RNA modules and protein complexes which assist pCharme recruitment and activity at specific genomic loci. Our evidence points to this interplay as a multi-step process in which nuclear RNA-binding proteins are necessary to stabilize a myogenic-specific chromatin milieu. However, the exact mechanism through which this is achieved is still unclear. Together with our previous CharmeKO animals, the availability of intron-1 CharmeDINT mutant mice already provide clues on the functional importance of intron-1 in vivo and will deliver relevant material for mechanistically clarify its contribution to pCharme epigenetic functions. In addition, critical tools and collaborations have been already established, which increases the feasibility and constitute an invaluable resource for the project. Although in myogenesis, several examples of functional lncRNAs have been identified, pCharme is the only example of epigenetic regulator which ablation in mouse results into a peculiar cardiac remodeling phenotype with alteration of heart size, structure, and shape. The existence of morphological similarities between CharmeDINT1 and CharmeKO hearts further reinforce the idea of lncRNA contribution into the molecular circuitries controlling muscle differentiation and suggest the importance of intron-1 into Charme function. The existence of an orthologous transcript in human, regulating the same subset of target genes, suggests an important and evolutionarily conserved function for Charme.
Understanding whether the regulation of epigenetic circuitries in skeletal and cardiac myogenesis also concerns to the human Charme transcript is of paramount importance, to advance our knowledge about the development of functional myocites in vivo.