Resumen de DNA Methylation Dynamics during Myogenesis
Elvira Carrió Gaspar
Myogenesis is the differentiation process which encompasses the formation of skeletal muscle during development, regeneration and tissue homeostasis throughout life. Arising from embryonic or adult stem cells, the myogenic process comprehends the acquisition of a specialized cell identity and the loss of pluri/multipotent and proliferative capacities. Starting with the hypothesis that DNA methylation, together with other epigenetic mechanisms and the transcription factors, orchestrates the transcriptional program, this thesis provides a comprehensive picture of DNA methylation dynamics during murine myogenic progression, addresses their regulatory implications, and identifies relevant differentially methylated regions that define muscle cell identity. Initially, we performed a genome-scale DNA methylation study comparing embryonic stem cells (ESCs), primary myoblasts (MBs), differentiated myotubes (MTs), and mature myofibers (MFs) using AIMS-seq method. We identified 1,000 differentially methylated regions during muscle-lineage determination and terminal differentiation, mainly located in gene bodies and intergenic regions. As a whole, muscle lineage commitment was characterized by a major gain of DNA methylation, while muscle differentiation was accompanied by loss of DNA methylation in CpG-poor regions. Notably, hypomethylated sequences were enriched in enhancer-type chromatin regions, suggesting the involvement of DNA methylation in the regulation of cell-type specific enhancers. Importantly, we detected a demethylated region overlapping the super-enhancer of the cell-identity factor Myf5. We showed that the activation of My5 super-enhancer took place upon DNA demethylation exclusively in muscle-committed cells resulting in gene expression. ChIP analyses showed that the binding of the Upstream stimulatory factor 1 (Usf1) to Myf5 locus was DNA demethylation-dependent in myogenic committed cells. Moreover, Usf1 binding site contained an embedded CpG conserved in humans and demethylated in human MBs but not in human ESCs, altogether reinforcing the hypothesis that DNA methylation regulates gene expression by modulating transcription factor binding accessibility. Next, we analyzed by sodium bisulphite sequencing the DNA methylation state of reported regulatory regions (with and without CpG island) of key genes implicated in myogenesis. After analyzing myogenic and non-myogenic cells we concluded that the muscle cell identity comprehends DNA demethylation of lineage-specific CpG-poor regulatory regions leading to a transcriptionally poised or activated state, while myogenic CpG island promoters are totally unmethylated during myogenesis and are regulated by histone modifications. A collaborative work with Charles Keller’s Lab (Oregon Health & Science University, USA) allowed us to conclude that Rhabdomyosarcoma cell lines present a spurious methylation pattern at usually unmethylated CpG islands, consequently with the aberrant methylation associated to tumorigenesis. Furthermore, the study of pluripotency gene promoters during myogenesis pointed that CpG-poor promoters are repressed during differentiation by DNA methylation and by Polycomb complex at CpG island promoters. Interested in deepen in the DNA demethylation dynamics, we started a collaboration with Rita Perlingeiro’s Lab (University of Minnesota, USA) to study the DNA methylation changes in the myogenic inducible Pax7 ESC-derived model. We showed that the ESC-derived myoblast precursors recreated the DNA methylation signature of in vivo isolated muscle stem cells, supporting this model as a bona fide strategy to generate myogenic precursors in vitro with therapeutic purposes. Finally, we addressed the involvement of an active demethylating mechanism during myogenesis. Apobec2 down-regulation in inducible ESC-derived myoblast precursors with shRNA strategies affected dramatically the myogenic differentiation by impairing DNA demethylation of the Myogenin promoter and abolishing the expression of Myogenin and MHC proteins. Based on these results, we proposed that Apobec2 might be involved in the active muscle specific DNA demethylation along myogenesis.
