Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/65983
Title: DNA Methylation Dynamics during Myogenesis
Author: Carrió Gaspar, Elvira
Director: Suelves Esteban, Mònica
Peinado Morales, Miguel Á. (Miguel Ángel)
Keywords: Epigenètica
Miogènesi
Metilació
ADN
Diferenciació cel·lular
Myogenesis
Methylation
DNA
Cell diferentiation
Issue Date: 19-Mar-2015
Publisher: Universitat de Barcelona
Abstract: [cat] Partint de la hipòtesi que la metilació de l’ADN, junt amb altres mecanismes epigenètics i els factors de transcripció, orquestra el programa transcripcional, aquesta tesi ofereix un estudi exhaustiu de les dinàmiques de l'ADN durant la progressió miogènica, aborda les seves possibles implicacions reguladores i identifica regions diferencialment metilades que defineixen la identitat de la cèl·lula muscular. L’anàlisi a escala genòmica els perfils de metilació en diferents estadis de la miogènesi va permetre identificar 1000 regions diferencialment metilades, localitzades principalment en regions intergèniques i intragèniques. La majoria de canvis observats eren guanys de metilació i ocorrien durant la determinació de llinatge. D’altra banda, certes regions amb perfils de cromatina associats a enhancers esdevenien desmetilades, suggerint que la metilació de l’ADN pot estar implicada en la regulació de enhancers específics de teixit. L’estudi de gens específics de múscul va mostrar que la identitat de la cèl·lula muscular requereix la desmetilació de l'ADN de regions reguladores pobres en CpGs, alhora que els gens miogènics amb illes CpG a la regió promotora es troben sempre desmetilats i són regulats per modificacions d’histones. Un exemple de la desmetilació específica de múscul és la regió super-enhancer de Myf5. Els assajos d'immunoprecipitació de la cromatina van demostrar que la unió del factor de transcripció Usf1 al locus Myf5 només es donava quan l’ADN estava desmetilat, reforçant la hipòtesi que la metilació de l'ADN regula l'expressió gènica mitjançant la modulació de l'accessibilitat dels factors de transcripció al seu lloc d’unió. Mitjançant l’estudi el perfil de metilació de l'ADN de gens miogènics en un model miogènic derivat de cèl·lules embrionàries, es va observar el mateix perfil observat en mioblasts primaris, indicant que aquest model és una bona estratègia per obtenir mioblasts in vitro amb finalitats terapèutiques. Finalment, el bloqueig de la deaminasa Apobec2 va afectar severament la diferenciació miogènica i la desmetilació de l'ADN del promotor de la Miogenina, indicant que la deaminasa Apobec2 podria estar implicada en la desmetilació activa de l'ADN.
[eng] 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.
URI: http://hdl.handle.net/2445/65983
Appears in Collections:Tesis Doctorals - Departament - Genètica

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