During the previous cycle of this RO1 grant, we have discovered an essential role of the SWI/SNF chromatin- remodeling sub-unit BAF60C in signal-mediated activation of muscle gene expression in muscle progenitors (Forcales et al. 2012, EMBO J) and hESCs (Albini et al. 2013 Cell Rep). We found that transcriptional repression of BAF60C prevents direct differentiation of hESC into skeletal muscle, and de-repression by developmental signals during embryoid body (EB) formation (Cai et al. 2013, Genes Dev) coincides with the competence of mesodermal cells to adopt the skeletal and cardiac myogenic lineage. This knowledge was instrumental to establish an efficient protocol of hESC direct conversion into skeletal muscles, based on the ectopic, sequential expression of BAF60C and MyoD. Under defined culture conditions BAF60C- and MyoD- expressing hESC form 3D contractile myospheres, which recapitulate biological features of human muscles and can be used as miniaturized muscles when derived from hiPSCs (Albini and Puri JoVE 2013). In this grant renewal application we will capitalize on these discoveries to move forward in two interconnected directions: we will exploit BAF60C/MyoD-directed conversion of hESC and hiPSC to elucidate the mechanism by which BAF60C pre-sets the epigenetic landscape conducive for MyoD-mediated activation of skeletal myogenesis in hESCs (Aim 1 - Identification of muscle enhancers and mapping nucleosome topography generated by BAF60C and MyoD in hESCs) and in hiPSC (Aim 2 - MyoD chromatin binding and nucleosome mapping in hiPSC-derived muscles). We will use the knowledge gained from Aims 1 and 2 to identify long-distance interactions established within the epigenetic landscape of hiPSC-derived muscle cells (Aim 3 - Mapping topological domains generated by during myogenic conversion of human fibroblasts and in hiPSC derivatives) and their dependence on dystrophin signalling (Aim 4 - Role of dystrophin signalling in the control of the epigenetic landscape of myospheres). As such, this application will apply state-of-art technologies to a recently developed model of stem cell-derived skeletal muscles, to investigate the determinants of the epigenetic landscape that forms during human skeletal myogenesis. We will use this knowledge to infer the role of dystrophin in the regulation of muscle epigenome during development and after contractile activity, by TALEN-mediated ablation of dystrophin. Thus, this study can shed light on a previously unrecognized link between genetic and epigenetic determinants of Duchenne Muscular Dystrophy (DMD) pathogenesis and will identify "epigenetic signatures" of disease history. Our study will also provide the rationale for the use of epigenetic drugs to restore the nuclear landscape of dystrophic muscles in the treatment of DMD. As dystrophin is ubiquitously expressed, and because DMD patients present extra-muscular pathology (i.e. cardiac dysfunction) that complicates disease progression and treatment, this study will open new perspectives in the interpretation of DMD pathogenesis and therapy.
In this project we will apply state-of-art technologies to a model of stem cell-derived skeletal muscles from hiPSCs that has been recently developed in Puri lab to reveal the mechanism that leads to the formation of the epigenetic landscape during human skeletal myogenesis and investigate relationship between dystrophin signaling and the dynamic modulation of the epigenetic landscape. As such, this study might reveal the contribution of epigenetic alterations to the pathogenesis of DMD and potential epigenetic signatures of disease history. Once extended to other diseases, the conceptual outputs from this study can help to understand the relationship between the primary gene mutations and the epigenetic pathogenesis of human genetic diseases.
|Latella, Lucia; Puri, Pier Lorenzo (2014) Redox or death: checking on fetal myogenesis. Dev Cell 29:373-4|
|Tierney, Matthew Timothy; Aydogdu, Tufan; Sala, David et al. (2014) STAT3 signaling controls satellite cell expansion and skeletal muscle repair. Nat Med 20:1182-6|
|Berghella, Libera; Puri, Pier Lorenzo (2014) Nitric oxide and histone acetylation-shaping craniofacial development. Chem Biol 21:565-6|
|Albini, Sonia; Puri, Pier Lorenzo (2014) Generation of myospheres from hESCs by epigenetic reprogramming. J Vis Exp :e51243|
|Saccone, Valentina; Consalvi, Silvia; Giordani, Lorenzo et al. (2014) HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles. Genes Dev 28:841-57|
|Giordani, Lorenzo; Puri, Pier Lorenzo (2013) Epigenetic control of skeletal muscle regeneration: Integrating genetic determinants and environmental changes. FEBS J 280:4014-25|
|Cai, Wenqing; Albini, Sonia; Wei, Ke et al. (2013) Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment. Genes Dev 27:2332-44|
|Ryan, Tammy; Shelton, Michael; Lambert, Jean-Philippe et al. (2013) Myosin phosphatase modulates the cardiac cell fate by regulating the subcellular localization of Nkx2.5 in a Wnt/Rho-associated protein kinase-dependent pathway. Circ Res 112:257-66|
|Kim, Changsung; Wong, Johnson; Wen, Jianyan et al. (2013) Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature 494:105-10|
|Mozzetta, Chiara; Consalvi, Silvia; Saccone, Valentina et al. (2013) Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice. EMBO Mol Med 5:626-39|
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