Dystrophin signaling and the epigenetic landscape of human iPSC-derived muscles

描述（由申请人提供）：在该RO 1资助的前一个周期中，我们发现了SWI/SNF染色质重塑亚基BAF 60 C在肌肉祖细胞（Forcales等人，2012，EMBO J）和hESC（Albini等人，2013 Cell Rep）中肌肉基因表达的信号介导激活中的重要作用。我们发现BAF 60 C的转录抑制阻止hESC直接分化为骨骼肌，并且在胚状体（EB）形成期间发育信号的去抑制（Cai等人，2013，Genes Dev）与中胚层细胞采用骨骼肌和心肌细胞谱系的能力一致。这一知识有助于建立一个有效的协议，人胚胎干细胞直接转化为骨骼肌，异位的基础上，顺序表达的BAF 60 C和MyoD。在确定的培养条件下，表达BAF 60 C和MyoD的hESC形成3D收缩性肌球，其重现了人肌肉的生物学特征，并且当源自hiPSC时可用作小型化肌肉（Albini和Puri JoVE 2013）。在这次资助更新申请中，我们将利用这些发现，朝着两个相互关联的方向前进：我们将利用BAF 60 C/MyoD介导的hESC和hiPSC的转化来阐明BAF 60 C预设有利于MyoD介导的hESC骨骼肌发生激活的表观遗传景观的机制（目的1 -在hESC中鉴定肌肉增强子并绘制由BAF 60 C和MyoD产生的核小体拓扑图）和在hiPSC中（目的2 -在hiPSC衍生的肌肉中MyoD染色质结合和核小体绘制）。我们将使用从目标1和2中获得的知识来鉴定在hiPSC衍生的肌细胞的表观遗传景观内建立的长距离相互作用（目标3 -在人成纤维细胞的肌原性转化期间和hiPSC衍生物中绘制拓扑结构域）及其对肌营养不良蛋白信号传导的依赖性（目标4 -肌营养不良蛋白信号传导在控制肌球的表观遗传景观中的作用）。因此，本申请将最先进的技术应用于最近开发的干细胞衍生骨骼肌模型，以研究人类骨骼肌发生过程中形成的表观遗传景观的决定因素。我们将使用这些知识来推断肌营养不良蛋白在发育过程中和收缩活动后的肌肉表观基因组调节中的作用，通过TALEN介导的肌营养不良蛋白消融。因此，这项研究可以揭示以前未被认识到的遗传和表观遗传决定因素之间的联系杜氏肌营养不良症（DMD）的发病机制，并将确定疾病史的“表观遗传签名”。我们的研究还将为在DMD治疗中使用表观遗传药物恢复营养不良肌肉的核景观提供理论基础。由于肌营养不良蛋白广泛表达，并且由于DMD患者存在使疾病进展和治疗变得复杂的肌外病理（即心功能障碍），因此这项研究将为解释DMD发病机制和治疗开辟新的视角。

DESCRIPTION (provided by applicant): 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.