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

肌营养不良蛋白信号传导和人类 iPSC 衍生肌肉的表观遗传景观

• 批准号: 8774130
• 负责人: Pier Lorenzo Puri
• 金额: $ 42.9万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8774130
• 关键词: Ablation; Adopted; Affect; Arts; Binding; Biological; Cardiac; Cells; Chromatin; Collaborations; Competence; Dependence; Development; Disease; Disease Progression; Duchenne muscular dystrophy; Dystrophin; Enhancers; Epigenetic Process; Event; Fibroblasts; Functional disorder; Funding; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Transcription; Grant; Hereditary Disease; Human; Human Genetics; Investigation; Knowledge; Light; Link; Maintenance; Maps; Mediating; Mesoderm Cell; Modeling; Molecular; Monitor; Muscle; Muscle Cells; Muscle Development; Muscular Dystrophies; Nuclear; Nucleosomes; Output; Pathogenesis; Pathology; Patients; Pharmaceutical Preparations; Protocols documentation; Recording of previous events; Recruitment Activity; Regulation; Repression; Research Personnel; Role; Signal Transduction; Skeletal Muscle; Stem cells; Technology; United States National Institutes of Health; base; chromatin remodeling; epigenome; gene repression; genome wide association study; genome-wide; human embryonic stem cell; induced pluripotent stem cell; miniaturize; myogenesis; prevent; progenitor; programs; public health relevance; skeletal

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.

描述(由申请人提供)：在RO1资助的前一个周期中，我们发现了SWI/SNF染色质重塑亚单位BAF60c在肌肉前体细胞中信号介导的肌肉基因表达激活中的重要作用(Forcales等人)。2012年，EMBO J)和hESCs(Albini等人)。2013 Cell Rep)。我们发现，BAF60c的转录抑制阻止hESC直接分化为骨骼肌，并在类胚体(EB)形成过程中通过发育信号抑制抑制(Cai等人。2013年，基因Dev)与中胚层细胞采用骨骼和心肌成肌谱系的能力不谋而合。这些知识有助于基于BAF60c和MyoD的异位顺序表达，建立一种有效的hESC直接转化为骨骼肌的方案。在特定的培养条件下，表达BAF60c和MyoD的hESC形成3D收缩肌球，它概括了人类肌肉的生物学特征，当从hPSCs中分离出来时，可以用作微型肌肉(Albini和Puri JoVE 2013)。在这次拨款续期申请中，我们将利用这些发现在两个相互关联的方向上向前推进：我们将利用BAF60c/MyoD指导的hESC和HiPSC的转换来阐明BAF60c预置有利于MyoD介导的hESCs中骨骼肌生成的表观遗传格局的机制(目标1-鉴定肌肉增强剂并绘制由BAF60c和MyoD在hESCs中产生的核小体拓扑图)和在hiPSC中(目标2-MyoD染色质结合和hPSC来源的肌肉中的核小体图谱)。我们将使用从AIMS 1和2获得的知识来确定在HiPSC来源的肌肉细胞的表观遗传格局中建立的长距离相互作用(目标3-映射在人成肌细胞和HIPSC衍生物中产生的拓扑域)以及它们对dystrophin信号的依赖(目标4-dystrophin信号在肌球表观遗传格局控制中的作用)。因此，这项应用将把最先进的技术应用于最近开发的干细胞来源的骨骼肌模型，以研究在人类骨骼肌发生过程中形成的表观遗传格局的决定因素。我们将利用这一知识来推断dystrophin在发育过程中和收缩活动后通过TALEN介导的dystrophin消融来调节肌肉表观基因组的作用。因此，这项研究可以阐明Duchenne肌营养不良症(DMD)发病的遗传和表观遗传决定因素之间以前未知的联系，并将识别病史的“表观遗传特征”。我们的研究还将为在DMD的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Dstrophin普遍表达，DMD患者存在肌外病变(即心功能障碍)，使疾病进展和治疗复杂化，本研究将为解释DMD的发病机制和治疗开辟新的视角。