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: 8897264
• 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/8897264
• 关键词: Ablation; Adopted; Affect; Arts; Binding; Biological; Cardiac; Cells; Chromatin; Collaborations; Competence; DNA Sequence Alteration; Dependence; Development; Disease; Disease Progression; Duchenne muscular dystrophy; Dystrophin; Enhancers; Epigenetic Process; Event; Fibroblasts; Funding; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Transcription; Grant; Health; Hereditary Disease; Human; Human Genetics; Investigation; Knowledge; Light; Link; Maintenance; Maps; Mediating; Mesoderm Cell; Modeling; Molecular; Monitor; Muscle; Muscle Cells; Muscle Development; Muscular Dystrophies; Myocardial dysfunction; 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; genome-wide analysis; human embryonic stem cell; induced pluripotent stem cell; miniaturize; myogenesis; prevent; progenitor; programs; skeletal; targeted sequencing

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资助的上一个周期中，我们发现了SWI/SNF染色质重塑亚基BAF60C在肌肉祖细胞（Forcales et al. 2012, EMBO J）和hESCs （Albini et al. 2013 Cell Rep）中信号介导的肌肉基因表达激活中的重要作用。我们发现BAF60C的转录抑制阻止hESC直接分化为骨骼肌，并且在胚状体（EB）形成过程中发育信号的去抑制（Cai etal . 2013, Genes Dev）与中胚层细胞采用骨骼和心肌谱系的能力相吻合。这一知识有助于建立基于BAF60C和MyoD异位序列表达的hESC直接转化为骨骼肌的有效方案。在规定的培养条件下，表达BAF60C-和MyoD-的hESC形成3D收缩肌球，再现了人类肌肉的生物学特征，当从hiPSCs中提取时，可以用作小型化肌肉（Albini和Puri JoVE 2013）。在这次拨款续期申请中，我们将利用这些发现向两个相互关联的方向推进：我们将利用BAF60C/MyoD导向的hESC和hiPSC的转化来阐明BAF60C预先设置有利于MyoD介导的hESCs骨骼肌生成激活的表观遗传景观的机制（目标1 -识别肌肉增强子并绘制hESCs中BAF60C和MyoD产生的核小体地形）和hiPSC（目标2 - MyoD染色质结合和hiPSC衍生肌肉中的核小体图谱）。我们将利用目标1和目标2中获得的知识来确定hiPSC衍生肌肉细胞表观遗传景观中建立的远距离相互作用（目标3 -绘制人类成纤维细胞和hiPSC衍生物在成肌转化过程中产生的拓扑结构域）及其对肌营养不良蛋白信号的依赖（目标4 -肌营养不良蛋白信号在控制肌球表观遗传景观中的作用）。因此，本应用程序将应用最先进的技术到最近开发的干细胞衍生骨骼肌模型中，以研究人类骨骼肌形成过程中形成的表观遗传景观的决定因素。我们将利用这些知识，通过talen介导的肌营养不良蛋白消融，推断肌营养不良蛋白在肌肉发育期间和收缩活动后对肌肉表观基因组的调节中的作用。因此，这项研究可以揭示以前未被认识到的杜氏肌营养不良（DMD）发病机制的遗传和表观遗传决定因素之间的联系，并将确定疾病史的“表观遗传特征”。我们的研究也将为在DMD治疗中使用表观遗传药物来恢复营养不良肌肉的核景观提供理论依据。由于肌营养不良蛋白是普遍表达的，并且由于DMD患者存在肌肉外病理（如心功能障碍），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。