MYOD Regulation of 3D Chromatin Structure

项目摘要 该建议旨在提供MYOD调节高阶染色质的机制见解 相互作用，定义骨骼肌细胞的3D核结构，通过利用 MYOD诱导的基因组相互作用的高分辨率矩阵的前所未有的可用性， 骨骼肌生成的初步研究。这项研究将填补一个关键的基础知识的差距， 控制转录和肌肉干细胞生物学，通过探索更高水平的复杂性， 空间维度引入的肌肉卫星细胞中基因表达的变化。具体目标是：目标1 肌肉祖细胞中MYOD指导的3D染色质结构调节的分子分析。1a至 确定特定的MYOD结构域对高序结构域的结构和功能改变的贡献。 调控基因表达的染色质相互作用。我们将监测MYOD突变体对3D 染色质结构和特定基因座的转录输出。1b为了确定辅助因子在MYOD中的作用， 调控基因表达的染色质相互作用的定向重新布线。我们将研究 结构蛋白（CTCF和cohesin）以及染色质重塑的特定组分 复合物和转录机制-包括BAF 60 C，聚合酶II（PolII），TAF 3，Bromodomain和 末端外结构域（BET）蛋白-在MYOD指导下的染色质相互作用的重新配置中 特定的核拓扑结构域。1c研究MYOD指导的高级染色质控制 卫星细胞的相互作用。我们将确定MYOD和辅助因子在3D染色质调控中的作用 分离自条件遗传小鼠模型卫星细胞中的相互作用组和转录输出 MyoD的消融。目的2 MYOD介导的信号依赖性染色质三维结构调控 基因表达调控2a.为了在功能上挑战MYOD调节的顺式调节元件， 调控基因表达。我们将利用基于Cas9的基因组编辑，基于RNAi的敲除， 药理学靶向关键表观遗传事件，以询问增强子激活的动力学， MYOD转化的IMR90成纤维细胞中对外源性信号反应的染色质相互作用。2b调查 MYOD调控的卫星细胞对再生信号反应的顺式调控元件。我们将使用鼠标 MyoD的卫星细胞条件性遗传消融模型或MyoD的信号响应组分， SWI/SNF染色质重塑复合物BAF 60 C，探讨MYOD， 染色质重塑和信号调节基因表达的染色质相互作用的变化。 这项研究的数据将为真核生物转录的空间控制提供基本的见解， 卫星细胞生物学将有利于再生医学从目前的治疗策略的过渡 影响全球基因表达的定制方法，基于基因组编辑技术， 选择性靶向暴露于患病肌肉致病线索的卫星细胞中的单个基因。

PROJECT SUMMARY This proposal aims at providing mechanistic insights into MYOD regulation of the high-order chromatin interactions that define the 3D nuclear architecture of skeletal muscle cells, by capitalizing on the unprecedented availability of high-resolution matrices of the genomic interactions induced by MYOD during skeletal myogenesis from our preliminary studies. This study will fill a critical gap of basic knowledge in the control of transcription and muscle stem cell biology, by exploring a higher level of complexity in the regulation of gene expression in muscle satellite cells introduced by the spatial dimension. The Specific Aims are: Aim 1 Molecular analysis of MYOD-directed regulation of 3D chromatin structure in muscle progenitors. 1a To determine the contribution of specific MYOD domains to structural and functional alterations of high-order chromatin interactions that regulate gene expression. We will monitor the effect of MYOD mutants on 3D chromatin structure and transcriptional output at specific loci. 1b To determine the role of co-factors in MYOD- directed rewiring of chromatin interactions that regulate gene expression. We will investigate the role of architectural proteins (CTCF and cohesin) as well as specific components of the chromatin-remodeling complex and the transcriptional machinery - including BAF60C, Polymerase II (PolII), TAF3, Bromodomain and Extra-Terminal Domain (BET) proteins - in MYOD-directed reconfiguration of chromatin interactions within specific nuclear topological domains. 1c To investigate MYOD-directed control of high-order chromatin interactions in satellite cells. We will determine the role of MYOD and co-factors on regulation of 3D chromatin interactome and transcriptional output in satellite cells isolated from a mouse model of conditional genetic ablation of MyoD. Aim 2 MYOD-directed regulation of 3D chromatin structure for signal-dependent control of gene expression 2a. To functionally challenge MYOD-regulated cis-regulatory elements for signal- regulated gene expression. We will exploit Cas9-based genome editing, RNAi-based knockdown and pharmacological targeting of key epigenetic events to interrogate the dynamics of enhancer activation and chromatin interactions in response to extrinsic signals in MYOD-converted IMR90 fibroblasts. 2b To investigate MYOD-regulated cis-regulatory elements for satellite cell response to regeneration cues. We will use mouse models of satellite cell conditional genetic ablation of MyoD or the signal-responsive component of the SWI/SNF chromatin-remodeling complex BAF60C, to investigate the functional relationship between MYOD, chromatin remodeling and changes in chromatin interactions for signal-regulated gene expression. Data from this study will provide fundamental insights into the spatial control of eukaryotic transcription and satellite cell biology that will favor the transition in regenerative medicine from current therapeutic strategies affecting global gene expression to tailored approaches, based on genome editing techniques, toward a selective targeting of individual genes in satellite cells exposed to pathogenic cues from diseased muscles.