MYOD Regulation of 3D Chromatin Structure

项目总结 本提案旨在提供对MYOD高阶染色质调控的机械性见解 定义骨骼肌细胞3D核结构的相互作用，通过利用 MYOD期间诱导的基因组相互作用的高分辨率矩阵前所未有的可用性 来自我们初步研究的骨骼肌发生。这项研究将填补中国在基础知识方面的一个关键空白 转录和肌肉干细胞生物学的控制，通过探索更高水平的调控复杂性 基因在肌肉卫星细胞中的表达受到空间维度的影响。具体目标是：目标1 MYOD对肌祖细胞3D染色质结构调控的分子分析。1a至 确定特定的MYOD结构域对高阶结构和功能改变的贡献 染色质相互作用调节基因表达。我们将监测MYOD突变对3D的影响 染色质结构和特定基因座的转录输出。1B确定辅助因素在MYOD中的作用- 调节基因表达的染色质相互作用的定向重新布线。我们将调查 结构蛋白(CTCF和粘附素)以及染色质重塑的特定成分 复合体和转录机制-包括BAF60c、聚合酶II(PolII)、TAF3、溴域和 末端外结构域(BET)蛋白--在MYOD引导的染色质相互作用的重组中 特定的核拓扑域。1C研究MYOD指导的高阶染色质控制 卫星细胞中的相互作用。我们将确定MYOD和辅助因素在3D染色质调控中的作用 条件性遗传性痴呆小鼠模型卫星细胞的相互作用组和转录输出 消融MyoD。目的2MYOD对信号依赖的3D染色质结构的调节 基因表达调控2a。从功能上挑战MYOD监管的顺式监管元素的信号- 调控的基因表达。我们将利用基于Cas9的基因组编辑、基于RNAi的击倒和 关键表观遗传事件的药理学靶向，以询问增强子激活和 MYOD转化的IMR90成纤维细胞中染色质的相互作用对外部信号的响应。2B进行调查 MYOD调控的卫星细胞对再生信号的顺式调控元件。我们将使用鼠标 卫星细胞条件性遗传消融MyoD或信号反应成分的模型 SWI/SNF染色质重塑复合体BAF60c，探讨MYOD、 信号调节基因表达的染色质重塑和染色质相互作用的变化。 这项研究的数据将为真核转录和空间调控提供基本的见解 卫星细胞生物学将有利于再生医学从目前的治疗策略过渡 基于基因组编辑技术的量身定制的方法，影响全球基因表达，从而实现 卫星细胞中个别基因的选择性靶向，暴露于来自患病肌肉的致病线索。

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.