Epigenetics & Signaling in hESC Commitment and Differentiation into Muscle

描述（由申请人提供）：该提案旨在阐明胚胎干细胞（ESC）对骨骼肌谱系的承诺和进一步成熟为分化肌管的表观遗传控制，以响应细胞外信号激活的p38途径。我们将阐明SWl/SNF染色质重塑组分BAF60c（由SMARCD3编码）使MyoD将ESCs转化为肌源性谱系的机制，并将通过两种不同的染色质修饰复合物（SWI/SNF和Polycomb group （PcG）复合物）的基因组重新分布，表征p38信号通路在esc来源的肌肉祖细胞中协调基因激活和抑制的分子基础。我们还将进行高通量筛选（HTS），以确定通过诱导内源性BAF60c表达使ESCs采用骨骼肌谱系敏感的化合物。以下是具体目标。目的1。阐明BAF60c使MyoD将ESC转化为肌肉祖细胞的机制（ESC BAF60c/MyoD）。1a-通过MyoD和BAF60c的异位表达，对具有肌源性谱系的hESCs进行基因表达谱和全基因组染色质分析。1 b。表征BAF60c在hESCs肌生成中的功能。目标2。表征ESC BAF60c/MyoD对p38信号的响应。2a-分析p38介导的SWI/SNF复合物在ESC BAF60c/MyoD肌肉分化基因染色质上的募集。2b分析p38依赖性Pax7在ESC BAF60c/MyoD中的抑制作用。目标3。鉴定通过BAF60c诱导实现ESC向肌源性谱系转化的化合物。3a-分析BAF60c基因座的表观遗传谱，表征hESC向肌肉祖细胞转化过程中BAF60c启动子的特征。3b-利用BAF60c启动子- gfp报告子和高通量筛选（HTS）鉴定ESCs中诱导BAF60c的化合物。我们预计，从这项研究中获得的知识将有助于确定实施基于干细胞的病变肌肉再生的策略。总的来说，我们提出的研究将揭示针对细胞内信号通路和下游表观遗传修饰之间的界面的新干预目标，用于肌肉干细胞的药理控制。公共卫生相关性：本研究旨在阐明控制人类胚胎干细胞（hESC）向骨骼肌谱系发展的表观遗传基础，它们进一步成熟为分化的肌肉，以及它们对细胞外信号的反应。这项提议的最终目标是确定新的目标策略，操纵干细胞再生病变肌肉。我们预测，从这项提议中收集到的结果将有助于填补我们目前对干细胞生物学的认识与干细胞根据外部信号对其基因组进行重编程的机制之间的空白。这一信息对于hESC的药理学操作产生组织祖细胞和制定再生医学策略至关重要，例如病变肌肉的干细胞再生。这一建议的另一个好处是可能发现hesc来源的肌肉祖细胞与成体肌肉干细胞（卫星细胞）对外部信号的不同反应的表观遗传学基础。这种比较将有助于优化外源（hESC）或内源（卫星细胞）肌肉祖细胞的再生策略。

DESCRIPTION (provided by applicant): This proposal aims at elucidating the epigenetic control of embryonic stem cell (ESC) commitment to the skeletal muscle lineage and further maturation into differentiated myotubes in response to the extra-cellular signal-activated p38 pathway. We will elucidate the mechanism by which the SWl/SNF chromatin remodeling component BAF60c (encoded by SMARCD3) enables MyoD to convert ESCs into the myogenic lineage and will characterize the molecular basis by which the p38 signaling coordinates gene activation and repression in ESC-derived muscle progenitors through the genome re-distribution of two distinct chromatin-modifying complexes - the SWI/SNF and the Polycomb group (PcG) complexes. We will also perform high-throughput screening (HTS) to identify compounds that sensitize ESCs to adopt the skeletal muscle lineage, via induction of endogenous BAF60c expression. The following are specific aims. Aim 1. To elucidate the mechanism by which BAF60c enables MyoD to convert ESC into muscle progenitors (ESC BAF60c/MyoD). 1a- Perform gene expression profiling and genome-wide chromatin analysis in hESCs committed to the myogenic lineage by the ectopic expression of MyoD and BAF60c. 1b. Characterize the function of BAF60c in the myogenic commitment of hESCs. Aim 2. To characterize the response of ESC BAF60c/MyoD to the p38 signaling. 2a- Analyze the p38-mediated recruitment of SWI/SNF complex to the chromatin of muscle differentiation genes in ESC BAF60c/MyoD. 2b Analyze the p38-dependent repression of Pax7 in ESC BAF60c/MyoD. Aim 3. To identify compounds that implement ESC conversion to the myogenic lineage, via BAF60c induction. 3a- Analyze the epigenetic profile of the BAF60c locus and characterize the BAF60c promoter during hESC transition to muscle progenitor cells. 3b- Use BAF60c promoter-GFP reporter and high- throughput screening (HTS) to identify compounds that induce BAF60c in ESCs. We anticipate that the knowledge gained from this research will contribute to identify strategies that implement stem cell-based regeneration of diseased muscles. Overall, our proposed research will reveal targets for novel interventions directed to the interface between intracellular signaling pathways and downstream epigenetic modifications, for a pharmacological control of muscle stem cells. PUBLIC HEALTH RELEVANCE: This proposal aims at elucidating the epigenetic basis that control human embryonic stem cell (hESC) commitment to the skeletal myogenic lineage, their further maturation into differentiated muscles, and their response to extra-cellular signals. The ultimate goal of this proposal is to identify novel targets for strategies that manipulate stem cells to regenerate diseased muscles. We predict that the results gathered from this proposal will contribute to fill the gap between our current knowledge of stem cell biology and the mechanism by which stem cells reprogram their genome toward specific cellular lineages in response to extrinsic signals. This information is critical for the pharmacological manipulation of hESC to generate tissue progenitors and devise strategies in regenerative medicine, such as stem cell-based regeneration of diseased muscles. An additional benefit that will derive from this proposal is the potential discovery of the epigenetic basis underlying the different responsiveness to external cues in hESC-derived muscle progenitors vs adult muscle stem cells (satellite cells). This comparison will help to optimize regenerative strategies from exogenous (hESC) or endogenous (satellite cells) sources of muscle progenitors.