Epigenetics & Signaling in hESC Commitment and Differentiation into Muscle

描述（由申请人提供）：本提案旨在阐明胚胎干细胞（ESC）定型为骨骼肌谱系并响应细胞外信号激活的p38通路进一步成熟为分化的肌管的表观遗传控制。我们将阐明SW 1/SNF染色质重塑组分BAF 60 c（由SMARD 3编码）使MyoD能够将ESC转化为肌源性谱系，并将表征p38信号传导通过两种不同的染色质修饰复合物-SWI/SNF和Polycomb组（PcG）的基因组重新分布协调ESC衍生的肌肉祖细胞中基因激活和抑制的分子基础。配合物我们还将进行高通量筛选（HTS），以确定通过诱导内源性BAF 60 c表达使ESC敏感以适应骨骼肌谱系的化合物。以下是具体目标。目标1.阐明BAF 60 c使MyoD能够将ESC转化为肌肉祖细胞（ESC BAF 60 c/MyoD）的机制。1a-在通过MyoD和BAF 60 c的异位表达而定型为肌源性谱系的hESC中进行基因表达谱分析和全基因组染色质分析。1b.表征BAF 60 c在hESC的肌源性定型中的功能。目标二。目的：研究人胚胎干细胞BAF 60 c/MyoD对p38信号的应答。2a-分析p38介导的SWI/SNF复合物向ESC BAF 60 c/MyoD中肌肉分化基因的染色质的募集。2b分析ESC BAF 60 c/MyoD中Pax 7的p38依赖性抑制。目标3：鉴定通过BAF 60 c诱导实现ESC向肌源性谱系转化的化合物。3a-分析BAF 60 c基因座的表观遗传谱并表征hESC向肌肉祖细胞转变期间的BAF 60 c启动子。3b-使用BAF 60 c启动子-GFP报告基因和高通量筛选（HTS）来鉴定在ESC中诱导BAF 60 c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患病肌肉再生的策略。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措施的目标，用于肌肉干细胞的药理学控制。公共卫生相关性：该提案旨在阐明控制人胚胎干细胞（hESC）向骨骼肌源性谱系的承诺，其进一步成熟为分化的肌肉，以及其对细胞外信号的反应的表观遗传基础。这项提议的最终目标是确定操纵干细胞再生患病肌肉的策略的新靶点。我们预测，从这个建议中收集的结果将有助于填补我们目前对干细胞生物学的知识与干细胞响应外部信号向特定细胞谱系重编程其基因组的机制之间的差距。这些信息对于hESC的药理学操作以产生组织祖细胞和设计再生医学中的策略（例如基于干细胞的患病肌肉再生）是至关重要的。从该提议中获得的另一个益处是可能发现hESC衍生的肌肉祖细胞与成体肌肉干细胞（卫星细胞）对外部线索的不同反应性的表观遗传基础。这种比较将有助于优化从肌肉祖细胞的外源性（hESC）或内源性（卫星细胞）来源的再生策略。

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.