Epigenetics & Signaling in hESC Commitment and Differentiation into Muscle

描述(申请人提供)：这项建议旨在阐明胚胎干细胞(ESC)对骨骼肌谱系的表观遗传控制，并进一步成熟为分化的肌管，以响应细胞外信号激活的p38途径。我们将阐明SWL/SNF染色质重塑组件BAF60c(由SMARCD3编码)使MyoD能够将ESCs转化为肌源性谱系的机制，并将描述p38信号通过两个不同的染色质修饰复合体-SWI/SNF和Polycomb group(PcG)复合体的基因组重新分布来协调ESC来源的肌肉前体细胞的基因激活和抑制的分子基础。我们还将进行高通量筛选(HTS)，以通过诱导内源性BAF60c表达来鉴定使ESCs采用骨骼肌谱系的化合物。以下是具体目标。目的1.阐明BAF60c使MyoD将ESC转化为肌祖细胞(ESC BAF60c/MyoD)的机制。通过异位表达MyoD和BAF60c，对致力于肌源性血统的hESCs进行基因表达谱分析和全基因组染色质分析。1B.鉴定BAF60c在hESCs肌源性承诺中的作用。目的2.研究ESC BAF60c/MyoD对p38信号通路的反应。2A-分析p38介导的SWI/SNF复合体对ESC BAF60c/MyoD肌肉分化基因染色质的募集。2B分析Pax7在ESC BAF60c/MyoD中的p38依赖性抑制。目的3.鉴定通过BAF60c诱导将胚胎干细胞转化为肌源性血统的化合物。3A-分析BAF60c基因座的表观遗传学特征，并鉴定hESC向肌祖细胞转化过程中BAF60c启动子的特征。3B-使用BAF60c启动子-GFP报告基因和高通量筛选(HTS)来鉴定在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。总体而言，我们提议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接口的新干预目标，用于肌肉干细胞的药物控制。公共卫生相关性：这项建议旨在阐明控制人类胚胎干细胞(HESC)对骨骼肌源性谱系的承诺的表观遗传学基础，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为操纵干细胞再生患病肌肉的策略确定新的目标。我们预测，从这一提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和干细胞根据外部信号对特定细胞谱系重新编程的机制之间的空白。这些信息对于hESC的药理学操作以产生组织前体细胞和在再生医学中设计策略至关重要，例如基于干细胞的疾病肌肉再生。这一提议的另一个好处是潜在地发现了hESC来源的肌肉前体细胞与成年肌肉干细胞(卫星细胞)对外部提示的不同反应背后的表观遗传学基础。这种比较将有助于优化来自外源性(HESC)或内源性(卫星细胞)来源的肌肉前体细胞的再生策略。

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.