Signal-dependent switch of SWI/SNF by miRNAs & control of muscle stem cell fate

描述（由申请人提供）：该申请是上级资助的自然延伸，题为“通过信号通路控制肌肉基因表达”（提供进展报告），旨在解读肌肉特异性mirna与SWI/SNF亚复合物形成之间的功能关系，该亚复合物含有相互排斥的BAF60变体，这些变体指导肌肉来源的多能细胞中的肌肉生成或纤维脂肪生成程序。这个miRNAs-SWI/SNF网络由组蛋白去乙酰化酶（HDACs）控制，并受到来自营养不良肌肉再生环境的外部信号的影响。因此，本研究的一个重要目标是确定营养不良肌肉在疾病进展的不同阶段产生的信号对一个新的调节轴的影响，该调节轴由HDAC、miRNA和BAF60变体组成，它构成了肌肉来源的多能细胞选择肌源性或脂肪性命运的决定的限制点。因此，这一建议将揭示一个意想不到的细胞内网络，该网络将再生信号与肌营养不良发病机制的表观遗传控制联系起来，并将阐明HDAC抑制剂在营养不良肌肉中的有益作用的机制。我们将使用多种方法，包括全基因组编码RNA和miRNA分析，基于ChIP和蛋白质组学的方法，来解卷积miRNA与染色质相关复合物相互作用的网络，这些复合物形成了营养不良肌肉间质细胞的表观基因组。我们还将使用不同的小鼠模型，进行旨在评估肌肉间质细胞和卫星细胞之间功能相互作用的实验，以及这些相互作用在共培养和细胞移植后的体内调节。这些实验将确定再生线索和HDAC阻断对肌肉间质细胞和卫星细胞之间相互作用的影响。由于肌肉间质细胞和卫星细胞是病变肌肉代偿性再生或脂肪浸润和纤维化的候选细胞决定因素，因此本研究收集的信息将填补骨骼肌疾病相关变化分子基础知识的关键空白。同样，这项研究可能会揭示“再生生态位”中不同细胞类型之间的相互作用，以及这些相互作用的破坏对衰老肌肉再生能力下降的贡献。具体目标是：目标1 -整合质谱/微阵列/芯片分析，破译Sca1+ mic中的HDAC-miRNA- SWI/SNF网络；目的2-通过体外靶向miRNA/BAF60网络调节Sca1+ mic的谱系承诺；目的3 -通过靶向miRNA/BAF60c网络在体内调节Sca1+ mic的谱系承诺；目的4 -确定再生激活的p38-BAF60c信号在激活Sca1+ mic的肌生成电位中的作用

DESCRIPTION (provided by applicant): This application is the natural extension of the parent grant, entitled ""Control of muscle gene expression by signaling pathways" (for which progress report is provided), and seeks to decipher the functional relationship between muscle-specific miRNAs and the formation of SWI/SNF sub-complexes containing mutually exclusive BAF60 variants that direct the myogenic or the fibro-adipogenic program in muscle-derived pluripotent cells. This miRNAs-SWI/SNF network is controlled by histone deacetylases - HDACs - and is influenced by extrinsic cues derived from the regeneration environment of dystrophic muscles. Thus, an important goal of this proposal is to determine the impact of signals generated by dystrophic muscles at different stages of disease progression on a novel regulatory axis - consisting of HDAC, miRNA and BAF60 variants - which constitutes the restriction point of the decision of muscle-derived pluripotent cells to adopt a myogenic or an adipogenic fate. As such, this proposal will shed light on an unanticipated intracellular network that links regeneration signals to the epigenetic control of muscular dystrophy pathogenesis, and will illustrate the mechanism underlying the beneficial effects of HDAC inhibitors in dystrophic muscles. We will use a combination of approaches, including genome-wide coding RNA and miRNA analysis, ChIP- based and proteomic approaches, to deconvolute the network of miRNA interactions with chromatin- associated complexes that shape the epigenome of muscle interstitial cells from dystrophic muscles. We will also perform experiments aimed at evaluating the functional interactions between muscle interstitial cells and satellite cells, and the regulation of these interactions in co-culture and in vivo upon cell transplantation, using different mouse models. These experiments will determine the impact of regeneration cues and HDAC blockade on interactions between muscle interstitial cells and satellite cells. Because muscle interstitial cells and satellite cells are candidate cellular determinants of compensatory regeneration or adipose infiltration and fibrosis in diseased muscles, the information gathered from this study will fill a critical gap of knowledge on the molecular basis of disease-associated changes in skeletal muscles. Likewise, this research might shed light on the interactions between different cell types within the "regeneration niche", and the contribution that disruption of these interactions has to the regeneration decline in aged muscles. Specific aims are: Aim 1 - Integration of Mass-spect/Microarray/ChIP analyses to decipher the HDAC-miRNA- SWI/SNF network in Sca1+ MICs; Aim 2- Modulation of lineage commitment of Sca1+ MICs by targeting miRNA/BAF60 networks in vitro; Aim 3 - Modulation of lineage commitment of Sca1+ MICs by targeting miRNA/BAF60c network in vivo; Aim 4 - Determine the contribution of regeneration-activated p38-BAF60c signaling to the activation of the myogenic potential of Sca1+ MICs