Exploring networks underlying muscle stem cell identity - Resubmission - 1

项目摘要 肌肉干细胞，也被称为卫星细胞(SC)，是需要补充的前体细胞群 肌肉因运动而受损，或因疾病或衰老而消瘦。产生肌肉的能力 胚胎干细胞(ESC)的祖细胞将代表着再生医学的重大进步。 然而，尽管最近取得了进展，但区分小鼠和人类的胚胎干细胞仍然非常具有挑战性。 肌肉祖细胞和SC代表肌肉组织中的一个非常小的群体。这些限制具有 严重阻碍了了解肌肉干细胞和关键调控过程的进展 管理SC身份的规则。转录因子Pax7在建立SC身份中起着关键作用，但给出了 由于与获得SC相关的限制，我们对其分子功能的了解仍然不完整。 我们已经利用了一种系统，在该系统中，ESC中Pax7的表达产生了能够 为了重新填充SC生态位，我们已经开始研究染色质景观和表观遗传特征 了解这一群体的独特属性。通过这个系统，我们已经证明了Pax7可以 调节增强子功能和基因组拓扑。我们的目标是了解特定的染色质是如何 环境使用最先进的基因组、生化和蛋白质组来确定卫星细胞的身份 接近了。在两个目标中，我们将：(I)研究Pax7如何重新建模基因组，包装染色质以 远程相互作用；(Ii)探索Pax7如何与其他因素合作建立基因表达和 在体外和体内肌源性前体细胞的基因组重新连接。我们将停用与Pax7关联的子集 因子、增强剂和识别的调控元件，并检查它们对基因组的功能影响 结构、基因表达、细胞特性和分化。 众所周知，卫星细胞在衰老过程中更新效率较低，而且考虑到肌肉肿瘤 (横纹肌肉瘤)异常表达Pax7融合蛋白以维持肌肉处于预分化状态， 我们的建议将对由异常的Pax7活性和干细胞引起的病理状态提供重要的解释 功能。

Project Summary Muscle stem cells, also known as satellite cells (SC), are a progenitor population required to replenish muscle damaged by exercise or wasting caused by disease or aging. The ability to generate muscle progenitors from embryonic stem cells (ESC) would represent a major advance in regenerative medicine. However, despite recent progress, it remains very challenging to differentiate mouse and human ESC into muscle progenitors, and SC represent a very small population within muscle tissue. These limitations have significantly hampered progress toward understanding muscle stem cells, and the critical regulatory processes that govern SC identity. The transcription factor, Pax7, plays a critical role in establishing SC identity, but given the limitations associated with obtaining SC, our understanding of its molecular function remains incomplete. We have taken advantage of a system in which Pax7 expression in ESC generates myogenic precursors able to repopulate the SC niche, and we have begun investigating the chromatin landscape and epigenetic features of this population to understand its unique properties. With this system, we have shown that Pax7 can modulate enhancer function and genome topology. Our goal is to understand how a specific chromatin environment specifies satellite cell identity using state-of-the-art genomic, biochemical, and proteomic approaches. In two Aims, we will: (I) investigate how Pax7 re-models the genome, packaging chromatin for long-range interactions; (II) explore how Pax7 collaborates with other factors to establish gene expression and genome re-wiring in myogenic precursors in vitro and in vivo. We will inactivate a subset of Pax7-associated factors, enhancers, and identified regulatory elements and examine their functional impact on genome architecture, gene expression, cell identity, and differentiation. It is known that satellite cells are less efficiently renewed during aging, and given that muscle tumors (rhabdomyosarcomas) aberrantly express Pax7 fusion proteins to maintain muscle in a pre-differentiated state, our proposal will shed important light on pathological states resulting from aberrant Pax7 activity and stem cell function.