Ubiquitination-coupled transcriptional control of muscle stem cell quiescence and activation

出生后，骨骼肌保留少量肌肉干细胞（musc），它们将退出细胞周期，获得静止状态（G0），但能够重新进入细胞周期。与胚胎发育中增殖祖细胞产生新肌肉不同，成年MuSCs必须在接收细胞外信号后迅速从G0过渡到重新进入细胞周期以维持组织。musc如何在静止和激活状态之间快速可逆地转换是一个基本问题，但人们对这个问题的理解仍然很少。我们最近发现，通过泛素蛋白酶体系统（UPS）和染色质重塑的转录控制耦合的综合机制是musc静止的必要条件。在MuSCs中抑制UPS可导致其自发激活。随后的分析表明，E3连接酶Nedd4l可能通过调节Wdr5和Baf53a的蛋白水平，在连接UPS和musc基因表达的转录控制中发挥核心作用。后两种蛋白分别是Ash2l/MLL组蛋白甲基转移酶（HMT）和SWI/SNF染色质重塑复合物的关键组分。我们假设，在再生过程中维持MuSCs的休眠和快速激活是通过一种机制实现的，在这种机制中，UPS对关键染色质重塑因子的主动回收和降解阻止了MuSCs进入细胞周期。因此，UPS可能需要保持musc处于“平衡”状态。在短期内，我们的目标是分子定义Nedd4l如何将UPS与染色质重塑联系起来，如下面的三个目标所定义。我们的长期目标是揭示MuSCs如何广泛地将UPS与转录机制整合以维持组织稳态。我期望从这个项目中获得的知识可以作为研究干细胞和其他组织类型稳态的模型。目的1：我们将结合FACS和LC-MS/MS来确定Nedd4L- cko和WT小鼠新鲜分选的musc中Nedd4L的蛋白质组学特征。Aim1是图5中所进行分析的扩展，将在多个生物和技术复制上进行。AIM2：我们将利用诱导的Nedd4Lf/f来确定Nedd4L在静止、自我更新和分化中的作用；Pax7CreERT2和本构性条件敲除，Nedd4Lf/f；Pax7Cre旨在研究nedd41基因暂时缺失和长期发育至成年晚期（8个月）的影响。我们将进行差异基因表达和通路分析，并通过RT-qPCR和免疫荧光染色对指长伸肌（EDL）肌纤维相关的MuSCs进行高优先级候选。AIM3：我们将通过对H3K4me3（由Ash2l/MLL/Wdr5沉积的活性标记）复合物和Baf53a的ChIP-seq进行转座酶可及染色质测序（ATAC-seq）和ChIP-seq来确定新分选的musc中Nedd4L基因缺失引起的表观遗传改变。

Summary Postnatally, skeletal muscle retains a small population of muscle stem cells (MuSCs) which will exit cell cycle, acquire a quiescent state (G0) but are capable of cell cycle reentry. Unlike embryonic development where proliferating progenitors give rise to new muscle, adult MuSCs must rapidly transition from G0 to reenter cell cycle upon receiving extracellular cues to maintain tissue. How MuSCs swiftly and reversibly transition between quiescent and activated states is a fundamental question which remains poorly understood. We have recently discovered that an integrated mechanism via coupling of ubiquitin proteasome system (UPS) and transcriptional control by chromatin remodelling is essential for MuSCs quiescence. Inhibition of UPS in MuSCs leads to their spontaneous activation. Subsequent analyses showed that Nedd4l, an E3 ligase may play a central role in bridging the UPS with transcriptional control of gene expression in MuSCs by modulating the protein level of Wdr5 and Baf53a. The latter proteins are critical components of the Ash2l/MLL histone methyltransferase (HMT) and the SWI/SNF chromatin remodeling complexes respectively. We hypothesize that maintenance of MuSCs quiescence and their rapid activation during regeneration is achieved by a mechanism in which active recycling and degradation of key chromatin remodeling factors by UPS blocks MuSCs from entering the cell cycle. Therefore, UPS may be required to keep MuSCs in a "poised" state. In short term, we aim to molecularly define how Nedd4l links UPS with chromatin remodelling as defined by the three aims below. Our long-term goal is to unravel how MuSCs broadly integrate UPS with transcriptional mechanisms to maintain tissue homeostasis. I anticipate that the knowledge gained from this program can be used as a model to study stem cells and the homeostasis of other tissue types. AIM1: We will combine FACS with LC-MS/MS to determine the proteomic signature of Nedd4L in freshly sorted MuSCs from Nedd4L-cKO and WT mice. Aim1 is the extension of the analysis performed in Figure 5 and will be carried out on multiple biological and technical replicates. AIM2: We will determine the role of Nedd4L in quiescence, self-renewal and differentiation using an inducible Nedd4Lf/f; Pax7CreERT2 and constitutive conditional knockout, Nedd4Lf/f; Pax7Cre to study the effect of loss of Nedd4l temporally and during long-term development into late adulthood (8-month-old). We will perform differential gene expression and pathway analysis and validate high-priority candidates by RT-qPCR and immunofluorescence staining on myofiber-associated MuSCs from the Extensor Digitorum Longus (EDL) muscle. AIM3: We will determine the epigenetic alterations caused by genetic deletion of Nedd4L in freshly sorted MuSCs by applying Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and ChIP-seq for H3K4me3 (active mark deposited by Ash2l/MLL/Wdr5) complex and by ChIP-seq of Baf53a.