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

出生后，骨骼肌保留了一小部分肌肉干细胞（MuSC），它们将退出细胞周期，获得静止状态（G 0），但能够重新进入细胞周期。与增殖祖细胞产生新肌肉的胚胎发育不同，成年MuSC必须在接受细胞外信号后迅速从G 0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变是一个基本问题，但仍然知之甚少。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整合机制是MuSC静止所必需的。抑制MuSC中的UPS导致其自发活化。随后的分析表明，E3连接酶Nedd 4l可能通过调节Wdr 5和Baf 53 a的蛋白水平，在MuSC中将UPS与基因表达的转录控制桥接中发挥核心作用。后者蛋白分别是Ash 2l/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的静止和快速激活是通过一种机制实现的，在该机制中，UPS对关键染色质重塑因子的主动回收和降解阻止MuSCs进入细胞周期。因此，可能需要UPS将MuSC保持在“平衡”状态。在短期内，我们的目标是从分子上定义Nedd 4l如何将UPS与染色质重塑联系起来，如下面三个目标所定义的。我们的长期目标是揭示MuSC如何广泛整合UPS与转录机制以维持组织稳态。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的稳态的模型。目标1：我们将联合收割机FACS与LC-MS/MS相结合，以确定来自Nedd 4L-cKO和WT小鼠的新鲜分选的MuSC中Nedd 4L的蛋白质组特征。Aim 1是图5中进行的分析的扩展，将对多个生物学和技术重复样本进行分析。目标2：我们将使用诱导型Nedd 4Lf/f; Pax 7 CreERT 2和组成性条件性敲除Nedd 4Lf/f; Pax 7 Cre来确定Nedd 4L在静止、自我更新和分化中的作用，以研究Nedd 4l暂时和长期发育至成年晚期（8个月大）期间的损失的影响。我们将进行差异基因表达和途径分析，并通过RT-qPCR和免疫荧光染色对来自趾长伸肌（EDL）的肌纤维相关MuSC进行高优先级候选人验证。目标3：我们将通过使用测序（ATAC-seq）和H3 K4 me 3（由Ash 2l/MLL/Wdr 5沉积的活性标记）复合物的ChIP-seq和Baf 53 a的ChIP-seq应用转座酶可降解染色质测定法来确定由Nedd 4L的遗传缺失在新鲜分选的MuSC中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.