Single-cell chromatin accessibility profiling reveals a self-renewing muscle satellite cell state

Successful skeletal muscle regeneration is primarily mediated by muscle stem cells or satellite cells which express Pax7. In homeostasis, these satellite cells exist in a ‘genuine quiescent’ state, although some satellite cells may also be primed. After an acute injury, satellite cells, enabled by several other niche cells in the muscle tissue, undergo a series of molecular and cellular changes such as activation, proliferation, and differentiation. These processes culminate in the generation of nascent muscle fibers or the restoration of worn-out muscle fibers, leading to the restoration of muscle function. To prevent the depletion of the quiescent satellite cell pool, some satellite cells must resist the ambient differentiation signals and undergo the self-renewal process. While the differentiation process has been well characterized, little is known about the mechanism of satellite cell self-renewal. Furthermore, the molecular identity of self-renewing satellite cells remains unknown. To fill this gap, we utilize single nuclei ATACseq with high temporal resolution to characterize the temporal dynamics of chromatin accessibility in satellite cells and other muscle niche cells during muscle regeneration. This enables us to identify for the first time the self-renewing trajectory undertaken by satellite cells for their long-term repopulation. We also validated these findings using single-cell RNAseq and other protein-level assays. We also utilize gene perturbation to show that SMAD4, a co-SMAD regulating the TGF-beta pathway regulates the cfate choice of self-renewal versus differentiation. 1) Mouse muscle injury (uninjured, 6, 12, 24, 48, 72 hours post injury, 7 days post injury) (2 replicates per sample) and 2) Smad4 f/f:Pax7 CreER/CreER :R26 YFP/YFP mice (i.e., Smad4 iKO) and Smad4 f/+:Pax7 CreER/CreER:R26 YFP/YFP mice (i.e. Smad4 Ctrl)

成功的骨骼肌再生主要由表达Pax7的卫星细胞（satellite cells）介导，此类细胞属于肌肉干细胞。在稳态条件下，这类卫星细胞处于“真性静息”状态，尽管部分卫星细胞也可能处于预激活状态。当发生急性损伤后，在肌肉组织内多种其他龛细胞（niche cells）的支持下，卫星细胞会经历一系列分子与细胞层面的变化，包括激活、增殖与分化。上述过程最终生成新生肌纤维或修复受损肌纤维，从而恢复肌肉功能。

为避免静息卫星细胞库耗竭，部分卫星细胞需抵抗周围的分化信号并完成自我更新过程。尽管分化过程已得到充分阐明，但卫星细胞自我更新的机制仍知之甚少。此外，自我更新型卫星细胞的分子特征仍未明确。

为填补这一研究空白，本研究采用高时间分辨率的单细胞核ATAC测序（single nuclei ATAC-seq）技术，对肌肉再生过程中卫星细胞与其他肌肉龛细胞的染色质可及性时序动态变化进行表征。这使得我们首次鉴定出卫星细胞为实现长期自我更新所经历的自我更新轨迹。本研究还通过单细胞RNA测序（single-cell RNA-seq）及其他蛋白质水平检测实验对上述发现进行了验证。

本研究通过基因扰动实验证实，调控转化生长因子-β（TGF-β）通路的协同SMAD蛋白SMAD4，可调控卫星细胞自我更新与分化间的细胞命运抉择。

1. 小鼠肌肉损伤模型：设置未损伤组，以及损伤后6、12、24、48、72小时和损伤后7天组，每组样本设置2个生物学重复；
2. 基因修饰小鼠模型：包括Smad4 flox/flox:Pax7-CreER:R26-YFP（黄色荧光蛋白，YFP）小鼠（即Smad4条件性敲除小鼠，Smad4 iKO），以及Smad4 flox/+:Pax7-CreER:R26-YFP小鼠（即Smad4野生型对照小鼠，Smad4 Ctrl）