seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 KO ChIP-seq]. seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 KO ChIP-seq]

There is an absolute requirement of Pax7 for the normal function of MuSCs during regenerative myogenesis in skeletal muscle at any stage of life. Here using RNA-seq, H3K27ac and Pax7 ChIP-seq, we discover PAM-1 (Pax7 Associated Muscle lncRNA) that is enriched in activated skeletal muscle satellite cells (ASCs) 24 and 48 hours after activation. Knockdown of PAM-1 reduces proliferating Pax7+Myod+ ASCs number, while overexpression of PAM-1 increases ASCs number. Mechanistically, PAM-1 is located on ASCs and myoblast specific super-enhancer (SE), and we categorize it as seRNA. Through a series of multiomics analysis of PAM-1 interactome in myoblast including PAM-1-DNA interaction by ChIRP-seq, PAM-1 SE-DNA interaction by 4C-seq, PAM-1-protein interaction by mass spectrometry and ChIP-seq, we identify a novel class of transcriptional regulation that seRNA PAM-1 interacts with RNA binding protein Ddx5 and tethers PAM-1 SE to regulate inter-chromosomal targets Timp2. Altogether, our findings identify PAM-1 is driven by Pax7 in ASC and myoblast to regulate myogenic activation through binding with Ddx5 and targeting Timp2. Overall design: H3K27ac ChIP-seq on PAM-1 knockout

在生命任一阶段的骨骼肌再生性肌发生过程中，Pax7是肌肉卫星细胞（Muscle Satellite Cells, MuSCs）发挥正常功能的绝对必需因子。本研究通过RNA测序（RNA-seq）、H3K27乙酰化染色质免疫共沉淀测序（H3K27ac ChIP-seq）以及Pax7染色质免疫共沉淀测序（Pax7 ChIP-seq），鉴定出PAM-1（Pax7相关肌肉长链非编码RNA，Pax7 Associated Muscle lncRNA），该分子在激活后24小时与48小时的激活态骨骼肌卫星细胞（Activated Skeletal Muscle Satellite Cells, ASCs）中富集表达。
敲低PAM-1会降低增殖型Pax7+Myod+ ASCs的数量，而过表达PAM-1则可增加ASCs的数量。
机制层面，PAM-1定位于ASCs与成肌细胞特异性超级增强子（Super-enhancer, SE）区域，因此我们将其归类为超级增强子相关RNA（Super-enhancer associated RNA, seRNA）。
我们针对成肌细胞中的PAM-1互作组开展了一系列多组学分析：包括通过染色质RNA纯化分离测序（ChIRP-seq）检测PAM-1与DNA的互作、通过染色体构象捕获测序（4C-seq）检测PAM-1与超级增强子DNA的互作、通过质谱分析与染色质免疫共沉淀测序（ChIP-seq）检测PAM-1与蛋白质的互作，最终鉴定出一类全新的转录调控模式：超级增强子相关RNA PAM-1可与RNA结合蛋白Ddx5结合，并锚定其所在的超级增强子区域，从而调控跨染色体靶基因Timp2。
综上，本研究发现PAM-1由Pax7在ASCs与成肌细胞中驱动表达，并通过结合Ddx5以及靶向Timp2来调控肌源性激活过程。
整体实验设计：PAM-1敲除样本的H3K27ac ChIP-seq测序。