Single-nucleus RNA-seq and FISH reveal coordinated transcriptional activity in mammalian myofibers

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. By snRNA-seq and snATAC-seq, we showed that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we showed that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential pane of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a unique mechanism of coordination of gene expression in a syncytium. Single nuclei RNA-seq and single nuclei ATAC-seq骨骼肌纤维为大型合胞体，但目前尚不明确其众多细胞核内的基因表达是否存在协同调控机制。本研究通过单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）与单细胞核转座酶可及性测序（single-nucleus assay for transposase-accessible chromatin sequencing, snATAC-seq）发现，慢肌、快肌、肌腱连接以及神经肌肉接头相关的肌细胞核各自具备独特的转录程序，且与不同的染色质状态及转录因子组合密切相关。在成年小鼠体内，已鉴定的肌纤维类型主要表达慢肌型肌球蛋白重链（Myosin heavy chain, MYH），或是三种快肌型肌球蛋白重链亚型之一；仅少数杂合纤维可同时表达多种MYH亚型。借助单细胞核RNA测序与荧光原位杂交（fluorescence in situ hybridization, FISH），我们发现肌纤维内的绝大多数肌细胞核处于同步状态，仅协同表达一种快肌Myh亚型，并偏好表达特定的肌肉特异性基因集。值得注意的是，这种表达协同性在出生后发育早期便已形成，且依赖于神经支配。本研究结果揭示了合胞体内基因表达协同调控的独特机制。单细胞核RNA测序与单细胞核转座酶可及性测序