The ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating enzymes (DUBs) in the muscle cell context. We performed a genetic screen to identify DUBs as potential regulators of muscle cell differentiation. Surprisingly, we observed that the depletion of ubiquitin-specific protease 18 (USP18) affected the differentiation of muscle cells. USP18 depletion first stimulated differentiation initiation. Later, during differentiation, the absence of USP18 expression abrogated myotube maintenance. USP18 enzymatic function typically attenuates the immune response by removing interferon-stimulated gene 15 (ISG15) from protein substrates. However, in muscle cells, we found that USP18, predominantly nuclear, regulates differentiation independent of ISG15 and the ISG response. Exploring the pattern of RNA expression profiles and protein networks whose levels depend on USP18 expression, we found that differentiation initiation was concomitant with reduced expression of the cell-cycle gene network and altered expression of myogenic transcription (co) factors. We show that USP18 depletion altered the calcium channel gene network, resulting in reduced calcium flux in myotubes. Additionally, we show that reduced expression of sarcomeric proteins in the USP18 proteome was consistent with reduced contractile force in an engineered muscle model. Our results revealed nuclear USP18 as a critical regulator of differentiation initiation and maintenance, independent of ISG15 and its role in the ISG response. Highlights USP18 knockdown results in transcriptome-wide changes that cause a switch from proliferation to muscle cell differentiation. Fully differentiated muscles cells (or simply ‘myotubes’) formed in absence of USP18 lack sarcomeric integrity (in both cell culture and engineered muscle models). An increase in USP18 expression during muscle cell differentiation is independent of its role in the interferon type 1 stimulated response. During normal muscle cell differentiation, USP18 accumulates in the cell nucleus regulating multiple cellular processes, including proliferation, differentiation initiation and muscle cell maturation. In the absence of USP18, muscle cells stop proliferating and undergo accelerated fusion as part of the differentiation process by altering the expression levels of myogenic regulatory (co-)transcription factors, whereas maturation is impaired due to reduced expression of calcium-flux genes and sarcomeric genes.
【저자키워드】 Neuroimmunology, differentiation,