The circadian E3 ligase FBXL21 regulates myoblast differentiation and sarcomere architecture via MYOZ1 ubiquitination and NFAT signaling

Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3 beta, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3 beta coexpression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wildtype mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Zline. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling. Author summary Skeletal muscle is a vital organ for our mobility, metabolism, and health. It is also highly dynamic at the molecular level, yet the underlying regulatory mechanisms are not well understood. Mounting evidence suggests a critical role of the biological timer, called circadian clock, in the molecular flux during the daily activity/rest cycle. Our previous research has revealed a circadian E3 ubiquitin ligase, FBXL21, as a regulator in the skeletal muscle. In the current study, we provide molecular and genetic evidence that FBXL21, with its upstream regulator GSK-3 beta, regulates degradation of a sarcomere protein, MYOZ1. Consistent with a key regulatory function, FBXL21 deficiency in Psttm mice and C2C12 cells led to marked abnormalities in muscle differentiation and function. Mechanistically, we show that FBXL21 and MYOZ1 regulate diurnal NFAT nuclear translocation and activity, a pathway important for muscle differentiation and development. Together, our work establishes a critical role of the circadian E3 ligase FBXL21 in muscle differentiation and sarcomere structure by regulating MYOZ1 and NFAT signaling.

Automated summary

This study illustrates the pivotal role of the circadian E3 ligase FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling, providing important insights into the regulatory mechanisms of the skeletal muscle biological clock.