A.M., V.; Zhang, Q.; Srivastava, S.; Koronowski, K. B.; Srivastava, A.

The circadian clock genes Bmal1 and Nr1d1/2 (REV-ERB/{beta}) regulate skeletal muscle metabolism and homeostasis, yet the precise genes and mechanisms involved remain incompletely understood. Here, we perform Weighted Gene Co-expression Network Analysis (WGCNA) on skeletal muscle circadian transcriptomes with varying Bmal1 operational status to identify genes central to muscle circadian function. The largest WGCNA module, potentially under Bmal1 regulation, contains clock and muscle-specific output genes governed hierarchically by hub genes including Igf2bp2, an RNA-binding protein involved in muscle progenitor growth and maintenance. Igf2bp2 expression is rhythmic in mouse and human muscle and functional experiments in muscle-specific Bmal1 knockout mice show that Igf2bp2 is upregulated by loss of Bmal1 at ZT8 and negatively correlated with Nr1d2, suggesting de-repression through REV-ERB{beta} as a regulatory mechanism. Luciferase reporter experiments in cultured myotubes show that REV-ERB{beta}, but not REV-ERB, represses Igf2bp2 transcription and that repression is mediated by non-canonical GCC motifs in the Igf2bp2 promoter region. Together, these findings uncover a circadian Nr1d2-Igf2bp2 regulatory axis linking transcriptional and post-transcriptional regulation in skeletal muscle, with implications for muscle homeostasis.