SummaryThe circadian clock network is an evolutionarily conserved mechanism that imparts temporal regulation to diverse biological processes. Brain and muscle Arnt-like 1 (Bmal1), an essential transcriptional activator of the clock, is highly expressed in skeletal muscle. However, whether this key clock component impacts myogenesis, a temporally regulated event that requires the sequential activation of myogenic regulatory factors, is not known. Here we report a novel function of Bmal1 in controlling myogenic differentiation through direct transcriptional activation of components of the canonical Wnt signaling cascade, a major inductive signal for embryonic and postnatal muscle growth. Genetic loss of Bmal1 in mice leads to reduced total muscle mass and Bmal1-deficient primary myoblasts exhibit significantly impaired myogenic differentiation accompanied by markedly blunted expression of key myogenic regulatory factors. Conversely, forced expression of Bmal1 enhances differentiation of C2C12 myoblasts. This cell-autonomous effect of Bmal1 is mediated by Wnt signaling as both expression and activity of Wnt components are markedly attenuated by inhibition of Bmal1, and activation of the Wnt pathway partially rescues the myogenic defect in Bmal1-deficient myoblasts. We further reveal direct association of Bmal1 with promoters of canonical Wnt pathway genes, and as a result of this transcriptional regulation, Wnt signaling components exhibit intrinsic circadian oscillation. Collectively, our study demonstrates that the core clock gene, Bmal1, is a positive regulator of myogenesis, which may represent a temporal regulatory mechanism to fine-tune myocyte differentiation.

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The molecular clock is intimately linked to metabolic regulation, and brown adipose tissue plays a key role in energy homeostasis. However, whether the cell-intrinsic clock machinery participates in brown adipocyte development is unknown. Here, we show that Bmal1 (also known as ARNTL), the essential clock transcription activator, inhibits brown adipogenesis to adversely affect brown fat formation and thermogenic capacity. Global ablation of Bmal1 in mice increases brown fat mass and cold tolerance, and adipocyteselective inactivation of Bmal1 recapitulates these effects and demonstrates its cell-autonomous role in brown adipocyte formation. Further loss-and gain-of-function studies in mesenchymal precursors and committed brown progenitors reveal that Bmal1 inhibits brown adipocyte lineage commitment and terminal differentiation. Mechanistically, Bmal1 inhibits brown adipogenesis through direct transcriptional control of key components of the TGF-b pathway together with reciprocally altered BMP signaling; activation of TGF-b or blockade of BMP pathways suppresses enhanced differentiation in Bmal1-deficient brown adipocytes. Collectively, our study demonstrates a novel temporal regulatory mechanism in finetuning brown adipocyte lineage progression to affect brown fat formation and thermogenic regulation, which could be targeted therapeutically to combat obesity.

Brown adipose tissue is a major thermogenic organ that plays a key role in maintenance of body temperature and whole-body energy homeostasis. Rev-erbα, a ligand-dependent nuclear receptor and transcription repressor of the molecular clock, has been implicated in the regulation of adipogenesis. However, whether Rev-erbα participates in brown fat formation is not known. Here we show that Rev-erbα is a key regulator of brown adipose tissue development by promoting brown adipogenesis. Genetic ablation of Rev-erbα in mice severely impairs embryonic and neonatal brown fat formation accompanied by loss of brown identity. This defect is due to a cell-autonomous function of Rev-erbα in brown adipocyte lineage commitment and terminal differentiation, as demonstrated by genetic loss- and gain-of-function studies in mesenchymal precursors and brown preadipocytes. Moreover, pharmacological activation of Rev-erbα activity promotes, whereas its inhibition suppresses brown adipocyte differentiation. Mechanistic investigations reveal that Rev-erbα represses key components of the TGF-β cascade, an inhibitory pathway of brown fat development. Collectively, our findings delineate a novel role of Rev-erbα in driving brown adipocyte development, and provide experimental evidence that pharmacological interventions of Rev-erbα may offer new avenues for the treatment of obesity and related metabolic disorders.

The SRF/MRTF and upstream signaling cascade play key roles in actin cytoskeleton organization and myocyte development. To date, how this signaling axis may function in brown adipocyte lineage commitment and maturation has not been delineated. Here we report that MRTF-SRF signaling exerts inhibitory actions on brown adipogenesis, and suppressing this negative regulation promotes brown adipocyte lineage development. During brown adipogenic differentiation, protein expressions of SRF, MRTFA/B and its transcription targets were down-regulated, and MRTFA/B shuttled from nucleus to cytoplasm. Silencing of SRF or MRTF-A/MRTF-B enhanced two distinct stages of brown adipocyte development, mesenchymal stem cell determination to brown adipocytes and terminal differentiation of brown adipogenic progenitors. We further demonstrate that the MRTF-SRF axis exerts transcriptional regulations of the TGF-β and BMP signaling pathway, critical developmental cues for brown adipocyte development. TGF-β signaling activity was significantly attenuated, whereas that of the BMP pathway augmented by inhibition of SRF or MRTF-A/MRTF-B, leading to enhanced brown adipocyte differentiation. Our study demonstrates the MRTF-SRF transcriptional cascade as a negative regulator of brown adipogenesis, through its transcriptional control of the TGF-β/BMP signaling pathways.