REV-ERBα controls muscle lipid and protein metabolism by directly repressing key regulators.

(A) Cotransfection of HEK-293T cells with empty control vector or mouse REV-ERBα expression plasmid, along with REV-ERB sensor (mBmal1::luc), containing 2 ROREs from the proximal promoter of Bmal1 linked to LUC. Data from 2 independent experiments are expressed as mean fold-change normalized to the empty vector (n = 6; ±SEM). (B) In vivo imaging of LUC activity in WT and mKO mouse TA muscles following electric pulse–mediated gene transfer of mBmal1::luc cotransfected either with empty vector or REV-ERBα expression plasmid. Imaging analysis was performed 3 d after gene transfer at ZT10 as described in Materials and methods. Pseudocolors overlaid on the image indicate the luminescence intensity from mBmal1::luc reporter gene activity as indicated by the scale bar radiance (photons/second/cm2/steradian). (C) Quantification of LUC activity in TA muscles normalized to mKate2, an exogenous spike-in control plasmid (mean ± SEM; n = 3 mice; *p p p t test). (D) Cotransfection of HEK-293T cells with empty control vector, mouse REV-ERBα expression plasmid, mouse GR expression plasmid, or c.a.FOXO3A expression plasmid, along with GR:FOXO sensor containing −5 kb or −1 kb promoter sequence from mouse MuRF-1 linked to LUC. Data are expressed as mean fold-change normalized to the empty vector (n = 2–3; ±SEM). (E) In vivo REV-ERBα, NCoR1, and HDAC3 occupancy at target sites in control and mKO gastrocnemius at ZT8 (mean fold-enrichment over IgG ± SEM; n = 4 independent biological replicates from 4 independent ChIP-qPCR experiments; Foxl2 promoter used as negative control). (F) Gene expression (RT-qPCR) of mKO TA muscles at ZT10 following electric pulse-mediated gene transfer of REV-ERBα expression plasmids or empty vector. Data normalized to 36B4 expression and expressed as fold-change relative to contralateral muscle containing empty vector (mean ± SEM, n = 4 mice, *p t test). Underlying data can be found in supporting file S1 Data. BMAL1, brain and muscle ARTN-like protein 1; c.a.FOXO3A, constitutively active forkhead box O3; ChIP-qPCR, chromatin immunoprecipitation–quantitative real-time PCR; CLOCK, circadian locomotor output cycles kaput; GR, glucocorticoid receptor; HDAC3, histone deacetylase 3; HEK-293T, human embryonic kidney 293T; IgG, immunoglobulin G; LUC, luciferase; mKO, myocyte-specific loss of BMAL1; NCor1, nuclear receptor corepressor 1; PDH, pyruvate dehydrogenase; ROR, RAR-related orphan receptor; RORE, ROR response element; RT-qPCR, quantitative reverse transcription PCR; TA, tibialis anterior; ZT, Zeitgeber time.