Rev-erb? heterozygosity produces a dose-dependent phenotypic advantage in mice.
ABSTRACT: Numerous mutational studies have demonstrated that circadian clock proteins regulate behavior and metabolism. Nr1d1(Rev-erb?) is a key regulator of circadian gene expression and a pleiotropic regulator of skeletal muscle homeostasis and lipid metabolism. Loss of Rev-erb? expression induces muscular atrophy, high adiposity, and metabolic syndrome in mice. Here we show that, unlike knockout mice, Nr1d1 heterozygous mice are not susceptible to muscular atrophy and in fact paradoxically possess larger myofiber diameters and improved neuromuscular function, compared to wildtype mice. Heterozygous mice lacked dyslipidemia, a characteristic of Nr1d1 knockout mice and displayed increased whole-body fatty-acid oxidation during periods of inactivity (light cycle). Heterozygous mice also exhibited higher rates of glucose uptake when fasted, and had elevated basal rates of gluconeogenesis compared to wildtype and knockout littermates. Rev-erb? ablation suppressed glycolysis and fatty acid-oxidation in white-adipose tissue (WAT), whereas partial Rev-erb? loss, curiously stimulated these processes. Our investigations revealed that Rev-erb? dose-dependently regulates glucose metabolism and fatty acid oxidation in WAT and muscle.
Project description:OBJECTIVE:The loss of skeletal muscle mass and strength are a central feature of traumatic injury and degenerative myopathies. Unfortunately, pharmacological interventions typically fail to stem the long-term decline in quality of life. Reduced Rev-Erb-mediated gene suppression in cultured C2C12 myoblasts has been shown to stimulate myoblast differentiation. Yet the mechanisms that allow Rev-Erb to pleiotropically inhibit muscle differentiation are not well understood. In this study, we sought to elucidate the role of Rev-Erb in the regulation of muscle differentiation and regeneration in vivo. METHODS:Using Rev-Erb?/? shRNAs, pharmacological ligands, and Rev-Erb? null and heterozygous mice, we probed the mechanism of Rev-Erb?/? regulation of muscle differentiation and muscle regeneration. RESULTS:ChIP seq analysis of Rev-Erb in differentiating myoblasts showed that Rev-Erb? did not transcriptionally regulate muscle differentiation through cognate Rev-Erb/ROR-response elements but through possible interaction with the cell fate regulator NF-Y at CCAAT-motifs. Muscle differentiation is stimulated by Rev-Erb release from CCAAT-motifs at promoter and enhancer elements of a number of myogenesis proteins. Partial loss of Rev-Erb expression in mice heterozygous for Rev-Erb? accelerated muscle repair in vivo whereas Rev-Erb knockout mice showed deficiencies in regenerative repair compared to wild type mice. These phenotypic differences between heterozygous and knockout mice were not apparently dependent on MRF induction in response to injury. Similarly, pharmacological disruption of Rev-Erb suppressive activity in injured muscle accelerated regenerative repair in response to acute injury. CONCLUSIONS:Disrupting Rev-Erb activity in injured muscle accelerates regenerative muscle repair/differentiation through transcriptional de-repression of myogenic programs. Rev-Erb, therefore, may be a potent therapeutic target for a myriad of muscular disorders.
Project description:The function of the nuclear receptor Rev-erb? (Nr1d1) in the brain is, apart from its role in the circadian clock mechanism, unknown. Therefore, we compared gene expression profiles in the brain between wild-type and Rev-erb? knock-out (KO) animals. We identified fatty acid binding protein 7 (Fabp7, Blbp) as a direct target of repression by REV-ERB?. Loss of Rev-erb? manifested in memory and mood related behavioral phenotypes and led to overexpression of Fabp7 in various brain areas including the subgranular zone (SGZ) of the hippocampus, where neuronal progenitor cells (NPCs) can initiate adult neurogenesis. We found increased proliferation of hippocampal neurons and loss of its diurnal pattern in Rev-erb? KO mice. In vitro, proliferation and migration of glioblastoma cells were affected by manipulating either Fabp7 expression or REV-ERB? activity. These results suggest an important role of Rev-erb? and Fabp7 in adult neurogenesis, which may open new avenues for treatment of gliomas as well as neurological diseases such as depression and Alzheimer.
Project description:The nuclear receptors REV-ERB? and REV-ERB? have been demonstrated to be core members of the circadian clock and participate in the regulation of a diverse set of metabolic functions. Due to their overlapping tissue expression patterns and gene expression profiles, REV-ERB? is thought to be redundant to REV-ERB?. Recent work has highlighted REV-ERB?'s role in the regulation of skeletal muscle oxidative capacity and mitochondrial biogenesis. Considering the similarity between the REV-ERBs and the hypothesized overlap in function, we sought to determine whether REV-ERB?-deficiency presented with a similar skeletal muscle phenotype as REV-ERB?-deficiency. Ectopic overexpression in C2C12 cells demonstrated that REV-ERB? drives mitochondrial biogenesis and the expression of genes involved in fatty acid oxidation. Intriguingly, knock down of REV-ERB? in C2C12 cultures also resulted in mitochondrial biogenesis and increased expression of genes involved in fatty acid ?-oxidation. To determine whether these effects occurred in vivo, we examined REV-ERB?-deficient mice and observed a similar increase in expression of genes involved in mitochondrial biogenesis and fatty acid ?-oxidation. Consistent with these results, REV-ERB?-deficient mice exhibited an altered metabolic phenotype compared to wild-type littermate controls when measured by indirect calorimetry. This likely compensated for the increased food consumption that occurred, possibly aiding in the maintenance of their weight over time. Since feeding behaviors are a direct circadian output, this study suggests that REV-ERB? may have more subtle effects on circadian behaviors than originally identified. Furthermore, these data implicate REV-ERB? in the control of skeletal muscle metabolism and energy expenditure and suggest that development of REV-ERB? versus REV-ERB? selective ligands may have therapeutic utility in the treatment of metabolic syndrome.
Project description:To investigate the role of the transcriptional repressor Rev-erb alpha in epididymal white adipose tissue, we performed a microarray analysis of gene expression in the epididymal white adipose tissue of wildtype and Rev-erb alpha knock-out mice. Examination of the transcriptome in epididymal white adipose tissue of Rev-erb alpha kockout mice compared to wildtype mice.
Project description:Circadian oscillation of body temperature is a basic, evolutionarily conserved feature of mammalian biology. In addition, homeostatic pathways allow organisms to protect their core temperatures in response to cold exposure. However, the mechanism responsible for coordinating daily body temperature rhythm and adaptability to environmental challenges is unknown. Here we show that the nuclear receptor Rev-erb? (also known as Nr1d1), a powerful transcriptional repressor, links circadian and thermogenic networks through the regulation of brown adipose tissue (BAT) function. Mice exposed to cold fare considerably better at 05:00 (Zeitgeber time?22) when Rev-erb? is barely expressed than at 17:00 (Zeitgeber time?10) when Rev-erb? is abundant. Deletion of Rev-erb? markedly improves cold tolerance at 17:00, indicating that overcoming Rev-erb?-dependent repression is a fundamental feature of the thermogenic response to cold. Physiological induction of uncoupling protein 1 (Ucp1) by cold temperatures is preceded by rapid downregulation of Rev-erb? in BAT. Rev-erb? represses Ucp1 in a brown-adipose-cell-autonomous manner and BAT Ucp1 levels are high in Rev-erb?-null mice, even at thermoneutrality. Genetic loss of Rev-erb? also abolishes normal rhythms of body temperature and BAT activity. Thus, Rev-erb? acts as a thermogenic focal point required for establishing and maintaining body temperature rhythm in a manner that is adaptable to environmental demands.
Project description:The goal of this study is to identify the cistrome of the transcriptional repressor Rev-erb alpha in epididymal white adipose tissue. Performing Rev-erb alpha ChIP-seq on epididymal white adipose tissue from wildtype mice at 5PM when Rev-erb alpha protein level peaks in wild type (WT) mice, we were able to globally determine the genomic regions undergoing Rev-erb alpha-dependent de-repression. Examination of Rev-erb alpha binding in epididymal white adipose tissue.
Project description:Several investigations suggested abnormalities in circadian rhythms are related to the pathophysiology of psychiatric disorders, including drug addiction. Recently, orphan nuclear receptor rev-erb alpha and glycogen synthase kinase-3 ? (GSK-3?) were shown to be important circadian components. In addition, the orphan nuclear receptor rev-erb alpha is a key negative feedback regulator of the circadian clock. These evidences indicate that rev-erb alpha gene (NR1D1) is a good candidate gene for the pathogenesis of methamphetamine dependence. To evaluate the association between NR1D1 and methamphetamine dependence, we conducted a case-control study of Japanese samples (215 methamphetamine dependence and 232 controls) with three tagging SNPs selected by HapMap database. Written informed consent was obtained from each subject. This study was approved by the ethics committees at Fujita Health University, Nagoya University Graduate School of Medicine and each participating member of the Institute of the Japanese Genetics Initiative for Drug Abuse (JGIDA). We did not detect an association between NR1D1 and Japanese methamphetamine dependence patients in allele/genotype-wise analysis, or the haplotype analysis. Our findings suggest that NR1D1 does not play a major role in the pathophysiology of methamphetamine dependence in the Japanese population.
Project description:Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERB? (NR1D1) and REV-ERB? (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERB? ligand-binding domain (LBD). We solved two crystal structures of REV-ERB? LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.
Project description:Bipolar disorder (BD) is characterized by disruptions in circadian rhythms such as sleep and daily activity that often normalize after lithium treatment in responsive patients. As lithium is known to interact with the circadian clock, we hypothesized that variation in circadian 'clock genes' would be associated with lithium response in BD. We determined genotype for 16 variants in seven circadian clock genes and conducted a candidate gene association study of these in 282 Caucasian patients with BD who were previously treated with lithium. We found that a variant in the promoter of NR1D1 encoding Rev-Erb? (rs2071427) and a second variant in CRY1 (rs8192440) were nominally associated with good treatment response. Previous studies have shown that lithium regulates Rev-Erb? protein stability by inhibiting glycogen synthase kinase 3? (GSK3?). We found that GSK3? genotype was also suggestive of a lithium response association, but not statistically significant. However, when GSK3? and NR1D1 genotypes were considered together, they predicted lithium response robustly and additively in proportion to the number of response-associated alleles. Using lymphoblastoid cell lines from patients with BD, we found that both the NR1D1 and GSK3? variants are associated with functional differences in gene expression. Our findings support a role for Rev-Erb? in the therapeutic mechanism of lithium and suggest that the interaction between Rev-Erb? and GSK3? may warrant further study.
Project description:The nuclear receptor Rev-erb-? modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-? is highly expressed in oxidative skeletal muscle and that its deficiency in muscle leads to reduced mitochondrial content and oxidative function, as well as upregulation of autophagy. These cellular effects resulted in both impaired mitochondrial biogenesis and increased clearance of this organelle, leading to compromised exercise capacity. On a molecular level, Rev-erb-? deficiency resulted in deactivation of the Lkb1-Ampk-Sirt1-Ppargc-1? signaling pathway. These effects were recapitulated in isolated fibers and in muscle cells after knockdown of the gene encoding Rev-erb-?, Nr1d1. In complementary experiments, Rev-erb-? overexpression in vitro increased the number of mitochondria and improved respiratory capacity, whereas muscle overexpression or pharmacological activation of Rev-erb-? in vivo increased exercise capacity. This study identifies Rev-erb-? as a pharmacological target that improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.