REV-ERB? and REV-ERB? function as key factors regulating Mammalian Circadian Output.
ABSTRACT: The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERB? and REV-ERB? are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erb?/Rev-erb? double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the double knockout of both Rev-erb? and Rev-erb? does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERB?/? deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2, are significantly upregulated. This study highlight the relevance of REV-ERBs as pivotal output mediators of the mammalian circadian clock.
Project description:The nuclear receptor Rev-erb? regulates circadian rhythm and metabolism, but its effects are modest and it has been considered to be a secondary regulator of the cell-autonomous clock. Here we report that depletion of Rev-erb? together with closely related Rev-erb? has dramatic effects on the cell-autonomous clock as well as hepatic lipid metabolism. Mouse embryonic fibroblasts were rendered arrhythmic by depletion of both Rev-erbs. In mouse livers, Rev-erb? mRNA and protein levels oscillate with a diurnal pattern similar to that of Rev-erb?, and both Rev-erbs are recruited to a remarkably similar set of binding sites across the genome, enriched near metabolic genes. Depletion of both Rev-erbs in liver synergistically derepresses several metabolic genes as well as genes that control the positive limb of the molecular clock. Moreover, deficiency of both Rev-erbs causes marked hepatic steatosis, in contrast to relatively subtle changes upon loss of either subtype alone. These findings establish the two Rev-erbs as major regulators of both clock function and metabolism, displaying a level of subtype collaboration that is unusual among nuclear receptors but common among core clock proteins, protecting the organism from major perturbations in circadian and metabolic physiology.
Project description:Description Loss of SCN REV-ERBs alters oscillator period but does not render the clock arrhythmic. Circadian disruption, as occurs in shift work, is associated with metabolic diseases often attributed to a discordance between internal clocks and environmental timekeepers. REV-ERB nuclear receptors are key components of the molecular clock, but their specific role in the SCN master clock is unknown. We report here that mice lacking circadian REV-ERB nuclear receptors in the SCN maintain free-running locomotor and metabolic rhythms, but these rhythms are notably shortened by 3 hours. When housed under a 24-hour light:dark cycle and fed an obesogenic diet, these mice gained excess weight and accrued more liver fat than controls. These metabolic disturbances were corrected by matching environmental lighting to the shortened endogenous 21-hour clock period, which decreased food consumption. Thus, SCN REV-ERBs are not required for rhythmicity but determine the free-running period length. Moreover, these results support the concept that dissonance between environmental conditions and endogenous time periods causes metabolic disruption.
Project description:The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-? and REV-ERB-? have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-? has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-? and REV-ERB-? genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-? and REV-ERB-? cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-? and Rev-erb-? function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-? and REV-ERB-? with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.
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:Circadian dysfunction is a common attribute of many neurodegenerative diseases, most of which are associated with neuroinflammation. Circadian rhythm dysfunction has been associated with inflammation in the periphery, but the role of the core clock in neuroinflammation remains poorly understood. Here we demonstrate that Rev-erb?, a nuclear receptor and circadian clock component, is a mediator of microglial activation and neuroinflammation. We observed time-of-day oscillation in microglial immunoreactivity in the hippocampus, which was disrupted in Rev-erb?-/- mice. Rev-erb? deletion caused spontaneous microglial activation in the hippocampus and increased expression of proinflammatory transcripts, as well as secondary astrogliosis. Transcriptomic analysis of hippocampus from Rev-erb?-/- mice revealed a predominant inflammatory phenotype and suggested dysregulated NF-?B signaling. Primary Rev-erb?-/- microglia exhibited proinflammatory phenotypes and increased basal NF-?B activation. Chromatin immunoprecipitation revealed that Rev-erb? physically interacts with the promoter regions of several NF-?B-related genes in primary microglia. Loss of Rev-erb? in primary astrocytes had no effect on basal activation but did potentiate the inflammatory response to lipopolysaccharide (LPS). In vivo, Rev-erb?-/- mice exhibited enhanced hippocampal neuroinflammatory responses to peripheral LPS injection, while pharmacologic activation of Rev-erbs with the small molecule agonist SR9009 suppressed LPS-induced hippocampal neuroinflammation. Rev-erb? deletion influenced neuronal health, as conditioned media from Rev-erb?-deficient primary glial cultures exacerbated oxidative damage in cultured neurons. Rev-erb?-/- mice also exhibited significantly altered cortical resting-state functional connectivity, similar to that observed in neurodegenerative models. Our results reveal Rev-erb? as a pharmacologically accessible link between the circadian clock and neuroinflammation.
Project description:The circadian clock acts at the genomic level to coordinate internal behavioral and physiologic rhythms via the CLOCK-BMAL transcriptional heterodimer. Although the nuclear receptors REV-ERB? and ? have been proposed to contribute to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential we generated comparative cistromes of both Rev-erb isoforms, which revealed shared recognition at over ~50% of their total sites and extensive overlap with the master clock regulator Bmal. While Rev-erb? has been shown to directly regulate Bmal expression, the cistromic analysis reveals a more profound connection between Bmal and Rev-erb? and ? regulatory circuits than previously suspected. Genes within the intersection of the Bmal and Rev-erb cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb?/? function by creating double-knockout mice (DKOs) profoundly disrupted circadian expression of core clock and lipid homeostatic genes. As a result, DKOs show strikingly altered circadian wheel-running behavior and deregulated lipid metabolism. These data reveal an integral role of Rev-erb?/? in clock function as well as provide a cistromic basis for the integration of circadian rhythm and metabolism. Total RNA was obtained from livers of wild-type and Liver-specific Reverb alpha/beta double knockout mice at ZT 0, 4, 8, 12, 16, and 20.
Project description:The cell-autonomous circadian clock regulates IgE- and IL-33-mediated mast cell activation, both of which are key events in the development of allergic diseases. Accordingly, clock modifiers could be used to treat allergic diseases, as well as many other circadian-related diseases, such as sleep and metabolic disorders. The nuclear receptors REV-ERB-? and -? (REV-ERBs) are crucial components of the circadian clockwork. Efforts to pharmacologically target REV-ERBs using putatively specific synthetic agonists, particularly SR9009, have yielded beneficial effects on sleep and metabolism. Here, we sought to determine whether REV-ERBs are functional in the circadian clockwork in mast cells and, if so, whether SR9009 affects IgE- and IL-33-mediated mast cell activation. Bone marrow-derived mast cells (BMMCs) obtained from wild-type mice expressed REV-ERBs, and SR9009 or other synthetic REV-ERBs agonists affected the mast cell clockwork. SR9009 inhibited IgE- and IL-33-mediated mast cell activation in wild-type BMMCs in association with inhibition of Gab2/PI3K and NF-?B activation. Unexpectedly, these suppressive effects of SR9009 were observed in BMMCs following mutation of the core circadian gene <i>Clock</i>. These findings suggest that SR9009 inhibits IgE- and IL-33-mediated mast cell activation independently of the functional circadian clock activity. Thus, SR9009 or other synthetic REV-ERB agonists may have potential for anti-allergic agents.
Project description:The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erb? and Rev-erb?, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erb? in osteoclast precursor cells enhanced receptor activator of nuclear factor-?B ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erb? leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erb? in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erb? interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverb? in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erb? negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.
Project description:Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-? and REV-ERB-? have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.
Project description:The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERBa and REV-ERBb are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erba/Rev-erbb double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the complete knockout of both Rev-erba and Rev-erbb does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERBa/b deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2, are significantly upregulated. This study emphasizes REV-ERBs function to form an essential link between the circadian clock and a wide variety of cellular physiological functions.