RESUMO
There is increasing demand to control circadian clock functions in a conditional manner for deeper understanding of the circadian system as well as for potential treatment of clock-related diseases. Small-molecule compounds provide powerful tools to reveal novel functions of target proteins in the circadian clock mechanism, and can be great therapeutic candidates. Here we describe the detailed methods of measuring cellular circadian rhythms in a high-throughput manner for chemical screening to identify compounds that affect circadian rhythms by targeting clock-related proteins.
Assuntos
Relógios Circadianos , Proteínas CLOCK , Relógios Circadianos/genética , Ritmo CircadianoRESUMO
CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.
Assuntos
Criptocromos/química , Isoformas de Proteínas/química , Animais , Sítios de Ligação , Relógios Circadianos , Criptocromos/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos Knockout , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/genética , TermodinâmicaRESUMO
The form and synaptic fine structure of melanopsin-expressing retinal ganglion cells, also called intrinsically photosensitive retinal ganglion cells (ipRGCs), were determined using a new membrane-targeted version of a genetic probe for correlated light and electron microscopy (CLEM). ipRGCs project to multiple brain regions, and because the method labels the entire neuron, it was possible to analyze nerve terminals in multiple retinorecipient brain regions, including the suprachiasmatic nucleus (SCN), olivary pretectal nucleus (OPN), and subregions of the lateral geniculate. Although ipRGCs provide the only direct retinal input to the OPN and SCN, ipRGC terminal arbors and boutons were found to be remarkably different in each target region. A network of dendro-dendritic chemical synapses (DDCSs) was also revealed in the SCN, with ipRGC axon terminals preferentially synapsing on the DDCS-linked cells. The methods developed to enable this analysis should propel other CLEM studies of long-distance brain circuits at high resolution.
Assuntos
Encéfalo/metabolismo , Células Ganglionares da Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Sinapses/metabolismo , Animais , Axônios/fisiologia , Encéfalo/patologia , Ritmo Circadiano/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Área Pré-Tectal/metabolismo , Área Pré-Tectal/patologia , Células Ganglionares da Retina/patologia , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/genética , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/patologiaRESUMO
Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are indispensable for non-image-forming visual responses that sustain under prolonged illumination. For sustained signaling of ipRGCs, the melanopsin photopigment must continuously regenerate. The underlying mechanism is unknown. We discovered that a cluster of Ser/Thr sites within the C-terminal region of mammalian melanopsin is phosphorylated after a light pulse. This forms a binding site for ß-arrestin 1 (ßARR1) and ß-arrestin 2. ß-arrestin 2 primarily regulates the deactivation of melanopsin; accordingly, ßαrr2-/- mice exhibit prolonged ipRGC responses after cessation of a light pulse. ß-arrestin 1 primes melanopsin for regeneration. Therefore, ßαrr1-/- ipRGCs become desensitized after repeated or prolonged photostimulation. The lack of either ß-arrestin attenuates ipRGC response under prolonged illumination, suggesting that ß-arrestin 2-mediated deactivation and ß-arrestin 1-dependent regeneration of melanopsin function in sequence. In conclusion, we discovered a molecular mechanism by which ß-arrestins regulate different aspects of melanopsin photoresponses and allow ipRGC-sustained responses under prolonged illumination.
Assuntos
Luz , Regeneração/efeitos da radiação , Opsinas de Bastonetes/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo , Adaptação Ocular/efeitos da radiação , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Comportamento Animal , Células CHO , Cricetinae , Cricetulus , Humanos , Transdução de Sinal Luminoso , Camundongos , Modelos Biológicos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Opsinas de Bastonetes/químicaRESUMO
Daily rhythms of physiological and behavioral processes such as sleep and arousal are controlled by the circadian clock. The expression of the clock genes oscillate rhythmically in daily manner, and this clock oscillator resides in almost all of the cells in the body. The circadian clock entrains to diurnal environmental changes by using light and food intake as external time cues. Timing of feeding and fasting strongly affects daily rhythms in the expression of circadian clock genes and key regulators of nutrient homeostasis. Understanding roles of the clock oscillator system and feeding-fasting cycles lets us recognize the importance of timing of feeding, ultimately to adjust the aging-related changes in circadian rhythms.
Assuntos
Envelhecimento , Ritmo Circadiano , Homeostase , Síndrome Metabólica/fisiopatologia , Animais , Comportamento Alimentar , Humanos , Obesidade/fisiopatologiaRESUMO
Mammals receive light information through the eyes, which perform two major functions: image forming vision to see objects and non-image forming adaptation of physiology and behavior to light. Cone and rod photoreceptors form images and send the information via retinal ganglion cells to the brain for image reconstruction. In contrast, nonimage-forming photoresponses vary widely from adjustment of pupil diameter to adaptation of the circadian clock. nonimage-forming responses are mediated by retinal ganglion cells expressing the photopigment melanopsin. Melanopsin-expressing cells constitute 1-2% of retinal ganglion cells in the adult mammalian retina, are intrinsically photosensitive, and integrate photic information from rods and cones to control nonimage-forming adaptation. Action spectra of ipRGCs and of melanopsin photopigment peak around 480 nm blue light. Understanding melanopsin function lets us recognize considerable physiological effects of blue light, which is increasingly important in our modern society that uses light-emitting diode. Misalignment of circadian rhythmicity is observed in numerous conditions, including aging, and is thought to be involved in the development of age-related disorders, such as depression, diabetes, hypertension, obesity, and cancer. The appropriate regulation of circadian rhythmicity by proper lighting is therefore essential. This perspective introduces the potential risks of excessive blue light for human health through circadian rhythm disruption and sleep deprivation. Knowing the positive and negative aspects, this study claims the importance of being exposed to light at optimal times and intensities during the day, based on the concept of the circadian clock, ultimately to improve quality of life to have a healthy and longer life.
RESUMO
Prevalence of myopia is increasing worldwide. Outdoor activity is one of the most important environmental factors for myopia control. Here we show that violet light (VL, 360-400nm wavelength) suppresses myopia progression. First, we confirmed that VL suppressed the axial length (AL) elongation in the chick myopia model. Expression microarray analyses revealed that myopia suppressive gene EGR1 was upregulated by VL exposure. VL exposure induced significantly higher upregulation of EGR1 in chick chorioretinal tissues than blue light under the same conditions. Next, we conducted clinical research retrospectively to compare the AL elongation among myopic children who wore eyeglasses (VL blocked) and two types of contact lenses (partially VL blocked and VL transmitting). The data showed the VL transmitting contact lenses suppressed myopia progression most. These results suggest that VL is one of the important outdoor environmental factors for myopia control. Since VL is apt to be excluded from our modern society due to the excessive UV protection, VL exposure can be a preventive strategy against myopia progression.
Assuntos
Luz , Miopia/diagnóstico , Miopia/terapia , Fototerapia , Adolescente , Animais , Linhagem Celular , Galinhas , Criança , Modelos Animais de Doenças , Progressão da Doença , Óculos , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Masculino , Miopia/etiologia , Refração Ocular , Luz Solar , Resultado do Tratamento , Raios UltravioletaRESUMO
Melanopsin photopigment expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) plays a crucial role in the adaptation of mammals to their ambient light environment through both image-forming and non-image-forming visual responses. The ipRGCs are structurally and functionally distinct from classical rod/cone photoreceptors and have unique properties, including single-photon response, long response latency, photon integration over time, and slow deactivation. We discovered that amino acid sequence features of melanopsin protein contribute to the functional properties of the ipRGCs. Phosphorylation of a cluster of Ser/Thr residues in the C-terminal cytoplasmic region of melanopsin contributes to deactivation, which in turn determines response latency and threshold sensitivity of the ipRGCs. The poorly conserved region distal to the phosphorylation cluster inhibits phosphorylation's functional role, thereby constituting a unique delayed deactivation mechanism. Concerted action of both regions sustains responses to dim light, allows for the integration of light over time, and results in precise signal duration.
Assuntos
Transdução de Sinal Luminoso/fisiologia , Células Ganglionares da Retina/fisiologia , Opsinas de Bastonetes/fisiologia , Animais , Células Cultivadas , Ritmo Circadiano/fisiologia , Locomoção/fisiologia , Camundongos , Mutação , Fosforilação , Estimulação Luminosa , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , XenopusRESUMO
Daily rhythms of many physiological and behavioral processes such as sleep and arousal are controlled by the circadian clock. The circadian clock entrains to environmental diurnal changes by using light and food intake as external time cues. Circadian photoentrainment is mediated by retinal ganglion cells expressing a blue-light sensitive photopigment mela- nopsin. Feeding and fasting drive daily rhythms in the expression of circadian clock genes and key regulators of nutrient homeostasis in peripheral tissues. Understanding melanopsin function and timing of feeding-fasting lets us recognize the importance of timing of blue light exposure and feeding based on the concept of the circadian clock, ultimately to adjust the age-related changes in daily rhythm.
Assuntos
Envelhecimento , Relógios Circadianos , Ritmo Circadiano , Sono , Envelhecimento/fisiologia , Animais , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Ingestão de Alimentos , Humanos , LuzRESUMO
Metabolism and physiology in animals show diurnal rhythm to adapt to the daily cycles of activity-rest and the associated rhythm in feeding and fasting. Accordingly, gene expression, protein activities, and numerous metabolites show daily rhythm in abundance. The significance of these rhythms in promoting healthy lifespan and preventing disease has recently come to light. Mice with genetic disruption of circadian rhythm, mice, and humans under shift-work paradigm, and mice fed high-fat diet ad libitum exhibit chronic disruption of feeding-fasting rhythm and dampened daily rhythms in physiology, metabolism, and gene expression. These dampened rhythms are associated with metabolic diseases. Conversely, time-restricted feeding, in which mice are fed for certain number of hours every day, restores rhythms and can prevent obesity and metabolic diseases even when mice are fed high-fat diet. These observations seek mechanistic explanations, which will require careful experiments in which feeding duration, genotype, nutrient, and feeding time relative to light:dark cycle will be manipulated and molecular changes in peripheral organs and a few brain regions will be assessed. This chapter will primarily focus on the use of mouse as an experimental animal and the experimental setup so that the molecular readouts can be better interpreted.
Assuntos
Ritmo Circadiano , Comportamento Alimentar , Animais , Dieta , Camundongos , Camundongos EndogâmicosRESUMO
The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.
Assuntos
Encéfalo/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Animais , Imageamento Tridimensional , Camundongos , Microscopia Eletrônica de Varredura/instrumentaçãoRESUMO
The robustness and limited plasticity of the master circadian clock in the suprachiasmatic nucleus (SCN) is attributed to strong intercellular communication among its constituent neurons. However, factors that specify this characteristic feature of the SCN are unknown. Here, we identified Lhx1 as a regulator of SCN coupling. A phase-shifting light pulse causes acute reduction in Lhx1 expression and of its target genes that participate in SCN coupling. Mice lacking Lhx1 in the SCN have intact circadian oscillators, but reduced levels of coupling factors. Consequently, the mice rapidly phase shift under a jet lag paradigm and their behavior rhythms gradually deteriorate under constant condition. Ex vivo recordings of the SCN from these mice showed rapid desynchronization of unit oscillators. Therefore, by regulating expression of genes mediating intercellular communication, Lhx1 imparts synchrony among SCN neurons and ensures consolidated rhythms of activity and rest that is resistant to photic noise.
Assuntos
Relógios Circadianos/genética , Síndrome do Jet Lag/genética , Proteínas com Homeodomínio LIM/genética , Neurônios/metabolismo , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/metabolismo , Fatores de Transcrição/genética , Animais , Comunicação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Síndrome do Jet Lag/metabolismo , Síndrome do Jet Lag/patologia , Proteínas com Homeodomínio LIM/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/patologia , Proteínas Circadianas Period/metabolismo , Fotoperíodo , Transdução de Sinais , Núcleo Supraquiasmático/patologia , Fatores de Transcrição/metabolismoRESUMO
Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image-forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several central nervous system responses, including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. New sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod- and cone-mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses, including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.
Assuntos
Transdução de Sinal Luminoso/efeitos dos fármacos , Opsinas de Bastonetes/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Humanos , Estrutura Molecular , Opsinas de Bastonetes/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
The central and peripheral nervous systems have the capacity of synthesizing steroids de novo from cholesterol, the so-called 'neurosteroids'. De novo synthesis of neurosteroids from cholesterol appears to be a conserved property across the subphylum vertebrata. Until recently, it was generally believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. However, our recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7α-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity of juvenile birds, connecting light-induced gene expression with locomotion. The other major pineal neurosteroid allopregnanolone is involved in Purkinje cell survival by suppressing the activity of caspase-3, a crucial mediator of apoptosis during cerebellar development. This review is an updated summary of the biosynthesis and biological actions of pineal neurosteroids.
Assuntos
Aves/fisiologia , Neurotransmissores/biossíntese , Neurotransmissores/fisiologia , Glândula Pineal/metabolismo , Animais , Animais Domésticos , Sobrevivência Celular/efeitos dos fármacos , Coturnix , Atividade Motora/efeitos dos fármacos , Neurotransmissores/farmacologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/fisiologiaRESUMO
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and ß-galactosidase, we demonstrated the lack of these SCN markers in all Six6-null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN.
Assuntos
Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/fisiologia , Transativadores/deficiência , Animais , Arginina Vasopressina/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fotoperíodo , Esqueleto , Núcleo Supraquiasmático/metabolismo , Transativadores/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , beta-Galactosidase/metabolismoRESUMO
De novo neurosteroidogenesis from cholesterol occurs in the brain of various avian species. However, the biosynthetic pathways leading to the formation of neurosteroids are still not completely elucidated. We have recently found that the avian brain produces 7α-hydroxypregnenolone, a novel bioactive neurosteroid that stimulates locomotor activity. Until recently, it was believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. However, our recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7α-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity of juvenile birds, connecting light-induced gene expression with locomotion. The other major pineal neurosteroid allopregnanolone is involved in Purkinje cell survival during development. This paper highlights new aspects of neurosteroid synthesis and actions in birds.
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Chronic sleep deprivation perturbs the circadian clock and increases susceptibility to diseases such as diabetes, obesity, and cancer. Increased inflammation is one of the common underlying mechanisms of these diseases, thus raising a hypothesis that circadian-oscillator components may regulate immune response. Here we show that absence of the core clock component protein cryptochrome (CRY) leads to constitutive elevation of proinflammatory cytokines in a cell-autonomous manner. We observed a constitutive NF-κB and protein kinase A (PKA) signaling activation in Cry1(-/-);Cry2(-/-) cells. We further demonstrate that increased phosphorylation of p65 at S276 residue in Cry1(-/-);Cry2(-/-) cells is due to increased PKA signaling activity, likely induced by a significantly high basal level of cAMP, which we detected in these cells. In addition, we report that CRY1 binds to adenylyl cyclase and limits cAMP production. Based on these data, we propose that absence of CRY protein(s) might release its (their) inhibition on cAMP production, resulting in elevated cAMP and increased PKA activation, subsequently leading to NF-κB activation through phosphorylation of p65 at S276. These results offer a mechanistic framework for understanding the link between circadian rhythm disruption and increased susceptibility to chronic inflammatory diseases.
Assuntos
Ritmo Circadiano , Criptocromos/metabolismo , Citocinas/biossíntese , Regulação da Expressão Gênica , Adenilil Ciclases/genética , Adenilil Ciclases/imunologia , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , Criptocromos/genética , Criptocromos/imunologia , AMP Cíclico/genética , AMP Cíclico/imunologia , AMP Cíclico/metabolismo , Citocinas/genética , Citocinas/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismoRESUMO
While diet-induced obesity has been exclusively attributed to increased caloric intake from fat, animals fed a high-fat diet (HFD) ad libitum (ad lib) eat frequently throughout day and night, disrupting the normal feeding cycle. To test whether obesity and metabolic diseases result from HFD or disruption of metabolic cycles, we subjected mice to either ad lib or time-restricted feeding (tRF) of a HFD for 8 hr per day. Mice under tRF consume equivalent calories from HFD as those with ad lib access yet are protected against obesity, hyperinsulinemia, hepatic steatosis, and inflammation and have improved motor coordination. The tRF regimen improved CREB, mTOR, and AMPK pathway function and oscillations of the circadian clock and their target genes' expression. These changes in catabolic and anabolic pathways altered liver metabolome and improved nutrient utilization and energy expenditure. We demonstrate in mice that tRF regimen is a nonpharmacological strategy against obesity and associated diseases.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos , Ingestão de Energia , Doenças Metabólicas/prevenção & controle , Adenilato Quinase/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/metabolismo , Adiposidade , Animais , Ácidos e Sais Biliares/biossíntese , Colesterol/sangue , Ritmo Circadiano , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas/genética , Citocinas/metabolismo , Metabolismo Energético , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Expressão Gênica , Glucose/metabolismo , Homeostase , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio , Fosforilação , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Aumento de PesoRESUMO
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-α and REV-ERB-ß 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-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-ß genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-ß cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-ß 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-α and REV-ERB-ß 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.