CIRCADIAN CLOCK

The Dana Foundation, Cerebrum
The Time of Your Life

Each morning we wake up from a night of sleep, and each day we eat our regularly timed meals, go through our normal routines, and fall asleep again for another night. This rhythm, so-called circadian—after the Latin words circa diem (“about a day”) —underlies a wide variety of human physiological functions, including sleep-wake cycles, body temperature, hormone secretion, locomotor activity, and feeding behavior. 

The Cyclic AMP Pathway, CREM and Melatonin Synthesis

A key question in the field of transcription is how extracellular signals, elicited for example by hormones and growth factors, lead to modulation in gene expression. The second messenger cAMP is critical in a large variety of neuronal, endocrine and metabolic responses. We discovered and characterized the CREM gene whose complex structure allows remarkable flexibility so as to generate a variety of transcription factors with multiple functions (Foulkes et al. 1991; de Groot et al. 1994). Among these, the CREM gene leads to the dynamic synthesis of an inducible cAMP early repressor (ICER) that is responsible for the transcriptional attenuation of cAMP-dependent early activation of many genes (Molina et al. 1993; Stehle et al. 1993). ICER protein stability is timed so to allow for a new transcriptional activation cycle, generating a transcriptional-translational feedback loop that controls several genes, with implications to neuronal, hormonal and endocrine responses (Foulkes et al. 1996). Among the genes controlled by the CREM-ICER couple, there is the one encoding the 5HT N-acetyltransferase, the rate-limiting enzyme in the melatonin synthesis pathway in the pineal gland (Foulkes et al. 1996a; 1996b). Thus, the CREM transcription factor modulates the oscillatory levels of the circadian hormone melatonin. 

A network of clocks and their interplay
The central clock in the suprachiasmatic nucleus (SCN) controls various endocrine and metabolic functions. Neurons of the SCN undergo oscillations in depolarization and gene expression. The SCN indirectly controls oscillations of humoral factors coming from other tissues, such as the pineal and adrenal cortex. Other tissues also maintain circadian output through positive and negative feedback loops within cells that make up different compartments of the tissue. Oscillations in humoral factors control the circadian release of factors from the periphery, such as ghrelin, leptin, insulin and glucose, and these in turn provide positive and negative feedback to the brain. Melatonin, which is released in a circadian fashion from the pineal gland, is involved in feed- back regulation of the SCN, where melatonin receptors are abundantly expressed. Thus, the periphery may influence brain functions, and specifically SCN neurons, through yet undefined feedback mechanisms. Additional elements, such as food intake and exercise, may uncouple peripheral tissues from the central clock. Indeed, time-restricted feeding (TRF) experiments, as well as time-specific exercise, have been shown to have fundamentally distinct metabolic effects on peripheral clocks.  

Peripheral Circadian Oscillators

Our studies on cAMP-dependent transcription lead us to discover the mechanism controlling circadian melatonin synthesis and introduced us to the field of circadian biology. Various contributions include the elucidation of signaling and transcriptional pathways and the discovery of key clock proteins post-translational modifications. By establishing the zebrafish as a valuable model for the study of circadian rhythms, we discovered the presence of peripheral oscillators that operate by using an intrinsic clock that is independent from the neuronal pacemaker (Whitmore et al. 1998). We went on to discover that peripheral organs and cells directly sense light and to decipher the signaling pathways involved in light transduction. These features were recapitulated in a cell-based system in culture (Whitmore et al. 2000; Pando et al. 2001). Based on these concepts, we explored whether in mammals the presence of independent clocks may be illustrated by applying cell grafting. Using a tissue implant methodology we uncovered the hierarchical organization of the circadian system in the mouse (Pando et al. 2002). These studies challenged the common knowledge of the field that for decades had accepted the view of a unique brain-based clock (Schibler and Sassone-Corsi 2002). The impact of these findings is extensive because of its implications in systems biology, endocrine control and physiology.

Molecular Features of the Circadian Clock 
The transcriptional-translational feedback loop (TTFL) of the circadian clock is elaborate, with external loops and posttranslational modifications, such as phosphorylation contributing to maintenance of the core oscillatory players. The CLOCK:BMAL1 activators bind E-box elements on promoters of clock-controlled genes (CCGs). The E-box is most common promoter element on the genome. Post-translational modifications of CRY and PER proteins play a critical role in their stability and degradation. Phosphorylation has been shown to be regulating the negative-feedback potential of these proteins on the CLOCK:BMAL1 complex. The transcription of Bmal1 is negatively regulated by the product of one of its own gene targets, Rev-erba, the process of which controls the amount of BMAL1 protein available for CLOCK binding. These interlocked loops control the expression of thousand genes, leading to clock outputs that govern a large array of physiological, metabolic and behavioral functions. 

The Circadian Clock Links Metabolism to Epigenetics

The circadian system transcriptionally controls a significant fraction of the genome, suggesting a role for the clock in chromatin remodeling. One features of clock proteins is their capacity of being dynamically modified post-translationally. Our laboratory has contributed to the field by illustrating that BMAL1 is for example phosphorylated (Tamaru et al. 2009), acetylated (Hirayama et al. 2007) and SUMOylated (Cardone et al. 2005). 

We first uncovered that indeed clock function is associated with chromatin remodeling (Crosio et al. 2000), and then went on to elucidate the molecular mechanisms by revealing the histone acetyltransferase function of the regulator CLOCK (Doi et al. Cell 2006). We then discovered the implication of the NAD+-dependent deacetylase SIRT1, which established a direct link of the clock with cellular metabolism and histone modifications (Nakahata et al 2008). This led to the discovery that SIRT1 enzymatic activity is cyclic because the NAD+levels oscillate through a direct control by the clock of the Namptgene. This gene encodes the nicotinamide (NAM) phosphoribosyltransferaseenzyme, which controls rate-limiting spet of the NAD+salvage pathway. Thus, the circadian clock links transcriptional regulation to enzymatic control (Nakahata et al. 2009). The implications of this finding are multiple as NAD+operates as a central element in energy metabolism and functions as coenzyme for several NAD+-consuming enzymes. Further studies demonstrated that SIRT1 is involved also in the control of Acetyl-CoA synthesis through the cyclic acetylation of the acetyl-coenzyme A synthase I enzyme (Sahar et al. 2014) and that another sirtuin, SIRT6, contributes to the partitioning of the circadian epigenome in a manner that is distinct from SIRT1 (Masri et al. 2014).

These findings prompted further studies by MS metabolomics to determine the oscillating metabolome in various tissues and to generate, in collaboration with the Institute for Genomics and Bioinformatics at UCI (https://www.igb.uci.edu/) a Biocomputing resource that integrates and links circadian metabolomics to transcriptomics (CircadiOmics; http://circadiomics.igb.uci.edu/) (Patel et al. 2012; Eckel-Mahan et al. 2012). This high-throughput metabolome approach, coupled with transcriptomics and epigenomics, has been instrumental to uncover the impact of nutrition, and other physiological regimes. Our findings have revealed that the clock can be reprogrammed through regulatory circuits that link cellular metabolism to epigenetic control.

Reprogramming of the Circadian Clock

We have revealed previously unforeseen pathways of circadian control that connect to nutrition, cancer and aging. Accumulating evidence shows that time of food is critical in the control of circadian metabolism (Asher and Sassone-Corsi 2005). These studies provide new leads towards therapeutic strategies for metabolic disorders.

We have revealed how nutritional challenges reprogram circadian homeostasis whether these are in the form of high-fat diet (Eckel-Mahan et al. 2013), ketogenic diet (Tognini et al. 2017) and fasting (Kinouchi et al. 2018). These studies have particular relevance with aging as we have demonstrated that claroic restriction connects directly with acetylation pathways in the control of liver metabolism (Sato et al. 2017). Importantly, analysis of the circadian acetylome had already pointed to unique metabolic pathways in energy metabolism (Masri et al. 2013). Reprogramming is also evident in response to time-specific exercise in both liver and muscle tissues (Sato et al. 2019). These studies provide new leads towards therapeutic strategies for metabolic disorders.

Linking the Clock to the NAD+ Salvage Pathway
The sirtuin SIRT1 links circadian rhythmicity to metabolism. The SIRT1:CLOCK:BMAL1 complex drives expression of Nampt, the rate-limiting enzyme in the salvage pathway for SIRT1’s own cofactor, NAD+. The two loops depicted in the figure demonstrate the mechanisms of both the transcriptional feedback circuit as well as the enzymatic feedback circuit. 

Communications of Circadian Clocks

The mammalian circadian clock system orchestrates daily rhythms in behavior and physiology, allowing animals to anticipate environmental changes and synchronize their internal processes accordingly. The discovery of peripheral oscillators nearly 20 years ago changed the perspective of the field; until then, the central oscillator, localized in suprachiasmatic nucleus (SCN) neurons, was considered the sole pacemaker directing all cyclic physiology and behavior. Subsequently, the concept of a ‘web of pacemakers’ has been largely adopted, where the SCN controls the tempo of oscillators in peripheral tissues and organs (Schibler and Sassone-Corsi, 2002). Additional work has also demonstrated the presence in the ventromedial hypothalamus of clock cells that operate independently of the SCN to control energy expenditure (Orozco-Solis et al. 2016).

Peripheral clocks respond to signals emanating from the SCN, which responds to light, through yet ill-defined pathways. By regulating the sleep-wake cycle, the SCN clock also indirectly evokes the feeding-fasting cycle, a synchronizer and driver of cycling transcripts in the periphery.These notions prompted the question of whether peripheral clocks may communicate with each other. Recent findings demonstrated system-wide metabolic coordination between mammalian clocks (Dyar et al., 2018).Dysregulation of clock function may also be a hallmark of various pathological states, as in the case of lung adenocarcinoma, which distally rewires circadian function in the liver (Masri et al., 2016). Identifying how peripheral clocks interplay will provide critical information on circadian physiology as well as clock disruption and disease.

Among peripheral clocks, the liver is central in controlling cyclic metabolism and adapts remarkably to changes in nutritional regimes. Liver-specific clock deficiency causes loss of circadian transcription and ultimately loss of oscillation in key glucose, lipid and oxidative pathways. Nevertheless, some cyclic transcripts persist in the absence of a functioning clock, suggesting alternative mechanisms contributing to fluctuations in the liver.

The presence of a circadian clock in virtually all cells begs the question of its dependence on external cyclic signals. The molecular clock consists of an oscillator, based on interlocked transcriptional-translational feedback loops. Rhythmic post-translational modification of clock proteins and daily chromatin remodeling also contribute to this canonical feedback loop. The extent to which this complex regulatory circuit is tissue-autonomous or requires external cues from other clocks remains unclear. 

Tissue-specific clock ablation has been instrumental in identifying the functions of peripheral clocks, however they have not allowed assessment of their degree of autonomy. In collaboration with the laboratory of Salvador Aznar Benitah (Barcelona) (Welz et al. 2019) we have developed a mouse model in which the liver clock is reconstituted in an otherwise BMAL1-deficient animal (Liver-RE) (Koronowski et al. 2019). This model is a tool to study whether and to what extent a peripheral clock operates independently from all other clocks. We demonstrate that the liver is intrinsically capable of clock function even in absence of functioning clocks in all other tissues. The independence of the liver clock illustrates a degree of autonomy at the tissue level, limited to a specific set of genes and metabolic pathways. Remarkably, the genome-wide capacity and specificity of reconstituted BMAL1 to bind chromatin is comparable to that of wild type mice, demonstrating that external clock-dependent inputs are required to elicit a full circadian program. Lastly, lack of circadian rhythms in liver-RE mice maintained under constant darkness reveals a potentially critical regulatory role of the light-dark cycle on tissue-autonomous function.


References

Asher G and Sassone-Corsi P. (2015) Time for Food: the intimate interplay between nutrition, metabolism and the circadian clock. Cell 161: 84-92.

Cardone, L., Hirayama, J., Giordano, F., Tamaru, T., Palvimo, J. J., Sassone-Corsi, P. (2005) Circadian clock control by SUMOylation of BMAL1. Science 309: 1390-1394.

Crosio C, Cermakian N, Allis CD, Sassone-Corsi P. (2000) Light induces chromatin modification in cells of the mammalian circadian clock. Nature Neurosci. 3: 1241-7.

de Groot, R. P., Ballou, L., Sassone-Corsi, P. (1994) Positive regulation of the cAMP-responsive activator CREM by the p70 S6 kinase: an alternative route to mitogen-induced gene expression. Cell 79: 81-91.

Doi M, Hirayama J, Sassone-Corsi P. (2006) Circadian regulator clock is a histone acetyltransferase. Cell 5: 497-508

Dyar KA, Lutter D, Artati A, Ceglia NJ, Liu Y, Armenta D, Jastroch M, Schneider S, de Mateo S, Cervantes M, Abbondante S, Tognini P, Orozco-Solis R, Kinouchi K, Wang C, Swerdloff R, Nadeef S, Masri S, Magistretti P, Orlando V, Borrelli E, Uhlenhaut NH, Baldi P, Adamski J, Tschöp MH, Eckel-Mahan K, Sassone-Corsi P. (2018) Atlas of circadian metabolism reveals system-wide coordination and communication between clocks. Cell 174: 1571-1585

Eckel-Mahan, K. L., Patel, V. R., de Mateo, S., Orozco-Solis, R., Ceglia, N. J., Sahar, S., Dilag-Penilla, S. A., Dyar, K. A., Baldi, P., Sassone-Corsi, P. (2013) Reprogramming of the circadian clock by nutritional challenge. Cell 155: 1464-78.

Eckel-Mahan KL, Patel VR, Mohney RP, Vignola KS, Baldi P, Sassone-Corsi P. (2012) Coordination of the transcriptome and metabolome by the circadian clock.Proc. Natl. Acad. Sci. USA 109: 5541-6

Foulkes NS, Borjigin J, Snyder SH, Sassone-Corsi P. (1997) Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Trends Neurosci. 20: 487-92.

Foulkes NS, Borjigin J, Snyder SH, Sassone-Corsi P. (1996) Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc. Natl. Acad. Sci. USA 93: 14140-5.

Foulkes NS, Duval G, Sassone-Corsi P. (1996) Adaptive inducibility of CREM as transcriptional memory of circadian rhythms. Nature 381: 83-5.

Foulkes, N. S, Borrelli, E., Sassone-Corsi, P. (1991) CREM gene: Use of alternative DNA binding domains generates multiple antagonists of cAMP-induced transcription. Cell 64: 739-749.

Hirayama J, Sahar S, Grimaldi B, Tamaru T, Takamatsu K, Nakahata Y, Sassone-Corsi P. (2007) CLOCK-mediated acetylation of BMAL1 controls circadian function. Nature 450:1086-90.

Kinouchi K, Magnan C, Ceglia N, Liu Y, Cervantes M, Pastore N, Huynh T, Ballabio A, Baldi P, Masri S, Sassone-Corsi P. (2018) Fasting Imparts a Switch to Alternative Daily Pathways in Liver and Muscle. Cell Rep. 25: 3299-3314.

Koronowski KB, Kinouchi K, Welz PS, Smith JG, Zinna VM, Shi J, Samad M, Chen S, Magnan CN, Kinchen JM, Li W, Baldi P, Benitah SA, Sassone-Corsi P. (2019) Defining the Independence of the Liver Circadian Clock. Cell 177: 1448-1462

Masri S., Papagiannakopoulos T., Kinouchi K., Liu Y., Cervantes M., Baldi P., Jacks T. and Sassone-Corsi P. (2016) Lung adenocarcinoma distally rewires hepatic circadian homeostasis. Cell 165: 896-909.

Masri, S., Rigor, P., Cervantes, M., Ceglia, N., Sebastian, C., Xiao, C., Roqueta-Rivera, M., Deng, C., Osborne, T. F., Mostoslavsky, R., Baldi, P., Sassone-Corsi, P. (2014) Partitioning circadian transcription by SIRT6 leads to segregated control of cellular metabolism. Cell 158: 659-672.  

Masri S, Patel VR, Eckel-Mahan KL, Peleg S, Forne I, Ladurner AG, Baldi P, Imhof A, Sassone-CorsiP. (2013) Circadian acetylome reveals regulation of mitochondrial metabolic pathways. Proc. Natl. Acad. Sci. USA 110: 3339-44.

Molina, C. A., Foulkes, N. S., Lalli, E., Sassone-Corsi, P. (1993) Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75: 875-886.

Nakahata, Y., Sahar, S., Astarita, G., Kaluzova, M., Sassone-Corsi, P. (2009) Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1. Science 324: 654-657.  

Nakahata, Y., Kaluzova, M., Grimaldi, B., Sahar, S., Hirayama, J., Chen, D., Guarente, L., Sassone-Corsi, P. (2008) The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control. Cell 134: 329-340.  

Orozco-Solis R., Aguilar-Arnal L., Murakami M., Peruquetti R., Ramadori G., Coppari R. and Sassone-Corsi P. (2016) The circadian clock in the ventromedial hypothalamus controls cyclic energy expenditure. Cell Metab. 23: 467-478.

Pando, M. P., Morse, D., Cermakian, N., Sassone-Corsi, P. (2002) Phenotypic rescue of a peripheral clock genetic defect via SCN hierarchical dominance. Cell 110: 107-17.

Pando MP, Pinchak AB, Cermakian N, Sassone-Corsi P. (2001) A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock. Proc. Natl. Acad. Sci. USA 98: 10178-83

Patel VR, Eckel-Mahan K, Sassone-Corsi P, Baldi P. (2012) CircadiOmics: integrating circadian genomics, transcriptomics, proteomics and metabolomics. Nat Methods 9: 772-3. 

Sahar S, Masubuchi S, Eckel-Mahan K, Vollmer S, Galla L, Ceglia N, Masri S, Barth TK, Grimaldi B, Oluyemi O, Astarita G, Hallows WC, Piomelli D, Imhof A, Baldi P, Denu JM, Sassone-Corsi P. (2014) Circadian control of fatty acid elongation by SIRT1 protein-mediated deacetylation of acetyl-coenzyme A synthetase 1. J. Biol. Chem. 289: 6091-7.

Sato S, Solanas G, Peixoto FO, Bee L, Symeonidi A, Schmidt MS, Brenner C, Masri S, Benitah SA, Sassone-Corsi P. (2017) Circadian Reprogramming in the Liver Identifies Metabolic Pathways of Aging. Cell 170: 664-677

Sato S, Basse AL, Schönke M, Chen S, Samad M, Altıntaş A, Laker RC, Dalbram E, Barrès R, Baldi P, Treebak JT, Zierath JR, Sassone-Corsi P. (2019) Time of Exercise Specifies the Impact on Muscle Metabolic Pathways and Systemic Energy Homeostasis.Cell Metab. 30: 92-110

Schibler U and Sassone-Corsi, P. (2002). A web of circadian pacemakers. Cell 111: 919-922

Stehle, J. H., Foulkes, N. S., Molina, C. A., Simonneaux, V., Pévet, P., Sassone-Corsi, P. (1993) Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 365: 314-320.

Tamaru T, Hirayama J, Isojima Y, Nagai K, Norioka S, Takamatsu K, Sassone-CorsiP. (2009) CK2alpha phosphorylates BMAL1 to regulate the mammalian clock. Nat. Struct. Mol. Biol. 16: 446-8.

Tognini P, Murakami M, Liu Y, Eckel-Mahan KL, Newman JC, Verdin E, Baldi P, Sassone-Corsi P. (2017) Distinct Circadian Signatures in Liver and Gut Clocks Revealed by Ketogenic Diet. Cell Metab. 26: 523-5

Welz PS, Zinna VM, Symeonidi A, Koronowski KB, Kinouchi K, Smith JG, Guillén IM, Castellanos A, Crainiciuc G, Prats N, Caballero JM, Hidalgo A, Sassone-Corsi P, Benitah SA. (2019) BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis. Cell 177: 1436-1447

Whitmore, D., Foulkes, N. S., Strähle, U., Sassone-Corsi, P. (1998) Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators. Nature Neuroscience 1: 701-708.

Whitmore, D., Foulkes, N. S., Sassone-Corsi, P. (2000) Light acts directly on organ cells in culture to set the vertebrate circadian clock. Nature 404: 87-91.