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Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms

Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms Bokai Zhu, Clifford C Dacso and Bert W O’Malley Journal of the Endocrine Society Endocrine Society Submitted: April 19, 2018 Accepted: June 01, 2018 First Online: June 06, 2018 Advance Articles are PDF versions of manuscripts that have been peer reviewed and accepted but not yet copyedited. The manuscripts are published online as soon as possible after acceptance and before the copyedited, typeset articles are published. They are posted "as is" (i.e., as submitted by the authors at the modification stage), and do not reflect editorial changes. No corrections/changes to the PDF manuscripts are accepted. Accordingly, there likely will be differences between the Advance Article manuscripts and the final, typeset articles. 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Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms 1 1,2,3 1,3 Bokai Zhu , Clifford C Dacso and Bert W O’Malley Department of Molecular and Cellular Biology Department of Medicine Dan L. Duncan Cancer Center Baylor College of Medicine, Houston, TX 77030, USA Received 19 April 2018. Accepted 01 June 2018. Musica Universalis is an ancient philosophical concept claiming the movements of celestial bodies follow mathematical equations and resonate to produce an inaudible harmony of music, and the harmonious sounds that men make were an approximation of this larger harmony of the universe (1). Besides music, electromagnetic waves such as light and electric signals also are presented as harmonic resonances. Despite the seemingly universal theme of harmonic resonance in various disciplines, it was not until recently that the same harmonic resonance was discovered also to exist in biological systems. Contrary to traditional belief that a biological system is either at steady state or cycles with a single frequency, it is now appreciated that most biological systems have no homeostatic “set point”, but rather oscillate as composite rhythms consisting of superimposed oscillations (2). These oscillations often cycle at different harmonics of the circadian rhythm, and among these, the ~12h oscillation is most prevalent (2). In this mini- review, we focus on these 12h oscillations, with special attention to their evolutionary origin, regulation and functions in mammals, as well as their relationship to the circadian rhythm. We further examine the potential roles of the 12h-clock in regulating hepatic steatosis, aging and the possibility of 12h-clock-based chronotherapy. Finally, we posit that biological rhythms are also “Musica Universalis”: while the circadian rhythm is synchronized to the 24h light/dark cycle coinciding with the Earth’s rotation, the mammalian 12h-clock may have evolved from the circatidal clock, which is entrained by the 12h tidal cues orchestrated by the moon. we focus on these 12h oscillations, with special attention to their evolutionary origin, regulation and functions in mammals, as well as their relationship to the circadian rhythm. . Search methods: For the search related to circatidal rhythms in coastal and estuarine animals, the keywords of circatidal and/or tidal rhythms were used and literatures between 1950 and present were reviewed. For reported ~12h rhythms in humans, the keywords of circasemidian and/or 12h rhythm were searched against literatures published between 1950 and present. For the 12h rhythms in mouse, due to the very limited number of publications on this topic, the authors are very acquainted with the handful of literatures and therefore no search was performed. Main text: The concept of harmonics in physics and their biological counterpart The term “harmonic” originates in musical instrument theory, where the wavelengths of the overtones of a vibrating string are derived from its fundamental wavelength. A harmonic of such Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 a wave has a frequency that is a positive integer multiple of this fundamental frequency, which is st st also called the 1 harmonic (3,4). For instance, as shown in Fig. 1A, the fundamental, or the 1 nd harmonic is a wave with a frequency of 200Hz, whereas the 2 harmonic is 400Hz (200Hz x 2=400Hz), and so on. Summing the harmonics generates a composite waveform that looks “distorted and noisy” (Fig. 1A bottom) but is actually much closer to perception of music than the individual harmonics. This is because pure oscillation with a single frequency rarely exists in nature. Rather, physical rhythms often present as superpositions of basic waves such as the harmonic resonances found in music, light and Kepler’s Law of planetary motion. In order to uncover all superimposed oscillations in an unbiased manner, we utilized a recently developed eigenvalue/pencil method (2,5-7) and disclosed that just like other forms of physical oscillations, biological oscillations also are composite rhythms consisting of oscillations of various frequencies (2). The eigenvalue/pencil method assumes that any oscillation can be approximated by a linear combination of exponentials (sine waves with a decay factor) plus noise and therefore can be applied to any situations where such assumptions are valid. Furthermore, unlike most of the current cycling transcripts-identification methodologies that require the user to define a narrow period range (8), the eigenvalue/pencil method does not pre- assign a period and thus permits the identification of all superimposed oscillations in an unbiased manner (2) [for a more detailed understanding of the strengths and limits of the eigenvalue/pencil method and its wider applications, please refer to (5)]. Oscillations disclosed by this analysis often cycle at the harmonics of the circadian rhythm (2). For instance, comprehensive analysis of a published mouse liver microarray dataset employing the eigenvalue/pencil method (where liver samples were taken every hour for a total of 48 hours under constant darkness condition) revealed that the majority of oscillations cycle at periods close to 24h, 12h, 8h and 4h, st nd rd th corresponding to the 1 , 2 , 3 and 6 harmonics of the circadian rhythm (Fig. 1B) (2,9). While the ~4h oscillations are near the Nyquist limit of sampling frequency and therefore may arise from technical artifacts (10), oscillations cycling with 12h and 8h periods are believed to be “real” biological oscillations (2). One example is glucokinase (Gck), which encodes for a rate- limiting enzyme involved in both glycogenesis and glycolysis. While Gck mRNA expression was originally thought to be solely under the circadian clock control (11), the new analysis revealed that it actually comprises of superimposed major oscillations cycling at 24h, 12h and 8h periods with comparable amplitudes (Fig. 1C) (2). Similar to gene expression oscillations, mammalian metabolic rhythms also are composite oscillations cycling at different harmonics of the circadian rhythm. Analyzing 1h resolution mouse hepatic metabolites profiling dataset (12) revealed that the majority of metabolites consist of superimposed oscillations cycling at 24h, 12h, 8h and 6h periods (Fig. 1D). This is consistent with the observed 24h, 12h and 8h harmonic oscillations of respiratory exchange ratio (RER) through indirect calorimetry measurement of energy expenditure for mice fed ad libitum (2). Taken together, these unbiased analyses strongly indicate that as proposed thousands of years ago, harmonic resonance is a common ‘universal’ theme that permeates from the physical world all the way to biological systems. The circatidal rhythm: the best characterized ~12h cycling oscillation The wide prevalence of 12h gene expression and metabolic rhythms observed in mouse liver is intriguing. In fact, more than 20% of all mouse hepatic mRNA and metabolites cycle with a ~12h period, of which 4.2% and 11.5% are dominant oscillations (~12h cycling amplitudes are the largest among all superimposed oscillations), respectively (2). The prevalent mammalian 12h Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 rhythm of gene expression and metabolism is reminiscent of the ~12h circatidal rhythms of coastal and estuarine animals that modulate their behavior in tune to the ~12.4 hour ebb and flow of the tides (13). Prominent examples include numerous crustaceans such as fiddler crab (Uca pugnax) (14,15), mudflat crab (Chiromantes haematocheir) (16), green shore crab (Carcinus maenas) (17,18), sea louse (Eurydice pulchra) (13,19,20); Dimorphostylis asiatica (21); Chelicerata horseshoe crab (Limulus polyphemus) (22); ragworm (Alitta virens) (23), Mollusca (24-27) and intertidal insects like the Asian mangrove cricket (Apteronemobius asahinai) (28,29). These animals either emerge at low tides to forage, mate and fight (such as Uca pugnax and Apteronemobius asahinai) or are more active during high tides when the incoming tide cover their forage and mating grounds (such as Carcinus maenas and Eurydice pulchra) (13). More importantly, when removed to the laboratory and divorced from tidal cues, they can still maintain their circatidal locomotive activities at times of expected low water or high water under constant conditions (13-15,17,24,28,29). Further, their circatidal rhythms also can be entrained by artificial vibrations or inundation cycles (20,24,28). This free running behavior combined with entrainability indicates the presence of circatidal clocks in these animals, which under natural conditions are synchronized to the phase of the tidal cycle encountered on their home environment (13). So how are these circatidal rhythms generated? Two mechanisms underlying tidal rhythms have been proposed. Some argue that a circatidal clock might be generated by two slightly longer (24.8-hour) circadian clocks acting anti-phase to produce 12.4h peaks in behaviors such as swimming and foraging (30). The second school of thought advocates that a dedicated circatidal clock with a period of ~12.4h is the basis for circatidal behavior. Superimposed on this tidal clock is a circadian oscillator that drives the day/night modulation observed in locomotor output of crustaceans and crickets (31). One of the most convincing studies supporting the latter hypothesis was published in Current Biology in 2013 (20). In this elegant study, Zhang et al. used two orthogonal approaches (RNAi against circadian clock genes and bright constant light to disrupt the circadian clock) to abolish the circadian clock in Eurydice pulchra and tested their effects on the circatidal swimming rhythms of E. pulchra. While both approaches disrupted the circadian clock as expected, neither influenced the tidal periodicity (20). Additional studies performed on the American horseshoe crab Limulus polyphemes and mangrove cricket (Apteronemobius asahinai) found similar results (31-34). Collectively, these studies convincingly demonstrate the existence of a dedicated circatidal pacemaker in marine animals that is distinct from the circadian clock and responsible for the establishment of circatidal rhythms. The prevalence of mammalian 12h rhythms The first study that reported the presence of circadian harmonics of gene expression in mammalian systems, including 12h rhythms, was published by John Hogenesch’s group (9). By comprehensively profiling temporal gene expression in mouse liver from animals maintained at constant darkness at high resolution (at 1h intervals for a total of 48h) using DNA microarray, the authors identified ~200 genes that cycle with a dominant 12h period using two statistical methods: Fisher’s G-test and COSOPT (9). Ingenuity pathway analysis of these 12h-cycling hepatic genes revealed enriched gene ontology terms including protein processing and endoplasmic reticulum (ER) homeostasis (9). Contrary to the evenly distributed phases (the time of peak) of circadian genes, the acrophases of 12h-cycling hepatic genes are more restricted to the dawn (CT0) and dusk (CT12) of a diurnal cycle, even though the underlying reason was then Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 unknown (9) [CT stands for circadian time. By convention, the onset of activity of diurnal organisms defines circadian time zero (CT 0), while the onset of activity of nocturnal organisms defines circadian time twelve (CT 12). Therefore, for nocturnal animals such as mice, they are active from CT12 to CT24]. In addition to the liver, they also detected 12h-cycling transcripts in many different tissues. For example, Hspa1b, a heat shock protein 70kD family member, showed clear 12h transcriptional rhythms in lung, kidney, adrenal gland, heart and even in hypothalamus of the central nervous system (CNS), indicating that the presence of circadian harmonics is not restricted to the liver (9). In addition, in all these tissues, the phases of peak Hspa1b expression are nearly identical, again at dawn and dusk. Since then, a number of studies have confirmed the presence of these 12h rhythms of gene expression, especially in mouse liver, using different cycling-transcripts identification methods (35-38), even though the total repertoire of identified 12h-cycling transcripts remained small. We reasoned that the ~200 originally identified 12h-cycling transcripts were a significant underestimation of the true prevalence of mammalian 12h rhythms of gene expression. Since the COSOPT/ Fisher’s G-test methods used to identify 12h-cycling transcripts in the original study require the user to pre-assign a period range, it favors the detection of genes with dominant 12h rhythms and is thereby biased against other 12h-cycling transcripts having superimposed oscillations with larger amplitudes. As expected, when applying the “eigenvalue/pencil” method on the same mouse microarray dataset, we identified more than 3,000 hepatic genes (approximately 20% of all hepatic transcripts) with 12h rhythms (2). As expected, the majority of them have superimposed 24h circadian rhythms and therefore evade detection by traditional methods. Among all ~3,000 genes, 760 of them are dominant 12h-cycling genes (2). Gene ontology analysis of either all or dominant 12h-cycling genes revealed that, in addition to the expected ER homeostasis and protein quality control pathways, various metabolism-related biological pathways are also highly enriched (2). While ER-related genes (such as Eif2ak3, Gfpt1 and Sec23b) have dominant 12h oscillations and lack superimposed circadian rhythms, 12 h- cycling metabolism genes often have superimposed circadian rhythms (including Gck, Acly and Fasn) (2). In addition to the wide-spread 12h cycling mature mRNAs in mouse liver, prevalent 12h- cycling hepatic protein (~15% of hepatic proteome) also was observed from a time series mass spectrometry dataset (2,39). A limited ~35% overlap with 12h cycling mRNAs indicates that both transcriptional and post-transcriptional controls contribute to the regulation of 12h rhythms of hepatic gene expression (2). GO analysis of 12h cycling proteins revealed strong enrichment of ER, and intriguingly, mitochondria-associated metabolism pathways, implicating potential coordinated actions of ER and mitochondria in maintaining systemic metabolic homeostasis and stress response (2). While unlike mature mRNA, the phases of 12h protein oscillations are distributed evenly throughout the diurnal cycle, and a clear phase separation of proteins involved in opposing metabolic pathways was found (2). The prevalent hepatic 12h cycling mRNA and proteins involved in metabolic regulation predict a 12h rhythm for hepatic metabolites, which is confirmed by post hoc analysis of two recently published metabolomics datasets (40,41). Eigenvalue/pencil analysis revealed that more than 20% of hepatic metabolites profiled in these two studies exhibited 12h oscillations (2). Further, joint pathway analysis using MetaboAnalyst (42,43) showed coordinated 12h rhythms of metabolites and gene expressions (Fig. 2A, B) (2). For instance, top enriched KEGG pathways exhibiting 12h rhythms of paired gene expression and metabolites include amino sugar and nucleotide sugar metabolism (Gfpt1/Gale: UDP-glucose/UDP-N-acetylamino sugars), Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 pyrimidine metabolism (Uck2: cytidine/CMP), purine metabolism (Ppat/Gart/Atic/Adsl/Ampd2/Nt5e/Gmps: inosine/adenosine/AMP/ADP/ATP/xanthine/guanosine/guanine/GDP), polyamine metabolism (Srm/spermidine), glycerophospholipid metabolism (Pcyt1a: CDP-choline; Lpcat3: 2-Lyso-PC), sphingolipid metabolism (Sphk2: sphingosine), pentose phosphate pathway (Rbks: ribose), fatty acid biosynthesis (Fasn/Elovl6: palmitoleate/vaccinate), and amino acid metabolism (P4ha1: proline/hydroxyproline), among many others (Fig. 2A, B) (2). These highly coupled 12h rhythms of hepatic metabolic gene expressions and metabolite oscillations strongly imply the presence of an endogenous 12h clock component that is responsible for the precisely-timed orchestration of metabolic flux by temporally regulating the expressions of metabolic enzymes. This hypothesis was confirmed experimentally and will be discussed in greater detail later. Of particular interest is the prevalent 12h hepatic ribonucleoside and ribonucleotide oscillations, which are building blocks for RNA synthesis (Fig. 2C). The acrophases of ribonucleosides and ribonucleotides oscillations are restricted at CT22 and CT34, preceding the two peaks of 12h mRNA transcription at CT26 and CT38, implying coordinated purine/pyrimidine metabolism and 12h mRNA transcription (2) (Fig. 2C). The 12h oscillations of metabolism are not restricted to liver tissues. Indirect calorimetry measurement of real-time mouse energy expenditure revealed the presence of 12h harmonics in respiratory exchange ratio oscillations (2), supporting the hypothesis that other peripheral tissues also possess a 12h clock that perhaps helps to synchronize 12h systemic metabolic homeostasis. While the prevalence of 12h rhythms of gene expression and metabolism in mouse is undisputable, due to the technical limitations and ethical constraint of obtaining time series tissue samples from humans, the evidence supporting the existence of 12h rhythms in human tissues in vivo is still largely anecdotal at this time. Nonetheless, in humans, using traditional spectrum analysis methods such as Fourier transform and wavelet analysis, rhythms of body temperature (44-49), blood pressure and heart rate variability (50-55), cognitive performance (49,56-62), migraine onsets and subsequent cerebrospinal fluid sodium level (63), circulating serum and urine metal levels (64), circulating hormone level (65-67) and sleep patterns (58,59,68-75) were all reported to exhibit a 12h rhythmic component. Of these reported 12h rhythms in humans, daily variation of temperature and sleep-wake cycles are two of the most interesting. As discussed later, temperature is a strong Zeitgeber (Zeitgeber is a German word for time giver, which refers to a synchronizing agent) for 12h rhythms in C. elegans (2,76). While it remains unknown whether temperature also can entrain the mammalian 12h clock, it is tempting to hypothesize that the 12h rhythm of mammalian gene expressions and metabolic rhythms are also in tune to the daily temperature oscillations, similar to what has been observed for the circadian clock (77-79). As to the 12h rhythms of sleep-wake patterns, in addition to the well-characterized peak of sleepiness during the subjective night, a second smaller peak of sleepiness during the early afternoon is a widespread phenomenon (also known as ‘siesta’ in Spanish-speaking countries). Moreover, since the afternoon sleep propensity occurred either under a constant routine (70,80) or without having lunch (81), it is highly suggested that it is a part of an endogenous biological rhythm. Currently, sleep regulation is conceptualized by the popular “two-process” model that posits that homoeostatic and circadian drives control sleep (82). The homoeostatic process is controlled by sleep pressure, which accumulates during the course of wakefulness and dissipates during sleep, and is tightly regulated by local changing levels of neuromodulators such as adenosine (83-85). On the other hand, the sleep–wake cycle during the day and night is regulated by the circadian clock, Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 independent of prior sleep or wake time (82). While it has long been thought that the afternoon sleepiness is under the homeostatic sleep control, several studies challenged this notion and suggested that the afternoon sleep propensity may instead reflect an endogenous 12h cycle of slow wave sleep (SWS) (which is also referred as non-rapid eye movement sleep or in more colloquial term ‘deep sleep’), independent of duration of prior wakefulness (62,70,75). Although it has been convincingly demonstrated that slow wave sleep can be strongly induced by local increased levels of adenosine in the basal forebrain through its adenosine A1 receptor and repressed by its antagonist caffeine (83,86), the mechanism of how the concentration of adenosine is regulated in the brain remain elusive. Intriguingly, adenosine and three of its main precursors ATP, AMP and SAH all exhibit robust 12h rhythms in mouse liver (Fig. 2A-B). Further, xanthine, a close relative of caffeine (which is a methylxanthine) also revealed a 12h rhythm (Fig. 2A). While it is premature to conclude that local intracellular and extracellular levels of adenosine and xanthine derivatives may also exhibit 12h rhythms in the CNS and is coupled to the 12h rhythms of slow wave activity cycle, these intriguing observations and correlations warrant future studies to either prove or refute this hypothesis. Evidences supporting the existence of a cell-autonomous mammalian 12h clock. Two pertinent questions arise regarding the mammalian 12h rhythms: 1) Are the 12h oscillations of gene expression and metabolism cell-autonomous? and 2) Are 12h rhythms established by a dedicated mammalian 12h clock or are they regulated by some variants of the well-characterized circadian clock? Theoretically, at least three possibilities exist. In the first scenario, the 12h rhythms are not cell-autonomous in nature. It is possible that one of the two peaks is established by the circadian clock and the second peak results from physiology-associated systemic cues only found in vivo. In the second scenario, the 12h rhythm is cell-autonomous. However, it is established by two circadian transcription activators or repressors appearing in anti-phase and therefore are dependent on the circadian clock (37). In the third scenario, the mammalian 12h rhythms are not only cell-autonomous, but also are established by a dedicated 12h-clock ‘separate’ from the circadian clock, a mechanism similar to the independent circatidal clock in certain crustaceans and intertidal insects (20,33,34). Early studies favor the first hypothesis (the 12h rhythms are not cell-autonomous and established by the combined effects of circadian clock and fasting-feeding cues) due to the following three major observations: 1) No statistically significant 12h rhythms of gene expression were detected in forskolin-synchronized NIH3T3 cells, implying that mammalian 12h rhythms are not cell-autonomous (Fig. 3B) (9); 2) 8h day-time restricted feeding significantly impairs the 12h rhythms of several hepatic gene expression. These genes still maintained peak expression at approximately CT26, coinciding with the new feeding time; however, the subjective evening peak was largely absent. This evidence suggests that at least one component ∆19 of the 12h rhythm is driven by feeding (9); and 3) Brain-specific rescue of Clock mutant mice converted hepatic 12h rhythms into 24h rhythms, suggesting that signaling via the central circadian oscillator may be required to generate one of the two daily peaks of expression (38). While these results could suggest the lack of a cell-autonomous mammalian 12h clock, in light of strong new evidences supporting the existence of a cell-autonomous mammalian 12h clock (2), alternative interpretations of these data are offered and will be elaborated on in greater detail below. An initial clue hinting at the possible existence of an independent mammalian 12h-clock comes from the observed mathematical orthogonal relationship between superimposed 12h and Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 circadian oscillations uncovered for most of the 12h-cycling ER homeostasis and metabolism genes (2). Subsequent post-hoc analysis of hepatic RNA-Seq data in wild-type and whole-body BMAL1 knock-out mice fed ad libitum [both conventional and adult BMAL1 deletion mice (87)] confirmed the independence of 12h rhythms of key ER and metabolism gene expressions from the circadian clock (2). Specifically, for 12h-cycling genes lacking superimposed circadian rhythms (such as Eif2ak3, Gfpt1, Creld2 and Sec23b), their 12h rhythms are almost identical between wild-type and BMAL1 knock-out mice under either a 12h/12h light/dark schedule or a constant darkness condition (2,87) (Fig. 3A). For 12h-cycling genes with superimposed circadian rhythms (such as Gck, Fasn, Dnajb4 and Chka), more perceptible 12h rhythms were observed in BMAL1 knock-out mice (2,87). The independent relationship between 12h and 24h rhythms for ∆19 these ER homeostasis and metabolism genes also was verified in Clock mutant mice (2,88). Can the 12h rhythm of gene expression be found in vitro or does its establishment require systemic cues only found in vivo as previously suggested (9,36)? In light of the newly found independence of 12h-cycling genes from the circadian clock, an alternative interpretation of the lack of observed 12h rhythms in forskolin-synchronzied NIH3T3 cells (9) is that forskolin is capable only of synchronizing the circadian clock but not the circadian clock-independent 12h clock. Consistent with this hypothesis, it was found that the circadian and 12h-cycling genes are responding to a different repertoire of external cues (Fig. 3B) (2). While the cyclic AMP (cAMP) inducer forskolin is only a strong synchronizer of the mammalian circadian clock (9), the ER stress inducer tunicamycin (which blocks UDP-GlcNAc-mediated UDP-N-linked glycosylation) can only synchronize the 12h-clock (2) (Fig. 3B). This is consistent with observed dominant circadian and 12h rhythms of cAMP and UDP-N-acetylamino sugars levels, respectively, in mouse liver (12) (Fig. 2B). Contrary to the discriminatory nature of forskolin and tunicamycin, dexamethasone and glucose depletion, on the other hand, can synchronize both the circadian and 12h-clocks (2) (Fig. 3B). More importantly, while siRNA-mediated depletion of Bmal1 abolishes dexamethasone and glucose depletion-synchronized circadian Per2 expression, it does not affect dexamethasone, glucose depletion nor tunicamycin-synchronized 12h rhythms of Eif2ak3 expression in mouse embryonic fibroblasts (MEFs) (2) (Fig. 3B). Moreover, by cloning the Eif2ak3 promoter before a destabilized green fluorescence protein (dGFP) and performing time- lapse imaging of dGFP intensity in single cells without any prior perturbation, robust 12h oscillations were observed in MEFs (2), which also was not affected by Bmal1 siRNA knockdown (Fig. 3C) (2). Lastly, 37 metabolites of the 137 examined also exhibited ~12h rhythms in dexamethasone-entrained human U2OS cells and 19 of them still maintained ~12h rhythms in the presence of Bmal1 knockdown (12) (Fig. 3D). Taken together, these observations strongly support the hypothesis that the 12h rhythm of gene expression, especially those involved in ER/metabolic pathways, and subsequent 12h rhythms of metabolism, is cell-autonomous and driven by a dedicated 12h pacemaker ‘distinct’ from the circadian clock in mammalian cells (2). While the 12h rhythms of gene expression and metabolism are cell-autonomous, they can be disrupted by altered fast/feeding cycles. Day-time restricted feeding negatively influences 12h rhythms of hepatic gene expression (9). Consistent with this finding, day-time restricted feeding also significantly impairs 12h oscillations of RER in the mouse (2). Thus, one also can envision how the indirect systemic interactions of the circadian and 12h-clocks work. For instance, manipulation/perturbation of the central circadian clock in the brain can lead to altered feeding/fasting and wake/sleep behaviors, which in turn may feedback to the 12h clock to alter 12h rhythms of gene expression and metabolism in peripheral tissues. In fact, this may very well account for the observed phase shift in several 12h-cycling hepatic genes as well as the varied Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 12h-cycling transcriptome reported in different circadian deficient mice models (2,36,87,88). In addition, since many of the 12h cycling genes have superimposed circadian rhythms, alteration of the circadian clock can have a major influence on the overall gene oscillations, without necessarily affecting the superimposed 12h rhythms. This could be the case for the observed conversion of certain hepatic 12h rhythms into 24h rhythms upon brain-specific rescue of ∆19 Clock mutant mice (38). While these new data support the existence of a cell-autonomous mammalian 12h-clock, they do not rule out the possibility that certain 12h rhythms are influenced by the circadian clock and/or the effects of certain external cues and are not cell-autonomous. Future studies are needed to profile the complete 12h transcriptome under the 12h-clock control, as well as to investigate the physiologic conditions by which these distinct clocks can interact systemically in multiple model organisms. The spliced form of XBP1 (XBP1s) transcriptionally regulates the 12h-clock. How is the mammalian 12h-clock regulated? Observed prevalent 12h rhythms of hepatic nascent mRNA and enhancer RNA (eRNA) expression indicate that the mammalian 12h-clock is regulated primarily at the transcriptional level (2,89). Consistent with the strong enrichment of ER stress and unfolded protein response (URP) pathways in 12h-cycling hepatic transcriptome [for a review of mammalian UPR, see (90-92)], 12h cycling of UPR transcription factor XBP1s and ATF4 was found in mouse liver under both 12h/12h light/dark schedule and constant darkness conditions with peak expressions found at CT10-12 and CT22-24, consistent with the acrophases of 12h-cycling hepatic transcriptome (2,36). Furthermore, 12h-cycling of XBP1s expression was found to be induced in tunicamycin-synchronized MEFs and is independent from the circadian clock (2). Intriguingly, both the total level of Xbp1 mRNA (the combined level of Xbp1s and Xbp1us) and its splicing efficiency exhibited robust 12h rhythms both in mouse liver in vivo and in MEFs in vitro (2,36). The 12h rhythms of Xbp1 splicing efficiency and XBP1s expression in vivo is further supported by the observed 12h rhythms of its upstream endoribonuclease IRE1α phosphorylation at Ser724 (36). 12h rhythms of nuclear XBP1s levels correlates with the 12h rhythms of chromatin recruitment of XBP1s to the promoters of several 12h-cycling genes both in mouse liver in vivo and in tunicamycin-synchronized MEFs in vitro (2). Knock-down of Xbp1 significantly inhibits both tunicamycin and glucose depletion-synchronized 12h rhythms of ER homeostasis (such as Eif2ak3, Ddit3, Sec23b and Herpud1) and metabolism (such as Gfpt1 and Acly) gene expression in MEFs (2) (Fig. 3B). In additional, knock-down of Xbp1 abolished cell-autonomous 12h rhythms of Eif2ak3 promoter-driven dGFP intensity in single MEF cell (Fig. 3C). In contrast, ablation of Xbp1 has no effects on dexamethasone and glucose depletion-synchronized circadian gene expression (2) (Fig. 3B). The transcriptional regulation of mammalian 12h transcriptome by XBP1s is further consistent with numerous past reports on the transcriptional regulation of the same repertoire of genes by XBP1s during pathological conditions of ER stress (93-96). This evidence suggests that similar to the circadian clock, the mammalian 12h-clock is also subject to major transcriptional control. It is worth mentioning that so far, the evidence supporting the role of Xbp1s in the regulation of mammalian 12h-clock is solely restricted to the study of a few 12h rhythmic genes in MEFs in vitro, future study using XBP1s knockout mice models are needed to comprehensively profile XBP1s-regulated 12h transcriptome in an unbiased manner. The mammalian 12h-clock is evolutionarily conserved and may be circatidal in origin. Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 From where does the mammalian 12h-clock originate from? Given the distinctions of the circadian clock from 12h-clock/circatidal clock in both mouse and E. pulchra (2,20) and the fact that mammals share common marine ancestors, it is logical to postulate that the mammalian 12h clock evolved from the ancient circatidal clock. Previous work revealed robust 12h circatidal mRNA rhythms for 10 mitochondrial DNA (mtDNA)-encoded protein-coding genes (Mt-Nd1~6, Mt-Cox1~3 and Cytb) that encode mitochondrial components of complexes I (NADH dehydrogenase) and IV (cytochrome c oxidase) in E. pulchra under free-running conditions, which are correlated with circatidal rhythms of oxygen consumption in the same animals (19,97). Post hoc analysis of two published mouse hepatic Gro-Seq (89) and Nascent-Seq (98) datasets that measure nascent RNA transcription rate revealed, surprisingly, conserved 12h rhythms of mtDNA-encoded gene transcription (2) These 12h rhythms of mtDNA-encoded gene expression were further confirmed at mature mRNA and protein levels in mouse liver (2,39,41). Further, cell-autonomous 12h rhythms of mtDNA-encoded gene expression were found in glucose depletion-synchronized MEFs in a BMAL1-independent manner (2). Also supporting the hypothesis that the mammalian 12h-clock evolves from a circatidal origin is the observed similar free-running periodicity of Eif2ak3 promoter-driven dGFP oscillation in MEFs (12.6h) with that of circatidal activity rhythms reported in E. pulchra after tidal cues entrainment (12.7h) and in the mangrove cricket A. asahinai (12.6h to 12.9h) (20,33,34). In addition to crustaceans and mammals, strikingly, mtDNA-encoded gene transcription was found in 12h/12h warm/cold temperature cycles-entrained as well as free-running C. elegans (76) (Fig. 4A). In fact, the vast majority of the C. elegans homologs of core 12h-cycling mammalian transcriptome involved in ER homeostasis and metabolism revealed robust 12h rhythms under the same conditions (2,76)(Fig. 4B). These include the C. elegans homologs of dominant 12h-cycling mammalian genes such as total Xbp1, Eif2ak3, Hspa1b, Gfpt1, Hspa5, Sec23b, Creld2, Dnaja1 and P4ha1, with Hspa1b/hsp-70 being the strongest oscillating gene in both species (Fig. 4B). It is significant that for mammalian 12h-cycling genes with superimposed dominant circadian rhythms (such as Pfkfb3, Por, Map3k5 and Upp2), 12h oscillations are the dominant ones in C. elegans (Fig. 4B). This could be explained by the absence of oscillations of the canonical circadian clock genes in C. elegans (2,76,99). Adding further to this point, these 12h rhythms oscillate much less robustly or are completely absent in free-running C. elegans that are previously entrained by the 12h/12h light/dark cycles (76). Together, this evidence indicates that the C. elegans 12h-clock is responding to temperature, rather than light cues, which is justified by the overall lack of exposure to light in their natural soil habitat. In addition to the nematode, 12h oscillations of ER homeostasis and metabolism gene expression also were found in light-entrained and free-running zebrafish (100) as well as in the liver of light-entrained baboons (101) (Fig. 4C). Most intriguingly, by thoroughly analyzing published time series -187 database, we further identified a total of 280 genes (with a p value of 1.30 x e for observing the same number of conserved 12h genes by chance) that exhibited conserved ~12h rhythms in temperature-entrained C. elegans (76), naturally-caught Cellana rota (a mollusc captured from its natural intertidal habitat and exhibiting a dominant circatidal clock) (24) and mouse liver (9) (Fig. 4E). As expected, gene ontology analysis of these 280 common ~12h-cycling genes reveals enriched biological pathways in metabolic control and ER homeostasis (Fig. 4F). While it is much appreciated that the molecular mechanisms for generating the circadian clock, namely, the transcriptional/translational feedback loop (TTFL), are conserved in multiple species, there is little overlap in the individual genes involved in the establishment of the circadian TTFL clockwork in different species. Therefore, many believed that the circadian clock Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 evolved separately in different lineages (102). However, the fact that the core 12h-clock genes remained conserved even in divergent species that cross phylum boundaries (Fig. 4C) strongly implies that the 12h-clock may even be more ancient and evolved earlier than the circadian clock. Further supporting this hypothesis is the conserved 12h oscillation of the proposed transcriptional regulator of the mammalian 12h-clock, Xbp1 in Cellana rota, whose oscillation is also in phase with the general circatidal transcriptome peaking at rising tides (24) (Fig. 4D,E). Additionally, computationally constructing predicted mammalian interactive network using these 280 genes via STRING (103) put XBP1 in the center of the network connecting different peripheral sub-hubs (Fig. 4G), consistent with the experimental results demonstrating the essential role of Xbp1 in regulating the 12h-clock in MEFs (Fig. 3). Notwithstanding the evidence, the causal relationships between the circatidal Xbp1/UPR-related gene oscillations and the circatidal behavior in these marine animals remain to be determined. The functions of the mammalian 12h-clock It has been widely accepted that all organisms adjust their biochemical, physiological, and behavioral processes in the most advantageous manner, in phase with predictable environmental changes (104). Alignment of endogenous clocks with environmental timing increases the organisms’ overall fitness and survival, while misalignment between the two features significantly reduce an organism’s fitness and can be a strong predisposing factor to multiple morbidities in almost all species examined (105-112). While this conclusion was based mostly upon studies on the circadian clock, it is no exception for the 12h-clock. Intertidal animals exhibiting dominant circatidal clocks tune their tidal rhythmicity to ~12h rhythmic cycles of inundation and exposure that give rise to rapid changes in temperature, salinity, hydrostatic pressure, osmotic pressure, food, and predation pressure (113). The reasons for the existence of a 12h-clock in mammals are, however, subtler. After all, it has been a long time since we “came out of the sea” and mammals no longer experience 12h environmental changes of salinity, temperature or hydrostatic pressure. Instead, we are constantly exposed to circadian changes of temperature, light and food availability and as a result, circadian clock has long been thought to be the only clock present in higher organisms including mammals. Nonetheless, if the theory that biological clocks are always in sync with environmental timing also holds true for the mammalian 12h-clock, then we must have either overlooked some 12h-cycling environmental factors to which the mammals are exposed to daily, or there is “something else” cycling with a 12h period that is indirectly caused by the circadian environmental changes of temperature, light or food commonly associated with land mammals. Here we favor the latter hypothesis and conjecture that the mammalian 12h clock is in tune to a 12h-cycling “stress cycle”, or more specifically, a 12h-cycling “metabolic stress cycle” that results from the innate misalignment between energy intake and energy expenditure in a diurnal cycle (Fig. 5A). The early clues suggesting the existence of such a 12h-cycling metabolic cycle is the unique dawn and dusk phase distribution of 12h-cycling mRNAs in mouse liver, both of which correspond to transition periods between fasting/feeding and sleep/wake (Fig. 2C) (2). At the subjective dawn (ZT10 to ZT12 for the nocturnal mouse; ZT stands for Zeitgeber time and ZT0 is the time of light-on and ZT12 is the time of light-off), the prolonged absence of energy intake from the subjective night combined with reduced but still significant energy expenditure [during sleep the brain and other parts of the body still consume significant amount of energy to consolidate memory, dispose of the metabolic waste and repair the body (114-116)] leads to a peak of energy ‘overdraft’ . By contrast, at the subjective dusk (ZT0 to ZT2 for nocturnal Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 mouse), sufficient energy intake during the subjective day plus significantly reduced energy expenditure gives rise to a peak of energy ‘excess’ (Fig. 5A). Both energy ‘overdraft’ and energy ‘excess’ create great metabolic stress for the mammalian cells. They activate the same stress response pathways to mitigate the original stress, including the two most well-known stress responses that evolved to cope with such metabolic stress: the classical ER-associated unfolded ER protein response (UPR ) (91) and a second stress response pathway reacting to unfolded mt proteins in the mitochondria coined “mitochondrial UPR” or UPR (117-120). For example, both fasting-associated hypoglycemia and feeding-associated hyperglycemia ER conditions can activate the UPR (121-124). Likewise, both increased and decreased mt mitochondria respiration capacity can activate UPR and other mitochondrial stress pathways ER mt (125). Therefore, it is not surprising that both UPR transcription factor xbp-1 and UPR transcription factor atfs-1 exhibit robust 12h rhythms of expression peaking at dawn (C10) and dusk (CT22), synchronized to the 12h rhythms of proposed cellular metabolic stress (Fig. 5A, D). While the true mammalian homolog of atfs-1 remains elusive at this time, one potential candidate Ddit3/Chop (118) does exhibit a robust 12h rhythm in mouse (2). Based on these findings, we propose that the mammalian 12h-clock may govern physiological 12h oscillations ER mt of UPR and UPR to maintain metabolic homeostasis. These oscillations are further synchronized to the energy imbalance-induced 12h metabolic stress cycle occurring in the ER and mitochondria (2). Therefore, we coined the term CREMA (Coordinated Rhythms of ER and Mitochondria Action) to reflect the coupled 12h rhythms of gene expression involved in both ER and mitochondria homeostasis (2). Supporting the existence of such a 12h metabolic stress cycle are observed 12h-cycling metabolites (such as ADP and inosine) in dexamethasone-synchronized human U2OS cells with or without BMAL1 knock-down (Fig. 3D) (12). Nevertheless, more studies utilizing real-time imaging of stress-related metabolites in single cells are needed to substantiate that such a 12h metabolic stress cycle indeed exists in a fully cell-autonomous manner as predicted. The potential roles of the 12h-clock in diseases and therapy In this section, we present early evidences supporting the potential roles of the 12h-clock in regulating human diseases and further discuss how we may exploit this knowledge to better implement chronotherapy. Due to the strong implications that the 12h-clock is key to the controlling of hepatic metabolism and stress responses, we focus specifically on two conditions that are profoundly influenced by these two pathways: nonalcoholic fatty liver diseases (NAFLD) and aging (126-135). Loss of a functional 12h-clock is strongly associated with NAFLD progression. It has been well-established that both nutritional challenge and chronic ER/mitochondrial stress are strong contributing factors to the pathogenesis of NAFLD (126,128,129,135-142). Given the newly discovered housekeeping functions of the 12h-clock in maintaining cycling metabolic stress response, we propose a new mechanism whereby pathological levels of metabolic stress impairs the hepatic 12h-clock and perturbs the metabolic homeostasis that eventually results in NAFLD (Fig. 6A). This hypothesis is reminiscent of the well-established causal relationships among chronic “jet lag stress”, and the perturbed circadian rhythm and metabolic syndrome in both animal models and humans (143-146). At least four pieces of evidence support this hypothesis. First, acute induction of severe ER stress by tunicamycin injection alters the 12h rhythmic gene expression in mouse liver (36). Secondly, nutritional challenge by high fat diet significantly disrupts the 12h oscillation of UPR and metabolic genes including Manf, Sec23b, Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Hspa5, Eif2ak3 and Gfpt1 in mouse liver. By contrast, circadian oscillations of all 17 core circadian clock genes including Bmal1, Per2 and Rev-erbα were affected either modestly, or not at all by the high fat diet challenge (Fig. 6B) (41). Thirdly, gene set enrichment analysis (GSEA) revealed that the downregulation of 12h gene expressions is associated strongly with progression to hepatic steatosis and nonalcoholic steatohepatitis (NASH) in humans (2,147). Last but not least, using both genetic and pharmacological models, it has been convincingly shown that activating ER stress-sensing or ER quality control pathways, including Xbp1s, are protective against hepatic steatosis (148-151). While this evidence strongly supports an association of 12h- clock disruption with progression to NAFLD, future studies using both genetic and pharmacological 12h-clock deficient animal models are needed to firmly establish the causal roles of the 12h-clock in disease development (Fig. 6A). Is the 12h-clock in essence an anti-aging hormetic response? Aging is a complex process characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability and eventually to death. In a comprehensive review published in 2013, a total of nine hallmarks representing the common denominator of the aging process in multiple organisms were proposed (134). Of these hallmarks, loss of proteostasis, deregulated nutrient sensing and mitochondria dysfunction are biological processes under strong 12h-clock regulation, suggesting a potential role of the 12h- clock in mediating aging, especially in the prevention of aging-related metabolic decline (2,130,134). In fact, NAFLD itself is commonly believed to be an age-related morbidity (135,152). Further supporting this hypothesis is the significantly disrupted 12h rhythm (but not circadian rhythm) of hepatic gene expression in old mice (Fig. 5C) (153), reminiscent of similarly disrupted hepatic 12h rhythms in mice fed a high fat diet (Fig. 5B). Intriguingly, genes known to regulate lifespan in C.elegans are highly enriched for 12h rhythms of gene expression (Fig. 5D) (76). These include positive regulators of longevity such as sir2.1 (homolog of mammalian Sirt1) (154-158), daf-16 (homolog of mammalian Foxo1) (159,160), aak-2 (homolog of mammalian Prkaa1/2, also known as Ampkα1/2) (161,162) and tcer 1 (homolog of mammalian Tcerg1) (163) as well as a significant number of negative regulators of C. elegans lifespan uncovered from a RNAi screen study (164) (Fig. 5D). More interestingly, Sirt1, Foxo1, Prkaa1 and Prkaa2 also exhibit 12h rhythms in mouse liver (Fig. 5D) (2). The potential mechanisms of the 12h-clock in mediating longevity and aging, in our opinion, may lie in the ancient concept of ‘hormesis’, which is the biological embodiment of the idiom “what doesn’t kill you makes you stronger”. In scientific terms, hormesis is an adaptive cellular response whereby exposure to low doses of stress (but not high dose) activates protective mechanisms that render the cell resistant to a subsequent challenge with higher doses of stress (165). The exact molecular mechanisms underlying hormesis are still unclear but have been previously attributed to the actions of both ER and mitochondria, which are termed ER hormesis and mitohormesis, respectively (125,166,167). In either case, the hormetic response is mediated ER either by xbp1-dependent UPR (for ER hormesis) or atfs-1/hsp-60 (Hspd1 in mammals)- mt dependent UPR (for mitohormesis) (125,166). Both ER hormesis and mitohormesis are positively associated with organismal development, stress resistance, and more importantly, longevity. For example, a study in C. elegans showed that expression of XBP1s in neurons was sufficient to increase longevity and induce ER hermetic response in distal, non-neuronal cell types via a cell-nonautonomous mechanism (168,169). Further, mitochondrial ribosomal protein mt knockdown-mediated lifespan extension also requires hsp60-dependent UPR in both C. elegans and mammalian cells (170). Considering the fact that both ER hormesis and Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 mitohormesis master regulators xbp-1 and atfs-1/hsp-60 exhibit conserved 12h rhythms and the central roles of the 12h-clock in stress response regulation, we conjecture that the hormetic response may exert its anti-aging effects partially through the amplification/boosting of the endogenous 12h-clock. Indeed, we found that only low dose, but not high dose of tunicamycin (lower than 30 ng/ml) is capable of synchronizing the 12h-clock in MEFs, consistent with the concept of hormesis ((2) and unpublished data). 12h-clock-based chronotherapy Chronotherapy is the concept that treatment of an illness or disorder should take the body’s natural rhythms and cycles into accounts (171). It has been shown that drugs that target rhythmic, high-amplitude circadian gene products represent a potential pathway for mechanism- driven chronotherapy (172-176). Here we propose that similarly, 12h rhythmic transcriptome and proteome can function as blueprint for future design of 12h-clock-based chronotherapy. To this end, we probed FDA-approved drugs for potential interactions with high amplitude 12h-cycling hepatic transcripts and proteins using the Drug Gene Interaction Database (DGIdb) (177,178) and found a number of hits against both 12h-cycling hepatic mRNA and/or their protein targets (Fig. 5E,F). For example, 6-mercaptopurine is a medication for treating a variety of blood cancer and autoimmune diseases. Among of its mechanisms of action is the inhibition of de novo purine synthesis by directly inhibiting the rate-limiting enzyme phosphoribosyl pyrophosphate amidotransferase encoded by the Ppat gene (179,180). Since, as we discussed previously, coordinated 12h rhythms of hepatic purine metabolism with RNA transcription is a strong feature of the mammalian 12h-clock (Fig. 2B-C) (2), it may be desirable to administer mercaptopurine (and other drugs targeting de novo purine and pyrimidine biosynthesis) at times of nadir purine/pyrimidine metabolism (at 3-4pm) to minimize hepatic toxicity. Due to the very early stage of the 12h rhythm field (and ultradian rhythms field as a whole), currently, very little is known about its definitive functions and implications in human diseases. To a large extent, we can only speculate on their functions and implications based upon the limited literature available so far. We hope this review/preview can attract more scientists into this nascent field, because as outlined below, we believe that only the tip of the iceberg has been uncovered thus far and that there is so much more to explore. Future directions Future work could be directly toward three major aims: 1) delineating the molecular and physiological mechanisms of 12h-clock regulation; 2) establishing the causal relationship between 12h-clock disruption and disease progression, and 3) translating the above knowledge into chronotherapy-based medical practice (Fig. 6A). For example, at the molecular and cellular levels: Is the 12h rhythm temperature-compensated? What is the full spectrum of the 12h-cycling transcriptome, proteome and metabolome under 12h-clock control? What are the negative transcriptional regulators of the mammalian 12h-clock? What are the co-regulators for XBP1s- dependent 12h-clock transcriptional control? Do some post-transcriptional and post-translational mechanism exist, if so, what are they? At the systemic and physiological level: How are the 12h rhythms in different tissues and organs coordinated and synchronized at the systemic level? Are there 12h-cycling hormonal factors coordinating different tissues? Maybe a more tempting question is: does the 12h-clock exist in the central nervous system and if so, where? More, 12h- clock deficient animal models are needed in the future to rigorously establish the causal relationship between 12h-clock dysfunction and multiple disease development. Our ultimate goal Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 should be to successfully translate the newly-learnt knowledge into chronotherapy-based medicine and disease prevention. Conclusion Based upon the current available literature, here we propose a model where combined actions of both the circadian and 12h-clock are required to ensure cellular homeostasis and promote organism integrity and overall fitness (Fig. 6B). Quite a number of genes in the mammalian genome are under dual circadian and 12h-clock, although through distinct molecular mechanisms. The circadian clock is transcriptionally regulated through a revised TTFL model that includes activation phase-facilitated repression mainly via the ROR response element (RORE) (Fig. 6B) (181), while the 12h-clock is transcriptionally regulated by 12h rhythms of XBP1s chromatin recruitment to ER response element (ERSE) (Fig. 6B) (2). Superimposition of both circadian and 12h rhythms of gene expression leads to a temporal gene expression pattern characterized by two unsymmetrical peaks within a diurnal cycle (Fig. 6B), which is commonly observed in metabolic genes such as Fasn and Gck (Fig. 1C) (2). Further, the circadian clock and the 12h-clock are synchronized to different environmental cues. The circadian clock is responding to the differential between the two opposite metabolic states: fast and feeding; whereas the 12h-clock is entrained by the common denominator associated with both fast and feeding: the peaking of metabolic stress (Fig. 6B). In addition to the circadian and 12h-clocks, it is very likely that additional clock components also exist in mammals, such as an 8h-clock (Fig. 1B-D). We reason that having multiple clock components endows organisms with more flexibility and the heightened ability to adapt to different environments, thus significantly increasing survival advantage. In theory, simply adjusting the relative phases and fine-tuning the amplitudes of different clocks can give rise to an infinite number of peak patterns to cope with a variety of daily environmental cycles. The co-existence of multiple harmonic biological rhythms in tune to the natural rhythms is reminiscent of the ancient philosophic concept of ‘musica universalis’, which was first proposed by the Greek philosopher Pythagoras over two thousand years ago (1). According to this theory, all natural appearing rhythms are in essence tuned to the rhythmic movements of celestial bodies (1). While the circadian rhythm is synchronized to the 24h light/dark cycle coinciding with the Earth’s rotation, our findings suggest that the 12h clock may have evolved from the ancient circatidal clock, which is in turn entrained by the 12h tidal cues orchestrated mainly by the moon. Thus, it appears after two thousand years, we may have finally found evidence for the “biological musica universalis” (Fig. 6B). American Diabetes Association http://dx.doi.org/10.13039/100000041, 1-18-JDF-025, Bokai Zhu; Brockman Foundation, Bert W O’Malley; Brockman Foundation, Clifford Dacso; Center for the Advancement of Science in Space http://dx.doi.org/10.13039/100011048, GA-2014-136, Clifford Dacso; Peter J. Fluor Family Fund, Clifford Dacso; Joyce Family Foundation, Clifford Dacso; Sonya and William Carpenter, Clifford Dacso; National Science Foundation http://dx.doi.org/10.13039/100000001, 11703170, Clifford Dacso; Center for the Advancement of Science in Space http://dx.doi.org/10.13039/100011048, GA-2014-136, Bert W O’Malley; Phillip J. Carroll, Jr. Professorship, Clifford Dacso Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Acknowledgment: We acknowledge all members of O’Malley’s lab for support for this project, with special thanks to Naomi Gonzalez, Dr. Brian York and Dr. Maricarmen Delia Planas-Silva. We also like to thank Drs. Oren Levy and Yisrael Schnytzer from Bar-Ilan University for kindly sharing the processed transcriptome data of C. rota. We apologize for omission of relevant works and citations due to space constraints. This research was supported by the Brockman Foundation to B.W.O. and C.C.D. In addition, this research also was supported by grants from the Center for Advancement of Science in Space, Peter J. Fluor Family Fund, Philip J. Carroll, Jr. Professorship, Joyce Family Foundation to C.C.D and the American Diabetes Association junior faculty development award 1-18-JDF-025 to B.Z. The authors declare no competing or financial interests. *Correspondence: berto@bcm.edu The authors declare no conflicts of interest. References: 1. Taruskin R. Music in the Western World: A History in Documents (2d ed.). Choral Journal 2008; 48:68-70 2. Zhu B, Zhang Q, Pan Y, Mace EM, York B, Antoulas AC, Dacso CC, O'Malley BW. A Cell-Autonomous Mammalian 12 hr Clock Coordinates Metabolic and Stress Rhythms. 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Coactivator-Dependent Oscillation of Chromatin Accessibility Dictates Circadian Gene Amplitude via REV-ERB Loading. Mol Cell 2015; 60:769-783 Figure 1. The concept of harmonics in physics and their biological counterpart. (A) A diagram showing the individual harmonics (200Hz, 400Hz, 600Hz, 800Hz and 1000Hz) as well as the composite waveform resulting from adding up all the harmonics. (B) The distribution of periods of all oscillations identified from the hepatic gene expression microarray dataset (9) via st nd the eigenvalue/pencil approach. The vast majority of oscillations cycle at the 1 (24h), 2 (12h) rd or 3 (8h) harmonic of the circadian rhythm (24h). (C) Representative deconvolution of Gck gene mRNA expression by the eigenvalue/pencil method. Gck expressions detected by two different probe sets are analyzed by the eigenvalue/pencil method. (i) Raw microarray data (solid line) and model fit (dashed line) for Gck hepatic expression as reported in (9). Superimposed harmonic oscillations revealed by the eigenvalue/pencil method for probe 1 (ii) and probe 2 (iii). (iv) Amplitudes, phases and periods of different oscillations for the two probes with the color matching the different oscillations depicted in (ii) and (iii). (D) The distribution of periods of all oscillations identified from the hepatic metabolites dataset (12) via the eigenvalue/pencil st nd rd th approach. The vast majority of oscillations cycle at the 1 (24h), 2 (12h), 3 (8h) or 4 (6h) harmonic of the circadian rhythm (24h). Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Figure 2. Coupled 12h rhythms of hepatic gene expression and metabolism. (A) Heatmap of mouse 12h-cycling hepatic metabolites identified by the eigenvalue/pencil method from published metabolomic dataset (12) after hierarchical clustering. Metabolites with dominant 12h oscillations are highlighted in red. (B) (Top) Gene-metabolite joint pathway analysis using using MetaboAnalyst (42,43) reveals top enriched biological pathways. Both enrichment as well as topology scores were shown for each pathway. (Bottom) Representative data showing paired gene expression and metabolite oscillations. (C) Distribution of acrophases of all dominant hepatic 12h-cycling mRNA and ribonuclesides/ribonucleotides. Figure 3. 12h rhythms of gene expression and metabolism are cell-autonomous, established by a dedicated 12h-clock and evolutionarily conserved. (A) Representative RPKM normalized hepatic circadian (top) as well as 12h-cycling (bottom) gene expression from WT and conventional BMAL1 KO mouse under 12h/12h light/dark conditions as reported in (87) . Data are graphed as the mean ± SEM (n = 4) and double plotted for better visualization. (B) Heatmap representation of oscillations of Eif2ak3 and Per2 mRNA level after dexamethasone, tunicamycin or glucose depletion shock treatment in MEFs. The heatmap is derived from qPCR data reported in (2). A summary of the conclusion is shown in the table below. (C) Representative recordings of single cell time lapse microscopy analysis of Eif2ak3 promoter- driven dGFP oscillation in scrambled siRNA, Bmal1 siRNA or Xbp1 siRNA transfected MEFs. (D) Heatmap of 12h-cycling metabolites in dexamethasone-synchronized human U2OS cells under both scrambled siRNA and Bmal1 siRNA transfection conditions compiled from (12). 12h-cycling metabolites are identified by the eigenvalue/pencil method (2). Figure 4. 12h rhythms of gene expression are evolutionarily conserved. (A) Heatmap of temporal mtDNA-encoded gene expression in temperature-entrained as well as free-running C.elegans compiled from (76). (B) Heatmap of core ER homeostasis and metabolism related 12h-cycling gene expression in both temperature-entrained as well as free-running C.elegans [left and compiled from (76)] and mouse liver under constant darkness [right and compiled from (9)]. Xbp1 is highlighted in red. (C) Phylogenetic tree and relative mRNA expression of Eif2ak3, Gfpt1 and Dnajb4 in Cellana rota [second row and reported in (24)], Caenorhabditis elegans [third row and reported in (76)], Danio rerio [fourth row and reported in (100)], Mus musculus [fifth row and reported in (9)] and Papio anubis [last row and reported in (101)] during a 48h interval. The status of the three genes were not reported for Eurydice pulchra in the study (19). The data for Papio anubis is double plotted for better visualization. (D) The mRNA level of Xbp1 ortholog in Cellana rota captured from different time of day from their natural intertidal habitat in the wild as compiled from (24). (E) Heatmap of all 280 evolutionarily 12h-cycling gene expression in temperature-entrained as well as free-running C.elegans [left and compiled from (76)], naturally caught C. rota in tune to 12h-cycling tidal cues under 12h/12h natural light condition [middle and compiled from (24)] and mouse liver under constant darkness [right and compiled from (9)]. (F) GO analysis of enriched KEGG pathways from these 280 genes. (G) Predicted interactive network construction of these 280 proteins using STRING (103) with XBP1 highlighted by the arrow. Figure 5. Mammalian 12h-clock in diseases and chronotherapy. (A) A diagram summarizing the origin, regulation, function and species conservation of 12h-clock. Please see the main text for detailed description of each section. (B) High fat diet disrupts the hepatic 12h-cycling, but not circadian gene expression in mouse. Heatmap (left) and representative log2 normalized Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 expression (right) of key 12h-cycling and circadian genes under normal chow and high fat diet conditions are compiled from (41). The data is double plotted for better visualization. (C) Disrupted hepatic 12h rhythms are associated with aging. Heatmap (top) and representative mRNA expression (bottom) of key 12h-cycling and circadian gene in young and old male mice are compiled from (153). (D) Aging-regulating genes are enriched for 12h rhythmicity in C.elegans. (Top) Heatmap of aging-related 12h-cycling gene expression in both temperature- entrained as well as free-running C.elegans as compiled from (76). Of 62 genes that increase worm lifespan by 10% when knocked down post-developmentally (164), 38 (62%) of them showed 12h rhythm in temperature-entrained and free-run worms as shown in the heatmap. In addition, positive regulators of aging including atfs-1, xbp-1, daf-16, aak-2, tcer-1 and sir-2.1 all exhibited 12h rhythms of gene expression. (Bottom) Heatmap of mammalian ortholog of sir-2.1 (Sirt1), daf-16 (Foxo1), aak-2 (Prkaa1 and Prkaa2) expression in mouse liver under constant darkness condition as compiled from (9). Atfs1 and Xbp1 are highlighted in red. (E, F) A 12h- clock based chronotherapy blueprint. Heatmap of temporal mRNA (E) and protein (F) expression of hepatic 12h-cycling genes with FDA-approved drug interactions as compiled from (9,39). The names of drug are indicated on the right. FEN1, FASN and PPAT also exhibit 12h rhythms of mRNA expression and are highlighted in red. Figure 6. The framework of 12h-clock study. (A) A diagram summarizing the potential causal roles of mammalian 12h-clock in disease development with future directions outlined below. 12h rhythms can be disrupted either by the dampening of the amplitude or alteration of the period. (B) A diagram summarizing the basis for biological system being part of “musica universalis”. 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Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms

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Abstract

Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms Bokai Zhu, Clifford C Dacso and Bert W O’Malley Journal of the Endocrine Society Endocrine Society Submitted: April 19, 2018 Accepted: June 01, 2018 First Online: June 06, 2018 Advance Articles are PDF versions of manuscripts that have been peer reviewed and accepted but not yet copyedited. The manuscripts are published online as soon as possible after acceptance and before the copyedited, typeset articles are published. They are posted "as is" (i.e., as submitted by the authors at the modification stage), and do not reflect editorial changes. No corrections/changes to the PDF manuscripts are accepted. Accordingly, there likely will be differences between the Advance Article manuscripts and the final, typeset articles. The manuscripts remain listed on the Advance Article page until the final, typeset articles are posted. At that point, the manuscripts are removed from the Advance Article page. DISCLAIMER: These manuscripts are provided "as is" without warranty of any kind, either express or particular purpose, or non-infringement. Changes will be made to these manuscripts before publication. Review and/or use or reliance on these materials is at the discretion and risk of the reader/user. In no event shall the Endocrine Society be liable for damages of any kind arising references to, products or publications do not imply endorsement of that product or publication. Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Unveiling “Musica Universalis” of the Cell: A Brief History of biological 12h Rhythms 1 1,2,3 1,3 Bokai Zhu , Clifford C Dacso and Bert W O’Malley Department of Molecular and Cellular Biology Department of Medicine Dan L. Duncan Cancer Center Baylor College of Medicine, Houston, TX 77030, USA Received 19 April 2018. Accepted 01 June 2018. Musica Universalis is an ancient philosophical concept claiming the movements of celestial bodies follow mathematical equations and resonate to produce an inaudible harmony of music, and the harmonious sounds that men make were an approximation of this larger harmony of the universe (1). Besides music, electromagnetic waves such as light and electric signals also are presented as harmonic resonances. Despite the seemingly universal theme of harmonic resonance in various disciplines, it was not until recently that the same harmonic resonance was discovered also to exist in biological systems. Contrary to traditional belief that a biological system is either at steady state or cycles with a single frequency, it is now appreciated that most biological systems have no homeostatic “set point”, but rather oscillate as composite rhythms consisting of superimposed oscillations (2). These oscillations often cycle at different harmonics of the circadian rhythm, and among these, the ~12h oscillation is most prevalent (2). In this mini- review, we focus on these 12h oscillations, with special attention to their evolutionary origin, regulation and functions in mammals, as well as their relationship to the circadian rhythm. We further examine the potential roles of the 12h-clock in regulating hepatic steatosis, aging and the possibility of 12h-clock-based chronotherapy. Finally, we posit that biological rhythms are also “Musica Universalis”: while the circadian rhythm is synchronized to the 24h light/dark cycle coinciding with the Earth’s rotation, the mammalian 12h-clock may have evolved from the circatidal clock, which is entrained by the 12h tidal cues orchestrated by the moon. we focus on these 12h oscillations, with special attention to their evolutionary origin, regulation and functions in mammals, as well as their relationship to the circadian rhythm. . Search methods: For the search related to circatidal rhythms in coastal and estuarine animals, the keywords of circatidal and/or tidal rhythms were used and literatures between 1950 and present were reviewed. For reported ~12h rhythms in humans, the keywords of circasemidian and/or 12h rhythm were searched against literatures published between 1950 and present. For the 12h rhythms in mouse, due to the very limited number of publications on this topic, the authors are very acquainted with the handful of literatures and therefore no search was performed. Main text: The concept of harmonics in physics and their biological counterpart The term “harmonic” originates in musical instrument theory, where the wavelengths of the overtones of a vibrating string are derived from its fundamental wavelength. A harmonic of such Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 a wave has a frequency that is a positive integer multiple of this fundamental frequency, which is st st also called the 1 harmonic (3,4). For instance, as shown in Fig. 1A, the fundamental, or the 1 nd harmonic is a wave with a frequency of 200Hz, whereas the 2 harmonic is 400Hz (200Hz x 2=400Hz), and so on. Summing the harmonics generates a composite waveform that looks “distorted and noisy” (Fig. 1A bottom) but is actually much closer to perception of music than the individual harmonics. This is because pure oscillation with a single frequency rarely exists in nature. Rather, physical rhythms often present as superpositions of basic waves such as the harmonic resonances found in music, light and Kepler’s Law of planetary motion. In order to uncover all superimposed oscillations in an unbiased manner, we utilized a recently developed eigenvalue/pencil method (2,5-7) and disclosed that just like other forms of physical oscillations, biological oscillations also are composite rhythms consisting of oscillations of various frequencies (2). The eigenvalue/pencil method assumes that any oscillation can be approximated by a linear combination of exponentials (sine waves with a decay factor) plus noise and therefore can be applied to any situations where such assumptions are valid. Furthermore, unlike most of the current cycling transcripts-identification methodologies that require the user to define a narrow period range (8), the eigenvalue/pencil method does not pre- assign a period and thus permits the identification of all superimposed oscillations in an unbiased manner (2) [for a more detailed understanding of the strengths and limits of the eigenvalue/pencil method and its wider applications, please refer to (5)]. Oscillations disclosed by this analysis often cycle at the harmonics of the circadian rhythm (2). For instance, comprehensive analysis of a published mouse liver microarray dataset employing the eigenvalue/pencil method (where liver samples were taken every hour for a total of 48 hours under constant darkness condition) revealed that the majority of oscillations cycle at periods close to 24h, 12h, 8h and 4h, st nd rd th corresponding to the 1 , 2 , 3 and 6 harmonics of the circadian rhythm (Fig. 1B) (2,9). While the ~4h oscillations are near the Nyquist limit of sampling frequency and therefore may arise from technical artifacts (10), oscillations cycling with 12h and 8h periods are believed to be “real” biological oscillations (2). One example is glucokinase (Gck), which encodes for a rate- limiting enzyme involved in both glycogenesis and glycolysis. While Gck mRNA expression was originally thought to be solely under the circadian clock control (11), the new analysis revealed that it actually comprises of superimposed major oscillations cycling at 24h, 12h and 8h periods with comparable amplitudes (Fig. 1C) (2). Similar to gene expression oscillations, mammalian metabolic rhythms also are composite oscillations cycling at different harmonics of the circadian rhythm. Analyzing 1h resolution mouse hepatic metabolites profiling dataset (12) revealed that the majority of metabolites consist of superimposed oscillations cycling at 24h, 12h, 8h and 6h periods (Fig. 1D). This is consistent with the observed 24h, 12h and 8h harmonic oscillations of respiratory exchange ratio (RER) through indirect calorimetry measurement of energy expenditure for mice fed ad libitum (2). Taken together, these unbiased analyses strongly indicate that as proposed thousands of years ago, harmonic resonance is a common ‘universal’ theme that permeates from the physical world all the way to biological systems. The circatidal rhythm: the best characterized ~12h cycling oscillation The wide prevalence of 12h gene expression and metabolic rhythms observed in mouse liver is intriguing. In fact, more than 20% of all mouse hepatic mRNA and metabolites cycle with a ~12h period, of which 4.2% and 11.5% are dominant oscillations (~12h cycling amplitudes are the largest among all superimposed oscillations), respectively (2). The prevalent mammalian 12h Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 rhythm of gene expression and metabolism is reminiscent of the ~12h circatidal rhythms of coastal and estuarine animals that modulate their behavior in tune to the ~12.4 hour ebb and flow of the tides (13). Prominent examples include numerous crustaceans such as fiddler crab (Uca pugnax) (14,15), mudflat crab (Chiromantes haematocheir) (16), green shore crab (Carcinus maenas) (17,18), sea louse (Eurydice pulchra) (13,19,20); Dimorphostylis asiatica (21); Chelicerata horseshoe crab (Limulus polyphemus) (22); ragworm (Alitta virens) (23), Mollusca (24-27) and intertidal insects like the Asian mangrove cricket (Apteronemobius asahinai) (28,29). These animals either emerge at low tides to forage, mate and fight (such as Uca pugnax and Apteronemobius asahinai) or are more active during high tides when the incoming tide cover their forage and mating grounds (such as Carcinus maenas and Eurydice pulchra) (13). More importantly, when removed to the laboratory and divorced from tidal cues, they can still maintain their circatidal locomotive activities at times of expected low water or high water under constant conditions (13-15,17,24,28,29). Further, their circatidal rhythms also can be entrained by artificial vibrations or inundation cycles (20,24,28). This free running behavior combined with entrainability indicates the presence of circatidal clocks in these animals, which under natural conditions are synchronized to the phase of the tidal cycle encountered on their home environment (13). So how are these circatidal rhythms generated? Two mechanisms underlying tidal rhythms have been proposed. Some argue that a circatidal clock might be generated by two slightly longer (24.8-hour) circadian clocks acting anti-phase to produce 12.4h peaks in behaviors such as swimming and foraging (30). The second school of thought advocates that a dedicated circatidal clock with a period of ~12.4h is the basis for circatidal behavior. Superimposed on this tidal clock is a circadian oscillator that drives the day/night modulation observed in locomotor output of crustaceans and crickets (31). One of the most convincing studies supporting the latter hypothesis was published in Current Biology in 2013 (20). In this elegant study, Zhang et al. used two orthogonal approaches (RNAi against circadian clock genes and bright constant light to disrupt the circadian clock) to abolish the circadian clock in Eurydice pulchra and tested their effects on the circatidal swimming rhythms of E. pulchra. While both approaches disrupted the circadian clock as expected, neither influenced the tidal periodicity (20). Additional studies performed on the American horseshoe crab Limulus polyphemes and mangrove cricket (Apteronemobius asahinai) found similar results (31-34). Collectively, these studies convincingly demonstrate the existence of a dedicated circatidal pacemaker in marine animals that is distinct from the circadian clock and responsible for the establishment of circatidal rhythms. The prevalence of mammalian 12h rhythms The first study that reported the presence of circadian harmonics of gene expression in mammalian systems, including 12h rhythms, was published by John Hogenesch’s group (9). By comprehensively profiling temporal gene expression in mouse liver from animals maintained at constant darkness at high resolution (at 1h intervals for a total of 48h) using DNA microarray, the authors identified ~200 genes that cycle with a dominant 12h period using two statistical methods: Fisher’s G-test and COSOPT (9). Ingenuity pathway analysis of these 12h-cycling hepatic genes revealed enriched gene ontology terms including protein processing and endoplasmic reticulum (ER) homeostasis (9). Contrary to the evenly distributed phases (the time of peak) of circadian genes, the acrophases of 12h-cycling hepatic genes are more restricted to the dawn (CT0) and dusk (CT12) of a diurnal cycle, even though the underlying reason was then Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 unknown (9) [CT stands for circadian time. By convention, the onset of activity of diurnal organisms defines circadian time zero (CT 0), while the onset of activity of nocturnal organisms defines circadian time twelve (CT 12). Therefore, for nocturnal animals such as mice, they are active from CT12 to CT24]. In addition to the liver, they also detected 12h-cycling transcripts in many different tissues. For example, Hspa1b, a heat shock protein 70kD family member, showed clear 12h transcriptional rhythms in lung, kidney, adrenal gland, heart and even in hypothalamus of the central nervous system (CNS), indicating that the presence of circadian harmonics is not restricted to the liver (9). In addition, in all these tissues, the phases of peak Hspa1b expression are nearly identical, again at dawn and dusk. Since then, a number of studies have confirmed the presence of these 12h rhythms of gene expression, especially in mouse liver, using different cycling-transcripts identification methods (35-38), even though the total repertoire of identified 12h-cycling transcripts remained small. We reasoned that the ~200 originally identified 12h-cycling transcripts were a significant underestimation of the true prevalence of mammalian 12h rhythms of gene expression. Since the COSOPT/ Fisher’s G-test methods used to identify 12h-cycling transcripts in the original study require the user to pre-assign a period range, it favors the detection of genes with dominant 12h rhythms and is thereby biased against other 12h-cycling transcripts having superimposed oscillations with larger amplitudes. As expected, when applying the “eigenvalue/pencil” method on the same mouse microarray dataset, we identified more than 3,000 hepatic genes (approximately 20% of all hepatic transcripts) with 12h rhythms (2). As expected, the majority of them have superimposed 24h circadian rhythms and therefore evade detection by traditional methods. Among all ~3,000 genes, 760 of them are dominant 12h-cycling genes (2). Gene ontology analysis of either all or dominant 12h-cycling genes revealed that, in addition to the expected ER homeostasis and protein quality control pathways, various metabolism-related biological pathways are also highly enriched (2). While ER-related genes (such as Eif2ak3, Gfpt1 and Sec23b) have dominant 12h oscillations and lack superimposed circadian rhythms, 12 h- cycling metabolism genes often have superimposed circadian rhythms (including Gck, Acly and Fasn) (2). In addition to the wide-spread 12h cycling mature mRNAs in mouse liver, prevalent 12h- cycling hepatic protein (~15% of hepatic proteome) also was observed from a time series mass spectrometry dataset (2,39). A limited ~35% overlap with 12h cycling mRNAs indicates that both transcriptional and post-transcriptional controls contribute to the regulation of 12h rhythms of hepatic gene expression (2). GO analysis of 12h cycling proteins revealed strong enrichment of ER, and intriguingly, mitochondria-associated metabolism pathways, implicating potential coordinated actions of ER and mitochondria in maintaining systemic metabolic homeostasis and stress response (2). While unlike mature mRNA, the phases of 12h protein oscillations are distributed evenly throughout the diurnal cycle, and a clear phase separation of proteins involved in opposing metabolic pathways was found (2). The prevalent hepatic 12h cycling mRNA and proteins involved in metabolic regulation predict a 12h rhythm for hepatic metabolites, which is confirmed by post hoc analysis of two recently published metabolomics datasets (40,41). Eigenvalue/pencil analysis revealed that more than 20% of hepatic metabolites profiled in these two studies exhibited 12h oscillations (2). Further, joint pathway analysis using MetaboAnalyst (42,43) showed coordinated 12h rhythms of metabolites and gene expressions (Fig. 2A, B) (2). For instance, top enriched KEGG pathways exhibiting 12h rhythms of paired gene expression and metabolites include amino sugar and nucleotide sugar metabolism (Gfpt1/Gale: UDP-glucose/UDP-N-acetylamino sugars), Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 pyrimidine metabolism (Uck2: cytidine/CMP), purine metabolism (Ppat/Gart/Atic/Adsl/Ampd2/Nt5e/Gmps: inosine/adenosine/AMP/ADP/ATP/xanthine/guanosine/guanine/GDP), polyamine metabolism (Srm/spermidine), glycerophospholipid metabolism (Pcyt1a: CDP-choline; Lpcat3: 2-Lyso-PC), sphingolipid metabolism (Sphk2: sphingosine), pentose phosphate pathway (Rbks: ribose), fatty acid biosynthesis (Fasn/Elovl6: palmitoleate/vaccinate), and amino acid metabolism (P4ha1: proline/hydroxyproline), among many others (Fig. 2A, B) (2). These highly coupled 12h rhythms of hepatic metabolic gene expressions and metabolite oscillations strongly imply the presence of an endogenous 12h clock component that is responsible for the precisely-timed orchestration of metabolic flux by temporally regulating the expressions of metabolic enzymes. This hypothesis was confirmed experimentally and will be discussed in greater detail later. Of particular interest is the prevalent 12h hepatic ribonucleoside and ribonucleotide oscillations, which are building blocks for RNA synthesis (Fig. 2C). The acrophases of ribonucleosides and ribonucleotides oscillations are restricted at CT22 and CT34, preceding the two peaks of 12h mRNA transcription at CT26 and CT38, implying coordinated purine/pyrimidine metabolism and 12h mRNA transcription (2) (Fig. 2C). The 12h oscillations of metabolism are not restricted to liver tissues. Indirect calorimetry measurement of real-time mouse energy expenditure revealed the presence of 12h harmonics in respiratory exchange ratio oscillations (2), supporting the hypothesis that other peripheral tissues also possess a 12h clock that perhaps helps to synchronize 12h systemic metabolic homeostasis. While the prevalence of 12h rhythms of gene expression and metabolism in mouse is undisputable, due to the technical limitations and ethical constraint of obtaining time series tissue samples from humans, the evidence supporting the existence of 12h rhythms in human tissues in vivo is still largely anecdotal at this time. Nonetheless, in humans, using traditional spectrum analysis methods such as Fourier transform and wavelet analysis, rhythms of body temperature (44-49), blood pressure and heart rate variability (50-55), cognitive performance (49,56-62), migraine onsets and subsequent cerebrospinal fluid sodium level (63), circulating serum and urine metal levels (64), circulating hormone level (65-67) and sleep patterns (58,59,68-75) were all reported to exhibit a 12h rhythmic component. Of these reported 12h rhythms in humans, daily variation of temperature and sleep-wake cycles are two of the most interesting. As discussed later, temperature is a strong Zeitgeber (Zeitgeber is a German word for time giver, which refers to a synchronizing agent) for 12h rhythms in C. elegans (2,76). While it remains unknown whether temperature also can entrain the mammalian 12h clock, it is tempting to hypothesize that the 12h rhythm of mammalian gene expressions and metabolic rhythms are also in tune to the daily temperature oscillations, similar to what has been observed for the circadian clock (77-79). As to the 12h rhythms of sleep-wake patterns, in addition to the well-characterized peak of sleepiness during the subjective night, a second smaller peak of sleepiness during the early afternoon is a widespread phenomenon (also known as ‘siesta’ in Spanish-speaking countries). Moreover, since the afternoon sleep propensity occurred either under a constant routine (70,80) or without having lunch (81), it is highly suggested that it is a part of an endogenous biological rhythm. Currently, sleep regulation is conceptualized by the popular “two-process” model that posits that homoeostatic and circadian drives control sleep (82). The homoeostatic process is controlled by sleep pressure, which accumulates during the course of wakefulness and dissipates during sleep, and is tightly regulated by local changing levels of neuromodulators such as adenosine (83-85). On the other hand, the sleep–wake cycle during the day and night is regulated by the circadian clock, Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 independent of prior sleep or wake time (82). While it has long been thought that the afternoon sleepiness is under the homeostatic sleep control, several studies challenged this notion and suggested that the afternoon sleep propensity may instead reflect an endogenous 12h cycle of slow wave sleep (SWS) (which is also referred as non-rapid eye movement sleep or in more colloquial term ‘deep sleep’), independent of duration of prior wakefulness (62,70,75). Although it has been convincingly demonstrated that slow wave sleep can be strongly induced by local increased levels of adenosine in the basal forebrain through its adenosine A1 receptor and repressed by its antagonist caffeine (83,86), the mechanism of how the concentration of adenosine is regulated in the brain remain elusive. Intriguingly, adenosine and three of its main precursors ATP, AMP and SAH all exhibit robust 12h rhythms in mouse liver (Fig. 2A-B). Further, xanthine, a close relative of caffeine (which is a methylxanthine) also revealed a 12h rhythm (Fig. 2A). While it is premature to conclude that local intracellular and extracellular levels of adenosine and xanthine derivatives may also exhibit 12h rhythms in the CNS and is coupled to the 12h rhythms of slow wave activity cycle, these intriguing observations and correlations warrant future studies to either prove or refute this hypothesis. Evidences supporting the existence of a cell-autonomous mammalian 12h clock. Two pertinent questions arise regarding the mammalian 12h rhythms: 1) Are the 12h oscillations of gene expression and metabolism cell-autonomous? and 2) Are 12h rhythms established by a dedicated mammalian 12h clock or are they regulated by some variants of the well-characterized circadian clock? Theoretically, at least three possibilities exist. In the first scenario, the 12h rhythms are not cell-autonomous in nature. It is possible that one of the two peaks is established by the circadian clock and the second peak results from physiology-associated systemic cues only found in vivo. In the second scenario, the 12h rhythm is cell-autonomous. However, it is established by two circadian transcription activators or repressors appearing in anti-phase and therefore are dependent on the circadian clock (37). In the third scenario, the mammalian 12h rhythms are not only cell-autonomous, but also are established by a dedicated 12h-clock ‘separate’ from the circadian clock, a mechanism similar to the independent circatidal clock in certain crustaceans and intertidal insects (20,33,34). Early studies favor the first hypothesis (the 12h rhythms are not cell-autonomous and established by the combined effects of circadian clock and fasting-feeding cues) due to the following three major observations: 1) No statistically significant 12h rhythms of gene expression were detected in forskolin-synchronized NIH3T3 cells, implying that mammalian 12h rhythms are not cell-autonomous (Fig. 3B) (9); 2) 8h day-time restricted feeding significantly impairs the 12h rhythms of several hepatic gene expression. These genes still maintained peak expression at approximately CT26, coinciding with the new feeding time; however, the subjective evening peak was largely absent. This evidence suggests that at least one component ∆19 of the 12h rhythm is driven by feeding (9); and 3) Brain-specific rescue of Clock mutant mice converted hepatic 12h rhythms into 24h rhythms, suggesting that signaling via the central circadian oscillator may be required to generate one of the two daily peaks of expression (38). While these results could suggest the lack of a cell-autonomous mammalian 12h clock, in light of strong new evidences supporting the existence of a cell-autonomous mammalian 12h clock (2), alternative interpretations of these data are offered and will be elaborated on in greater detail below. An initial clue hinting at the possible existence of an independent mammalian 12h-clock comes from the observed mathematical orthogonal relationship between superimposed 12h and Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 circadian oscillations uncovered for most of the 12h-cycling ER homeostasis and metabolism genes (2). Subsequent post-hoc analysis of hepatic RNA-Seq data in wild-type and whole-body BMAL1 knock-out mice fed ad libitum [both conventional and adult BMAL1 deletion mice (87)] confirmed the independence of 12h rhythms of key ER and metabolism gene expressions from the circadian clock (2). Specifically, for 12h-cycling genes lacking superimposed circadian rhythms (such as Eif2ak3, Gfpt1, Creld2 and Sec23b), their 12h rhythms are almost identical between wild-type and BMAL1 knock-out mice under either a 12h/12h light/dark schedule or a constant darkness condition (2,87) (Fig. 3A). For 12h-cycling genes with superimposed circadian rhythms (such as Gck, Fasn, Dnajb4 and Chka), more perceptible 12h rhythms were observed in BMAL1 knock-out mice (2,87). The independent relationship between 12h and 24h rhythms for ∆19 these ER homeostasis and metabolism genes also was verified in Clock mutant mice (2,88). Can the 12h rhythm of gene expression be found in vitro or does its establishment require systemic cues only found in vivo as previously suggested (9,36)? In light of the newly found independence of 12h-cycling genes from the circadian clock, an alternative interpretation of the lack of observed 12h rhythms in forskolin-synchronzied NIH3T3 cells (9) is that forskolin is capable only of synchronizing the circadian clock but not the circadian clock-independent 12h clock. Consistent with this hypothesis, it was found that the circadian and 12h-cycling genes are responding to a different repertoire of external cues (Fig. 3B) (2). While the cyclic AMP (cAMP) inducer forskolin is only a strong synchronizer of the mammalian circadian clock (9), the ER stress inducer tunicamycin (which blocks UDP-GlcNAc-mediated UDP-N-linked glycosylation) can only synchronize the 12h-clock (2) (Fig. 3B). This is consistent with observed dominant circadian and 12h rhythms of cAMP and UDP-N-acetylamino sugars levels, respectively, in mouse liver (12) (Fig. 2B). Contrary to the discriminatory nature of forskolin and tunicamycin, dexamethasone and glucose depletion, on the other hand, can synchronize both the circadian and 12h-clocks (2) (Fig. 3B). More importantly, while siRNA-mediated depletion of Bmal1 abolishes dexamethasone and glucose depletion-synchronized circadian Per2 expression, it does not affect dexamethasone, glucose depletion nor tunicamycin-synchronized 12h rhythms of Eif2ak3 expression in mouse embryonic fibroblasts (MEFs) (2) (Fig. 3B). Moreover, by cloning the Eif2ak3 promoter before a destabilized green fluorescence protein (dGFP) and performing time- lapse imaging of dGFP intensity in single cells without any prior perturbation, robust 12h oscillations were observed in MEFs (2), which also was not affected by Bmal1 siRNA knockdown (Fig. 3C) (2). Lastly, 37 metabolites of the 137 examined also exhibited ~12h rhythms in dexamethasone-entrained human U2OS cells and 19 of them still maintained ~12h rhythms in the presence of Bmal1 knockdown (12) (Fig. 3D). Taken together, these observations strongly support the hypothesis that the 12h rhythm of gene expression, especially those involved in ER/metabolic pathways, and subsequent 12h rhythms of metabolism, is cell-autonomous and driven by a dedicated 12h pacemaker ‘distinct’ from the circadian clock in mammalian cells (2). While the 12h rhythms of gene expression and metabolism are cell-autonomous, they can be disrupted by altered fast/feeding cycles. Day-time restricted feeding negatively influences 12h rhythms of hepatic gene expression (9). Consistent with this finding, day-time restricted feeding also significantly impairs 12h oscillations of RER in the mouse (2). Thus, one also can envision how the indirect systemic interactions of the circadian and 12h-clocks work. For instance, manipulation/perturbation of the central circadian clock in the brain can lead to altered feeding/fasting and wake/sleep behaviors, which in turn may feedback to the 12h clock to alter 12h rhythms of gene expression and metabolism in peripheral tissues. In fact, this may very well account for the observed phase shift in several 12h-cycling hepatic genes as well as the varied Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 12h-cycling transcriptome reported in different circadian deficient mice models (2,36,87,88). In addition, since many of the 12h cycling genes have superimposed circadian rhythms, alteration of the circadian clock can have a major influence on the overall gene oscillations, without necessarily affecting the superimposed 12h rhythms. This could be the case for the observed conversion of certain hepatic 12h rhythms into 24h rhythms upon brain-specific rescue of ∆19 Clock mutant mice (38). While these new data support the existence of a cell-autonomous mammalian 12h-clock, they do not rule out the possibility that certain 12h rhythms are influenced by the circadian clock and/or the effects of certain external cues and are not cell-autonomous. Future studies are needed to profile the complete 12h transcriptome under the 12h-clock control, as well as to investigate the physiologic conditions by which these distinct clocks can interact systemically in multiple model organisms. The spliced form of XBP1 (XBP1s) transcriptionally regulates the 12h-clock. How is the mammalian 12h-clock regulated? Observed prevalent 12h rhythms of hepatic nascent mRNA and enhancer RNA (eRNA) expression indicate that the mammalian 12h-clock is regulated primarily at the transcriptional level (2,89). Consistent with the strong enrichment of ER stress and unfolded protein response (URP) pathways in 12h-cycling hepatic transcriptome [for a review of mammalian UPR, see (90-92)], 12h cycling of UPR transcription factor XBP1s and ATF4 was found in mouse liver under both 12h/12h light/dark schedule and constant darkness conditions with peak expressions found at CT10-12 and CT22-24, consistent with the acrophases of 12h-cycling hepatic transcriptome (2,36). Furthermore, 12h-cycling of XBP1s expression was found to be induced in tunicamycin-synchronized MEFs and is independent from the circadian clock (2). Intriguingly, both the total level of Xbp1 mRNA (the combined level of Xbp1s and Xbp1us) and its splicing efficiency exhibited robust 12h rhythms both in mouse liver in vivo and in MEFs in vitro (2,36). The 12h rhythms of Xbp1 splicing efficiency and XBP1s expression in vivo is further supported by the observed 12h rhythms of its upstream endoribonuclease IRE1α phosphorylation at Ser724 (36). 12h rhythms of nuclear XBP1s levels correlates with the 12h rhythms of chromatin recruitment of XBP1s to the promoters of several 12h-cycling genes both in mouse liver in vivo and in tunicamycin-synchronized MEFs in vitro (2). Knock-down of Xbp1 significantly inhibits both tunicamycin and glucose depletion-synchronized 12h rhythms of ER homeostasis (such as Eif2ak3, Ddit3, Sec23b and Herpud1) and metabolism (such as Gfpt1 and Acly) gene expression in MEFs (2) (Fig. 3B). In additional, knock-down of Xbp1 abolished cell-autonomous 12h rhythms of Eif2ak3 promoter-driven dGFP intensity in single MEF cell (Fig. 3C). In contrast, ablation of Xbp1 has no effects on dexamethasone and glucose depletion-synchronized circadian gene expression (2) (Fig. 3B). The transcriptional regulation of mammalian 12h transcriptome by XBP1s is further consistent with numerous past reports on the transcriptional regulation of the same repertoire of genes by XBP1s during pathological conditions of ER stress (93-96). This evidence suggests that similar to the circadian clock, the mammalian 12h-clock is also subject to major transcriptional control. It is worth mentioning that so far, the evidence supporting the role of Xbp1s in the regulation of mammalian 12h-clock is solely restricted to the study of a few 12h rhythmic genes in MEFs in vitro, future study using XBP1s knockout mice models are needed to comprehensively profile XBP1s-regulated 12h transcriptome in an unbiased manner. The mammalian 12h-clock is evolutionarily conserved and may be circatidal in origin. Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 From where does the mammalian 12h-clock originate from? Given the distinctions of the circadian clock from 12h-clock/circatidal clock in both mouse and E. pulchra (2,20) and the fact that mammals share common marine ancestors, it is logical to postulate that the mammalian 12h clock evolved from the ancient circatidal clock. Previous work revealed robust 12h circatidal mRNA rhythms for 10 mitochondrial DNA (mtDNA)-encoded protein-coding genes (Mt-Nd1~6, Mt-Cox1~3 and Cytb) that encode mitochondrial components of complexes I (NADH dehydrogenase) and IV (cytochrome c oxidase) in E. pulchra under free-running conditions, which are correlated with circatidal rhythms of oxygen consumption in the same animals (19,97). Post hoc analysis of two published mouse hepatic Gro-Seq (89) and Nascent-Seq (98) datasets that measure nascent RNA transcription rate revealed, surprisingly, conserved 12h rhythms of mtDNA-encoded gene transcription (2) These 12h rhythms of mtDNA-encoded gene expression were further confirmed at mature mRNA and protein levels in mouse liver (2,39,41). Further, cell-autonomous 12h rhythms of mtDNA-encoded gene expression were found in glucose depletion-synchronized MEFs in a BMAL1-independent manner (2). Also supporting the hypothesis that the mammalian 12h-clock evolves from a circatidal origin is the observed similar free-running periodicity of Eif2ak3 promoter-driven dGFP oscillation in MEFs (12.6h) with that of circatidal activity rhythms reported in E. pulchra after tidal cues entrainment (12.7h) and in the mangrove cricket A. asahinai (12.6h to 12.9h) (20,33,34). In addition to crustaceans and mammals, strikingly, mtDNA-encoded gene transcription was found in 12h/12h warm/cold temperature cycles-entrained as well as free-running C. elegans (76) (Fig. 4A). In fact, the vast majority of the C. elegans homologs of core 12h-cycling mammalian transcriptome involved in ER homeostasis and metabolism revealed robust 12h rhythms under the same conditions (2,76)(Fig. 4B). These include the C. elegans homologs of dominant 12h-cycling mammalian genes such as total Xbp1, Eif2ak3, Hspa1b, Gfpt1, Hspa5, Sec23b, Creld2, Dnaja1 and P4ha1, with Hspa1b/hsp-70 being the strongest oscillating gene in both species (Fig. 4B). It is significant that for mammalian 12h-cycling genes with superimposed dominant circadian rhythms (such as Pfkfb3, Por, Map3k5 and Upp2), 12h oscillations are the dominant ones in C. elegans (Fig. 4B). This could be explained by the absence of oscillations of the canonical circadian clock genes in C. elegans (2,76,99). Adding further to this point, these 12h rhythms oscillate much less robustly or are completely absent in free-running C. elegans that are previously entrained by the 12h/12h light/dark cycles (76). Together, this evidence indicates that the C. elegans 12h-clock is responding to temperature, rather than light cues, which is justified by the overall lack of exposure to light in their natural soil habitat. In addition to the nematode, 12h oscillations of ER homeostasis and metabolism gene expression also were found in light-entrained and free-running zebrafish (100) as well as in the liver of light-entrained baboons (101) (Fig. 4C). Most intriguingly, by thoroughly analyzing published time series -187 database, we further identified a total of 280 genes (with a p value of 1.30 x e for observing the same number of conserved 12h genes by chance) that exhibited conserved ~12h rhythms in temperature-entrained C. elegans (76), naturally-caught Cellana rota (a mollusc captured from its natural intertidal habitat and exhibiting a dominant circatidal clock) (24) and mouse liver (9) (Fig. 4E). As expected, gene ontology analysis of these 280 common ~12h-cycling genes reveals enriched biological pathways in metabolic control and ER homeostasis (Fig. 4F). While it is much appreciated that the molecular mechanisms for generating the circadian clock, namely, the transcriptional/translational feedback loop (TTFL), are conserved in multiple species, there is little overlap in the individual genes involved in the establishment of the circadian TTFL clockwork in different species. Therefore, many believed that the circadian clock Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 evolved separately in different lineages (102). However, the fact that the core 12h-clock genes remained conserved even in divergent species that cross phylum boundaries (Fig. 4C) strongly implies that the 12h-clock may even be more ancient and evolved earlier than the circadian clock. Further supporting this hypothesis is the conserved 12h oscillation of the proposed transcriptional regulator of the mammalian 12h-clock, Xbp1 in Cellana rota, whose oscillation is also in phase with the general circatidal transcriptome peaking at rising tides (24) (Fig. 4D,E). Additionally, computationally constructing predicted mammalian interactive network using these 280 genes via STRING (103) put XBP1 in the center of the network connecting different peripheral sub-hubs (Fig. 4G), consistent with the experimental results demonstrating the essential role of Xbp1 in regulating the 12h-clock in MEFs (Fig. 3). Notwithstanding the evidence, the causal relationships between the circatidal Xbp1/UPR-related gene oscillations and the circatidal behavior in these marine animals remain to be determined. The functions of the mammalian 12h-clock It has been widely accepted that all organisms adjust their biochemical, physiological, and behavioral processes in the most advantageous manner, in phase with predictable environmental changes (104). Alignment of endogenous clocks with environmental timing increases the organisms’ overall fitness and survival, while misalignment between the two features significantly reduce an organism’s fitness and can be a strong predisposing factor to multiple morbidities in almost all species examined (105-112). While this conclusion was based mostly upon studies on the circadian clock, it is no exception for the 12h-clock. Intertidal animals exhibiting dominant circatidal clocks tune their tidal rhythmicity to ~12h rhythmic cycles of inundation and exposure that give rise to rapid changes in temperature, salinity, hydrostatic pressure, osmotic pressure, food, and predation pressure (113). The reasons for the existence of a 12h-clock in mammals are, however, subtler. After all, it has been a long time since we “came out of the sea” and mammals no longer experience 12h environmental changes of salinity, temperature or hydrostatic pressure. Instead, we are constantly exposed to circadian changes of temperature, light and food availability and as a result, circadian clock has long been thought to be the only clock present in higher organisms including mammals. Nonetheless, if the theory that biological clocks are always in sync with environmental timing also holds true for the mammalian 12h-clock, then we must have either overlooked some 12h-cycling environmental factors to which the mammals are exposed to daily, or there is “something else” cycling with a 12h period that is indirectly caused by the circadian environmental changes of temperature, light or food commonly associated with land mammals. Here we favor the latter hypothesis and conjecture that the mammalian 12h clock is in tune to a 12h-cycling “stress cycle”, or more specifically, a 12h-cycling “metabolic stress cycle” that results from the innate misalignment between energy intake and energy expenditure in a diurnal cycle (Fig. 5A). The early clues suggesting the existence of such a 12h-cycling metabolic cycle is the unique dawn and dusk phase distribution of 12h-cycling mRNAs in mouse liver, both of which correspond to transition periods between fasting/feeding and sleep/wake (Fig. 2C) (2). At the subjective dawn (ZT10 to ZT12 for the nocturnal mouse; ZT stands for Zeitgeber time and ZT0 is the time of light-on and ZT12 is the time of light-off), the prolonged absence of energy intake from the subjective night combined with reduced but still significant energy expenditure [during sleep the brain and other parts of the body still consume significant amount of energy to consolidate memory, dispose of the metabolic waste and repair the body (114-116)] leads to a peak of energy ‘overdraft’ . By contrast, at the subjective dusk (ZT0 to ZT2 for nocturnal Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 mouse), sufficient energy intake during the subjective day plus significantly reduced energy expenditure gives rise to a peak of energy ‘excess’ (Fig. 5A). Both energy ‘overdraft’ and energy ‘excess’ create great metabolic stress for the mammalian cells. They activate the same stress response pathways to mitigate the original stress, including the two most well-known stress responses that evolved to cope with such metabolic stress: the classical ER-associated unfolded ER protein response (UPR ) (91) and a second stress response pathway reacting to unfolded mt proteins in the mitochondria coined “mitochondrial UPR” or UPR (117-120). For example, both fasting-associated hypoglycemia and feeding-associated hyperglycemia ER conditions can activate the UPR (121-124). Likewise, both increased and decreased mt mitochondria respiration capacity can activate UPR and other mitochondrial stress pathways ER mt (125). Therefore, it is not surprising that both UPR transcription factor xbp-1 and UPR transcription factor atfs-1 exhibit robust 12h rhythms of expression peaking at dawn (C10) and dusk (CT22), synchronized to the 12h rhythms of proposed cellular metabolic stress (Fig. 5A, D). While the true mammalian homolog of atfs-1 remains elusive at this time, one potential candidate Ddit3/Chop (118) does exhibit a robust 12h rhythm in mouse (2). Based on these findings, we propose that the mammalian 12h-clock may govern physiological 12h oscillations ER mt of UPR and UPR to maintain metabolic homeostasis. These oscillations are further synchronized to the energy imbalance-induced 12h metabolic stress cycle occurring in the ER and mitochondria (2). Therefore, we coined the term CREMA (Coordinated Rhythms of ER and Mitochondria Action) to reflect the coupled 12h rhythms of gene expression involved in both ER and mitochondria homeostasis (2). Supporting the existence of such a 12h metabolic stress cycle are observed 12h-cycling metabolites (such as ADP and inosine) in dexamethasone-synchronized human U2OS cells with or without BMAL1 knock-down (Fig. 3D) (12). Nevertheless, more studies utilizing real-time imaging of stress-related metabolites in single cells are needed to substantiate that such a 12h metabolic stress cycle indeed exists in a fully cell-autonomous manner as predicted. The potential roles of the 12h-clock in diseases and therapy In this section, we present early evidences supporting the potential roles of the 12h-clock in regulating human diseases and further discuss how we may exploit this knowledge to better implement chronotherapy. Due to the strong implications that the 12h-clock is key to the controlling of hepatic metabolism and stress responses, we focus specifically on two conditions that are profoundly influenced by these two pathways: nonalcoholic fatty liver diseases (NAFLD) and aging (126-135). Loss of a functional 12h-clock is strongly associated with NAFLD progression. It has been well-established that both nutritional challenge and chronic ER/mitochondrial stress are strong contributing factors to the pathogenesis of NAFLD (126,128,129,135-142). Given the newly discovered housekeeping functions of the 12h-clock in maintaining cycling metabolic stress response, we propose a new mechanism whereby pathological levels of metabolic stress impairs the hepatic 12h-clock and perturbs the metabolic homeostasis that eventually results in NAFLD (Fig. 6A). This hypothesis is reminiscent of the well-established causal relationships among chronic “jet lag stress”, and the perturbed circadian rhythm and metabolic syndrome in both animal models and humans (143-146). At least four pieces of evidence support this hypothesis. First, acute induction of severe ER stress by tunicamycin injection alters the 12h rhythmic gene expression in mouse liver (36). Secondly, nutritional challenge by high fat diet significantly disrupts the 12h oscillation of UPR and metabolic genes including Manf, Sec23b, Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Hspa5, Eif2ak3 and Gfpt1 in mouse liver. By contrast, circadian oscillations of all 17 core circadian clock genes including Bmal1, Per2 and Rev-erbα were affected either modestly, or not at all by the high fat diet challenge (Fig. 6B) (41). Thirdly, gene set enrichment analysis (GSEA) revealed that the downregulation of 12h gene expressions is associated strongly with progression to hepatic steatosis and nonalcoholic steatohepatitis (NASH) in humans (2,147). Last but not least, using both genetic and pharmacological models, it has been convincingly shown that activating ER stress-sensing or ER quality control pathways, including Xbp1s, are protective against hepatic steatosis (148-151). While this evidence strongly supports an association of 12h- clock disruption with progression to NAFLD, future studies using both genetic and pharmacological 12h-clock deficient animal models are needed to firmly establish the causal roles of the 12h-clock in disease development (Fig. 6A). Is the 12h-clock in essence an anti-aging hormetic response? Aging is a complex process characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability and eventually to death. In a comprehensive review published in 2013, a total of nine hallmarks representing the common denominator of the aging process in multiple organisms were proposed (134). Of these hallmarks, loss of proteostasis, deregulated nutrient sensing and mitochondria dysfunction are biological processes under strong 12h-clock regulation, suggesting a potential role of the 12h- clock in mediating aging, especially in the prevention of aging-related metabolic decline (2,130,134). In fact, NAFLD itself is commonly believed to be an age-related morbidity (135,152). Further supporting this hypothesis is the significantly disrupted 12h rhythm (but not circadian rhythm) of hepatic gene expression in old mice (Fig. 5C) (153), reminiscent of similarly disrupted hepatic 12h rhythms in mice fed a high fat diet (Fig. 5B). Intriguingly, genes known to regulate lifespan in C.elegans are highly enriched for 12h rhythms of gene expression (Fig. 5D) (76). These include positive regulators of longevity such as sir2.1 (homolog of mammalian Sirt1) (154-158), daf-16 (homolog of mammalian Foxo1) (159,160), aak-2 (homolog of mammalian Prkaa1/2, also known as Ampkα1/2) (161,162) and tcer 1 (homolog of mammalian Tcerg1) (163) as well as a significant number of negative regulators of C. elegans lifespan uncovered from a RNAi screen study (164) (Fig. 5D). More interestingly, Sirt1, Foxo1, Prkaa1 and Prkaa2 also exhibit 12h rhythms in mouse liver (Fig. 5D) (2). The potential mechanisms of the 12h-clock in mediating longevity and aging, in our opinion, may lie in the ancient concept of ‘hormesis’, which is the biological embodiment of the idiom “what doesn’t kill you makes you stronger”. In scientific terms, hormesis is an adaptive cellular response whereby exposure to low doses of stress (but not high dose) activates protective mechanisms that render the cell resistant to a subsequent challenge with higher doses of stress (165). The exact molecular mechanisms underlying hormesis are still unclear but have been previously attributed to the actions of both ER and mitochondria, which are termed ER hormesis and mitohormesis, respectively (125,166,167). In either case, the hormetic response is mediated ER either by xbp1-dependent UPR (for ER hormesis) or atfs-1/hsp-60 (Hspd1 in mammals)- mt dependent UPR (for mitohormesis) (125,166). Both ER hormesis and mitohormesis are positively associated with organismal development, stress resistance, and more importantly, longevity. For example, a study in C. elegans showed that expression of XBP1s in neurons was sufficient to increase longevity and induce ER hermetic response in distal, non-neuronal cell types via a cell-nonautonomous mechanism (168,169). Further, mitochondrial ribosomal protein mt knockdown-mediated lifespan extension also requires hsp60-dependent UPR in both C. elegans and mammalian cells (170). Considering the fact that both ER hormesis and Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 mitohormesis master regulators xbp-1 and atfs-1/hsp-60 exhibit conserved 12h rhythms and the central roles of the 12h-clock in stress response regulation, we conjecture that the hormetic response may exert its anti-aging effects partially through the amplification/boosting of the endogenous 12h-clock. Indeed, we found that only low dose, but not high dose of tunicamycin (lower than 30 ng/ml) is capable of synchronizing the 12h-clock in MEFs, consistent with the concept of hormesis ((2) and unpublished data). 12h-clock-based chronotherapy Chronotherapy is the concept that treatment of an illness or disorder should take the body’s natural rhythms and cycles into accounts (171). It has been shown that drugs that target rhythmic, high-amplitude circadian gene products represent a potential pathway for mechanism- driven chronotherapy (172-176). Here we propose that similarly, 12h rhythmic transcriptome and proteome can function as blueprint for future design of 12h-clock-based chronotherapy. To this end, we probed FDA-approved drugs for potential interactions with high amplitude 12h-cycling hepatic transcripts and proteins using the Drug Gene Interaction Database (DGIdb) (177,178) and found a number of hits against both 12h-cycling hepatic mRNA and/or their protein targets (Fig. 5E,F). For example, 6-mercaptopurine is a medication for treating a variety of blood cancer and autoimmune diseases. Among of its mechanisms of action is the inhibition of de novo purine synthesis by directly inhibiting the rate-limiting enzyme phosphoribosyl pyrophosphate amidotransferase encoded by the Ppat gene (179,180). Since, as we discussed previously, coordinated 12h rhythms of hepatic purine metabolism with RNA transcription is a strong feature of the mammalian 12h-clock (Fig. 2B-C) (2), it may be desirable to administer mercaptopurine (and other drugs targeting de novo purine and pyrimidine biosynthesis) at times of nadir purine/pyrimidine metabolism (at 3-4pm) to minimize hepatic toxicity. Due to the very early stage of the 12h rhythm field (and ultradian rhythms field as a whole), currently, very little is known about its definitive functions and implications in human diseases. To a large extent, we can only speculate on their functions and implications based upon the limited literature available so far. We hope this review/preview can attract more scientists into this nascent field, because as outlined below, we believe that only the tip of the iceberg has been uncovered thus far and that there is so much more to explore. Future directions Future work could be directly toward three major aims: 1) delineating the molecular and physiological mechanisms of 12h-clock regulation; 2) establishing the causal relationship between 12h-clock disruption and disease progression, and 3) translating the above knowledge into chronotherapy-based medical practice (Fig. 6A). For example, at the molecular and cellular levels: Is the 12h rhythm temperature-compensated? What is the full spectrum of the 12h-cycling transcriptome, proteome and metabolome under 12h-clock control? What are the negative transcriptional regulators of the mammalian 12h-clock? What are the co-regulators for XBP1s- dependent 12h-clock transcriptional control? Do some post-transcriptional and post-translational mechanism exist, if so, what are they? At the systemic and physiological level: How are the 12h rhythms in different tissues and organs coordinated and synchronized at the systemic level? Are there 12h-cycling hormonal factors coordinating different tissues? Maybe a more tempting question is: does the 12h-clock exist in the central nervous system and if so, where? More, 12h- clock deficient animal models are needed in the future to rigorously establish the causal relationship between 12h-clock dysfunction and multiple disease development. Our ultimate goal Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 should be to successfully translate the newly-learnt knowledge into chronotherapy-based medicine and disease prevention. Conclusion Based upon the current available literature, here we propose a model where combined actions of both the circadian and 12h-clock are required to ensure cellular homeostasis and promote organism integrity and overall fitness (Fig. 6B). Quite a number of genes in the mammalian genome are under dual circadian and 12h-clock, although through distinct molecular mechanisms. The circadian clock is transcriptionally regulated through a revised TTFL model that includes activation phase-facilitated repression mainly via the ROR response element (RORE) (Fig. 6B) (181), while the 12h-clock is transcriptionally regulated by 12h rhythms of XBP1s chromatin recruitment to ER response element (ERSE) (Fig. 6B) (2). Superimposition of both circadian and 12h rhythms of gene expression leads to a temporal gene expression pattern characterized by two unsymmetrical peaks within a diurnal cycle (Fig. 6B), which is commonly observed in metabolic genes such as Fasn and Gck (Fig. 1C) (2). Further, the circadian clock and the 12h-clock are synchronized to different environmental cues. The circadian clock is responding to the differential between the two opposite metabolic states: fast and feeding; whereas the 12h-clock is entrained by the common denominator associated with both fast and feeding: the peaking of metabolic stress (Fig. 6B). In addition to the circadian and 12h-clocks, it is very likely that additional clock components also exist in mammals, such as an 8h-clock (Fig. 1B-D). We reason that having multiple clock components endows organisms with more flexibility and the heightened ability to adapt to different environments, thus significantly increasing survival advantage. In theory, simply adjusting the relative phases and fine-tuning the amplitudes of different clocks can give rise to an infinite number of peak patterns to cope with a variety of daily environmental cycles. The co-existence of multiple harmonic biological rhythms in tune to the natural rhythms is reminiscent of the ancient philosophic concept of ‘musica universalis’, which was first proposed by the Greek philosopher Pythagoras over two thousand years ago (1). According to this theory, all natural appearing rhythms are in essence tuned to the rhythmic movements of celestial bodies (1). While the circadian rhythm is synchronized to the 24h light/dark cycle coinciding with the Earth’s rotation, our findings suggest that the 12h clock may have evolved from the ancient circatidal clock, which is in turn entrained by the 12h tidal cues orchestrated mainly by the moon. Thus, it appears after two thousand years, we may have finally found evidence for the “biological musica universalis” (Fig. 6B). American Diabetes Association http://dx.doi.org/10.13039/100000041, 1-18-JDF-025, Bokai Zhu; Brockman Foundation, Bert W O’Malley; Brockman Foundation, Clifford Dacso; Center for the Advancement of Science in Space http://dx.doi.org/10.13039/100011048, GA-2014-136, Clifford Dacso; Peter J. Fluor Family Fund, Clifford Dacso; Joyce Family Foundation, Clifford Dacso; Sonya and William Carpenter, Clifford Dacso; National Science Foundation http://dx.doi.org/10.13039/100000001, 11703170, Clifford Dacso; Center for the Advancement of Science in Space http://dx.doi.org/10.13039/100011048, GA-2014-136, Bert W O’Malley; Phillip J. Carroll, Jr. Professorship, Clifford Dacso Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Acknowledgment: We acknowledge all members of O’Malley’s lab for support for this project, with special thanks to Naomi Gonzalez, Dr. Brian York and Dr. Maricarmen Delia Planas-Silva. We also like to thank Drs. Oren Levy and Yisrael Schnytzer from Bar-Ilan University for kindly sharing the processed transcriptome data of C. rota. We apologize for omission of relevant works and citations due to space constraints. This research was supported by the Brockman Foundation to B.W.O. and C.C.D. In addition, this research also was supported by grants from the Center for Advancement of Science in Space, Peter J. Fluor Family Fund, Philip J. Carroll, Jr. Professorship, Joyce Family Foundation to C.C.D and the American Diabetes Association junior faculty development award 1-18-JDF-025 to B.Z. The authors declare no competing or financial interests. *Correspondence: berto@bcm.edu The authors declare no conflicts of interest. References: 1. Taruskin R. Music in the Western World: A History in Documents (2d ed.). Choral Journal 2008; 48:68-70 2. Zhu B, Zhang Q, Pan Y, Mace EM, York B, Antoulas AC, Dacso CC, O'Malley BW. A Cell-Autonomous Mammalian 12 hr Clock Coordinates Metabolic and Stress Rhythms. 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Coactivator-Dependent Oscillation of Chromatin Accessibility Dictates Circadian Gene Amplitude via REV-ERB Loading. Mol Cell 2015; 60:769-783 Figure 1. The concept of harmonics in physics and their biological counterpart. (A) A diagram showing the individual harmonics (200Hz, 400Hz, 600Hz, 800Hz and 1000Hz) as well as the composite waveform resulting from adding up all the harmonics. (B) The distribution of periods of all oscillations identified from the hepatic gene expression microarray dataset (9) via st nd the eigenvalue/pencil approach. The vast majority of oscillations cycle at the 1 (24h), 2 (12h) rd or 3 (8h) harmonic of the circadian rhythm (24h). (C) Representative deconvolution of Gck gene mRNA expression by the eigenvalue/pencil method. Gck expressions detected by two different probe sets are analyzed by the eigenvalue/pencil method. (i) Raw microarray data (solid line) and model fit (dashed line) for Gck hepatic expression as reported in (9). Superimposed harmonic oscillations revealed by the eigenvalue/pencil method for probe 1 (ii) and probe 2 (iii). (iv) Amplitudes, phases and periods of different oscillations for the two probes with the color matching the different oscillations depicted in (ii) and (iii). (D) The distribution of periods of all oscillations identified from the hepatic metabolites dataset (12) via the eigenvalue/pencil st nd rd th approach. The vast majority of oscillations cycle at the 1 (24h), 2 (12h), 3 (8h) or 4 (6h) harmonic of the circadian rhythm (24h). Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 Figure 2. Coupled 12h rhythms of hepatic gene expression and metabolism. (A) Heatmap of mouse 12h-cycling hepatic metabolites identified by the eigenvalue/pencil method from published metabolomic dataset (12) after hierarchical clustering. Metabolites with dominant 12h oscillations are highlighted in red. (B) (Top) Gene-metabolite joint pathway analysis using using MetaboAnalyst (42,43) reveals top enriched biological pathways. Both enrichment as well as topology scores were shown for each pathway. (Bottom) Representative data showing paired gene expression and metabolite oscillations. (C) Distribution of acrophases of all dominant hepatic 12h-cycling mRNA and ribonuclesides/ribonucleotides. Figure 3. 12h rhythms of gene expression and metabolism are cell-autonomous, established by a dedicated 12h-clock and evolutionarily conserved. (A) Representative RPKM normalized hepatic circadian (top) as well as 12h-cycling (bottom) gene expression from WT and conventional BMAL1 KO mouse under 12h/12h light/dark conditions as reported in (87) . Data are graphed as the mean ± SEM (n = 4) and double plotted for better visualization. (B) Heatmap representation of oscillations of Eif2ak3 and Per2 mRNA level after dexamethasone, tunicamycin or glucose depletion shock treatment in MEFs. The heatmap is derived from qPCR data reported in (2). A summary of the conclusion is shown in the table below. (C) Representative recordings of single cell time lapse microscopy analysis of Eif2ak3 promoter- driven dGFP oscillation in scrambled siRNA, Bmal1 siRNA or Xbp1 siRNA transfected MEFs. (D) Heatmap of 12h-cycling metabolites in dexamethasone-synchronized human U2OS cells under both scrambled siRNA and Bmal1 siRNA transfection conditions compiled from (12). 12h-cycling metabolites are identified by the eigenvalue/pencil method (2). Figure 4. 12h rhythms of gene expression are evolutionarily conserved. (A) Heatmap of temporal mtDNA-encoded gene expression in temperature-entrained as well as free-running C.elegans compiled from (76). (B) Heatmap of core ER homeostasis and metabolism related 12h-cycling gene expression in both temperature-entrained as well as free-running C.elegans [left and compiled from (76)] and mouse liver under constant darkness [right and compiled from (9)]. Xbp1 is highlighted in red. (C) Phylogenetic tree and relative mRNA expression of Eif2ak3, Gfpt1 and Dnajb4 in Cellana rota [second row and reported in (24)], Caenorhabditis elegans [third row and reported in (76)], Danio rerio [fourth row and reported in (100)], Mus musculus [fifth row and reported in (9)] and Papio anubis [last row and reported in (101)] during a 48h interval. The status of the three genes were not reported for Eurydice pulchra in the study (19). The data for Papio anubis is double plotted for better visualization. (D) The mRNA level of Xbp1 ortholog in Cellana rota captured from different time of day from their natural intertidal habitat in the wild as compiled from (24). (E) Heatmap of all 280 evolutionarily 12h-cycling gene expression in temperature-entrained as well as free-running C.elegans [left and compiled from (76)], naturally caught C. rota in tune to 12h-cycling tidal cues under 12h/12h natural light condition [middle and compiled from (24)] and mouse liver under constant darkness [right and compiled from (9)]. (F) GO analysis of enriched KEGG pathways from these 280 genes. (G) Predicted interactive network construction of these 280 proteins using STRING (103) with XBP1 highlighted by the arrow. Figure 5. Mammalian 12h-clock in diseases and chronotherapy. (A) A diagram summarizing the origin, regulation, function and species conservation of 12h-clock. Please see the main text for detailed description of each section. (B) High fat diet disrupts the hepatic 12h-cycling, but not circadian gene expression in mouse. Heatmap (left) and representative log2 normalized Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Journal of the Endocrine Society; Copyright 2018 DOI: 10.1210/js.2018-00113 expression (right) of key 12h-cycling and circadian genes under normal chow and high fat diet conditions are compiled from (41). The data is double plotted for better visualization. (C) Disrupted hepatic 12h rhythms are associated with aging. Heatmap (top) and representative mRNA expression (bottom) of key 12h-cycling and circadian gene in young and old male mice are compiled from (153). (D) Aging-regulating genes are enriched for 12h rhythmicity in C.elegans. (Top) Heatmap of aging-related 12h-cycling gene expression in both temperature- entrained as well as free-running C.elegans as compiled from (76). Of 62 genes that increase worm lifespan by 10% when knocked down post-developmentally (164), 38 (62%) of them showed 12h rhythm in temperature-entrained and free-run worms as shown in the heatmap. In addition, positive regulators of aging including atfs-1, xbp-1, daf-16, aak-2, tcer-1 and sir-2.1 all exhibited 12h rhythms of gene expression. (Bottom) Heatmap of mammalian ortholog of sir-2.1 (Sirt1), daf-16 (Foxo1), aak-2 (Prkaa1 and Prkaa2) expression in mouse liver under constant darkness condition as compiled from (9). Atfs1 and Xbp1 are highlighted in red. (E, F) A 12h- clock based chronotherapy blueprint. Heatmap of temporal mRNA (E) and protein (F) expression of hepatic 12h-cycling genes with FDA-approved drug interactions as compiled from (9,39). The names of drug are indicated on the right. FEN1, FASN and PPAT also exhibit 12h rhythms of mRNA expression and are highlighted in red. Figure 6. The framework of 12h-clock study. (A) A diagram summarizing the potential causal roles of mammalian 12h-clock in disease development with future directions outlined below. 12h rhythms can be disrupted either by the dampening of the amplitude or alteration of the period. (B) A diagram summarizing the basis for biological system being part of “musica universalis”. Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: JES Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: ADVANCE ARTICLE: ADVANCE ARTICLE: Endocrinology Endocrinology JES Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: ADVANCE ARTICLE: ADVANCE ARTICLE: Endocrinology Endocrinology JES Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: ADVANCE ARTICLE: ADVANCE ARTICLE: Endocrinology Endocrinology JES Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: ADVANCE ARTICLE: ADVANCE ARTICLE: Endocrinology Endocrinology JES Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: ADVANCE ARTICLE: ADVANCE ARTICLE: Endocrinology Endocrinology JES Downloaded from https://academic.oup.com/jes/advance-article-abstract/doi/10.1210/js.2018-00113/5033317 by Ed 'DeepDyve' Gillespie user on 08 June 2018 ADVANCE ARTICLE JOURNAL OF THE ENDOCRINE SOCIETY ADVANCE ARTICLE: ADVANCE ARTICLE: ADVANCE ARTICLE: Endocrinology Endocrinology JES

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Journal of the Endocrine SocietyOxford University Press

Published: Jun 6, 2018

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