Circadian regulation of macromolecular complex turnover and proteome renewal.

Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different pulse-labelling strategies, here we gain direct insight into the relationship between protein synthesis and abundance proteome-wide. We show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results in daily consolidation of proteome renewal whilst minimising changes in composition. Coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, particularly the ribosome, the most abundant species of complex in the cell. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythms in the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of protein turnover.

Potassium rhythms couple the circadian clock to the cell cycle.

Circadian (~24 h) rhythms are a fundamental feature of life, and their disruption increases the risk of infectious diseases, metabolic disorders, and cancer1-6. Circadian rhythms couple to the cell cycle across eukaryotes7,8 but the underlying mechanism is unknown. We previously identified an evolutionarily conserved circadian oscillation in intracellular potassium concentration, [K+]i9,10. As critical events in the cell cycle are regulated by intracellular potassium11,12, an enticing hypothesis is that circadian rhythms in [K+]i form the basis of this coupling. We used a minimal model cell, the alga Ostreococcus tauri, to uncover the role of potassium in linking these two cycles. We found direct reciprocal feedback between [K+]i and circadian gene expression. Inhibition of proliferation by manipulating potassium rhythms was dependent on the phase of the circadian cycle. Furthermore, we observed a total inhibition of cell proliferation when circadian gene expression is inhibited. Strikingly, under these conditions a sudden enforced gradient of extracellular potassium was sufficient to induce a round of cell division. Finally, we provide evidence that interactions between potassium and circadian rhythms also influence proliferation in mammalian cells. These results establish circadian regulation of intracellular potassium levels as a primary factor coupling the cell- and circadian cycles across diverse organisms.

NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction.

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR).

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Cell autonomous regulation of herpes and influenza virus infection by the circadian clock.

Viruses are intracellular pathogens that hijack host cell machinery and resources to replicate. Rather than being constant, host physiology is rhythmic, undergoing circadian (∼24 h) oscillations in many virus-relevant pathways, but whether daily rhythms impact on viral replication is unknown. We find that the time of day of host infection regulates virus progression in live mice and individual cells. Furthermore, we demonstrate that herpes and influenza A virus infections are enhanced when host circadian rhythms are abolished by disrupting the key clock gene transcription factor Bmal1. Intracellular trafficking, biosynthetic processes, protein synthesis, and chromatin assembly all contribute to circadian regulation of virus infection. Moreover, herpesviruses differentially target components of the molecular circadian clockwork. Our work demonstrates that viruses exploit the clockwork for their own gain and that the clock represents a novel target for modulating viral replication that extends beyond any single family of these ubiquitous pathogens.

Daily magnesium fluxes regulate cellular timekeeping and energy balance.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

Effects of caffeine on the human circadian clock in vivo and in vitro.

Caffeine's wakefulness-promoting and sleep-disrupting effects are well established, yet whether caffeine affects human circadian timing is unknown. We show that evening caffeine consumption delays the human circadian melatonin rhythm in vivo and that chronic application of caffeine lengthens the circadian period of molecular oscillations in vitro, primarily with an adenosine receptor/cyclic adenosine monophosphate (AMP)-dependent mechanism. In a double-blind, placebo-controlled, ~49-day long, within-subject study, we found that consumption of a caffeine dose equivalent to that in a double espresso 3 hours before habitual bedtime induced a ~40-min phase delay of the circadian melatonin rhythm in humans. This magnitude of delay was nearly half of the magnitude of the phase-delaying response induced by exposure to 3 hours of evening bright light (~3000 lux, ~7 W/m(2)) that began at habitual bedtime. Furthermore, using human osteosarcoma U2OS cells expressing clock gene luciferase reporters, we found a dose-dependent lengthening of the circadian period by caffeine. By pharmacological dissection and small interfering RNA knockdown, we established that perturbation of adenosine receptor signaling, but not ryanodine receptor or phosphodiesterase activity, was sufficient to account for caffeine's effects on cellular timekeeping. We also used a cyclic AMP biosensor to show that caffeine increased cyclic AMP levels, indicating that caffeine influenced a core component of the cellular circadian clock. Together, our findings demonstrate that caffeine influences human circadian timing, showing one way that the world's most widely consumed psychoactive drug affects human physiology.

Class IIa histone deacetylases are conserved regulators of circadian function.

Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca(2+) and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery.

Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse.

The suprachiasmatic nucleus (SCN) coordinates circadian rhythms that adapt the individual to solar time. SCN pacemaking revolves around feedback loops in which expression of Period (Per) and Cryptochrome (Cry) genes is periodically suppressed by their protein products. Specifically, PER/CRY complexes act at E-box sequences in Per and Cry to inhibit their transactivation by CLOCK/BMAL1 heterodimers. To function effectively, these closed intracellular loops need to be synchronized between SCN cells and to the light/dark cycle. For Per expression, this is mediated by neuropeptidergic and glutamatergic extracellular cues acting via cAMP/calcium-responsive elements (CREs) in Per genes. Cry genes, however, carry no CREs, and how CRY-dependent SCN pacemaking is synchronized remains unclear. Furthermore, whereas reporter lines are available to explore Per circadian expression in real time, no Cry equivalent exists. We therefore created a mouse, B6.Cg-Tg(Cry1-luc)01Ld, carrying a transgene (mCry1-luc) consisting of mCry1 elements containing an E-box and E'-box driving firefly luciferase. mCry1-luc organotypic SCN slices exhibited stable circadian bioluminescence rhythms with appropriate phase, period, profile, and spatial organization. In SCN lacking vasoactive intestinal peptide or its receptor, mCry1 expression was damped and desynchronized between cells. Despite the absence of CREs, mCry1-luc expression was nevertheless (indirectly) sensitive to manipulation of cAMP-dependent signaling. In mPer1/2-null SCN, mCry1-luc bioluminescence was arrhythmic and no longer suppressed by elevation of cAMP. Finally, an SCN graft procedure showed that PER-independent as well as PER-dependent mechanisms could sustain circadian expression of mCry1. The mCry1-luc mouse therefore reports circadian mCry1 expression and its interactions with vasoactive intestinal peptide, cAMP, and PER at the heart of the SCN pacemaker.

Histone methyltransferase MLL3 contributes to genome-scale circadian transcription.

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

The essential role of cAMP/Ca2+ signalling in mammalian circadian timekeeping.

Approximately daily, or circadian, rhythms are ubiquitous across eukaryotes. They are manifest in the temporal co-ordination of metabolism, physiology and behaviour, thereby allowing organisms to anticipate and synchronize with daily environmental cycles. Although cellular rhythms are self-sustained and cell-intrinsic, in mammals, the master regulator of timekeeping is localized within the hypothalamic SCN (suprachiasmatic nucleus). Molecular models for mammalian circadian rhythms have focused largely on transcriptional-translational feedback loops, but recent data have revealed essential contributions by intracellular signalling mechanisms. cAMP and Ca2+ signalling are not only regulated by the cellular clock, but also contribute directly to the timekeeping mechanism, in that appropriate manipulations determine the canonical pacemaker properties of amplitude, phase and period. It is proposed that daily auto-amplification of second messenger activity, through paracrine neuropeptidergic coupling, is necessary and sufficient to account for the increased amplitude, accuracy and robustness of SCN timekeeping.

Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri.

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.

Healthy clocks, healthy body, healthy mind.

Circadian rhythms permeate mammalian biology. They are manifested in the temporal organisation of behavioural, physiological, cellular and neuronal processes. Whereas it has been shown recently that these approximately 24-hour cycles are intrinsic to the cell and persist in vitro, internal synchrony in mammals is largely governed by the hypothalamic suprachiasmatic nuclei that facilitate anticipation of, and adaptation to, the solar cycle. Our timekeeping mechanism is deeply embedded in cell function and is modelled as a network of transcriptional and/or post-translational feedback loops. Concurrent with this, we are beginning to understand how this ancient timekeeper interacts with myriad cell systems, including signal transduction cascades and the cell cycle, and thus impacts on disease. An exemplary area where this knowledge is rapidly expanding and contributing to novel therapies is cancer, where the Period genes have been identified as tumour suppressors. In more complex disorders, where aetiology remains controversial, interactions with the clockwork are only now starting to be appreciated.

cAMP-dependent signaling as a core component of the mammalian circadian pacemaker.

The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3',5'-monophosphate (cAMP) signaling constitutes an additional, bona fide component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase, and period. This role is general and is evident in peripheral mammalian tissues and cell lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.

Synchronization and maintenance of timekeeping in suprachiasmatic circadian clock cells by neuropeptidergic signaling.

Circadian timekeeping in mammals is driven by transcriptional/posttranslational feedback loops that are active within both peripheral tissues and the circadian pacemaker of the suprachiasmatic nuclei (SCN). Spontaneous synchronization of these molecular loops between SCN neurons is a primary requirement of its pacemaker role and distinguishes it from peripheral tissues, which require extrinsic, SCN-dependent cues to impose cellular synchrony. Vasoactive intestinal polypeptide (VIP) is an intrinsic SCN factor implicated in acute activation and electrical synchronization of SCN neurons and coordination of behavioral rhythms. Using real-time imaging of cellular circadian gene expression across entire SCN slice cultures, we show for the first time that the Vipr2 gene encoding the VPAC2 receptor for VIP is necessary both to maintain molecular timekeeping within individual SCN neurons and to synchronize molecular timekeeping between SCN neurons embedded within intact, organotypical circuits. Moreover, we demonstrate that both depolarization and a second SCN neuropeptide, gastrin-releasing peptide (GRP), can acutely enhance and synchronize molecular timekeeping in Vipr2-/- SCN neurons. Nevertheless, transiently activated and synchronized Vipr2-/- cells cannot sustain synchrony in the absence of VIP-ergic signaling. Hence, neuropeptidergic interneuronal signaling confers a canonical property upon the SCN: spontaneous synchronization of the intracellular molecular clockworks of individual neurons.