[Harmonic oscillations of circadian rhythms come out of the shadows]. / Les oscillations harmoniques des rythmes circadiens sortent de l'ombre.

About 40 % of the liver transcriptome display a circadian expression. Recently, harmonic oscillations of the circadian rhythm and independent of the circadian clock have been identified. Transcripts oscillating with a 12h period are involved in fundamental and ubiquitous cellular mechanisms such as proteostasis, lipid metabolism or RNA metabolism. A 12h ultradian oscillator involving the UPR response regulator XBP1, the coactivator SRC-3 and the speckle protein SON has been uncovered. The XBP1 oscillator and the 12h ultradian transcriptome are highly conserved suggesting an early emergence that may date back to a time when the Earth's day was much shorter than 24h.

Title: Les oscillations harmoniques des rythmes circadiens sortent de l'ombre. Abstract: Environ 40 % du transcriptome hépatique a une expression circadienne. Récemment, des oscillations harmoniques du rythme circadien, indépendantes de l'horloge circadienne, ont été identifiées. Les transcrits oscillant avec une période de douze heures sont impliqués dans des mécanismes cellulaires fondamentaux et ubiquitaires, tels que la protéostase, le métabolisme des lipides ou le métabolisme des ARN. Un oscillateur ultradien de douze heures impliquant le régulateur de la réponse UPR XBP1, le coactivateur SRC-3 et la protéine des speckles SON, commence à être décrypté. L'oscillateur XBP1 et le transcriptome ultradien de douze heures sont très conservés, suggérant une émergence précoce qui pourrait remonter à une époque où le jour terrestre était bien inférieur à vingt-quatre heures.

Cycle dynamics and synchronization in a coupled network of peripheral circadian clocks.

The intercellular interactions between peripheral circadian clocks, located in tissues and organs other than the suprachiasmatic nuclei of the hypothalamus, are still very poorly understood. We propose a theoretical and computational study of the coupling between two or more clocks, using a calibrated, reduced model of the circadian clock to describe some synchronization properties between peripheral cellular clocks. Based on a piecewise linearization of the dynamics of the mutual CLOCK:BMAL1/PER:CRY inactivation term, we suggest a segmentation of the circadian cycle into six stages, to help analyse different types of synchronization between two clocks, including single stage duration, total period and maximal amplitudes. Finally, our model reproduces some recent experimental results on the effects of different regimes of time-restricted feeding in liver circadian clocks of mice.

KLF10 integrates circadian timing and sugar signaling to coordinate hepatic metabolism.

The mammalian circadian timing system and metabolism are highly interconnected, and disruption of this coupling is associated with negative health outcomes. Krüppel-like factors (KLFs) are transcription factors that govern metabolic homeostasis in various organs. Many KLFs show a circadian expression in the liver. Here, we show that the loss of the clock-controlled KLF10 in hepatocytes results in extensive reprogramming of the mouse liver circadian transcriptome, which in turn alters the temporal coordination of pathways associated with energy metabolism. We also show that glucose and fructose induce Klf10, which helps mitigate glucose intolerance and hepatic steatosis in mice challenged with a sugar beverage. Functional genomics further reveal that KLF10 target genes are primarily involved in central carbon metabolism. Together, these findings show that in the liver KLF10 integrates circadian timing and sugar metabolism-related signaling, and serves as a transcriptional brake that protects against the deleterious effects of increased sugar consumption.

A detailed map of coupled circadian clock and cell cycle with qualitative dynamics validation.

BACKGROUND: The temporal coordination of biological processes by the circadian clock is an important mechanism, and its disruption has negative health outcomes, including cancer. Experimental and theoretical evidence suggests that the oscillators driving the circadian clock and the cell cycle are coupled through phase locking. RESULTS: We present a detailed and documented map of known mechanisms related to the regulation of the circadian clock, and its coupling with an existing cell cycle map which includes main interactions of the mammalian cell cycle. The coherence of the merged map has been validated with a qualitative dynamics analysis. We verified that the coupled circadian clock and cell cycle maps reproduce the observed sequence of phase markers. Moreover, we predicted mutations that contribute to regulating checkpoints of the two oscillators. CONCLUSIONS: Our approach underlined the potential key role of the core clock protein NR1D1 in regulating cell cycle progression. We predicted that its activity influences negatively the progression of the cell cycle from phase G2 to M. This is consistent with the earlier experimental finding that pharmacological activation of NR1D1 inhibits tumour cell proliferation and shows that our approach can identify biologically relevant species in the context of large and complex networks.

MCD diet-induced steatohepatitis generates a diurnal rhythm of associated biomarkers and worsens liver injury in Klf10 deficient mice.

A large number of hepatic functions are regulated by the circadian clock and recent evidence suggests that clock disruption could be a risk factor for liver complications. The circadian transcription factor Krüppel like factor 10 (KLF10) has been involved in liver metabolism as well as cellular inflammatory and death pathways. Here, we show that hepatic steatosis and inflammation display diurnal rhythmicity in mice developing steatohepatitis upon feeding with a methionine and choline deficient diet (MCDD). Core clock gene mRNA oscillations remained mostly unaffected but rhythmic Klf10 expression was abolished in this model. We further show that Klf10 deficient mice display enhanced liver injury and fibrosis priming upon MCDD challenge. Silencing Klf10 also sensitized primary hepatocytes to apoptosis along with increased caspase 3 activation in response to TNFα. This data suggests that MCDD induced steatohepatitis barely affects the core clock mechanism but leads to a reprogramming of circadian gene expression in the liver in analogy to what is observed in other experimental disease paradigms. We further identify KLF10 as a component of this transcriptional reprogramming and a novel hepato-protective factor.

Transcription-based circadian mechanism controls the duration of molecular clock states in response to signaling inputs.

The molecular oscillator of the mammalian circadian clock consists in a dynamical network of genes and proteins whose main regulatory mechanisms occur at the transcriptional level. From a dynamical point of view, the mechanisms leading to an oscillatory solution with an orderly protein peak expression and a clear day/night phase distinction remain unclear. Our goal is to identify the essential interactions needed to generate phase opposition between the activating CLOCK:BMAL1 and the repressing PER:CRY complexes and to better distinguish these two main clock molecular phases relating to rest/activity and fast/feeding cycles. To do this, we develop a transcription-based mathematical model centered on linear combinations of the clock controlled elements (CCEs): E-box, R-box and D-box. Each CCE is responsive to activators and repressors. After model calibration with single-cell data, we explore entrainment and period tuning via interplay with metabolism. Variation of the PER degradation rate γp, relating to the tau mutation, results in asymmetric changes in the duration of the different clock molecular phases. Time spent at the state of high PER/PER:CRY decreases with γp, while time spent at the state of high BMAL1 and CRY1, both proteins with activity in promoting insulin sensitivity, remains constant. This result suggests a possible mechanism behind the altered metabolism of tau mutation animals. Furthermore, we expose the clock system to two regulatory inputs, one relating to the fast/feeding cycle and the other to the light-dependent synchronization signaling. We observe the phase difference between these signals to also affect the relative duration of molecular clock states. Simulated circadian misalignment, known to correlate with insulin resistance, leads to decreased duration of BMAL1 expression. Our results reveal a possible mechanism for clock-controlled metabolic homeostasis, whereby the circadian clock controls the relative duration of different molecular (and metabolic) states in response to signaling inputs.

Mathematical modelling reveals unexpected inheritance and variability patterns of cell cycle parameters in mammalian cells.

The cell cycle is the fundamental process of cell populations, it is regulated by environmental cues and by intracellular checkpoints. Cell cycle variability in clonal cell population is caused by stochastic processes such as random partitioning of cellular components to progeny cells at division and random interactions among biomolecules in cells. One of the important biological questions is how the dynamics at the cell cycle scale, which is related to family dependencies between the cell and its descendants, affects cell population behavior in the long-run. We address this question using a "mechanistic" model, built based on observations of single cells over several cell generations, and then extrapolated in time. We used cell pedigree observations of NIH 3T3 cells including FUCCI markers, to determine patterns of inheritance of cell-cycle phase durations and single-cell protein dynamics. Based on that information we developed a hybrid mathematical model, involving bifurcating autoregression to describe stochasticity of partitioning and inheritance of cell-cycle-phase times, and an ordinary differential equation system to capture single-cell protein dynamics. Long-term simulations, concordant with in vitro experiments, demonstrated the model reproduced the main features of our data and had homeostatic properties. Moreover, heterogeneity of cell cycle may have important consequences during population development. We discovered an effect similar to genetic drift, amplified by family relationships among cells. In consequence, the progeny of a single cell with a short cell cycle time had a high probability of eventually dominating the population, due to the heritability of cell-cycle phases. Patterns of epigenetic heritability in proliferating cells are important for understanding long-term trends of cell populations which are either required to provide the influx of maturing cells (such as hematopoietic stem cells) or which started proliferating uncontrollably (such as cancer cells).

Maternal eating behavior is a major synchronizer of fetal and postnatal peripheral clocks in mice.

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring.

Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis.

Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell cycle when the culture medium is enriched with growth factors, and the absence of such acceleration in confluent cells. In order to explain these observations, we study a possible entrainment of the circadian clock by the cell cycle through a regulation of clock genes around the mitosis phase. We develop a computational model and a formal specification of the observed behavior to investigate the conditions of entrainment in period and phase. We show that either the selective activation of RevErb-α or the selective inhibition of Bmal1 transcription during the mitosis phase, allow us to fit the experimental data on both period and phase, while a uniform inhibition of transcription during mitosis seems incompatible with the phase data. We conclude on the arguments favoring the RevErb-α up-regulation hypothesis and on some further predictions of the model.

Sexual Dimorphism in Circadian Physiology Is Altered in LXRα Deficient Mice.

The mammalian circadian timing system coordinates key molecular, cellular and physiological processes along the 24-h cycle. Accumulating evidence suggests that many clock-controlled processes display a sexual dimorphism. In mammals this is well exemplified by the difference between the male and female circadian patterns of glucocorticoid hormone secretion and clock gene expression. Here we show that the non-circadian nuclear receptor and metabolic sensor Liver X Receptor alpha (LXRα) which is known to regulate glucocorticoid production in mice modulates the sex specific circadian pattern of plasma corticosterone. Lxrα(-/-) males display a blunted corticosterone profile while females show higher amplitude as compared to wild type animals. Wild type males are significantly slower than females to resynchronize their locomotor activity rhythm after an 8 h phase advance but this difference is abrogated in Lxrα(-/-) males which display a female-like phenotype. We also show that circadian expression patterns of liver 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and Phosphoenolpyruvate carboxykinase (Pepck) differ between sexes and are differentially altered in Lxrα(-/-) animals. These changes are associated with a damped profile of plasma glucose oscillation in males but not in females. Sex specific alteration of the insulin and leptin circadian profiles were observed in Lxα(-/-) females and could be explained by the change in corticosterone profile. Together this data indicates that LXRα is a determinant of sexually dimorphic circadian patterns of key physiological parameters. The discovery of this unanticipated role for LXRα in circadian physiology underscores the importance of addressing sex differences in chronobiology studies and future LXRα targeted therapies.

A hybrid model of cell cycle in mammals.

Time plays an essential role in many biological systems, especially in cell cycle. Many models of biological systems rely on differential equations, but parameter identification is an obstacle to use differential frameworks. In this paper, we present a new hybrid modeling framework that extends René Thomas' discrete modeling. The core idea is to associate with each qualitative state "celerities" allowing us to compute the time spent in each state. This hybrid framework is illustrated by building a 5-variable model of the mammalian cell cycle. Its parameters are determined by applying formal methods on the underlying discrete model and by constraining parameters using timing observations on the cell cycle. This first hybrid model presents the most important known behaviors of the cell cycle, including quiescent phase and endoreplication.

The hepatic circadian clock regulates the choline kinase α gene through the BMAL1-REV-ERBα axis.

The circadian timing system adapts most of the mammalian physiology and behaviour to the 24 h light/dark cycle. This temporal coordination relies on endogenous circadian clocks present in virtually all tissues and organs and implicated in the regulation of key cellular processes including metabolism, transport and secretion. Environmental or genetic disruption of the circadian coordination causes metabolic imbalance leading for instance to fatty liver, dyslipidaemia and obesity, thereby contributing to the development of a metabolic syndrome state. In the liver, a key metabolic organ, the rhythmic regulation of lipid biosynthesis is known, yet the molecular mechanisms through which the circadian clock controls lipogenesis, in particular, that of phospholipids, is poorly characterised. In this study, we show that the wild-type mice display a rhythmic accumulation of hepatic phosphatidylcholine with a peak at ZT 22-0 while clock-deficient Bmal1(-/-) mice show elevated phosphatidylcholine levels in the liver associated with an atherogenic lipoprotein profile. Profiling of the mRNA expression of enzymes from the Kennedy and phosphatidylethanolamine N-methyltransferase pathways which control the production of hepatic phosphatidylcholine revealed a robust circadian pattern for Chkα while other mRNA showed low amplitude (Chkß and Pemt) or no rhythm (Cctα and Chpt1). Chkα mRNA expression was increased and no longer rhythmic in the liver from clock-deficient Bmal1(-/-) mice. This change resulted in the upregulation of the CHKα protein in these animals. We further show that the robust circadian expression of Chkα is restricted to the liver and adrenal glands. Analysis of the Chkα gene promoter revealed the presence of a conserved response element for the core clock transcription factors REV-ERB and ROR. Consistent with the antiphasic phase relationship between Chkα and Rev-erbα expression, in cotransfection experiments using HepG2 cells we show that RORα4-dependent transactivation of this element is repressed by REV-ERBα· Correspondingly, Rev-erbα(-/-)mice displayed higher Chkα mRNA levels in liver at ZT 12. Collectively, these data establish that hepatic phosphatidylcholine is regulated by the circadian clock through a Bmal1-Rev-erbα-Chkα axis and suggest that an intact circadian timing system is important for the temporal coordination of phospholipid metabolism.

Coupling between the Circadian Clock and Cell Cycle Oscillators: Implication for Healthy Cells and Malignant Growth.

Uncontrolled cell proliferation is one of the key features leading to cancer. Seminal works in chronobiology have revealed that disruption of the circadian timing system in mice, either by surgical, genetic, or environmental manipulation, increased tumor development. In humans, shift work is a risk factor for cancer. Based on these observations, the link between the circadian clock and cell cycle has become intuitive. But despite identification of molecular connections between the two processes, the influence of the clock on the dynamics of the cell cycle has never been formally observed. Recently, two studies combining single live cell imaging with computational methods have shed light on robust coupling between clock and cell cycle oscillators. We recapitulate here these novel findings and integrate them with earlier results in both healthy and cancerous cells. Moreover, we propose that the cell cycle may be synchronized or slowed down through coupling with the circadian clock, which results in reduced tumor growth. More than ever, systems biology has become instrumental to understand the dynamic interaction between the circadian clock and cell cycle, which is critical in cellular coordination and for diseases such as cancer.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.

Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Functional genomics identify Birc5/survivin as a candidate gene involved in the chronotoxicity of cyclin-dependent kinase inhibitors.

The circadian timing system orchestrates most of mammalian physiology and behavior in synchrony with the external light/dark cycle. This regulation is achieved through endogenous clocks present in virtually all body cells, where they control key cellular processes, including metabolism, transport, and the cell cycle. Consistently, it has been observed in preclinical cancer models that both the efficacy and toxicity of most chemotherapeutic drugs depend on their time of administration. To further explore the molecular basis underlying the link between the circadian timing system and the cellular response to anticancer drugs, we investigated the circadian transcriptome and CDK inhibitor toxicity in colon mucosa cells. We first show here that among 181 circadian transcripts, approximately 30% of them drive the cell cycle in the healthy mouse colon mucosa, with a majority peaking during the early resting phase. The identification of 26 mitotic genes within this cluster further indicated that the transcriptional coordination of mitosis by the circadian clock participates in the gating of cell division in this tissue. Subsequent selective siRNA-mediated silencing of these 26 targets revealed that low expression levels of the mitotic and anti-apoptotic gene Birc5/survivin significantly and specifically increased the sensitivity of colon epithelial cells to CDK inhibitors. By identifying Birc5/survivin as a potential determinant for the circadian modulation of CDK inhibitor toxicity, these data provide a mechanistic basis for the preclinical development of future CDK inhibitor-based chronotherapeutic strategies.

A circadian clock transcription model for the personalization of cancer chronotherapy.

Circadian timing of anticancer medications has improved treatment tolerability and efficacy several fold, yet with intersubject variability. Using three C57BL/6-based mouse strains of both sexes, we identified three chronotoxicity classes with distinct circadian toxicity patterns of irinotecan, a topoisomerase I inhibitor active against colorectal cancer. Liver and colon circadian 24-hour expression patterns of clock genes Rev-erbα and Bmal1 best discriminated these chronotoxicity classes, among 27 transcriptional 24-hour time series, according to sparse linear discriminant analysis. An 8-hour phase advance was found both for Rev-erbα and Bmal1 mRNA expressions and for irinotecan chronotoxicity in clock-altered Per2(m/m) mice. The application of a maximum-a-posteriori Bayesian inference method identified a linear model based on Rev-erbα and Bmal1 circadian expressions that accurately predicted for optimal irinotecan timing. The assessment of the Rev-erbα and Bmal1 regulatory transcription loop in the molecular clock could critically improve the tolerability of chemotherapy through a mathematical model-based determination of host-specific optimal timing.

Age-structured cell population model to study the influence of growth factors on cell cycle dynamics.

Cell proliferation is controlled by many complex regulatory networks. Our purpose is to analyse, through mathematical modeling, the effects of growth factors on the dynamics of the division cycle in cell populations. Our work is based on an age-structured PDE model of the cell division cycle within a population of cells in a common tissue. Cell proliferation is at its first stages exponential and is thus characterised by its growth exponent, the first eigenvalue of the linear system we consider here, a growth exponent that we will explicitly evaluate from biological data. Moreover, this study relies on recent and innovative imaging data (fluorescence microscopy) that make us able to experimentally determine the parameters of the model and to validate numerical results. This model has allowed us to study the degree of simultaneity of phase transitions within a proliferating cell population and to analyse the role of an increased growth factor concentration in this process. This study thus aims at helping biologists to elicit the impact of growth factor concentration on cell cycle regulation, at making more precise the dynamics of key mechanisms controlling the division cycle in proliferating cell populations, and eventually at establishing theoretical bases for optimised combined anticancer treatments.