Effects of Different PER Translational Kinetics on the Dynamics of a Core Circadian Clock Model

Living beings display self-sustained daily rhythms in multiple biological processes, which persist in the absence of external cues since they are generated by endogenous circadian clocks. The period (per) gene is a central player within the core molecular mechanism for keeping circadian time in most animals. Recently, the modulation PER translation has been reported, both in mammals and flies, suggesting that translational regulation of clock components is important for the proper clock gene expression and molecular clock performance. Because translational regulation ultimately implies changes in the kinetics of translation and, therefore, in the circadian clock dynamics, we sought to study how and to what extent the molecular clock dynamics is affected by the kinetics of PER translation. With this objective, we used a minimal mathematical model of the molecular circadian clock to qualitatively characterize the dynamical changes derived from kinetically different PER translational mechanisms. We found that the emergence of self-sustained oscillations with characteristic period, amplitude, and phase lag (time delays) between per mRNA and protein expression depends on the kinetic parameters related to PER translation. Interestingly, under certain conditions, a PER translation mechanism with saturable kinetics introduces longer time delays than a mechanism ruled by a first-order kinetics. In addition, the kinetic laws of PER translation significantly changed the sensitivity of our model to parameters related to the synthesis and degradation of per mRNA and PER degradation. Lastly, we found a set of parameters, with realistic values, for which our model reproduces some experimental results reported recently for Drosophila melanogaster and we present some predictions derived from our analysis.     

Deterministic Versus Stochastic Models for Circadian Rhythms

Circadian rhythms which occur with a period close to 24 h in nearly all living organisms originate from the negative autoregulation of gene expression.Deterministic models based on genetic regulatory processes account for theoccurrence of circadian rhythms in constant environmental conditions (e.g.constant darkness), for entrainment of these rhythms by light-dark cycles, and for their phase-shifting by light pulses. At low numbers of protein and mRNA molecules, it becomes necessary to resort to stochastic simulations to assess the influence of molecular noise on circadian oscillations. We address the effect of molecular noise by considering two stochastic versions of a core model for circadian rhythms. The deterministic version of this core modelwas previously proposed for circadian oscillations of the PER protein in Drosophila and of the FRQ protein in Neurospora. In the first, non-developed version of the stochastic model, we introduce molecular noise without decomposing the deterministic mechanism into detailed reaction steps while in the second, developed version we carry out such a detailed decomposition. Numerical simulations of the two stochastic versions of the model are performed by means of the Gillespie method. We compare the predictions of the deterministic approach with those of the two stochastic models, with respect both to sustained oscillations of the limit cycle type and to the influence of the proximity of a bifurcation point beyond which the system evolves to a stable steady state. The results indicate that robust circadian oscillations can occur even when the numbers of mRNA and nuclear protein involved in the oscillatory mechanism are reduced to a few tens orhundreds, respectively. The non-developed and developed versions of the stochastic model yield largely similar results and provide good agreement with the predictions of the deterministic model for circadian rhythms.     

PER-TIM Interactions with the Photoreceptor Cryptochrome Mediate Circadian Temperature Responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.     

Circadian PER2 protein oscillations do not persist in cycloheximide-treated mouse embryonic fibroblasts in culture

It is not known whether the endogenous mammalian core clock proteins sustain measurable oscillations in cells in culture where de novo translation is pharmacologically inhibited. We studied here the mammalian core clock protein PER2, which undergoes robust circadian oscillations in both abundance and phosphorylation. With a newly developed antibody that enables tracing the endogenous PER2 protein oscillations over circadian cycles with cultured mouse embryonic fibroblast cells, we provide evidence that PER2 does not persist noticeable circadian rhythms when translation is inhibited.     

Are competing intermolecular and intramolecular interactions of PERIOD protein important for the regulation of circadian rhythms in Drosophila?

Genetic analysis is revealing molecular components of circadian rhythms. The gene products of the period gene in Drosophila and the frequency gene in Neurospora oscillate with a circadian rhythm. A recent paper⁽¹⁾ has shown that the PERIOD protein can undergo both intermolecular and intramolecular interactions in vitro. The effects of temperature and two period mutations on these molecular interactions were compared to the effects of the mutations and temperature on the in vivo period length of circadian rhythms. The results suggest that the molecular interactions may compete to maintain a rhythm with a constant period over a wide temperature range.     

Cooperative Interaction between Phosphorylation Sites on PERIOD Maintains Circadian Period in Drosophila

Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 “phospho-occupied” serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per⁰¹ arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period.     

A temperature-compensated model for circadian rhythms that can be entrained by temperature cycles

From the viewpoint that reaction rates will change with temperature, we present a general method to build circadian clock models that generate circadian oscillations with almost constant period under different constant ambient temperature, and propose an algorithm estimating the parameter condition for compensated period against the change of temperature based on the PER single-feedback loop model of Goldbeter [1995. A model for circadian oscillations in the Drosophila period protein (PER). Proc. R. Soc. London Ser. B 261, 319-324] for Drosophila. We show that the model with derived parameters can realize the temperature compensation over a wide range of temperature, and simultaneously can realize the entrainment to temperature cycles.     

Organization of the Circadian System in Insects

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system. (Chronobiology International, 15(6), 567-594, 1998)     

Circadian Rhythms of Leaf and Stomatal Movements in Gymnosperm Species

It is generally accepted that various physiological, morphological and gene expression phenomena are under the control of a circadian clock, and that this time keeping mechanism is universally present. Although such endogenously regulated phenomena have first been documented in plants more than 250 years ago and much work has been accumulated particularly in the past 70 years, it was not obvious from the literature whether such time keeping mechanisms exist in gymnosperms. Two prominent parameters were investigated in several gymnosperm species which have been demonstrated to be under the control of a circadian clock in many plants: (i) leaf movement and (ii) stomata movement. In young plants of Pinus sylvestris, Picea abies, Taxus baccata, Araucaria angustifolia, Araucaria heterophylla and Ginkgo biloba leaf oscillations could be recorded for about 5 days. However, compared to angiosperm plants, the amplitude was small. The period length under free running conditions (constant temperature and continuous light) was characteristic for the species. Stomatal movement was observed in Ginkgo biloba leaves by electron microscopy. Stomata were open at noon and closed at midnight under normal day/night conditions (LD) as well as under constant light conditions (LL), indicating that stomatal aperture is under circadian control in the gymnosperm Ginkgo biloba. Online recordings of stomata conductance however, exhibited diurnal but not circadian oscillations of net CO₂-exchange in G. biloba leaves. Our results show that a circadian clock controls leaf and stomatal movements in gymnosperm species indicating that endogenous time keeping mechanisms are present.     

Circadian gene methylation in rotating-shift nurses: a cross-sectional study

Investigating the methylation status of the circadian genes may contribute to a better understanding of the shift work-related circadian disruption in individuals exposed to artificial light at night. In the present study, we determined the methylation status of the circadian genes associated with a shift work pattern among nurses and midwives participating in a cross-sectional study in Lodz, Poland.

Quantitative methylation polymerase chain reaction assays were used to assess promoter CpG methylation in PER1, PER2, PER3, CRY1, CRY2, BMAL1, CLOCK, and NPAS2 in genomic DNA from whole blood of 347 women having a rotating-shift work schedule and 363 women working days only. The percentage of methylated reference (PMR) was assessed using fluorescent probes for PER1, PER2, PER3, CRY1, and NPAS2, and the percentage of gene methylation, as the methylation index (MI), using two sets of primers for BMAL1, CLOCK, and CRY2.

We tested the possible association between current and lifetime rotating night-shift work characteristics and circadian gene methylation by using proportional odds regression model with blood DNA methylation, categorized into tertiles, and adjusted for age, current smoking status, folate intake and blood collection time. The findings indicated that CpG methylation in PER2 promoter was significantly decreased (P < 0.004) among nurses and midwives currently working rotating shifts, as compared with day-working nurses and midwives. The lower percentage of PER2 methylation was associated with a higher monthly frequency of current night duties (2–7 night shifts, and eight or more night shifts per month) (P = 0.012) and was associated at borderline significance (P = 0.092) with the lifetime duration of shift work (>10 ≤ 20 years and >20 ≤ 43 years of rotating-shift work) among nurses and midwives (N = 710). Moreover, women with a longer lifetime duration of shift work presented a lower status of PER1 methylation (P = 0.040) than did the women with up to 10 years of rotating-shift work. Long lifetime duration of shift work (> 10 years) among current rotating night-shift workers (N = 347) was associated with BMAL1 hypomethylation (P = 0.013).

Among eight of the investigated circadian genes, only PER1, PER2, and BMAL1 showed differential methylation attributable to the rotating-shift work of nurses and midwives. The findings on blood-based DNA methylation in the circadian genes may provide a better insight into the mechanistic principles underlying the possible health effects of night-shift work but these should be verified in further studies recruiting larger populations of shift workers.     

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry^b. The period mutant per^L manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per^L flies, because a per^L; cry^b strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.     

Human Intestinal Circadian Clock: Expression of Clock Genes in Colonocytes Lining the Crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per‐1, per‐2, and clock mRNA were detected by real‐time RT‐PCR. The three‐dimensional distributions of PER‐1, PER‐2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per‐1, per‐2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER‐1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER‐1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.     

Phylogeny and oscillating expression of period and cryptochrome in short and long photoperiods suggest a conserved function in Nasonia vitripennis

Photoperiodism, the ability to respond to seasonal varying day length with suitable life history changes, is a common trait in organisms that live in temperate regions. In most studied organisms, the circadian system appears to be the basis for photoperiodic time measurement. In insects this is still controversial: while some data indicate that the circadian system is causally involved in photoperiodism, others suggest that it may have a marginal or indirect role. Resonance experiments in the parasitic wasp Nasonia vitripennis have revealed a circadian component in photoperiodic time measurement compatible with a mechanism of internal coincidence where a two components oscillator system obtains information from dawn and dusk, respectively. The identity of this oscillator (or oscillators) is still unclear but possible candidates are the oscillating molecules of the auto-regulatory feedback loops in the heart of the circadian system. Here, we show for the first time the circadian oscillation of period and cryptochrome mRNAs in the heads of Nasonia females kept under short and long photoperiods. Period and cryptochrome mRNA levels display a synchronous oscillation in all conditions tested and persist, albeit with reduced amplitude, during the first day in constant light as well as constant darkness. More importantly, the signal for the period and cryptochrome oscillations is set by the light-on signal. These results, together with phylogenetic analyses, indicate that Nasonia’s period and cryptochrome display characteristics of homologous genes in other hymenopteran species.     

Effect of photoperiod on clock gene expression and subcellular distribution of PERIOD in the circadian clock neurons of the blow fly Protophormia terraenovae

We examined the effect of photoperiod on the expression of circadian clock genes period (per) and timeless (tim), using quantitative real-time polymerase chain reaction (PCR), and the effect of photoperiod on subcellular distribution of PERIOD (PER), using immunocytochemistry, in the blow fly, Protophormia terraenovae. Under both short-day and long-day conditions, the mRNA levels of per and tim in the brain oscillated, and their peaks and troughs occurred around lights-off and lights-on, respectively. The oscillations persisted even under constant darkness. In the large ventral lateral neurons (l-LN_vs), small ventral lateral neurons (s-LN_vs), dorsal lateral neurons (LN_ds), and medial dorsal neurons (DN_ms), the subcellular distribution of PER-immunoreactivity changed with time. The number of cells with PER-immunoreactivity in the nucleus was highest 12 h after lights-off and lowest 12 h after lights-on, regardless of photoperiod, suggesting that PER nuclear translocation entrains to photoperiod. When temporal changes in the nuclear localization of PER were compared, the neurons could be classified into 2 groups: the l-LN_vs were similar to the s-LN_vs, and the LN_ds were similar to DN_ms. In LN_ds and DN_ms, decreasing rates of the number of cells with PER immunoreactivity in the nucleus per brain from the maximum were large as compared with those in l-LN_vs and s-LN_vs under short-day conditions. These results suggest that photoperiodic information is reflected in the expression patterns of circadian clock genes per and tim and in the subcellular distribution of PER. This observation suggests that the 2 different groups of clock neurons respond to photoperiod in slightly different manners.