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Factors interacting with core circadian clock components are essential to achieve transcriptional feedback necessary for metazoan clocks. Here we show that all three members of the Drosophila Behavior Human Splicing (DBHS) family of RNA-binding proteins play a role in the mammalian circadian oscillator, abrogating or altering clock function when overexpressed or depleted in cells. Although these proteins are members of so-called nuclear paraspeckles, depletion of paraspeckles themselves via silencing of the structural non-coding RNA (ncRNA) Neat1 did not affect overall clock function, suggesting that paraspeckles are not required for DBHS-mediated circadian effects. Instead, we show that the proteins bound to circadian promoter DNA in a fashion that required the PERIOD (PER) proteins, and potently repressed E box-mediated transcription but not CMV promoter-mediated transcription when exogenously recruited. Nevertheless, mice with one or both copies of these genes deleted show only small changes in period length or clock gene expression in vivo. Data from transient transfections show that each of these proteins can either repress or activate depending on the context. Taken together, our data suggest that all of the DBHS family members serve overlapping or redundant roles as transcriptional cofactors at circadian clock-regulated genes.

Antarctic krill, Euphausia superba, shapes the structure of the Southern Ocean ecosystem. Its central position in the food web, the ongoing environmental changes due to climatic warming, and increasing commercial interest on this species emphasize the urgency of understanding the adaptability of krill to its environment. Krill has evolved rhythmic physiological and behavioral functions which are synchronized with the daily and seasonal cycles of the complex Southern Ocean ecosystem. The mechanisms, however, leading to these rhythms are essentially unknown. Here, we show that krill possesses an endogenous circadian clock that governs metabolic and physiological output rhythms. We found that expression of the canonical clock gene cry2 was highly rhythmic both in a light-dark cycle and in constant darkness. We detected a remarkable short circadian period, which we interpret as a special feature of the krill's circadian clock that helps to entrain the circadian system to the extreme range of photoperiods krill is exposed to throughout the year. Furthermore, we found that important key metabolic enzymes of krill showed bimodal circadian oscillations (~9–12 h period) in transcript abundance and enzymatic activity. Oxygen consumption of krill showed ~9–12 h oscillations that correlated with the temporal activity profile of key enzymes of aerobic energy metabolism. Our results demonstrate the first report of an endogenous circadian timing system in Antarctic krill and its likely link to metabolic key processes. Krill's circadian clock may not only be critical for synchronization to the solar day but also for the control of seasonal events. This study provides a powerful basis for the investigation into the mechanisms of temporal synchronization in this marine key species and will also lead to the first comprehensive analyses of the circadian clock of a polar marine organism through the entire photoperiodic cycle.

Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1) as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock.

In den meisten Organismen tickt eine zirkadiane Uhr mit einer Periode von ungefähr 24 Stunden. Sie ermöglicht ihnen die Zeitmessung ohne äußere Signale. Viele physiologische und zelluläre Prozesse unterliegen zirkadianer Regulation. Die molekulare Uhr besteht aus gekoppelten intrazellulären Rückkopplungen: Die Produkte der Uhrgene regulieren ihre eigene Bildung direkt oder indirekt und erzeugen so molekulare Oszillationen. In dieser Arbeit werden bestehende und neue mathematische Modelle des zirkadianen Oszillators verwendet, um die Bedeutung struktureller Merkmale - insbesondere der Rückkopplungen - für grundlegende zirkadiane Eigenschaften zu untersuchen. In einem Modell (Goodwin-Modell), mit einer negativen Rückkopplung erleichtert sättigende Kinetik in einem Abbauterm, nicht aber in einem Produktionsterm, Oszillationen. Ein neues Modell mit zusätzlicher positiver Rückkopplung erzeugt korrekte Phasen zwischen den Komponenten. Es reproduziert die Phänotypen von zirkadianen Mutanten. Das Modell synchronisiert mit dem Licht/Dunkel-Rhythmus. Die positive Rückkopplung beeinflußt die Robustheit der Oszillationen gegenüber Parametervariationen nur gering. Dies erklärt den rhythmischen Phänotyp von Rev-erb alpha-/- Mäusen, die die positive Rückkopplung nicht besitzen. Die überraschende Wiederherstellung von zirkadianen Oszillationen in der Per2Brdm1/Cry2-/- Doppelmutante kann mit dem Modell erklärt werden. Die Wiederherstellung zirkadianer Oszillationen in der arhythmischen Per2Brdm1 Mutante durch zusätzliche Mutation von Rev-erb alpha-/- wird vorausgesagt. Durch das Einfügen von Rev-erb alpha in das Modell entsteht eine weitere Rückkopplung. Mit diesem neuen Modell koennen Phänotypen von Mutanten reproduziert werden. Zuletzt werden Modelle verschiedener molekularer Oszillatoren und allgemeine Modelle, die aus einer positiven oder negativen Rückkopplung unterschiedlicher Kettenlänge bestehen, bezüglich ihrer Robustheit bei Parametervariationen verglichen. Die strukturell, In many organisms the circadian clock ticks with a period of approximately 24 hours, enabling the organisms to keep track of time without any environmental time cues. Many physiological and cellular processes underlie circadian regulation. The molecular clock is a network of intracellular feedback loops: The clock gene products directly or indirectly regulate their own transcription, which results in molecular oscillations. In this thesis, existing and new mathematical models of the circadian oscillator are used to investigate the meaning of structural features - in particular of the feedback loops - for fundamental circadian characteristics. In a simple model (Goodwin model) with one negative feedback loop, saturating kinetics in a degradation term, but not in a production term, support oscillations. A new model containing an additional positive feedback loop shows circadian oscillations with the correct phases between the clock components. The phenotype of several clock mutants can be reproduced. The model synchronizes with the light/dark cycle. Assuming restricted light-input (gating), its phase can be fixed to either light onset or light offset with varying day lengths. In this model, the positive feedback has only a minor influence on the robustness of the circadian oscillations towards parameter variations. This explains the rhythmic phenotype of Rev-erb alpha-/- mutant mice that lack a positive feedback. The model can also explain the unexpected regeneration of circadian oscillations in Per2Brdm1/ Cry2-/- double mutant mice. The regeneration of circadian oscillations in the arrhythmic Per2Brdm1 by additional mutation of Rev-erb alpha is predicted. By including Rev-erb alpha explicitly into the model, another negative feedback loop is added: This model reproduces the phenotypes of several clock mutants. Finally, models describing different molecular oscillators and general models with positive or negative feedback loops of varying chain length are compared wit

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.

Background: Virtually all living organisms have evolved a circadian (~24 hour) clock that controls physiological and behavioural processes with exquisite precision throughout the day/night cycle. The suprachiasmatic nucleus (SCN), which generates these ~24 h rhythms in mammals, consists of several thousand neurons. Each neuron contains a gene-regulatory network generating molecular oscillations, and the individual neuron oscillations are synchronised by intercellular coupling, presumably via neurotransmitters. Although this basic mechanism is currently accepted and has been recapitulated in mathematical models, several fundamental questions about the design principles of the SCN remain little understood. For example, a remarkable property of the SCN is that the phase of the SCN rhythm resets rapidly after a 'jet lag' type experiment, i.e. when the light/dark (LD) cycle is abruptly advanced or delayed by several hours. Results: Here, we describe an extensive parameter optimization of a previously constructed simplified model of the SCN in order to further understand its design principles. By examining the top 50 solutions from the parameter optimization, we show that the neurotransmitters' role in generating the molecular circadian rhythms is extremely important. In addition, we show that when a neurotransmitter drives the rhythm of a system of coupled damped oscillators, it exhibits very robust synchronization and is much more easily entrained to light/dark cycles. We were also able to recreate in our simulations the fast rhythm resetting seen after a 'jet lag' type experiment. Conclusion: Our work shows that a careful exploration of parameter space for even an extremely simplified model of the mammalian clock can reveal unexpected behaviours and non-trivial predictions. Our results suggest that the neurotransmitter feedback loop plays a crucial role in the robustness and phase resetting properties of the mammalian clock, even at the single neuron level.

The suprachiasmatic nuclei (SCN) host a robust, self-sustained circadian pacemaker that coordinates physiological rhythms with the daily changes in the environment. Neuronal clocks within the SCN form a heterogeneous network that must synchronize to maintain timekeeping activity. Coherent circadian output of the SCN tissue is established by intercellular signaling factors, such as vasointestinal polypeptide. It was recently shown that besides coordinating cells, the synchronization factors play a crucial role in the sustenance of intrinsic cellular rhythmicity. Disruption of intercellular signaling abolishes sustained rhythmicity in a majority of neurons and desynchronizes the remaining rhythmic neurons. Based on these observations, the authors propose a model for the synchronization of circadian oscillators that combines intracellular and intercellular dynamics at the single-cell level. The model is a heterogeneous network of circadian neuronal oscillators where individual oscillators are damped rather than self-sustained. The authors simulated different experimental conditions and found that: (1) in normal, constant conditions, coupled circadian oscillators quickly synchronize and produce a coherent output; (2) in large populations, such oscillators either synchronize or gradually lose rhythmicity, but do not run out of phase, demonstrating that rhythmicity and synchrony are codependent; (3) the number of oscillators and connectivity are important for these synchronization properties; (4) slow oscillators have a higher impact on the period in mixed populations; and (5) coupled circadian oscillators can be efficiently entrained by light-dark cycles. Based on these results, it is predicted that: (1) a majority of SCN neurons needs periodic synchronization signal to be rhythmic; (2) a small number of neurons or a low connectivity results in desynchrony; and (3) amplitudes and phases of neurons are negatively correlated. The authors conclude that to understand the orc, Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published