CRY associated proteins modulate circadian transcription by regulating CRY stability

Thesis

Reference

CRY associated proteins modulate circadian transcription by regulating CRY stability

ANDERSIN, Teemu

Abstract

Circadian clocks have evolved in all light sensitive organisms from cyanobacteria to mammals. These timing systems allow the organism to adjust their physiology and behavior to the geo-physical time. In mammals the circadian clocks exist in virtually all body cells, but the system function in a hierarchical manner in which the master pacemaker in the suprachiasmatic nucleus (SCN) sets the pace of the subsidiary peripheral oscillators. SCN is a small, approximately 10 000-20 000 cells, neuroendocrine gland in the hypothalamus. The SCN receives a direct photic input from the retina through the retino-hypothalamic tract and transmits this information to the oscillators in the periphery using neuronal and humoral signals. The molecular clock both in the SCN and in the peripheral oscillators is thought to consist of negative transcriptional and translational feedback loops. The PAS domain basic helix-loop-helix transcription factors BMAL1 and CLOCK bind to the promoters and transactivate two cryptochrome (cry) and two period (per) genes. Once CRY and PER proteins reach the threshold concentration they translocate into the [...]

ANDERSIN, Teemu. CRY associated proteins modulate circadian transcription by regulating CRY stability. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4142

URN : urn:nbn:ch:unige-51547

DOI : 10.13097/archive-ouverte/unige:5154

Available at:

http://archive-ouverte.unige.ch/unige:5154

Disclaimer: layout of this document may differ from the published version.

UNIVERSITE DE GENEVE FACULTE DES SCIENCES Professeur U. Schibler Département de Biologie Moléculaire

________________________________________________________________________

CRY Associated Proteins Modulate Circadian Transcription by Regulating CRY Stability

THESE

Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique

par

TEEMU ANDERSIN de

Finland

Thése N° 4142

GENÈVE

Atelier d’impression ReproMail 2009

Acknowledgments

I would like to thank the current and former members of the Schibler laboratory for their help and friendship.

I would like to thank Dr. Mehdi Tahti and Dr. Robbie Loewith for accepting to be the referees of this thesis work.

Finally, I would like to thank Ueli Schibler for accepting me as a Ph.D. student in his laboratory, for his inspiring mentoring and for providing an excellent environment for scientific research.

Table of Contents

Résumé en Français………4

Abstract………5

Introduction……….7

Results and Discussion………41

Conclusions and Perspectives……….77

Part I

Résumé en Français

Les horloges circadiennes ont évolué dans tous les organismes photosensibles, des cyanobactéries jusqu’aux mammifères. Ces systèmes d’horloges permettent aux

organismes d’ajuster leur physiologie et leur comportement à l’heure géophysique. Chez les mammifères, une horloge circadienne existe dans quasiment chaque cellule du corps, mais le système fonctionne de manière hiérarchique ou l’horloge centrale résidant dans les noyaux suprachiasmitiques ( SNC) impose la phase des rythmes aux horloges périphériques. Le SCN est une petite région de l’hypothalamus composée d’environ 10’000 à 20’000 cellules. Le SCN reçoit les signaux lumineux de la rétine à travers le tractus rétinol-hypothalamique et transmet cette information aux oscillateurs

périphériques à l’aide de signaux neuronaux et hormonaux. Il est entendu que l’horloge moléculaire dans le SCN et les oscillateurs circadiens dans les organes périphériques sont constitués de boucles négatives d’expression génique. BMAL1 and CLOCK, deux

facteurs de transcription appartenant à la famille de facteurs de transcription dites protéines PAS domain-basic helix–loop-helix, se lient aux promoteurs et transactivent deux gènes cytochrome (Cry1 et Cry2) et deux gènes période (Per1 et Per2).

Quand CRY et PER atteignent des concentrations critiques, ils sont transportées dans le noyau où ils inhibent l’activité de l’hétérodimère BMAL1-CLOCK et répriment ainsi leur propre expression. De plus, le gène Bmal1 est activé et réprimé par les récepteurs

nucléaires orphelins RORs et REV-ERBα respectivement. Les modifications post­

traductionnelles telles que la phosphorylation, l’actétylation, la sumolylation et l’ubiquitination jouent un rôle important dans la régulation fine de l’oscillateur pour atteindre une période de 24 heures. La régulation de la stabilité et de l’accumulation de la protéine CRY est particulièrement importante pour assurer une bonne fonction de

l’oscillateur circadien. Les protéines CRY sont ubiquitinées par l’ubiquitine SCF-FBXL3 E3-ligase de manière rythmique pour ensuite être dégrades par le protéasome.

Pour mieux comprendre le filet d’interactions de CRY, j’ai développé des lignés

cellulaires transgénique exprimant des protéines CRY1 et CRY2 « affinity-tagged ». J’ai

utilisé ces lignés cellulaires pour la purification des complexes protéique contenants CRY1 et CRY2 et pour l’identification les protéines associées par spéctromètrie de masse. Les protéines identifiées comprennent des protéines se liant à des ARNs tels que RBM5 et PSF, les régulateurs transcriptionnels TRAP150, BTF et KRIP1 et des protéines impliquées dans l’ubiquitination et dans la régulation de la stabilité protéique tels que SKP1 et OTUD4.

La déplétion de SKP1, un membre du complexe SCF ubiquitaire E3-ligase, par l’interférence d’ARN, résulte en une élongation de la période circadienne de manière similaire au phénotype observé chez la souris Fbxl3 mutante. De plus les observations suggèrent qu’OTUD4, une protéine contenant un domaine otu, est une dé-ubiquitinase.

Initialement OTUD4 n’avaient été trouvé que dans le complexe CRY1, mais par co­

immunoprécipitation j’ai pu montrer qu’OTUD4 interagit également avec CRY2. De plus, la déplétion de OTUD4 par un shARN abolie l’oscillation circadien de Bmal1, Per2, RevErbα, et Dbp dans des cellules NIH-3T3. Cette arythmie est accompagnée par un taux élevé de CRY1 et CRY2 suggère un mécanisme plausible pour la perte de l’oscillation circadienne.

Abstract

Circadian clocks have evolved in most light-sensitive organisms from cyanobacteria to mammals. These timing systems allow the organism to adjust their physiology and behavior to geophysical time. In mammals the circadian clocks exist in virtually all body cells, but the system functions in a hierarchical manner in that the master pacemaker in the suprachiasmatic nucleus (SCN) sets the pace of the subsidiary peripheral oscillators.

SCN is a small, neuroendocrine gland in the ventral hypothalamus, composed of

approximately 10 000-20 000 cells. The SCN receives direct photic inputs from the retina through the retino-hypothalamic tract and transmits this information to the oscillators in the periphery using neuronal and humoral signals. The molecular clock in both the SCN and peripheral cell types is thought to consist of negative transcriptional and translational feedback loops. The PAS domain basic helix-loop-helix transcription factors BMAL1 and CLOCK bind to the promoters and transactivate two cryptochrome (Cry1 and Cry2) and two period (Per1 and Per2) genes. Once CRY and PER proteins reach critical

threshold concentrations (or activities) they are translocated into the nucleus where they inhibit the activity of BMAL1-CLOCK heterodimers and thereby repress their own genes. In addition, the Bmal1 gene is rhythmically activated and repressed by the orphan nuclear receptors RORs and REV-ERBα, respectively, in an interconnecting loop. Post­

translational modifications such as phosphorylation, acetylation, sumoylation, and ubiquitination play an important role in the fine-tuning of the oscillator to generate an approximately 24 hour periodicity. The regulation of CRY protein stability and accumulation is particularly important for a functional circadian oscillator. CRYs are ubiquitinated by the SCF-FBXL3 ubiquitin E3-ligase in a temporally regulated manner and subsequently degraded by the proteasome.

In order to gain more insight into the CRY interaction network, I developed transgenic cell-lines expressing affinity-tagged CRY1 and CRY2 proteins. I used these cell lines to purify the CRY1 and CRY2 protein complexes and, in collaboration with Dr Francis Vilbois, identified the associated factors by mass-spectrometry. Identified proteins included RNA-binding proteins such as RBM5 and PSF, transcription regulators such as TRAP150, BTF and KRIP1 and proteins involved in ubiquitination and regulation of protein stability, such as SKP1 and OTUD4.

Depletion of SKP1, a member of SCF ubiquitin E3-ligase complex, by RNA interference (RNAi) resulted in the lengthening of the circadian period in NIH-3T3 cells, similar to the phenotype observed in Fbxl3 mutant mice. Interestingly, OTUD4 an otu-domain containing protein, is a putative de-ubiquitinase. While initially OTUD4 was only found in the CRY1 complex, I showed by co-immunoprecipitation that OTUD4 also interacted with CRY2. Furthermore, OTUD4 depletion by shRNA abolished circadian oscillations in the expression of Bmal1, Per2, Reverbα, and Dbp in NIH-3T3 cells. This

arrhythmicity was accompanied by elevated CRY1 and CRY2 levels, and this provided a plausible mechanism for the loss of circadian oscillations.

Introduction

Circadian rhythms

The 24 hour solar cycle of our planet causes changes in the environment that have an impact on every organism. It is therefore not surprising that the physiology of most organisms, from cyanobacteria to humans, displays circadian rhythms. “Circadian” is derived from latin words circa diem, which means “about a day”, and circadian rhythms thus have a period length (τ) of approximately 24 hours. Among the daily reappearing phenomena, the most obvious one is photosynthesis in plants, algae, and photosynthetic bacteria, which can only take place during the day when sunlight is available. In these organisms, the circadian clock helps to optimize photosynthesis by anticipating the expression of relevant genes, so that their products are already available before sunrise.

However, animal behavior displays great circadian variations too. For example, nocturnal rodents, such as mice, are active during the night when the risk of getting caught by their predators is smallest. Predators on the other hand hunt when their visual (or olfactive) acuity is most appropriate for it. Circadian rhythms are not just acute responses to

environmental changes but are driven by an endogenous circadian clock. Hence they can anticipate daily reappearing physiological changes, rather than just react to them.

In humans, sleep-wake cycles are the most obvious circadian behavioral phenomenon, and in most individuals sleeping time is concentrated to the night. Sleep in humans is a homeostatic process which is thought to be synchronized to the circadian clock (Borbely, 1982). In other species, the temporal sleep and wake windows are much more spread throughout the day, but the overall rest-activity cycles are still coupled to the circadian timing system. In addition to the sleep-wake cycles, many physiological processes vary in a circadian fashion. Blood pressure and heart beat as well as body temperature fluctuate during the day. In addition, the endocrine system displays circadian fluctuation:

melatonin for example is secreted during the night whereas the plasma concentration of glucocorticoids peaks during the early morning (in humans) (Hastings, 1991). The rest- activity cycles directly impose feeding patterns, which therefore also show daily

variations. Consequently, it is not surprising that processes involved in food metabolism are also circadian. These include the activity of the digestive tract, renal activity, and

liver metabolism. The benefits of circadian expression of liver enzymes might be that enzymatic reactions which produce potentially harmful side products only take place when they are needed. For example, cytochrome p450 monooxidases, which play pivotal functions in endo- and xenobiotic detoxification, generate genotoxic reactive oxygen species (ROS) when their substrates for hydroxylations are absent or scarce.

Circadian clocks

A striking property of circadian rhythms is that they persist in the absence of

environmental changes, in other words they are self-sustained. For example, laboratory mice display a highly rhythmic wheel-running activity which is normally correlated to the environmental light-dark cycle. However, in constant darkness this rhythmicity is

maintained indefinitely, with a period length close to 24 hours. Since the period length is not exactly 24 hours, the rhythm gradually drifts away from geophysical time. For this reason, circadian clocks have to be re-entrained periodically by external stimuli, light is being the most dominant timing cue (Zeitgeber). In addition the period length is an endogenous property of the clock, since mutations affecting the period length are strictly hereditable. Being able to predict rather than just respond to environmental changes is thought to give an organism a great benefit, although in laboratory conditions this has been convincingly shown for only two organisms, the cyanobacterium Synechococcus elongatus and the plant Arabidopsis thaliana. When wild-type cyanobacteria were mixed with mutant strains with different period lengths, the strain whose oscillator resonated with environmental light-dark cycles outgrew the one with a nonresonating clock (Ouyang et al., 1998; Woelfle et al., 2004). The reciprocity of this experiment rules out the possibility that the clock mutation reduced the fitness of the mutant strain; when the light-dark cycle was adapted to the period length of the mutant bacteria, these were now able to outgrow the wild-type bacteria. Arabidopsis thaliana also benefits from a

resonating circadian clock. Thus, the plants with a clock period matched to that of the imposed light-dark cycle contained more chlorophyll, fixed more carbon, grew faster, and survived better than plants with clock periods differing from the light-dark cycle (Dodd et al., 2005). Although compelling experimental evidence for the benefit of circadian clocks in other organisms, especially in mammals, is still missing, it would be naive to argue

that they are without virtue. Such a complicated genetic network circuitry would never have been conserved during hundreds of millions of years. In fact, many clock

components of the Drosophila clockwork have highly conserved orthologs in mammalian systems. One can thus speculate with confidence on the importance of various

physiological aspects that are gated to different time windows by mammalian circadian timekeepers. As mentioned above liver metabolism is under circadian control (Furukawa et al., 1999; Lavery et al., 1999). Enzymes such as cytochrome P450 mono-oxygenase are important for detoxification of xenobiotics, but the reaction creates reactive oxygen species as a by-product. It is therefore likely that limiting the expression of P450 enzymes to the absorptive phase is highly beneficial for the organism.

Another interesting property of circadian clocks is that they are temperature compensated. Whereas the rate of most biochemical reactions depends on the

temperature, the period length of a circadian clock remains nearly the same at different temperatures. This temperature compensation is especially important for poikilothermic organisms, such as fish and reptiles, to be able to predict the dusk or dawn irrespective of ambient temperature. Surprisingly, even mammalian circadian oscillators are temperature compensated (Izumo et al., 2003). The processes underlying this temperature

compensation remains elusive, but a recent report which showed that mouse fibroblast oscillators are insensitive to overall changes in transcription also suggest that the Period1 gene is involved in both temperature and transcriptional compensation (Dibner et al., 2009).

Molecular model of the Oscillator

An oscillator can be divided into three interconnected parts (FIG.1). In the center lies the oscillator itself that generates the periodicity. Since the oscillator is only capable of measuring time approximately, it has to be reset periodically to geophysical time by input pathways. In most cases, light is the dominant resetting cue, and it has been shown that a light pulse given during the night can change the phase of an oscillator. The timing of the light pulse determines whether a phase advance or phase delay results. A light pulse delivered in the early part of the night engenders a phase delay, and a light pulse given in the late part of the night advances the oscillator. The human circadian clock is only able

to phase shift about an hour per day, therefore long distance travelers who cross several time zones in a short period of time suffer from a “jetlag”. As the third part, the circadian oscillator is coupled to the downstream output pathways which use the temporal

information to drive overt rhythms in gene expression, physiology, and behavior.

While studies done in cyanobacteria have shown that circadian oscillators exist even in unicellular organism, studies done in mammals revealed a hierarchical circadian timing system. The “master pace maker” resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. Lesions of these small neuroendocrine glands result in complete arrhythmia in all examined circadian species (Moore and Eichler, 1972; Ralph et al., 1990; Ralph and Menaker, 1988; Silver et al., 1996). The SCN is composed of approximately 20 000 cells and, as insinuated by its name, is located above the optic chiasm to receive periodic photic inputs from the retina. Interestingly, circadian

oscillators have been found in nearly all peripheral tissues, even in cultured fibroblasts in vitro (Balsalobre et al., 1998; Yamazaki et al., 2000). Therefore, it is believed that the role of the SCN is to keep peripheral oscillators in phase with each other through neuronal and humoral signals. Before describing the mammalian circadian oscillator, I briefly review the circadian timing systems of two intensely studied model organisms; the clock of prokaryotic cyanobacteria S. elongatus and the clock of D. melanogaster. While there is no apparent sequence conservation between the clock components of prokaryotes and eukaryotes, the basic principle of generating circadian rhythms by feedback loops is conceptually similar.

Figure 1. Simplified model of an oscillator. (Williams and Sehgal, 2001)

The circadian clock of Cyanobacteria

The cyanobacterium S. elongatus represents the simplest organism to posses a circadian timing system, and virtually all genes are transcribed in a circadian fashion. This

timekeeper is the best understood oscillator, since it has been reconstituted in vitro (Nakajima et al., 2005). It consists of three proteins KaiA, KaiB and KaiC whose genes are transcribed from two operons (Ishiura et al., 1998). Inactivation of any of the kai genes results in arrhythmia. KaiC is both an auto-kinase and an auto-phosphatase. It has two phosphorylation sites at S431 and T432, and the protein exists in all four different phosphorylation states, i.e. fully phosphorylated, phosphorylated at S431 only,

phosphorylated at T432 only, and in an entirely unphosphorylated form (Nishiwaki et al., 2004; Xu et al., 2004). During a circadian cycle, the phosphorylation state of KaiC proceeds in an orderly manner (FIG. 2). Starting form unphosphorylated KaiC, T432 is the first residue to be phosphorylated, followed by phosphorylation at S431, creating the fully phosphorylated form (Nishiwaki et al., 2007). Dephosphorylation occurs first on T432 and then on S431.The role of KaiA is to stimulate the auto-kinase activity of KaiC, by repeatedly associating with it. KaiB preferentially binds to the S431 phosphorylated form of KaiC, thereby forming a ternary complex with KaiA. Presumably, this shifts the equilibrium towards the auto-phosphatase activity of KaiC, ensuing the

dephosphorylation of KaiC and the beginning of a new cycle. The assembly and

disassembly of clock protein complexes follows a circadian pattern. Though it is possible to generate a cyanobacterial oscillator in the absence of transcription, the abundance of KaiB and KaiC does oscillate robustly in vivo, suggesting that the cyanobacterial oscillator consists of both transcriptional and post-translational elements. It is not clear how oscillation of Kai protein complexes or oscillation of KaiC phosphorylation status result in circadian gene expression, since none of the Kai proteins possesses a DNA- binding domain. There is some evidence that DNA topology is involved in the generation of rhythmic gene expression (Xu et al., 2000). Thus, the cyanobacterial chromosome compacts during the subjective day and relaxes during the subjective night (Smith and Williams, 2006). This compaction rhythm continues in constant conditions and is

dependent on KaiC. It is therefore plausible to speculate that the clock controls the rhythmic gene expression by affecting the DNA topology and thereby the accessibility of the promoters.

Figure 2. A model of rhythmic Kai-C phosphorylation. (Dong and Golden, 2008)

Eukaryotic oscillators

The transcriptional/translational feedback loop model

Whereas the cyanobacterial oscillator relies strongly on post-translational modification, the eukaryotic oscillators seem to have developed a different mechanism. In eukaryotes, especially in mice, most of the clock genes are rhythmically transcribed and many of them encode transcription factors. This provides the basis of the currently held model, where transcription plays a central role in the generation of 24 hour rhythmicity. In this model, genes specifying positive elements drive the expression of genes encoding the negative elements (FIG 3). These negatively acting proteins undergo multiple post­

translational modifications, such as phosphorylation and multiprotein complex assembly.

These events are thought to regulate the nuclear entry and the stability of these proteins.

Once they are translocated to the nucleus, the negative protein complexes interfere with the positive elements and thereby repress the expression of their own genes. Since the half-life of the negative elements is only a few hours, their concentration soon falls below

the threshold level for auto-repression and a new cycle can begin. The kinetic rates of transcription, translation, mRNA decay, and protein degradation and the post­

translational modifications have evolved so as to generate an approximately 24 hour rhythm. In this model the post-translational events are essential in creating a delay

between the activation of the negative elements and the auto-repression, since without the delay the oscillator would fall into equilibrium, and the oscillation would dampen.

Figure 3. General model for circadian rhythm generation by delayed negative feedback loop of gene expression.

Circadian clock of the fruit fly Drosophila melanogaster

Owing to its powerful genetics, the fruit fly Drosophila melanogaster has been an

excellent model organism in the molecular dissection of circadian rhythms. Physiological manifestations of circadian rhythms in Drosophila are rest-activity cycles, olfactory

responses, egg laying, and pupal eclosion. The adult fruit flies emerge from their pupal cases in the early morning, and this is regulated by the circadian clock. This phenomenon was used in a screen of chemically mutagenized fly populations to identify X-linked circadian mutations that caused the animals to hatch with a different circadian phase or throughout the day (Konopka and Benzer, 1971). The mutants retrieved from this screen had different period lengths or were arrhythmic, and the mutated genetic locus was therefore named period (per). Since then many other clock genes have been identified (Allada et al., 1998; Hardin et al., 1990; Rutila et al., 1998; Sehgal et al., 1994). Two basic helix-loop-helix transcription factors CLOCK (CLK) and CYCLE (CYC) form the positive loop of the oscillator (FIG. 4). They form a heterodimer and bind to the E-box (CACGTG) elements within the promoters of the genes specifying the negative factors period (per) and timeless (tim) and thereby activate the expression of these genes. PER and TIM proteins then accumulate in the cytoplasm, heterodimerize, and translocate to the nucleus. Heterodimerization of PER with TIM protects PER form degradation (see below) and is indeed a prerequisite for nuclear translocation. PER protein degradation in the cytoplasm is controlled by the protein kinase Doubletime (DBT) (Kloss et al., 1998;

Price et al., 1998). DBT phosphorylates PER, and the phosphorylated form of PER is recognized by the ubiquitin ligase SLMB E3. The protein phosphatase 2A (PP2A) opposes the action of DBT by dephosphorylating PER (Sathyanarayanan et al., 2004).

SLMB E3 ubiquitinates PER and targets it to proteasomal degradation (Grima et al., 2002; Ko et al., 2002). The result of these events is that there is a lag of about four hours between maximal per mRNA and PER accumulation. When TIM levels are rising, TIM associates with the PER-DBT complex and protects PER from degradation. This complex is then a target of another kinase, SHAGGY (SGG) (Martinek et al., 2001).

Phosphorylation of TIM by SGG allows the complex to move to the nucleus. Once in the nucleus PER-TIM-DBT heterodimers associate with CLK-CYC complexes, block their activity and thereby repress per and tim transcripton. The exact mechanism of how PER- TIM-DBT complexes repress CLK-CYC heterodimer is still elusive. DBT has been suggested to phosphorylate CLK, and this leads to its proteasomal degradation (Kim and Edery, 2006). This model has been recently challenged by a study showing that the catalytic activity of DBT is not required for CLK phosphorylation (Yu et al., 2009). The

auto-repression of per and tim leads to a fall in PER and TIM levels, allowing a new cycle of transcription to start. As discussed earlier, the purpose of these post-translational modifications lies in the generation of a delay between the activation and the

autorepression of the negative elements.

Figure 4. Model of the PER and TIM feedback loop (Hardin, 2005).

Two transcription factors VRILLE (VRI) and PAR domain protein 1 (PDP1) form a second feed-back loop by controlling the cyclic expression of CLK (FIG.5) (Cyran et al., 2003). VRI acts as a transcriptional repressor on clk, whereas PDP1 as a transactivator.

Vri is also a target of CLK-CYC and as a consequence of PER-TIM (Glossop et al., 2003). This leads to an antiphasic accumulation of clk mRNA.

Figure 5. Model of the CLK feedback loop (Hardin, 2005).

Light Entrainment of the Drosophila Clock

The light entrainment in Drosophila is mediated by CRYPTOCHROME (CRY), a blue light photoreceptor homologue of photolyases (FIG.6) (Emery et al., 1998; Stanewsky et al., 1998). Upon exposure to light, CRY associates with TIM and promotes its

proteasomal degradation. The precise mechanism through which CRY triggers TIM degradation is not known, but recent studies have shown that an F-box protein JETLAG (JET), which is a component of the SCF E3-ligase complex, can promote TIM

degradation (Koh et al., 2006; Peschel et al., 2006). Since TIM stabilizes PER,

degradation of TIM also leads to PER degradation. Light pulses have different effects, depending on what time of the day they occur at. Thus, in the early evening when PER and TIM levels are increasing, a light pulse will result in a phase delay, whereas a light pulse during a later part of the night, when PER and TIM levels are falling, leads to a phase advance.

In addition to light entrainment, CRY has been suggested to play a role in the core clock, but only in the peripheral cells (i.e. not in the central pacemaker neurons (Ivanchenko et

al., 2001; Krishnan et al., 2001). The exact molecular function that CRY may adopt in the peripheral oscillator remains unknown.

Figure 6. The light entrainment pathway in Drosophila (Hardin, 2005)

Circadian clocks of mammals

In mammals most physiological and behavioral processes are under circadian control.

These include sleep-wake cycles, heart beat and blood pressure, renal activity, visual acuity, motility and activity of the digestive tract, secretion of hormones such as glucocorticoids and melatonin and liver metabolism (Hastings, 1997). Since circadian rhythms manifest themselves in such a wide repertoire of physiology, it is not surprising that disruption of these rhythms can have detrimental effects for our well-being.

Hierarchical organization of the mammalian circadian system

In mammals, nearly all cells possess a circadian oscillator (Yamazaki et al., 2000; Yoo et al., 2004), and even immortalized fibroblasts harbor such a timing device (Balsalobre et al., 1998). These clocks are sell-sustained and cell-autonomous, so that individual cells maintain their circadian oscillations when taken out from their natural environment (Nagoshi et al., 2004; Yoo et al., 2004). However, different organs seem to adopt

different period lengths and phases when isolated form the organism (Yoo et al., 2004).

The master oscillator that keeps individual organs in synchrony resides in the suprachiasmatic nuclei (SCN) of the hypothalamus. Elegant lesion and transplant experiments showed that SCN lesioned animals became completely arrhythmic and that the rhythmicity can be restored with an SCN transplant (Ralph et al., 1990). In addition, these experiments showed that after the transplantation, the period length of the animal was the one of the donor, identifying the SCN as the master regulator (Ralph et al., 1990).

As mentioned before, the individual SCN neurons are synchronized to geophysical time by light input through the retino-hypothalamic tract. The SCN then synchronizes the peripheral oscillators by mechanisms that are still poorly understood, but which probably involve both neuronal and humoral signaling (van Esseveldt et al., 2000).

The mammalian molecular oscillator

The identification of mammalian clock genes has profited enormously from genetic studies in Drosophila. The negative components, two Period (Per) genes and two Cryptochrome (Cry) genes were identified by their homology to the Drosophila factors (Shearman et al., 1997; Thresher et al., 1998; Vitaterna et al., 1999; Zheng et al., 1999).

Targeted deletion of per1 in mice results in a shortened period length, but the animals remain rhythmic in constant darkness (Cermakian et al., 2001; Zheng et al., 2001). On the other hand mice homozygous for a mutation that disrupts the PAS domain of mPer2 also show a short period phenotype, but over time become completely arrhythmic in constant darkness (Zheng et al., 1999). Mice deficient for both Per1 and Per2 do not exhibit circadian rhythms (Zheng et al., 2001). There is also a third period homolog in mice, dubbed Per3, but its role in the core clock function is unclear (Bae et al., 2001; Shearman et al., 2000; Zheng et al., 1999). Whereas in Drosophila the primary role of CRY proteins lies in photoperception (i.e. clock input), the mammalian counterparts CRY1 and CRY2 are essential parts of the core clock (Kume et al., 1999; van der Horst et al., 1999;

Vitaterna et al., 1999). The two CRY isoforms seem to have at least partially redundant

roles since only animals with disruptions in both genes become arrhythmic. It is important to mention here that Cry1 single mutants have different phenotypes. CRY1 knock-out mice exhibit a short period phenotype; whereas Cry2 deletion lengthens the period (van der Horst et al., 1999). These different phenotypes may arise from differing affinities to other clock components or from different expression levels. A mammalian homolog of TIM also exists, but it is not clear whether this protein is part of the core clock (Gotter et al., 2000; Zylka et al., 1998a). Two basic helix-loop-helix PAS-domain containing transcription factors CLOCK and BMAL1 represent the mammalian orthologs of dCLOCK and dCYCLE, respectively. The transcription factor CLOCK (for circadian locomotor output cycles kaput) was first identified in mice through a heroic forward genetic screen of behavioral mutants in ENU-mutagenized mice (King et al., 1997;

Vitaterna et al., 1994). Mammals have an additional CLOCK paralog, NPAS2, and it seems that CLOCK and NPAS2 have partially redundant roles in the oscillator. Whereas CLOCK is essential for peripheral oscillators, it can be replaced by its paralog NPAS2 in the SCN (DeBruyne et al., 2007a, b). BMAL1 (also called MOP3) is the mouse ortholog of dCYCLE. It is an essential clock component, and Bmal1 knock-out mice are

completely and immediately arrhythmic when exposed to constant darkness (Bunger et al., 2000). In addition, WDR5, a component of a histone methyltransferase complex, and the RNA binding protein NONO have been shown to associate with PER1 and to

modulate core clock function (Brown et al., 2005).

The current model of the mammalian oscillator (simplified in FIG7) describes an

autoregulatory transcriptional/post-translational feedback loop, in which the transcription factors CLOCK and BMAL1 form a heterodimer, bind to E-box sequences, and trans- activate the key genes of the negative limb, the Per and Cry genes. The PER and CRY proteins form heteromeric repressive complexes with unknown stoichiometry, and once the concentration (or activity) of these complexes has reached a critical threshold level in the cell nucleus, they interact with CLOCK/BMAL1 heterodimers and suppress the activation potential of these transcription factors. As a consequence Per and Cry mRNA and protein levels decrease, and once the nuclear concentration of these repressive

complexes has fallen below the concentrations needed for autorepression a new cycle can begin. Many additional mechanisms are involved in generation of a robust oscillator. An

orphan nuclear receptor REV-ERBα and its homolog REV-ERBβ interconnect the negative and positive loops (Guillaumond et al., 2005; Preitner et al., 2002). The

transcription of Rev-Erbα is controlled by the same mechanism as that responsible for the oscillaton of Per and Cry transcription. REV-ERBα itself functions as a repressor and binds to retinoid-related receptor response elements (RORE) in the promoter of Bmal1, where it competes with retinoid-related orphan receptors (RORs) that activate Bmal1 transcription. The resulting cyclic expression of REV-ERBα ensues the rhythmic repression of Bmal1 and leads to circadian accumulation of Bmal1 mRNA. As BMAL1 protein has a relatively long half-life, the amplitude of its cyclic accumulation is

considerably attenuated when compared to that of Bmal1 mRNA expression.

Figure 7. Simplified model of the mammalian circadian oscillator. (Gallego and Virshup, 2007)

Similar to what has been observed in Drosophila, post-translational modifications also play important roles in generating circadian oscillations in mammals. For example the closely related casein kinases 1ε (CKIε) and casein kinase 1δ (CKIδ), which are homologs of Drosophila DBT, have been shown to phosphorylate PER, CRY and BMAL1 proteins (Eide et al., 2002; Eide and Virshup, 2001; Lee et al., 2001), and a

mutant allele of Ck1ε (called tau) has been shown to cause a drastic period shortening in Syrian hamsters (Lowrey et al., 2000). The roles of CKIε and CKIδ are further

underscored by the observations that a mutation in the human Per2 that inactivates a phosphoacceptor site S659, which serves as a priming site for further phosphorylations by CKIε/δ, results in familial advanced sleep phase syndrome (FASP) (Toh et al., 2001;

Vanselow et al., 2006). CKIδ has also been shown to associate with PER-CRY

complexes and may serve similar function (Lee et al., 2001). Very recently, two groups have actually shown that the disruption of Ck1δ, but Ck1ε in mice, causes dramatic period length phenotypes (Weaver, 2009; Andrew Loudon, personal communication).

Hence, in retrospect it appears that the Ck1εTau allele (Lowrey et al., 2000) acted as a dominant negative for Ck1δ, rather than as a hypomorph as originally suggested. Indeed, a mutation in humans with FASP has been mapped to the Ck1δgene (Xu et al., 2005).

Recently, it has also been shown that casein kinase 2 (CKII) phosphorylates BMAL1 and PER2 and therey also plays a role in the generation of circadian rhythmicity (Maier et al., 2009; Tamaru et al., 2009). Finally, glycogen synthase kinase 3β (GSK3β), the

mammalian ortholog of Drosophila SHAGGY, phosphorylates PER2 and CRY2 (Harada et al., 2005; Iitaka et al., 2005). Phosphorylation is obviously a reversible process, and indeed several phosphatases have been identified that are involved in core clock function.

Protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein phosphatase 2B (PP2B) have all been shown to regulate the activity of (but see comments on CKIε/δ above) by removing an inhibitory phosphate group from the C-terminus of the kinase (Cegielska et al., 1998; Partch et al., 2006). In addition, PP1 interacts with PER2 and regulates its stability (Gallego et al., 2006). How do these rather complicated

phosphorylation events translate into circadian rhythmicity? Many of these modifications alter the stability of the targets, often that of PER2. It is not easy to draw a

straightforward model on how particular modifications affect protein stability, since the events seem to take place in an orderly and cooperative manner. In other words, one modification mark may serve as a target for the next modification.

PER stability is controlled by the F-box protein beta-transducin repeat proteins 1 and 2 (βTRCP1/2) (Eide et al., 2005; Reischl et al., 2007; Shirogane et al., 2005), which is a

component of one of the more than 70 ubiquitin E3 ligase complexes that generally mark proteins for proteasomal degradation. The recognition of PER by βTRCP1/2 is dependent on prior phosphorylations by CKIε. Interestingly, PER2 stability was recently shown to be regulated by Sirtuin1 (SIRT1), an NAD⁺-dependent protein de-acetylase (Asher et al., 2008). The authors observed elevated PER2 protein levels in cells of Sirt1 knock-out animals and showed that PER2 is an acetylated protein that can be de-acetylated by SIRT1 in vivo and in vitro. Since both acetyl and ubiquitin groups are targeted to lysine residues, it is conceivable that PER2 has higher metabolic stability due to an acetylation of a lysine moiety needed for ubiquitination. The enzyme responsible for acetylation of PER2 is not known, but one candidate is CLOCK which has been reported to possess acetyl-transferase activity (Hirayama et al., 2007). Increased PER stability results in elevated nuclear concentrations of the repressive complex, and therefore in enhanced repression on CLOCK/BMAL1 target genes and overall lengthening of the period.

Regulated protein degradation has also been reported for cryprochromes. Thus, three independent groups identified the F-box protein FBXL3 as the one responsible for

appropriate degradation of CRYs (Busino et al., 2007; Godinho et al., 2007; Siepka et al., 2007). Busino et al. purified the FBXL3 complex and identified CRY1 and CRY2 as associated factors. Godinho et al. and Siepka et al. used a genetic approach, searching for circadian behavioral mutants from progeny of ENU-mutagenized mice. Both groups identified mutants with long free-running periods and in both cases the mutation mapped to the Fbxl3 locus. Although these results point to the importance of regulated CRY degradation, there are still some open questions. First, the signals marking CRYs for ubiquitination and degradation remain elusive. These could be phosphorylation marks like in the case of PER proteins. Second, at least in liver CRY2 protein levels fluctuate with high circadian amplitude although the amplitude of the mRNA is modest (Preitner et al., 2002). This could be due to daily differences in protein synthesis rates, but could also result from temporal regulation of CRY2 degradation.

The crucial importance of these post-translational modifications comes from the fact that transcriptional-translational feedback loops generally generate cycles of only a few hours (Barkai and Leibler, 2000). In order to maintain 24 hour oscillations of clock proteins, a delay between activation and repression of transcription is required. This delay is

probably achieved by multiple post-translational modifications which affect the nuclear entry, protein complex formation, and the degradation of proteins.

Although most of the above mentioned modifications affect the stability of clock proteins, some modifications also regulate their activity. BMAL1 is phosphorylated by CK1ε/δ and mitogen activated protein kinase (MAPK) (Eide et al., 2002; Sanada et al., 2002). Phosphorylation by CK1ε/δ activates BMAL1, since inhibition of the kinases reduces BMAL1 phosphorylation and decreases CLOCK/BMAL1-dependent

transactivation. In contrast, MAPK mediated phosphorylation decreases the

transcriptional activity of CLOCK/BMAL1 heterodimers. BMAL1 has also been reported to be a target for sumoylation by SUMO1 and SUMO2/3. These modifications not only regulate the activity of the CLOCK/BMAL1 complex, but also target BMAL1 for proteasomal degradation (Cardone et al., 2005; Lee et al., 2008).

.

Input Pathways

In contrast to Drosophila where virtually all oscillating cells are photosensitive and thus capable of entraining their circadian oscillators directly by light, in mammals photic cues can only be transmitted to circadian clocks via the retina of the eye (FIG 8). Therefore, enucleated animals are not only visually blind, but are also unable to synchronize their circadian clocks (Yamazaki et al., 1999). Similarly, mice lacking the optic nerve cannot entrain their circadian clocks to the environmental light-dark cycles (Wee et al., 2002).

Surprisingly, however, a mutation (rd) provoking the loss of both rods and cones in mice, does not affect their circadian entrainment (Freedman et al., 1999; Yoshimura and

Ebihara, 1998). These results showed that some photoreceptors for circadian entrainment must lie elsewhere in the retina.

Several groups have identified melanopsin as this circadian photoreceptor. It is expressed in retinal ganglion cells (RGC) of the inner retina, and the melanopsin-containing RGCs have direct projections to the master pacemaker in the SCN (Hattar et al., 2002;

Provencio et al., 1998). Melanopsin knock-out mice are able to entrain to the circadian photoperiod and only display mild phase-shifting phenotypes (Panda et al., 2002b; Ruby et al., 2002). However, when these mice are crossed with rd mice they completely lose circadian photoentrainement and free-run in normal light-dark conditions (Hattar et al.,

2003; Panda et al., 2003). These results clearly demonstrate that circadian photoreception in mammals is performed by multiple cell types; rod and cones in the outer retina layer and RGCs in the inner retina layer. Importantly, all rods and cones targeting the SCN via the retinohypothalamic tract first project to melanopsin-containing RGCs, which make up a small minority of all RGCs. When melanopsin-containing cells are genetically ablated, the mice have a normal vision, but are incapable of synchronizing their SCN master clock (Guler et al., 2008).

In Drosophila cryptochrome is the major circadian photoreceptor (Emery et al., 1998;

Stanewsky et al., 1998). It is an FAD- and folate-containing blue light receptor with high similarity to DNA photolyases. In mammals the two cryptochromes, CRY1 and CRY2 have adopted a role as repressors of the CLOCK/BMAL1 heterodimer (van der Horst et al., 1999). Since mCry1/mCry2 double knock-out mice are arrhythmic it is not obvious to assess their role in photoreception. Despite this difficulty there is some evidence for the involvement of CRYs in photoreception. First, Cry-deficient mice present some defects in light induced Per induction in the SCN (Vitaterna et al., 1999). Second, mice devoid of both rods and cones, and CRYs are nearly arrhythmic in normal light-dark environment (Selby et al., 2000). Third, mice lacking vitamin A are visually blind but present normal circadian photoreception (Thompson et al., 2001). Finally, mice lacking CRYs have reduced pupillary responses, a photoadaptive mechanism closely related to photic entrainment of circadian clocks (Van Gelder et al., 2003).

Figure 8. Morphological organization of the mammalian retina (Cermakian and Sassone- Corsi, 2002)

Signal transduction

The light input is transmitted form the eye to the SCN through the retino-hypothalamic tract. These neurons use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as principal neurotransmitters. Micro-injection of either glutamate or PACAP into the SCN induces phase shifts similar to those elicited by light pulses (Harrington et

al., 1999; Mintz et al., 1999). The SCN responds to light by activating different signaling pathways, which activate cAMP response element-binding protein (CREB) and turn on the transcription of immediate early genes (Morris et al., 1998). These genes include c- Fos, Fos-B and Jun-B but also Per1 and Per2 (Albrecht et al., 1997; Shearman et al., 1997). Interestingly these genes are also induced in cultured fibroblast upon serum stimulation (Balsalobre et al., 1998). Importantly, the activation of CREB is under the control of the circadian clock and it can only be activated during the dark phase (Ginty et al., 1993). Light induced expression of Per genes provides a mechanism for how light pulses can cause both phase delays and advances. Induction of PERs in the first half of the day results in delayed clearance of PERs and leads to phase delay. On the other hand, light pulses and subsequent PER induction in the later part of the night causes PERs to accumulate in advance and hence result in phase advances.

The SCN synchronizes peripheral clocks

Peripheral organs possess robust clockworks that can sustain circadian oscillations for several weeks (Yoo et al., 2004). Organs ex vivo, even from the same animal, exhibit different circadian phases, and in vivo they must therefore be synchronized by the SCN.

Since most peripheral tissues do not receive direct neuronal signals from the SCN, the nature of synchronizing signals is likely to be humoral, although synaptic pathways linking the SCN into the autonomic nervous system are likely to play a role as well (Arendt and Skene, 2005).

Circadian oscillations can be observed in cultured cells after stimulation with a wide variety of chemical cues. These include glucocorticoids, retinoic acid, glucose and compounds inducing signaling pathways involving the activation of protein kinase A (PKA), protein kinase C (PKC), and mitogen activated protein kinase (MAPK) (Akashi and Nishida, 2000; Balsalobre et al., 2000a; Balsalobre et al., 2000b; Hirota et al., 2002;

McNamara et al., 2001; Yagita and Okamura, 2000; Yagita et al., 2001). Circadian gene expression in tissue culture cells can also be induced by temperature cycles which correspond to natural body temperature rhythms (Brown et al., 2002). Most of these synchronization signals induce Per gene expression. For example, glucocorticoid

receptor response elements (GREs) have been identified within the promoter and the first

intron of Per1 (Le Minh et al., 2001; Yamamoto et al., 2005). Since such a plethora of chemical compounds and physical signals can synchronize circadian oscillations in vitro, it is likely that the phase entrainment of peripheral clocks in vivo is both complex and redundant. Feeding time is probably the most dominant timing cue to entrain the phase of oscillators in peripheral organs. Forcing nocturnal animals, such as mice, to eat only during the day gradually shifts the phase in peripheral organs, without affecting the phase of the SCN (Damiola et al., 2000). However when food is offered ad libitum, the SCN rapidly entrains the peripheral oscillator to the normal mode (Le Minh et al., 2001). Since the restricted feeding-induced uncoupling of peripheral organs from the SCN takes longer (7-10 days) than their re-synchronization, it is likely that the SCN uses more direct

mechanism than imposing feeding time. Glucocorticoid signaling could be one of the mechanisms, since circadian corticosterone production is controlled by the SCN.

Moreover, glucocorticoid signaling has been shown to affect the uncoupling process. In mice with a liver-specific glucocorticoid receptor (GR) knock-out, the phase inversion of liver oscillators by restricted day time feeding proceeds much faster than in wild-type animals (Le Minh et al., 2001).

Finally, Kornmann et al. established a mouse strain with conditionally active liver clocks to study how systemic cues regulate the peripheral oscillators. Since REV-ERBα is a strong repressor of the essential clock gene Bmal1, these authors designed a tet-off regulated system where the over-expression of REV-ERBα shuts down Bmal1 transcription. The addition of the tetracycline analog doxycycline inactivates the Rev- erbαtransgene and hence re-establishes Bmal1 expression and oscillator function. Using this model combined with genome-wide transcriptome profiling, the authors of this study identified a set of genes which are expressed in a circadian manner even when the clock was shut down (Kornmann et al., 2007). Intriguingly, Per2 was among these genes, along with several genes whose expression is sensitive to temperature, such as heat shock protein (HSP) genes and genes encoding cold-inducible RNA-binding proteins. They further showed that Per2 expression can be induced by a heat shock and found several binding sites for heat shock transcription factor (HSF) on the Per2 promoter. These observations provide a plausible mechanism for the temperature-dependent phase entrainment of peripheral oscillators. Interestingly, it was recently shown that the DNA­

binding activity of HSF1 is indeed highly circadian, although its mRNA and protein levels remain flat throughout the day (Reinke et al., 2008). Taken together these results suggest that activation of HSF1 could be one of the ways through which the SCN resets peripheral clocks.

Outputs and Circadian Physiology

Many aspects of mammalian physiology and behavior are under the control of the circadian timing system. The temporally gated physiological functions in peripheral organs can be driven by either cyclic systemic cues or by the oscillator of these organs themselves. Global transcriptome-profiling studies by several groups have provided considerable insight into the contribution of peripheral oscillators in gene expression. For example, these studies have revealed that in liver approximately 2-10 % of all mRNAs display circadian expression pattern (Akhtar et al., 2002; Duffield et al., 2002; Kornmann et al., 2007; Panda et al., 2002a; Storch et al., 2002). Two general aspects of the circadian timing system became apparent from these studies. First, most circadian transcripts are expressed in a tissue-specific manner, indicating that the circadian clock controls different functions in different organs. Second, circadian transcripts in a given tissue accumulate according to different phases. In this way the oscillator can separate

chemically incompatible reactions, like glycogen anabolism and catabolism in the liver.

Genes with circadian expression can be divided into two groups; clock controlled genes (ccgs) which are direct targets of CLOCK/BMAL1 complex, and indirect target genes which are activated by downstream transcription factors.

Analysis of the circadian liver transcriptome revealed that many genes involved in major hepatic functions are regulated by the circadian clock (Akhtar et al., 2002; Hughes et al., 2009; Panda et al., 2002a; Storch et al., 2002). Metabolism of endo- and xenobiotic compounds, production of co-factors and ligands, and the synthesis of proteins involved in the immune system are all under circadian control. Importantly, many rate limiting enzymes involved in energy homeostasis are expressed in a circadian fashion. For example, hmg CoA reductase and cyp7a, the rate limiting enzymes of cholesterol synthesis and utilization, respectively, display robust circadian oscillations (Boll et al., 1999).

The three proline- and acid rich (PAR) basic leucine zipper (bZip) transcription factors, albumin-D-site binding protein (DBP), hepatic leukemia factor (HLF) and thryrotroph embryonic factor (TEF) are well studied circadian output regulators that translate core clock oscillations into overt rhythms in physiology. All three factors accumulate with high circadian amplitude and their transcription is controlled directly by core clock transcription factors (Gachon et al., 2004; Ripperger et al., 2000). Genetic loss-of-

function studies showed that mice with still one of these factors display mild phenotypes.

However, mice with all three genes deleted have a dramatically shortened lifespan. Triple knock-out animals are prone to sound-induced epileptic seizures during the first three months of their life, and surviving animals show signs of accelerated aging. The seizures are believed to be caused by a decreased expression of pyridoxal kinase (PDXK).

Although the PAR bZip proteins are expressed in a circadian manner in the SCN, their expression displays only low-amplitude oscillations in most other brain regions.

Transcriptome profiling revealed that PDXK is a target of PAR bZIP transcription factors both in the liver and the brain, and as expected its expression is cyclic in the liver but nearly flat in the brain. PDXK converts vitamin B6 derivatives into pyridoxal phosphate (PLP), which is a co-enzyme for many enzymes involved in neurotransmitter and amino acid metabolism. PAR bZip deficient mice not only have decreased levels of PLP, but also lower levels of serotonin and dopamine. Since such changes in neurotransmitters can provoke lethal seizures, strong oscillations in PDXK levels in brain would likely be harmful. On the contrary, circadian amino acid metabolism in the liver is likely to be beneficial to the animal.

PAR bZip proteins also regulate a set of genes that are involved in detoxification and drug metabolism (Gachon et al., 2006). These include cytochrome P450 enzymes, P450­

oxidoreductase, aminolevulinic acid synthase (ALAS1), glutathione-S-transferase (GST), members of drug transporter families and constitutive androstane receptor (CAR). CAR is a member of the nuclear receptor superfamily that senses xenobiotics and activates transcription of genes involved in the detoxification and clearance of drugs and xenobiotic compounds (Ueda et al., 2002). Accordingly, mice lacking PAR bZip transcription factors are more sensitive to the anticancer drugs mitoxantrone and cyclophosphamide. Interestingly, CLOCK mutant mice also show hypersensitivity to

cyclophosphamide (Gorbacheva et al., 2005), and this might be due to reduced levels of PAR bZip proteins in these animals (Ripperger et al., 2000). Taken together, these results revealed a fundamental role of the circadian timing system in modulating drug toxicity.

Further insight into circadian detoxification may enable clinicians to improve the efficacy of drug prescriptions by taking into account the timing of drug administration (Levi and Schibler, 2007).

Thesis Project

In mammals the repressive complex is a heteropolymeric complex formed by

Cryptochrome 1 and 2, Period 1 and 2, and additional proteins. Although the interaction of CRYs with PERs is well understood, the mechanism by which the complexes

containing these proteins repress BMAL1/CLOCK-mediated transcription remains elusive. Purification of PER1 associated proteins revealed two new proteins WDR5 and NONO involved in the generation of circadian rhythms (Brown et al., 2005). To learn more about proteins associated with CRYs I generated NIH-3T3 fibroblast cell lines that stably express epitope-tagged Cryptochromes. For this purpose I used lentivectors which are ideal for the generation of stable cell lines, due to their high transducing rate in NIH­

3T3 cells. The transgenes contained the coding sequence of either CRY1 or CRY2 with an N-terminal 6-histidine sequence and a C-terminal Flag sequence (His-CRY1/2-Flag).

The CRY1 transgene was expressed at comparable levels to endogenous CRY1, whereas the CRY2 levels were strongly overexpressed when compared to endogenous CRY2 levels. Interestingly, the endogenous CRY proteins were almost undetectable in these cell lines. This suggests that the constitutively expressed transgene produces functional proteins capable for autorepression. We also observed that the expression of clock genes were dampened in these cell lines, probably due to constant CRY overexpression, a phenomenon that had been reported previously (Ueda et al., 2005). I then prepared whole cell extracts and purified CRY1- and CRY2-containing complexes from these cells and, in collaboration with Dr. Francis Vilbois, identified the associated proteins by mass- spectrometry. Among these proteins was PER1, a known CRY-interacting protein. The identified proteins also included two RNA-binding proteins (RBM5 and PSF), three factors involved in transcription regulation (TRAP150, a mediator complex component,

BTF. and the corepressor KRIP1), and two proteins involved in ubiquitination and possibly deubiquitination pathways (SKP1 and OTUD4). OTUD4 is an otu-domain containing putative deubiquitinase, which interacts with both CRY1 and CRY2. Short hairpin RNA (shRNA) mediated depletion of OTUD4 leads to stabilized CRY proteins and impairs circadian transcription in NIH-3T3 cells.

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