the circadian clock component period2: from molecular to

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From: Jürgen A. Ripperger and Urs Albrecht, The Circadian Clock Component PERIOD2: From Molecular to Cerebral Functions. In Andries Kalsbeek,

Martha Merrow, Till Roenneberg and Russell G. Foster, editors: Progress in Brain Research, Vol. 199,

Amsterdam: The Netherlands, 2012, pp. 233-245. ISBN: 978-0-444-59427-3

© Copyright 2012 Elsevier BV. Elsevier

A. Kalsbeek, M. Merrow, T. Roenneberg and R. G. Foster (Eds.)Progress in Brain Research, Vol. 199ISSN: 0079-6123Copyright � 2012 Elsevier B.V. All rights reserved.

Author's personal copy

CHAPTER 14

The circadian clock component PERIOD2: Frommolecular to cerebral functions

Jürgen A. Ripperger* and Urs Albrecht

Department of Biology, Unit of Biochemistry, University of Fribourg, Fribourg, Switzerland

Abstract: The circadian clock is based on a molecular oscillator, which simulates the external day withinnearly all of a body’s cells. This “internalized” day then defines activity and rest phases for the cells and theorganism by generating precise rhythms in the metabolism, physiology, and behavior. In its perfect state,this timing system allows for the synchronization of an organism to its environment and this may optimizeenergy handling and responses to daily recurring challenges. However, nowadays, we believe thatdesynchronization of an organism due to its lifestyle or problems with its circadian clock not only causesdiscomfort but also may aggravate conditions such as depression, metabolic syndrome, addiction, or cancer.

In this review, we focus on one simple cogwheel of the mammalian circadian clock, the PERIOD2(PER2) protein. Originally identified as an integral part of the molecular mechanism that yields overtrhythms of about 24 h, more recently multiple other functions have been identified. In essence, thePER proteins, in addition to their important function within the molecular oscillator, can be seen notonly as integrators on the input side of the circadian clock but also as mediators of clock output. Thisdiversity in their function is possible, because the PER proteins can interact with a multitude of otherproteins transferring oscillator timing information to the latter. In this fashion, the circadian clocksynchronizes many rhythmic processes.

Keywords: metabolism; mood; synchronization; Per2.

Introduction

The circadian clock generates a projection of theexternal day into the individual cells of the body

*Corresponding author.Tel.: þ41-26-300-8674E-mail: [email protected]

233http://dx.doi.org/10.1016/B978-0-444-59427-3.00014-9

(Dibner et al., 2010; Ko and Takahashi, 2006; Liuet al., 2007). However, under normal conditionswe may not become aware of the existence of sucha timing device in us. The reason for this is that thecircadian clock is responsive to external timingcues, the so-called Zeitgebers (german for “time-giver”), such as the light/dark cycle. Hence, theeffects of the circadian clock are generally masked

http://dx.doi.org/10.1016/B978-0-444-59427-3.00014-9

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by external factors. To observe the characteristicproperties of a circadian clock, it is necessary to placean organism into conditions without externalZeitgebers (e.g., constant darkness or constant lightof constant light intensity). In these free-runningconditions, solely the circadian clock governs therhythmic metabolism, physiology, and behavior ofan organism. The advantage of such a process isobvious. It provides a temporal network allowingsynchronization of many metabolic pathways thatotherwise would run each on their own pace, justdriven by substrate availability, for example. Dailyseparation of metabolic pathways yielding or con-suming energy is already observed in simpleorganisms such as cyanobacteria (Dong et al., 2010;Johnson et al., 2011). Experiments performed withcyanobacteria also provide compelling evidence forthe biological advantage of possessing such timingsystems: the growth and survival rate of strainswith mutations in their circadian clock are generallynot affected unless these mutants are kept incoculture with another strain that is in better reso-nance with the environmental light/dark cycle(Ouyang et al., 1998). Under the latter conditions,the better adjusted one completely wipes out the lessadjusted strain. Hence, there is a substantialbiological advantage to possess a well functioningtiming system, and it is therefore not surprising thatcircadian clocks are found throughout the bacteria,plant, and animal kingdoms (Hut and Beersma,2011; Yerushalmi and Green, 2009). However, arecent field study with wild type and circadianrhythm-affected Period2 (Per2) knockout mice didnot reveal a strong selective disadvantage of havingan impaired function of the circadian clock inmammals (Daan et al., 2011). Here, we would liketo elaborate the basicmechanisms of themammaliancircadian clock and then focus on the functions ofthe circadian clock component PER2 in liver metab-olism and the brain.

General mechanism of circadian clocks

Circadian clocks arose probably independentlyduring evolution in different taxa such as cyano-bacteria, plants, and animals (Rosbash, 2009).

However, interesting similarities and common con-cepts can be found over the borders of the taxa, suchas the occurrence of posttranslational circadianoscillations of the redox state of peroxiredoxins,which are observed in human red blood cells(O’Neill and Reddy, 2011) and also the green algaeOstreococcus tauri (O’Neill et al., 2011). The mam-malian circadian oscillator is related to the onefound in the fruit fly Drosophila melanogaster(Dunlap, 1999). The basic concept is quite simpleand is found in essentially all cells of an organism.At the center of the clock is a negative transcrip-tional feedback loop. A pair of transcriptionalactivators increases the transcription of a set ofrepressors. The corresponding repressor proteinsaccumulate until their concentration is sufficientto shut down the transcription of their own genes.Consequently, transcription and translation of therepressors cease and the activity of the repressorproteins declines. As soon as the transcription blockis relieved, a new cycle of about 24 h restarts, yield-ing robust oscillations with a periodicity of abouta day. Obviously, such a simple mechanism wouldnot be sufficient to produce all of the robust rhythmsobserved in the organisms. There would be therisk that the oscillations generated by the feedbackloop dampen rapidly, nearly reaching equilibriumconditions (Ripperger and Brown, 2010). Hence,there exist regulatory posttranslational mechanismsand an associated network of other feedback loopsthat stabilizes and reinforces the circadian rhythms.This network also provides input information to thecore circadian oscillator to allow synchronization ofthe self-sustaining loop to the environment. On theother side, most of the primary output generatedby the circadian oscillator is mediated primarily byrhythmic transcriptional regulation of target genes.

The history of Period

In the early 1970s, Konopka and Benzer performeda mutagenesis screen inD. melanogaster to identifymutations in its circadian timing system (Konopkaand Benzer, 1971). Surprisingly, the identifiedmutations provoking shortening or lengthening of

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the free-running period length or completearrhythmicity in constant darkness mapped all tothe same gene, which was baptized Period (Per)(Konopka and Benzer, 1971; Reddy et al., 1984;Zehring et al., 1984). Nearly 20 years after the iden-tification of the Permutants, it was realized that Perregulated the accumulation of its own mRNA andthe concept of the feedback loop generating overtrhythms of about 24 h was born (Hardin et al.,1990). However, it was not until about 10 years laterthat the mammalian Per genes were identified andcloned (Albrecht et al., 1997; Shearman et al.,1997; Sun et al., 1997; Tei et al., 1997), and thatthe function of these Per genes for the mammaliancircadian oscillator (at least of Per1 and Per2)

Bmal1

Clock

P

Fig. 1. The network of the mammalian molecular oscillator. The coof about 24 h, is composed of activation of the Per genes by BMAL1gene expression by the accumulation of its own gene product. Tposttranslational modifications and interaction with the CRY proteErba gene is also activated by BMAL1 and CLOCK (not shown)REV-ERBa starts to inhibit transcription of the Bmal1 and ClocERBa (or PPARa, not shown) to regulate the Bmal1 gene. Tsynchronization of the core and stabilizing loops. Extended and ada

was verified genetically by analyzing the corre-sponding knockout mouse strains (Bae et al., 2001;Cermakian et al., 2001; Zheng et al., 1999, 2001).At about the same time the mammalian transcrip-tional activators, CLOCK (Gekakis et al., 1998;King et al., 1997) and BMAL1/MOP3 (Bungeret al., 2000; Hogenesch et al., 1998), and a pair ofadditional repressors, the CRYPTOCHROME(CRY) 1 and 2 proteins were also found (Griffinet al., 1999; Kume et al., 1999; van der Horst et al.,1999). These proteins cooperate and make up thecore loop of the molecular oscillator (Fig. 1). Asmentioned above, all of the mammalian genes havetheir counterparts in Drosophila, maybe with theexceptions that the mammalian CRY proteins

er2

Rev-Erbα

re loop (red shaded circle), responsible for generating rhythms(red) and CLOCK (blue) and the increasing repression of Perhe feedback inhibition by the PER proteins is delayed byins (not shown). In the stabilizing loop (blue circle), the Rev-and later on repressed by the PER proteins but immediatelyk genes and of its own gene. PER2 can interact with REV-he overall organization of the network allows for a tightpted from Ko and Takahashi (2006).

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are no (or less) important light-sensitive compo-nents of the circadian oscillator (Griffin et al.,1999; Okamura et al., 1999), and the function ofthe Drosophila Timeless protein (which functionssimilar as the mammalian CRY proteins) in themammalian circadian clock is less pronounced(Gotter et al., 2000).

CRYs or PERs?

Ever since the discovery of the main repressormolecules of the mammalian circadian oscillator,the question was posed, which of both kinds ofmolecules were the more important ones. Thegenetics were not really helpful to unravel theprecise functions of these supposed repressormolecules. Mutations that affect the free-runningperiod of the circadian oscillator are thought tobe integral parts of the clock mechanism or areaffecting its integral parts. Single knockout micedeficient for Per1 (Bae et al., 2001; Cermakianet al., 2001; Zheng et al., 2001), Per2 (Bae et al.,2001; Zheng et al., 1999), or Cry1 (van der Horstet al., 1999; Vitaterna et al., 1999) had shorterfree-running circadian oscillators and mice defi-cient for Cry2 (van der Horst et al., 1999;Vitaterna et al., 1999) had a longer free-runningcircadian oscillator, while the knockout for Per3did not affect circadian rhythmicity (Bae et al.,2001). Interestingly, depending on the strain,Per2 single knockout mice had a shorter free-running period length but became arrhythmicafter 1–2 weeks in constant darkness, suggestingthat PER2 has a more prominent functionfor the circadian clock than PER1. Hence, mostof these individual components have an impacton the clock mechanism. The combination ofknockout mice for Per1 and Per2 (Zheng et al.,2001) and the combination of knockout mice forCry1 and Cry2 (van der Horst et al., 1999) hadnearly the same phenotype, yielding arrhythmicbehavior immediately after placing the mice inconstant conditions. Consequently, both classesof proteins appear to be equally important to

maintain circadian oscillator function. Interestingly,the combination of any Per knockout with any Cryknockout yielded a whole plethora of phenotypesranging from a rescue of the Per2’s arrhythmic phe-notype in Per2:Cry2 double knockout mice tocompletely arrhythmic Per2:Cry1 double knockoutmice (Oster et al., 2002, 2003a,b). These geneticinteractions suggest that the PERandCRYproteinsform multiple different complexes with each otherto fulfill their function(s). Interestingly, all ofthe mammalian clock components are redundant(i.e., there exist Bmal1 and Bmal2, Clock andNpas2, Per1 and Per2, Cry1 and Cry2) (Rippergeret al., 2011). A reason for this is unknown yet. Arethere two oscillators running in parallel in the samecell or do the possible combinations mediate, forexample, tissue-specific gene regulation? At leastfor CLOCK and Npas2, it was established thatCLOCK may be more important in the periphery,and Npas2 may be more important in the brain(DeBruyne et al., 2007a,b).

Function of PER proteins on the core loop ofthe circadian oscillator

Considering the role of Per within the Drosophilacircadian oscillator (Hardin et al., 1990), it wastempting to speculate that the mammalian PERproteins fulfilled a similar function to create a tran-scriptional feedback loop. However, cotransfectionexperiments in vitro revealed that, by contrast tothe CRY proteins (Kume et al., 1999), the impactof the PER proteins on BMAL1 and CLOCK-mediated transcription was quite subtle (Kumeet al., 1999). This was surprising because the bio-chemical analysis clearly indicated that PER andCRY proteins can be coimmunoprecipitated andconsequently they are in the same (repressor?)complex (Brown et al., 2005; Duong et al., 2011;Lee et al., 2001). Indeed, the regions within bothclasses of proteins mediating the interaction werebiochemically mapped (Fig. 2) (Langmesser et al.,2008). Nevertheless, nonrhythmic overexpressionof PER2 but not CRY1 strongly interfered with

H3N COOH

HLH

PAC

PAS B

PAS A

NLSCLD

NES 3NES1 NES 2CoRNR

LXXLL LXXLL

1 1257

Pro-rich Coiled- coil

CKIGSK3β Cry, E4BP4β−TrCP

Fig. 2. Protein–protein interaction regions of PER2. The protein belongs to the class of PAS domain-containing factors. The PAS Aand B domains and the associated PAC domain mediate the interaction with other PER proteins and the BMAL1 and CLOCKheterodimer. The region is flanked by binding sites for the regulatory kinase GSK3b on the left and the ubiquitin ligase b-TrCPon the right. Both enzymatic activities affect the half-life of PER2. The basic helix-turn-helix (HLH) motif resembles a DNA-binding motif but is probably not functional. The protein has three nuclear-export sites (NES), one nuclear-localization site(NLS) and a cytoplasm-localization domain (CLD), which influence the nuclear localization of the protein. Interaction with theCRY proteins (and E4BP4) is mediated by the C-terminus. The protein contains a sequence of casein kinase 1 (CKI)phosphorylation sites organized as a relay, from which the priming site is affected in FASPS. PER2 also harbors some potentialmotifs, which resemble binding sites for nuclear receptors (LXXLL and CoRNR), but only for the most N-terminal LXXLLmotif an interaction with nuclear receptors was identified. Finally, the protein contains potential interaction motifs resemblingproline-rich (Pro-rich) motifs and coiled-coil interaction motifs. Interactions with further kinds of proteins have been described,such as with WDR5 (an adaptor for the histone methyl transferase Set1), NONO and SFPQ1, and histone demethylases of theJarid1 family; however, these were mainly identified in complexes containing also PER1 and a direct interaction has not beendemonstrated yet. Adapted from Albrecht et al. (2007).

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normal circadian oscillator function, which is astrong indicator for a prominent function of thePER2 protein for the circadian oscillator (Chenet al., 2009). The more detailed analysis suggestedthat the PER proteins form a kind of scaffold forthe CRY proteins to bind to their target sequencesin the genome (Chen et al., 2009). Interestingly,both kinds of proteins can interact with otherproteins that repress transcription via the modifica-tion of the chromatin structure. For CRYs, theseinclude histone deacetlyases (HDAC) (Naruseet al., 2004), which attenuate local transcriptionby removing specific acetyl groups from 50-terminiof histones, and for PER deacetylases (Duong

et al., 2011), but also proteins that remove methylgroups from (DiTacchio et al., 2011) or add methylgroups to 50-termini of histones (Brown et al.,2005; Etchegaray et al., 2006). However, it isnecessary to decipher the precise biochemicalfunction(s) and their impact on the molecularoscillator of both classes of repressor molecules inmore detail. Taken together, the available litera-ture on the function of PER proteins in vivo andin vitro suggests that it is not the protein itself thatacts as a regulator within the circadian oscillatormechanism, but it is the interaction of PERproteins with other proteins, which mediates theeffect(s).

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Role of PER2 in the stabilizing loop

Initially, the characterization of the available cir-cadian oscillator mutant and knockout miceuncovered a surprise (Shearman et al., 2000).The PER2 protein, but not PER1, was suggestedas activator of circadian Bmal1 transcription. Thispuzzle resolved itself about 2 years later, when anadditional feedback loop was discovered based onthe transcriptional repressor REV-ERBa (Pre-itner et al., 2002; Ueda et al., 2002). Hence, themodel of the circadian transcriptional feedbackloop was extended by the stabilizing loop (Fig. 1).In the core loop, BMAL1 and CLOCK activatetranscription of the Per genes until the PERproteins (together with the CRY proteins) feedback onto their own synthesis. To achieve this,there is a typical delay in the action of the repres-sor molecules as a prerequisite for the near 24 hoscillations. In parallel, BMAL1 and CLOCKalso activate transcription of Rev-Erba. Theaccumulating REV-ERBa subsequently repressestranscription of the Bmal1 and also the Clockgenes by replacing the transcriptional activatorRORa (Akashi and Takumi, 2005; Sato et al.,2004) or PPARa (Canaple et al., 2006), depen-dent on the cell type, and also by recruitingHDAC3 activity to the target genes (Feng et al.,2011). Because the action of REV-ERBa doesnot involve a delay mechanism typical for theaction of the PER proteins, the phases of tran-scription of the Per and Rev-Erba genes onone hand and of the Bmal1 and Clock genes onthe other hand are separated by about 12 h. Asconclusion, loss of repression of Bmal1 transcrip-tion as observed in Per2 knockout mice was prob-ably due to the reduction of REV-ERBa and aconcomitant increase in Bmal1 expression. Thestory became even more complicated about2 years ago. It was found that PER2 rather thanPER1 directly interacted with both REV-ERBaand PPARa, contributing to about 10% of therepression by REV-ERBa and 20–30% of activa-tion by PPARa of the Bmal1 gene in the liver(Schmutz et al., 2010). This interaction was due

to a specific amino acid motif in PER2, which res-embles a typical nuclear receptor/coregulatorbinding site (Fig. 2). However, the functionalextension of PER2 due to its interaction withnuclear receptors appears not to be restricted tothe core circadian oscillator alone (see below).

The rhythmic metabolism of the liver

As revealed by DNA-microanalysis experiments,many metabolic processes in the liver occurrhythmically (Miller et al., 2007; Panda et al.,2002; Storch et al., 2002; Vollmers et al., 2009).At the center of this regulation is glucose homeo-stasis to keep the blood glucose concentration at aconstant level (Lamia et al., 2008). Hence, overthe day, the liver has to switch steadily from glu-cose uptake from the blood and the storage ofglucose as glycogen to the mobilization of glucosefrom its glycogen stores and its secretion intothe blood stream. About 15% of the transcriptsfound in the liver accumulate in rhythmic fashion(Vollmers et al., 2009); however, only about 10%of those rhythmic transcripts can be assigned tothe circadian oscillator. This means that the bulkof rhythmic transcripts is induced solely by thepresence of their substrates. What is the advantageof either way of regulation by induction throughsubstrates or by the circadian clock? Inductionthrough substrates necessitates the presence ofsubstrates, but the response is normally fairly pro-portional to the substrate concentration. Hence, itis a quite economical way of gene regulationaccording to the needs. On the other hand, the cir-cadian clock allows the expression of genes beforethe substrates become available, obviously withthe drawback that the gene is expressed evenif the substrates never come. However, the antici-pation of daily recurring events may be the mainbiological advantage of possessing a circadianclock as evidenced by the increase of fitness of cya-nobacteria that are in better resonance with theenvironment. The circadian oscillator provides sta-ble rhythms of about 24 h, but over the course of a

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year the photoperiod (i.e., the day/night ratio)changes with an impact on the time when the ani-mal gets active and starts eating (Schultz and Kay,2003). Consequently, there exist mechanisms inthe liver that uncouple anticipation mechanismsfrom the circadian oscillator to keep the anticipa-tion potential intact (Stratmann et al., 2010). Asexample, some detoxification enzymes of theP450 family are rhythmically regulated by the cir-cadian transcription factor CAR, which itself isregulated by the circadian transcription factors ofthe PAR-Zip family (Gachon et al., 2006). Thereason may be that those detoxification enzymes,in the absence of their substrates, produce radicaloxygen species, which may cause damage to thecell. Hence, basal expression of these enzymesis restricted to a phase when the occurrence ofsubstrates is probable to provide some basic pro-tection. In addition, the genes of these enzymescan be super-induced by the nuclear receptorCAR according to the presence of their substrates.Consequently, the risk of damage to the cell isoptimally reduced. The expression of CAR isadjusted to the photoperiod by the PAR-Zipfactors, demonstrating that the precise temporalaction of these regulators and their target genesrelative to the activity phase of the animals isimportant (Stratmann et al., 2010). In conclusion,temporal organization of metabolic processes inthe liver optimizes not only energy expenditurebut also protects the organ from excessive damage.The circadian clock represents an elegant meansto fulfill such a coordinating function. Interest-ingly, the analysis of Per knockout mice revealeda function of PER proteins in the concertedaction of DNA damage repair enzymes andproto-oncogenes, rendering the Per knockoutanimals more prone to tumor formation undercertain circumstances (Fu et al., 2002).

Role of PER2 in the liver metabolism

Many of the metabolic processes in the liver areregulated by members of the nuclear receptor

superfamily (Yang et al., 2006). There are 49members of this family, of which about one halfis regulated in a circadian fashion. In essence,rhythmic expression of the transcriptional regu-lators of the nuclear receptor family suffices to reg-ulate their target genes in a rhythmic fashion. Inaddition, many of these nuclear receptors requirespecific ligands for their activity. Some of these lig-ands are produced and secreted in a rhythmic fash-ion. Under normal conditions, this system couldstably regulate rhythmic processes but appears tobe circuitous and inflexible to respond rapidly tochanges of the circadian clock. The extendedcapacity of PER2 to directly interact with thesenuclear receptors provides an elegant way to trans-mit circadian clock information to the nuclearreceptor regulated target genes (Fig. 3) (Schmutzet al., 2012). Nuclear receptors involved in the reg-ulation of metabolic pathways and able to interactwith PER2 in vitro include REV-ERBa, PPARaand PPARg, and HNF4a and affect the lipidand glucose metabolism, respectively. Interest-ingly, PER2 acts as nuclear receptor coregulator,context-dependent as coactivator or corepressor.However, further experiments are required tofully understand these differences in its action.Nevertheless, PER2 transmits circadian oscillatorinformation directly to nuclear receptor-mediatedmetabolic pathways.

Function of PER2 in the brain

Per2 expression was initially observed in thesuprachiasmatic nuclei in the ventral part of thehypothalamus and various other brain regions aswell as in peripheral tissues (Albrecht et al.,1997). Because of its inducibility by light, foodrestriction, and temperature pulses, Per2 wasrecognized soon as a link between signals fromthe environment (the input) and the clock mecha-nism. Per2 acts as a responder to environmentalsignals and is a component of the core clock mech-anism, but it can also affect physiological pathwaysdownstream of the clock (the output, Fig. 3).

Input

Light

Food

Nurr1 Dopaminergic system,inflammation

Glucose metabolism

Temperature

Fatty acid metabolism

Circadian timing

Others

Hnf4a

TRa

PPARa

Rev-Erba

?

PER2

Clock Output

NR homo/hetero

dimers

Target gene

promoters

Fig. 3. Transmission of circadian clock information to nuclear receptor-target genes by PER2. PER2 mediates primary output fromthe molecular oscillator. By direct interaction with the nuclear receptor (NR) homo- or heterodimers, it can affect the correspondingtarget gene promoters and metabolic or physiological processes. The activity of PER2 may be modulated by the input (e.g., light orfood) to the circadian oscillator. Adapted from Schmutz et al. (2010).

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Hence, it appeared that Per2 is an importantplayer in determining the transcription status ofthe genome in a specific environmental context.Therefore, it was not astonishing to find thatPer2 plays a role in many brain related functions.The reward system regulating addiction appearedto be at least partially affected when the Per2gene was mutated (Abarca et al., 2002; Hamppet al., 2008; Spanagel et al., 2005). In particular,it appeared that the rate-limiting step in dopa-mine degradation involving the monoamineoxidase A was under the control of the circadianclock mechanism including the Per2 gene(Hampp et al., 2008). In line with this view isthe observation that PER2 variation in humansis associated with depression vulnerability(Lavebratt et al., 2010; Partonen et al., 2007).Recent findings indicate that the reward systemand addiction processes share neurobiologicalmechanisms with overeating and obesity (Simerly,2006). Interestingly, a mutation in the Per2 geneof mice resulted in the loss of food anticipatory

activity, when animals were kept under a timedfeeding schedule (Feillet et al., 2006). Takentogether, it appears that the Per2 gene is at thecrossroads of the neurobiological circuitry that iscommon to feeding signals and drugs of abuse.In support of this view is the fact that Per2 isexpressed in regions of the midbrain such as thearcuate nucleus and the ventral tegmental area,which regulate food uptake and reward pro-cessing, respectively. Many of the metabolic pro-cesses in the brain involve also nuclear receptorssuch as Nurr1, which can physically interact withPER2 (Ripperger et al., 2010; Schmutz et al.,2010), and hence, brain function is at least par-tially regulated by this avenue.

Function of PER2 in sleep

Per2 appears to play a role in various parametersof sleep. Loss of Per2 affected proper sleep timing(Kopp et al., 2002), which is also evidenced in

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advanced sleep-phase syndrome (Jones et al.,1999). Per2 may affect parameters of sleephomeostasis as well (Franken et al., 2007), espe-cially sleep deprivation and recovery sleep appearto involve PER2. However, whether this functionof PER2 is only attributable to its role in thebrain is not known, because metabolic demandsin the liver involving this protein may also impacton sleep mechanisms (Albrecht, 2011). Sleepappears to be regulated by remodeling of neuro-nal connectivity either strengthening or weaken-ing synaptic connections. The finding that PER2is involved in gating the light/dark informationto vesicular glutamate transporter 1 (vGLUT1)content on synaptic vesicles (Yelamanchili et al.,2006) was of great interest, because the numberof vGLUT1 molecules on the synaptic vesiclecorrelates with the glutamate filling state of thisvesicle and hence affects glutamate release poten-tial, which may impinge on synaptic strength.Hence, light may impact via PER2 on brainfunction and therefore, alterations in lightingschedule as experienced in jet-lag and shift workmay affect behavior.

Posttranslational modifications of PER2

How is the interaction potential of PER2 withother proteins regulated? One possibility is theaccumulation of the PER2 protein over the circa-dian cycle (Lee et al., 2001). The peaks of tran-scription and translation of the Per genes arecharacteristically separated by a couple of hours.In addition, the accumulation of the PER proteinsin the nucleus occurs with high amplitude everyday and consequently, the interaction of PERproteins with other proteins would follow theiraffinity of interaction (i.e., first the high affinitybinders and then the low affinity binders).Exchange of interaction partners would occur bysimple competition mechanisms. This simple affin-ity model may be extended by posttranslationalmodifications (Vanselow and Kramer, 2010). Inthe extended model, the dynamic combination of

phosphorylation of the PER2 protein reflectsdirectly oscillator time and each phosphorylationsite may represent a docking site for a specificprotein. Specific phosphorylation of a particularsite is achieved by equilibrium of the action ofkinases on one side and of phosphatases on theother side. For PER2, about 21 phosphorylationsites have been identified in fibroblasts althoughthe kinetics of phosphorylation of the individualsites remains to be established (Maier et al., 2009).Some sites when phosphorylated by casein kinase1 (CK1) d or e serve as binding sites for b-TrCP1/2,which add ubiquitination marks to the PER2 pro-tein, rendering it unstable and finally provoke deg-radation by the proteasome (Reischl et al., 2007;Shirogane et al., 2005). An interesting case is thefamilial advanced sleep-phase syndrome (FASPS)(Jones et al., 1999). Individuals with specificmutations in either CK1d (Xu et al., 2005) or theCK1d binding site on PER2 display sleep rhythmsadvanced by several hours (Toh et al., 2001) (a sim-ilar mutation in CK1e is provoking the familialdelayed sleep-phase syndrome, (Takano et al.,2004)). Detailed analysis revealed that the ratio ofkinase to substrate was important for the free-running period length of the molecular oscillator(Xu et al., 2007). On the phosphatase side, regu-latory subunits of the protein phosphatase 1 wereshown to regulate the nuclear-localization PER2and the free-running period length (Lee et al.,2011; Schmutz et al., 2011). Are the interactions ofnuclear receptors affected by reversible phosphory-lation? At the moment, there is no direct evidencein favor of this hypothesis. The interaction motifappears not to be adjacent to a known phosphoryla-tion side. However, it could be that phosphor-ylation at a distant side affects the accessibility ofthe interaction motif. As speculation, the regionbetween the two PAS domains is flexible todynamically shift from a conformation facilitatinginteraction with BMAL1 and CLOCK to another,which confers interaction with nuclear receptors.Phosphorylation mediating interaction with PER2could also be present on the nuclear receptors, pos-sibly regulated by the presence of specific ligands.

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Conclusions

Synchronization of the individual branches of thecircadian oscillator and the different routes ofinput and output might necessitate more effortthan simple transcriptional feedback loops canprovide. Temporally controlled protein–proteininteractions enable the coupling of the high-precision timer of the circadian clock to, forexample, nuclear receptor-target genes. The highflexibility of these interactions allows PER2 toact as coactivator or corepressor depending onthe regulatory context and hence to fine-tunethe activity of these nuclear receptors. To fullyunderstand the interaction potential of PER2 andits regulation by posttranslational modifications,however, requires further experiments. Theseinteractions may form the base of the many addi-tional functions of PER2 in the metabolism andthe brain. Other members of the core circadianoscillator can interact with a variety of otherproteins and regulators, and it is tempting to spec-ulate that the overall function(s) of theseinteractions are similar to those of PER2. How-ever, it is also tempting to speculate that PER2can directly interact with many more classes oftranscriptional regulators.

Acknowledgments

We thank for the support of our laboratory by theState of Fribourg and the Swiss National ScienceFoundation.

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