10.1177/0748730404269151JOURNALOFTakahashi / FINDING BIOLOGICALRHYTHMS NEW CLOCK COMPONENTS / October 2004 PERSPECTIVE

Finding New Clock Components: Past and Future

Joseph S. Takahashi1 Howard Hughes Medical Institute, Department of Neurobiology & Physiology, Northwestern University, Evanston, IL

Abstract The molecular mechanism of circadian clocks has been unraveled pri- marily by the use of phenotype-driven (forward) genetic analysis in a number of model systems. We are now in a position to consider what constitutes a clock component, whether we can establish criteria for clock components, and whether we have found most of the primary clock components. This perspective discusses clock genes and how genetics, molecular biology, and biochemistry have been used to find clock genes in the past and how they will be used in the future.

Key words circadian clock components, clock genes, phenotype, forward genetics

The modern era of “clock genetics” began in 1971 plete genome sequences for most of the important with the discovery by Ron Konopka and Seymour model organisms. Benzer (Fig. 1) of the period (per) locus in Drosophila In the circadian field, we have identified cell- (Konopka and Benzer, 1971). At the time, even the autonomous circadian pathways that share a con- existence of single-gene mutations affecting a process served transcriptional autoregulatory feedback mech- as complex as circadian rhythms was met with great anism across 2 domains of life (Dunlap, 1999; Young skepticism. The eminent phage geneticist, Max and Kay, 2001; Reppert and Weaver, 2002; Lowrey and Delbruck, is purported to have said, “No, no, no. It is Takahashi, 2004). And we can already anticipate that impossible,” when told of Konopka’s results. Benzer’s circadian clock proteins will ultimately yield to struc- reply: “But Max, you don’t understand. They’ve tural biology (Uzumaki et al., 2004; Vakonakis et al., already been found” (Greenspan, 1990). Much has 2004; Ye et al., 2004). So where will this end, and what changed since the golden age of phage genetics, but issues remain? In a sense, this is where much has much has also stayed the same. Thirty-three years stayed the same. We are on the 50th anniversary of later, we have dozens of examples of single genes that Pittendrigh’s analysis of temperature compensation affect behavior (Kyriacou and Hall, 1994; Takahashi of circadian oscillations in Drosophila pseudoobscura et al., 1994; Bucan and Abel, 2002). We have lived (Pittendrigh, 1954), yet we know very little more through decades when it was hard to clone a gene— about this mechanism today. (Ironically, the D. pseudo- now, we can manipulate genes at will. We have com- obscura genome is being sequenced. Even Pittendrigh

1. To whom all correspondence should be addressed: Joseph S. Takahashi, Howard Hughes Medical Institute, Department of Neurobiology & Physiology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3520; e-mail: j-takahashi@ northwestern.edu. JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 19 No. 5, October 2004 339-347 DOI: 10.1177/0748730404269151 © 2004 Sage Publications 339 340 JOURNAL OF BIOLOGICAL RHYTHMS / October 2004

1986). The sevenless mutant defined a developmental pathway for photoreceptor determination (Banerjee et al., 1987; Hafen et al., 1987). And recent mutants such as bubblegum and methuselah have uncovered genes affecting neurodegeneration and life span (Lin et al., 1998; Min and Benzer, 1999). So what is my point here? The discovery of circadian rhythm genes rests largely (indeed, almost exclusively) on the use of for- ward genetic screens to isolate circadian mutants, which in turn were used to identify the causative gene. Including per (Bargiello et al., 1984; Zehring et al., 1984), almost every important circadian locus has been defined by so-called clock mutants. This list includes frequency (frq), timeless (tim), Clock, KaiABC, Figure 1. Photograph of Ron Konopka (left) and Seymour Benzer and double-time, which were first identified by posi- (right) taken in October 2000 in Pasadena, California. Photo cour- tesy of Howard Hughes Medical Institute, Holiday Lectures on tional cloning (McClung et al., 1989; Myers et al., 1995; Science, December 2000, Clockwork Genes: Discoveries in Bio- Antoch et al., 1997; King et al., 1997a; Ishiura et al., logical Time. 1998; Kloss et al., 1998). A 2nd list includes cycle and tau, which provided the primary phenotypic evidence that the underlying gene was a “core component” (see would not have expected this.) That is not to say that below) (Rutila et al., 1998; Lowrey et al., 2000). we haven’t learned much. To the contrary, we are approaching a complete catalog (“parts list”) of circa- dian oscillator components in a number of organisms, PHENOTYPE, PHENOTYPE, PHENOTYPE and we even think we know how many of these parts work and interact with one another. But a rigorous In retrospect, a compelling “phenotypic profile” description of clock mechanism that includes an un- can be described to define what we now consider derstanding of kinetics and stoichiometry still eludes “core clock genes.” First and foremost is an extreme us. Moreover, we need validation of such models in phenotype: All of these key loci can be defined by vivo (not in transfected cells!). Finally, we have no mutations that alter period length by more than 15% deep understanding of how cell-autonomous circa- (3-4 h) or lead to a complete loss of circadian rhythms. dian oscillators interact in multicellular organisms When multiple alleles are available, period-shortening ultimately to regulate behavior (Low-Zeddies and and -lengthening mutations are observed, as well as Takahashi, 2001). loss-of-rhythm mutants. Ironically, per met all of these criteria from the beginning. Subsequently, frq, tim, and KaiABC also produced this extreme range of pheno- IN THE BEGINNING THERE WAS per types. In the absence of multiple alleles or in cases in which null mutations are lethal, an extreme period Benzer’s (1973) bold foray into the genetics of brain phenotype appears to be sufficiently compelling to and behavior in Drosophila remains one of the most define a clock gene of interest. The mouse Clock, remarkable achievements in biology—by unraveling Drosophilia double-time, and hamster tau mutants fall ≥ complex systems using systematic screens focused on into this category in which mutations caused 4-h a judicious array of related phenotypes (Benzer, 1973). period changes and null mutations were not available. The per gene story remains the exemplar for genetic One could of course argue that this analysis is dissection of behavior (Hall, 2003); however, one biased—we have only gone after the extreme muta- should not forget the significance of the other Benzer tions, and so these are the ones that we know about. mutants. The Shaker mutant defined the 1st potassium This is fair, but if we look more broadly at targeted channel at the molecular level (Kaplan and Trout, mutations in which we begin with the gene and then 1969; Kamb et al., 1987; Papazian et al., 1987). The ask what the phenotype might be, the “extreme dunce mutant provided an entrée into the genetics of phenotypic profile” still appears to hold, especially if learning and memory (Dudai et al., 1976; Chen et al., we allow the inclusion of double mutants for para- Takahashi / FINDING NEW CLOCK COMPONENTS 341 logous genes such as Period and Cryptochrome in mice. it is difficult to make definitive statements here, but With the exception of the Bmal1 (also known as Mop3) some generalizations apply. Inputs to the circadian knockout, which has an extreme loss-of-rhythm phe- oscillator can exert significant phase-shifting and notype by itself (Bunger et al., 2000), most gene-tar- period effects; however, loss-of-function mutations geted null mutations have produced less extreme should not lead to arrhythmicity. Rather, such mu- (more subtle) circadian phenotypes. The next best tations should lead to defects in entrainment as knockout is Per2, which causes a 1.5-h period shorten- seen with the cryptochromeb mutation in Drosophila ing and eventual loss of rhythms in the majority of (Stanewsky et al., 1998) or with compound mutations mice in constant darkness (Zheng et al., 1999). Knock- of the mammalian visual system involving melanop- outs of Per1 and Per3 fall into the subtle (0-1 hour sin (Hattar et al., 2003; Panda et al., 2003). Output period changes) category at best (Bae et al., 2001). For- mutations can produce overt arrhythmicity, but tunately, the Per1/Per2 double-knockout has strong strong effects on period or phase should not occur. effects, causing complete loss of rhythms in constant Molecular readouts of core oscillator components darkness (Bae et al., 2001; Zheng et al., 2001). The (such as Per) can then be used to assess whether the cir- Cryptochrome knockouts are also interesting. The Cry1 cadian oscillator is intact. Good examples of the effects null mutation causes a relatively subtle 1-h shortening of loss-of-function mutations on output components of period, while the Cry2 null mutation causes a 1-h are Dbp, a direct target of CLOCK-BMAL1 (Lopez- lengthening of period (van der Horst et al., 1999; Molina et al., 1997; Ripperger et al., 2000), and Pdf,a Vitaterna et al., 1999). Again, the Cry1/Cry2 double peptide output signal in Drosophila (Renn et al., 1999). knockout has a profound phenotype with a complete Caution is warranted, however, because feedback loss of circadian rhythms in constant darkness (van effects of output pathways such as locomotor activ- der Horst et al., 1999; Vitaterna et al., 1999). ity can exert phase-shifting effects, subtle period In contrast to this strong class of mutant circadian changes, and modulation of clock gene expression phenotypes, there are also examples of circadian (Van Reeth and Turek, 1989; Edgar et al., 1991; genes that are involved in the clock mechanism but are Maywood et al., 1999). not essential for the generation of circadian rhythms. In spite of the complexities addressed above, we This second “nonessential category” is exemplified by can define a set of criteria for primary (core or essen- Rev-erbα (Preitner et al., 2002). This gene is activated tial) clock components. The strongest case can be by the CLOCK-BMAL1 complex and is in turn regu- made when there are “strong” (i.e., 3-4 h) period- lated by PER/CRY–mediated repression. REV-ERBα shortening and period-lengthening mutations, as well then feeds back on Bmal1 (and Clock) to drive cycles of as arrhythmicity for null mutations. Drosophila per and transcription that are out of phase with Per and Cry tim are the prototypical examples (Konopka and (Preitner et al., 2002). This pathway forms a second Benzer, 1971; Konopka et al., 1994; Sehgal et al., 1994; feedback loop in the circadian network that has been Rothenfluh et al., 2000). When homozygous null labeled as a “positive feedback” loop—which we now mutations are lethal, as in the case of double-time in know is the result of 2 inhibitory steps. Null mutations Drosophila, the spectrum of extreme period mutations of Rev-erbα abolish the feedback effects of this path- may be compelling (Price et al., 1998). However, the way on Bmal1, and transcription of Bmal1 is constitu- lack of a loss-of-function null mutation tempers our tively elevated. However, circadian rhythm genera- ability to conclude that the gene is essential in the clas- tion persists in the absence of REV-ERBα, and there sic genetic sense. In mammals, in which paralogous are only subtle effects on the period and stability of genes are common, compound null mutations are circadian rhythms in the behavior of knockout mice required to achieve a strong loss-of-rhythm pheno- (Preitner et al., 2002). This specific example illustrates type (van der Horst et al., 1999; Vitaterna et al., 1999; the quandary that exists when we define clock compo- Bae et al., 2001; Zheng et al., 2001). Finally, as seen for nents: REV-ERBα is clearly part of the native circadian Clock and tau, substantially abnormal period mutants oscillator network, yet it is not essential for the gener- (caused by antimorphic mutations) may be sufficient ation of circadian oscillations. to implicate the gene in circadian function in the Additional examples of genes that would not be ex- absence of null mutations (Vitaterna et al., 1994; King pected to produce extreme phenotypes include other et al., 1997a, 1997b; Lowrey et al., 2000). In addition to elements of the circadian oscillator system, such as period phenotypes, Clock and Per2 mutant mice also those involved in input and output pathways. Again, become progressively arrhythmic in constant dark- 342 JOURNAL OF BIOLOGICAL RHYTHMS / October 2004 ness, which are interesting examples of mutant alleles dian system can have circadian patterns (Kornhauser that have a combined period and loss-of-rhythm phe- et al., 1990; Ginty et al., 1993; Emery et al., 1998; notype (Vitaterna et al., 1994; Zheng et al., 1999). Heintzen et al., 2001; Merrow et al., 2001). If an allelic series is not available, mutant pheno- Perturbation analysis has been considered the gold types alone may not be sufficient to define core clock standard for the identification of clock components. components. However, elucidating the specific bio- The classical view has been that if a putative compo- chemical functions of the proteins combined with a nent is part of the oscillator mechanism, then pertur- strong circadian phenotype can be ultimately compel- bation of the expression level or activity of the com- ling. For example, in mammals, CLOCK was shown to ponent must change the steady-state phase of the interact with BMAL1 to activate transcriptionally the oscillator for pulse treatments, or may cause period Per and Cry genes (Gekakis et al., 1998; Kume et al., changes for continuous treatments. This criterion is ε 1999), and Casein kinase 1 epsilon (CK1 ) was shown to valid for distinguishing output elements but has phosphorylate the PERIOD, CRYPTOCHROME, and never been able to discriminate input pathways from BMAL1 proteins (Lowrey et al., 2000; Eide et al., 2002). effects on the oscillator itself. We now have clear Thus, a 2nd set of criteria (beyond an extreme circa- examples of input components that oscillate and dian phenotype in mutants) involves defining the bio- could fulfill the perturbation test but do not appear to chemical function of putative clock components be clock components (Emery et al., 1998; Heintzen within circadian pathways. et al., 2001). In addition, we have examples of output pathways such as locomotor activity that can feedback and influence the period and phase of circadian sys- WHAT ARE CLOCK tems (albeit with relatively weak phenotypic effects). COMPONENTS ANYWAY? So, unfortunately,the complexity of real circadian sys- tems has compromised our desire to have simple Traditionally, circadian expression patterns or cir- criteria to define oscillator components. cadian oscillations in “activity” of a putative clock So how do we define core clock components? Is component have been thought to be critical criteria for this a valid concept, or does the complexity of circa- inclusion (Aronson et al., 1994). However, real exam- dian networks as in the case of genetic regulatory net- ples have clearly shown that this assumption only works allow too many degrees of freedom to exist applies to a subset of core clock components. Theory (Greenspan, 2001; Roenneberg and Merrow, 2003)? I has defined state variables of limit-cycle oscillators as would argue that the situation is not hopeless. We can clock components, but under this definition, only the distinguish among different levels of importance of feedback elements of the oscillator would fulfill this clock components. As discussed above, it appears that criterion. (Two examples of mathematical models of only a limited number of genes can be mutated to the circadian oscillator incorporating recent molecu- cause strong (3-4 h) period changes for missense mu- lar information have appeared recently; Forger and tations and loss-of-rhythm phenotypes for null muta- Peskin, 2003; Leloup and Goldbeter, 2003.) While state tions. This allelic series definition incorporates both variables obviously oscillate and clock components the classic genetic loss-of-function criterion for neces- such as PER, TIM, and CRY match this definition, sity as well as the perturbation criterion when applied other critical elements as defined above do not neces- to strong period changes. Defining the position of sarily oscillate in an obvious manner. Clock and tau are a putative component in a regulatory network (classi- 2 examples in which circadian expression may not be cally known as defining a pathway) describes how necessary for their function—although for these spe- the component interacts with other elements. More- cific cases one could enter into a semantic discussion over, describing the biochemical function of the puta- concerning whether the “activity” of CLOCK or CK1ε tive element can further define its role within a circa- may be circadian. In any case, we know that circadian dian network. Some elements of the network may be expression patterns are neither restrictive nor compel- essential—their removal (loss-of-function mutations) ling since literally hundreds of downstream-target will lead to a loss-of-rhythm phenotype. Other ele- genes exhibit circadian patterns (Akhtar et al., 2002; ments of the network may not be essential either Panda et al., 2002; Storch et al., 2002; Ueda et al., 2002), because there are alternate paths for the network to and many components of input pathways to the circa- function or there may be true cases of redundancy Takahashi / FINDING NEW CLOCK COMPONENTS 343 or overlapping function. In these cases, the loss-of- the basis of our ignorance of gene annotation and function phenotypes will be subtle, and compound function. (2) There are a significant number of candi- mutations may be required to produce strong pheno- date clock genes that have been proposed to be com- types. If a strong phenotype cannot be produced by ponents of the circadian clock mechanism that require compound loss-of-function mutations for a para- genetic evidence of strong circadian phenotypes logous gene family, then these elements would be before they can be admitted to the “inner circle.” An considered nonessential. example is the recent description of the Dec1(Stra13) Ultimately, we would want to apply another level and Dec2 genes, which have circadian expression pat- of perturbation tests for core clock components. This terns in the SCN and have strong inhibitory effects on would require conditional expression of clock compo- CLOCK-BMAL1 activation in cell transfection/ nents. We would want to apply this test for a number reporter gene assays (Honma et al., 2002; Grechez- of reasons. First, it is important to demonstrate that the Cassiau et al., 2004). What is still missing, however, is phenotypic effects seen in classical mutants are be- in vivo evidence that mutations can produce strong cause of the “ongoing” or cycle-to-cycle effects of the circadian phenotypes (loss-of-function or strong mutation rather than some developmental effect period mutants). (3) Quantitative genetic experiments (since classical mutants develop in this context). Sec- analyzing circadian phenotypes in different inbred ond, conditional experiments can test whether rhyth- strains of mice reveal that the majority of quantitative mic or constitutive expression is required for the com- traitlocidonotmaptoknowncircadiangenes ponent. Third, conditional expression in a wild-type (Shimomura et al., 2001). These allelic variants con- background can be used to apply perturbation tests tribute to striking differences in circadian behavior in (phase shifts or period changes). Finally, conditional different inbred strains of mice and suggest that a experiments can be used in a tissue-specific manner to large number of circadian modifier genes remain to be test for cell autonomy, hierarchical regulation, and identified. (4) Veryfew forward genetic screens for cir- other types of interactions. To date, only a few clock cadian mutations have reached saturation. In mice, we components have been tested in this way. Aronson have barely scratched the surface. Indeed, in a recent et al. (1994) applied this test early on in the field. In ret- pilot screen in which 2 circadian mutants were recov- rospect, the only deficit in this analysis was definitive ered, 1 mutant was a new allele of a known locus, and measurement of the expression levels of the condi- a 2nd mutant defined a new circadian gene because no tionally expressed genes, which was performed later known circadian genes coincide with its map location. (Merrow et al., 1997). Similar analyses have been New large-scale recessive mutagenesis screens in accomplished in Cyanobacteria (Ishiura et al., 1998). mice promise to deliver many new circadian mutants Tissue-specific and constitutive expression patterns (Moldin et al., 2001). Furthermore, because saturation have also been tested in Drosophila (Yang and Sehgal, mutagenesis can only be assessed empirically by 2001; Zhao et al., 2003). Thus far, no critical tests of this recovery of multiple, new alleles only at previously type have been performed in mammals, particularly known loci, it is likely that this level of screening has in the context of the intact animal. We are still a long only been accomplished in Cyanobacteria (Kondo et al., way from critical testing of known clock components, 1994; Ishiura et al., 1998). In addition, even saturation especially in mammals. mutagenesis is ultimately limiting because it may not be possible to recover circadian mutants for “all” genes. We can already anticipate this result in the case ARE THERE MORE of paralogous genes such as Per, in which compound NEW CLOCK COMPONENTS? mutations may be required to produce strong circa- dian phenotypes. (5) Microarray experiments have The answer is a definite “yes.” There are many rea- uncovered literally hundreds of cycling genes (Akhtar sons for this opinion. (1) Even though we have com- et al., 2002; Duffield et al., 2002; Panda et al., 2002; plete genome sequences in humans, mice, and rats Storch et al., 2002; Ueda et al., 2002). In mice, about (Lindblad-Toh, 2004), we are still far from understand- 10% of all detectable transcripts in any tissue express ing the function of a large fraction (~40%) of genes. We circadian profiles. Asignificant set of genes appears to would be naively egotistical to think that we have cycle in the majority of tissues, and most of these genes already found all the core circadian clock genes just on have not been previously assigned a function in the 344 JOURNAL OF BIOLOGICAL RHYTHMS / October 2004 circadian network. It seems likely that at least a few of plete description of circadian clock components in a these cycling genes could define additional circadian number of model systems. And we think we know clock genes. In addition, more comprehensive micro- how the core autoregulatory transcriptional feedback arrays interrogating the entire transcriptome are now loop operates. However, we are still far from a quanti- available, so additional cycling genes will undoubt- tative understanding of any circadian oscillator with edly be uncovered (Su et al., 2004). (6) Biochemical in- respect to kinetics, stoichiometry, and cell biology in teraction screens have identified a large number of the context of real cells and organisms. We still have proteins that can interact with known circadian clock little knowledge concerning how this molecular oscil- proteins. For example, using chromatin immuno- lator regulates the large number of pathways in the precipitation assays on the Dbp promoter, Ripperger cell that are under circadian control—much less how et al. (2000) found many proteins associated in a com- these cells communicate circadian information more plex with CLOCK, most of which have not been iden- broadly. Aclear view has emerged that virtually every tified. Yeast 2-hybrid screens have also yielded about cell in the body has the capability of expressing circa- 20 bona fide CLOCK-interacting proteins that remain dian oscillations (Balsalobre et al., 1998; Yamazaki to be explored (Gekakis et al., 1998). Such protein- et al., 2000; Yamaguchi et al., 2003; Yoo et al., 2004). interaction screens have only begun to be pursued in Now we need to know why. Are circadian rhythms so earnest, and so much remains to be explored in this fundamental to metabolic efficiency that we cannot do arena using both traditional biochemistry as well as without them (Rutter et al., 2002)? Or are cellular circa- modern proteomics approaches. (7) Finally, many dian rhythms a vestige of a unicellular past in which large-scale approaches such as genomewide siRNA metabolism and environmental insult are more proxi- screens or overexpression screens with genomewide mal to survival (Ouyang et al., 1998; Johnson and cDNA collections are underway and are likely to re- Golden, 1999)? We can only speculate. Recent links veal additional circadian clock genes. Thus, the future between circadian rhythms and cancer (Fu and Lee, is bright. A daunting array of tools is being applied to 2003) and the cell cycle (Matsuo et al., 2003) in mice circadian gene discovery, and each of these is likely to suggest that the relationship is more than vestigial. yield new insight into the circadian mechanism. How has this molecular view of circadian rhythms changed us? For a while, some of us may have lost some of our focus on systems and integrative ques- THE PARADIGM SHIFT: tions (while we engaged in sprints to find new genes). WHAT HAVE WE LOST But with new tools such as circadian promoter::reporter AND WHAT HAVE WE GAINED? gene technology and the knowledge that we are composed of populations of circadian oscillators The application of genetics, biochemistry, molecu- (Millar et al., 1992; Plautz et al., 1997; Balsalobre et al., 1998; Yamazaki et al., 2000; Yamaguchi et al., 2003; Yoo lar biology, and genomics to the study of circadian et al., 2004), a renaissance in circadian systems biology biology in the past decade has certainly led to a para- is on the horizon. Lost forever is that black box that digm shift in the field. We have moved from analysis once represented the circadian clock. of circadian rhythms primarily at the formal level to a much more concrete mechanistic level. Much like the transition from classical to molecular genetics, the ACKNOWLEDGMENT field of circadian rhythms has flourished while still maintaining a common language and set of problems. I thank Jeff Hall for insightful comments on this Perhaps it is somewhat ironic that genetics on one paper. hand and circadian rhythms on the other hand are so complementary. Circadian rhythms stand as one of the great success stories of classical genetic ap- REFERENCES proaches to the study of behavior, while genetics and molecular biology have been the key to unraveling the network of genes that compose the circadian oscillator Akhtar RA, Reddy AB, Maywood ES, Clayton JD, King VM, Smith AG, Gant TW, Hastings MH, and Kyriacou CP system. (2002) Circadian cycling of the mouse liver trans- So where are we today? As was stated at the begin- criptome, as revealed by cDNA microarray, is driven by ning of this article, we are well on our way to a com- the suprachiasmatic nucleus. Curr Biol 12:540-550. Takahashi / FINDING NEW CLOCK COMPONENTS 345

Antoch MP, Song EJ, Chang AM, Vitaterna MH, Zhao Y, Forger DB and Peskin CS (2003) Adetailed predictive model Wilsbacher LD, Sangoram AM, King DP, Pinto LH, and of the mammalian circadian clock. Proc Natl Acad Sci Takahashi JS (1997) Functional identification of the U S A 100:14806-14811. mouse circadian Clock gene by transgenic BAC rescue. Fu L and Lee CC (2003) The circadian clock: Pacemaker and Cell 89:655-667. tumour suppressor. Nat Rev Cancer 3:350-361. Aronson BD, Johnson KA, Loros JJ, and Dunlap JC (1994) Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher Negative feedback defining a circadian clock: Autoregu- LD, King DP,TakahashiJS, and WeitzCJ (1998) Role of the lation of the clock gene frequency. Science 263:1578-1584. CLOCK protein in the mammalian circadian mechanism. Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, and Science 280:1564-1569. Weaver DR (2001) Differential functions of mPer1, Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo mPer2, and mPer3 in the SCN circadian clock. Neuron KE, TakahashiJS, and Greenberg ME (1993) Regulation of 30:525-536. CREB phosphorylation in the suprachiasmatic nucleus Balsalobre A, Damiola F, and Schibler U (1998) A serum by light and a circadian clock. Science 260:238-241. shock induces circadian gene expression in mammalian Grechez-Cassiau A, Panda S, Lacoche S, Teboul M, Azmi S, tissue culture cells. Cell 93:929-937. Laudet V,Hogenesch JB, Taneja R, and Delaunay F (2004) Banerjee U, Renfranz PJ, Pollock JA, and Benzer S (1987) The transcriptional repressor STRA13 regulates a subset Molecular characterization and expression of sevenless, of peripheral circadian outputs. J Biol Chem 279:1141- a gene involved in neuronal pattern formation in the 1150. Drosophila eye. Cell 49:281-291. Greenspan RJ (1990) The emergence of neurogenetics. Semin Bargiello TA, Jackson FR, and Young MW (1984) Restoration Neurosci 2:145-157. of circadian behavioural rhythms by gene transfer in Greenspan RJ (2001) The flexible genome. Nat Rev Genet Drosophila. Nature 312:752-754. 2:383-387. Benzer S (1973) Genetic dissection of behavior. Sci Am Hafen E, Basler K, Edstroem JE, Rubin GM (1987) Sevenless, 229:24-37. a cell-specific homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase Bucan M and Abel T (2002) The mouse: Genetics meets domain. Science 236:55-63. behaviour. Nat Rev Genet 3:114-123. Hall JC (2003) Genetics and molecular biology of rhythms in Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Drosophila and other insects. Adv Genet 48:1-280. Radcliffe LA, Hogenesch JB, Simon MC, Takahashi JS, Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, and Bradfield CA (2000) Mop3 is an essential component Hankins MW, Lem J, Biel M, Hofmann F, Foster RG, et al. of the master circadian pacemaker in mammals. Cell (2003) Melanopsin and rod-cone photoreceptive systems 103:1009-1017. account for all major accessory visual functions in mice. Chen CN, Denome S, and Davis RL (1986) Molecular analy- Nature 424:76-81. sis of cDNA clones and the corresponding genomic cod- Heintzen C, Loros JJ, and Dunlap JC (2001) The PAS protein ing sequences of the Drosophila dunce+ gene, the struc- VIVID defines a clock-associated feedback loop that tural gene for cAMP phosphodiesterase. Proc Natl Acad represses light input, modulates gating, and regulates Sci U S A 83:9313-9317. clock resetting. Cell 104:453-464. Dudai Y, Jan YN, Byers D, Quinn WG, and Benzer S (1976) Honma S, Kawamoto T, Takagi Y, Fujimoto K, Sato F, dunce, a mutant of Drosophila deficient in learning. Proc Noshiro M, Kato Y, and Honma K (2002) Dec1 and Dec2 Natl Acad Sci U S A 73:1684-1688. are regulators of the mammalian molecular clock. Nature Duffield GE, Best JD, Meurers BH, Bittner A, Loros JJ, and 419:841-844. Dunlap JC (2002) Circadian programs of transcriptional Ishiura M, Kutsuna S, Aoki S, Iwasaki H, Andersson CR, activation, signaling, and protein turnover revealed by Tanabe A, Golden SS, Johnson CH, and Kondo T (1998) microarray analysis of mammalian cells. Curr Biol Expression of a gene cluster kaiABC as a circadian feed- 12:551-557. back process in cyanobacteria. Science 281:1519-1523. Dunlap JC (1999) Molecular bases for circadian clocks. Cell Johnson CH and Golden SS (1999) Circadian programs in 96:271-290. cyanobacteria: Adaptiveness and mechanism. Annu Rev Edgar DM, Martin CE, and Dement WC (1991) Activity feed- Microbiol 53:389-409. back to the mammalian circadian pacemaker: Influence Kamb A, Iverson LE, and Tanouye MA (1987) Molecular on observed measures of rhythm period length. J Biol characterization of Shaker, a Drosophila gene that Rhythms 6:185-199. encodes a potassium channel. Cell 50:405-413. Eide EJ, Vielhaber EL, Hinz WA, and Virshup DM (2002) The Kaplan WD, Trout WE, 3rd (1969) The behavior of four neu- circadian regulatory proteins BMAL1 and crypto- rological mutants of Drosophila. Genetics 61:399-409. chromes are substrates of casein kinase Iepsilon. J Biol King DP, Vitaterna MH, Chang AM, Dove WF, Pinto LH, Chem 277:17248-17254. Turek FW, and Takahashi JS (1997a) The mouse Clock Emery P, So WV, Kaneko M, Hall JC, and Rosbash M (1998) mutation behaves as an antimorph and maps within the CRY, a Drosophila clock and light-regulated crypto- W19H deletion, distal of Kit. Genetics 146:1049-1060. chrome, is a major contributor to circadian rhythm reset- King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, ting and photosensitivity. Cell 95:669-679. Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, 346 JOURNAL OF BIOLOGICAL RHYTHMS / October 2004

Lowrey PL, et al. (1997b) Positional cloning of the mouse McClung CR, Fox BA, and Dunlap JC (1989) The Neurospora circadian clock gene. Cell 89:641-653. clock gene frequency shares a sequence element with the Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Drosophila clock gene period. Nature 339:558-562. and Young MW (1998) The Drosophila clock gene double- Merrow M, Franchi L, Dragovic Z, Gorl M, Johnson J, time encodes a protein closely related to human casein Brunner M, Macino G, and Roenneberg T (2001) Circa- kinase Iepsilon. Cell 94:97-107. dian regulation of the light input pathway in Neurospora Kondo T, Tsinoremas NF, Golden SS, Johnson CH, Kutsuna crassa. EMBO J 20:307-315. S, and Ishiura M (1994) Circadian clock mutants of Merrow MW, Garceau NY, and Dunlap JC (1997) Dissection cyanobacteria. Science 266:1233-1236. of a circadian oscillation into discrete domains. Proc Natl Konopka RJ and Benzer S (1971) Clock mutants of Drosophila Acad Sci U S A 94:3877-3882. melanogaster. Proc Natl Acad Sci U S A 68:2112-2116. Millar AJ, Short SR, Chua NH, Kay SA (1992) A novel circa- Konopka RJ, Hamblen-Coyle MJ, Jamison CF, and Hall JC dian phenotype based on firefly luciferase expression in (1994) An ultrashort clock mutation at the period locus of transgenic plants. Plant Cell 4:1075-1087. Drosophila melanogaster that reveals some new features of Min KT and Benzer S (1999) Preventing neurodegeneration the fly’s circadian system. J Biol Rhythms 9:189-216. in the Drosophila mutant bubblegum. Science 284:1985- Kornhauser JM, Nelson DE, Mayo KE, and Takahashi JS 1988. (1990) Photic and circadian regulation of c-fos gene ex- Moldin SO, Farmer ME, Chin HR, and Battey JF (2001) pression in the hamster suprachiasmatic nucleus. Neu- Trans-NIH neuroscience initiatives on mouse phenotyp- ron 5:127-134. ing and mutagenesis. Mamm Genome 12:575-581. Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin Myers MP, Wager-Smith K, Wesley CS, Young MW, and X, Maywood ES, Hastings MH, and Reppert SM (1999) Sehgal A (1995) Positional cloning and sequence analysis mCRY1 and mCRY2 are essential components of the neg- of the Drosophila clock gene, timeless. Science 270:805- ative limb of the circadian clock feedback loop. Cell 808. 98:193-205. Ouyang Y,Andersson CR, Kondo T, Golden SS, and Johnson Kyriacou CP and Hall JC (1994) Genetic and molecular anal- CH (1998) Resonating circadian clocks enhance fitness in ysis of Drosophila behavior. Adv Genet 31:139-186. cyanobacteria. Proc Natl Acad Sci U S A 95:8660-8664. Leloup JC and Goldbeter A(2003) Toward a detailed compu- Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume tational model for the mammalian circadian clock. Proc M, Schultz PG, Kay SA, Takahashi JS, and Hogenesch JB Natl Acad Sci U S A 100:7051-7056. (2002) Coordinated transcription of key pathways in the Lin YJ, Seroude L, and Benzer S (1998) Extended life-span mouse by the circadian clock. Cell 109:307-320. and stress resistance in the Drosophila mutant methuselah. Panda S, Provencio I, Tu DC, Pires SS, Rollag MD, Castrucci Science 282:943-946. AM, Pletcher MT, Sato TK, Wiltshire T, Andahazy M, Lindblad-Toh K (2004) Genome sequencing: three’s com- et al. (2003) Melanopsin is required for non-image- pany. Nature 428:475-476 forming photic responses in blind mice. Science 301:525- Lopez-Molina L, Conquet F, Dubois-Dauphin M, and 527. Schibler U (1997) The DBP gene is expressed according to Papazian DM, Schwarz TL, Tempel BL, Jan YN, and Jan LY a circadian rhythm in the suprachiasmatic nucleus and (1987) Cloning of genomic and complementary DNA influences circadian behavior. EMBO J 16:6762-6771. from Shaker, a putative potassium channel gene from Low-Zeddies SS and Takahashi JS (2001) Chimera analysis Drosophila. Science 237:749-753. of the Clock mutation in mice shows that complex cellu- Pittendrigh CS (1954) On temperature independence in the lar integration determines circadian behavior. Cell clock system controlling emergence in Drosophila. Proc 105:25-42. Natl Acad Sci U S A 40:2697-2701. Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Plautz JD, Kaneko M, Hall JC, Kay SA (1997) Independent Zemenides PD, Ralph MR, Menaker M, and Takahashi JS photoreceptive circadian clocks throughout Drosophila. (2000) Positional syntenic cloning and functional charac- Science 278:1632-1635. terization of the mammalian circadian mutation tau. Sci- Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule ence 288:483-492. D, Albrecht U, and Schibler U (2002) The orphan nuclear Lowrey PL and Takahashi JS (2004) Mammalian circadian receptor REV-ERBalpha controls circadian transcription biology: Elucidating genome-wide levels of temporal within the positive limb of the mammalian circadian organization. Annu Rev Genomics Hum Genet 5:407-441. oscillator. Cell 110:251-260. Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, and Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, and Okamura H (2003) Control mechanism of the circadian Young MW (1998) double-time is a novel Drosophila clock clock for timing of cell division in vivo. Science 302:255- gene that regulates PERIOD protein accumulation. Cell 259. 94:83-95. Maywood ES, Mrosovsky N, Field MD, and Hastings MH Renn SC, Park JH, Rosbash M, Hall JC, and Taghert PH (1999) Rapid down-regulation of mammalian period (1999) A pdf neuropeptide gene mutation and ablation of genes during behavioral resetting of the circadian clock. PDF neurons each cause severe abnormalities of behav- Proc Natl Acad Sci U S A 96:15211-15216. ioral circadian rhythms in Drosophila. Cell 99:791-802. Takahashi / FINDING NEW CLOCK COMPONENTS 347

Reppert SM and Weaver DR (2002) Coordination of circa- van der Horst GTJ, Muijtjens M, Kobayashi K, Takano R, dian timing in mammals. Nature 418:935-941. Kanno S, Takao M, de Wit J, Verkerk A, Eker APM, van Ripperger JA, Shearman LP, Reppert SM, and Schibler U Leenen D, et al. (1999) Mammalian Cry1 and Cry2 are (2000) CLOCK, an essential pacemaker component, con- essential for maintenance of circadian rhythms. Nature trols expression of the circadian transcription factor DBP. 398:627-630. Genes Dev 14:679-689. Van Reeth O and Turek FW (1989) Stimulated activity medi- Roenneberg T and Merrow M (2003) The network of time: ates phase shifts in the hamster circadian clock induced Understanding the molecular circadian system. Curr Biol by dark pulses or benzodiazepines. Nature 339:49-51. 13:R198-R207. Vitaterna MH, King DP,Chang AM, Kornhauser JM, Lowrey Rothenfluh A, Abodeely M, Price JL, and Young MW (2000) PL, McDonald JD, Dove WF, Pinto LH, Turek FW, and Isolation and analysis of six timeless alleles that cause Takahashi JS (1994) Mutagenesis and mapping of a short- or long-period circadian rhythms in Drosophila. mouse gene, Clock, essential for circadian behavior. Sci- Genetics 156:665-675. ence 264:719-725. Rutila JE, Suri V,Le M, So WV,Rosbash M, and Hall JC (1998) Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C, CYCLE is a second bHLH-PAS clock protein essential for Fruechte EM, Hitomi K, Thresher RJ, Ishikawa T, circadian rhythmicity and transcription of Drosophila Miyazaki J, et al. (1999) Differential regulation of mam- period and timeless. Cell 93:805-814. malian period genes and circadian rhythmicity by cryp- Rutter J, Reick M, and McKnight SL (2002) Metabolism and tochromes 1 and 2. Proc Natl Acad Sci U S A 96:12114- the control of circadian rhythms. Annu Rev Biochem 12119. 71:307-331. Yamaguchi S, Isejima H, Matsuo T, Okura R, Yagita K, Sehgal A, Price JL, Man B, and Young MW (1994) Loss of cir- Kobayashi M, and Okamura H (2003) Synchronization of cadian behavioral rhythms and per RNA oscillations in cellular clocks in the suprachiasmatic nucleus. Science the Drosophila mutant timeless. Science 263:1603-1606. 302:1408-1412. Shimomura K, Low-Zeddies SS, King DP, Steeves TD, Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda Whiteley A, Kushla J, Zemenides PD, Lin A, Vitaterna M, Block GD, Sakaki Y, Menaker M, and Tei H (2000) MH, Churchill GA, et al. (2001) Genome-wide epistatic Resetting central and peripheral circadian oscillators in interaction analysis reveals complex genetic determi- transgenic rats. Science 288:682-685. nants of circadian behavior in mice. Genome Res 11:959- Yang Z and Sehgal A (2001) Role of molecular oscillations in 980. generating behavioral rhythms in Drosophila.Neuron Stanewsky R, Kaneko M, Emery P,Beretta B, Wager-Smith K, 29:453-467. Kay SA, Rosbash M, and Hall JC (1998) The cryb mutation Ye S, VakonakisI, Ioerger TR, LiWang AC, and Sacchettini JC identifies cryptochrome as a circadian photoreceptor in (2004) Crystal structure of circadian clock protein KaiA Drosophila. Cell 95:681-692. from Synechococcus elongatus. J Biol Chem 279:20511- Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, 20518. Wong WH, and Weitz CJ (2002) Extensive and divergent Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, circadian gene expression in liver and heart. Nature Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, et al. 417:78-83. (2004) PERIOD2::LUCIFERASE real-time reporting of Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, circadian dynamics reveals persistent circadian oscilla- Zhang J, Soden R, Hayakawa M, Kreiman G, et al. (2004) tions in mouse peripheral tissues. Proc Natl Acad Sci A gene atlas of the mouse and human protein-encoding U S A 101:5339-5346. transcriptomes. Proc Natl Acad Sci U S A101:6062-6067. Young MW and Kay SA (2001) Time zones: A comparative Takahashi JS, Pinto LH, and Vitaterna MH (1994) Forward genetics of circadian clocks. Nat Rev Genet 2:702-715. and reverse genetic approaches to behavior in the mouse. Zehring WA, Wheeler DA, Reddy P, Konopka RJ, Kyriacou Science 264:1724-1733. CP,Rosbash M, and Hall JC (1984) P-element transforma- Ueda HR, Chen W, Adachi A, Wakamatsu H, Hayashi S, tion with period locus DNA restores rhythmicity to TakasugiT, Nagano M, Nakahama K, Suzuki Y,Sugano S, mutant, arrhythmic Drosophila melanogaster. Cell 39:369- et al. (2002) A transcription factor response element for 376. gene expression during circadian night. Nature 418:534- Zhao J, Kilman VL, Keegan KP,Peng Y,Emery P,Rosbash M, 539. and Allada R (2003) Drosophila clock can generate ectopic Uzumaki T, Fujita M, Nakatsu T, Hayashi F, Shibata H, Itoh circadian clocks. Cell 113:755-766. N, Kato H, and Ishiura M (2004) Crystal structure of the Zheng BH, Albrecht U, Kaasik K, Sage M, Lu WQ, Vaishnav C-terminal clock-oscillator domain of the cyanobacterial S, Li Q, Sun ZS, Eichele G, Bradley A, et al. (2001) KaiA protein. Nat Struct Mol Biol 11:623-631. Nonredundant roles of the mPer1 and mPer2 genes in the Vakonakis I, Sun J, Wu T, Holzenburg A, Golden SS, and mammalian circadian clock. Cell 105:683-694. LiWangAC (2004) NMR structure of the KaiC-interacting Zheng BH, Larkin DW, Albrecht L, Sun ZS, Sage M, Eichele C-terminal domain of KaiA, a circadian clock protein: G, Lee CC, and Bradley A(1999) The mPer2 gene encodes Implications for KaiA-KaiC interaction. Proc Natl Acad a functional component of the mammalian circadian Sci U S A 101:1479-1484. clock. Nature 400:169-173.