Circadian rhythms: molecular basis of the clock Lisa D WiIsbacher* and Joseph S Takahashit
Much progress has been made during the past year in the Iocalizition of PICK :uid of ‘I’lhl is hlockcd in fi/uol kind molecular dissection of the circadian clock. Recently identified /w/01 flies. rcspcctivelY [X.9]. PER physically interacts circadian genes in mouse, Drosophila, and cyanobacteria \vith ‘I’lhI \.i;i its i)ZS domain [l] both i/l .c,ifm [lZ\ and in demonstrate the universal nature of negative feedback cell cultlirc 11.31. Finally, light dcgradcs the ‘I’IiLl protein, regulation as a circadian mechanism; furthermore, the mouse which provides ;I mcchanicm for photic entrainment of and Drosophila genes are structurally and functionally the PI-R-‘I’IRI molecular cycle [X-lO,ll]. Integration of conserved. In addition, the discovery of brain-independent these 0l)scrwtions results in the follok\Ang model clocks promises to revolutionize the study of circadian biology. (b‘igurc 1 ): the pr and /i/r/ gciics ;irc transcribed diiring the subjccti\c day (peak kit circadian time [(:‘I‘] 12-14)
Addresses (Figure la): PER and ‘I’Ihl wcumulatc slowly- until a *Department of Neurobiology and Physiology, +Howard Hughes threshold Icvcl of ‘1‘11\1 is I-cached and stabilizcc PER Medical Institute, Northwestern University, 2153 N. Campus Drive, (12igurc 1 b,c). I’l Table 1 Circadian clock gene expression, function and effects I*. Gene Circadian phenotype Circadian Acute light Reference expressiont response Mammals Clock 28 hour period, arrhythmicity No n.d.: [18”,19”,21”] Per1 n.d. Yes ?mRNA [21”,22”,26’] Per2 n.d. Yes ?mRNA [23’1 [24’,25”] Per3 n.d. Yes No [25”] Bmall n.d. No n.d [28’,29”,30] Drosophila per Many alleles, including 16 hour period, Yes No [31 19 hour period, 28 hour period, arrhythmicity tim Arrhythmicity Yes Protein [31 degradation dClock (Jrk) Arrhythmicity; low per, tim expression Yes n.d. [36”,38”] (light-dark cycle) cycle (dbmall) Arrhythmicity; low per, tim expression n.d. n.d. [37”,38”1 double-time 18 hour period, 27 hour period, arrhythmicity No No [40”,41”1 Neurospora frq Many alleles, including short period, Yes ?mRNA long period, arrhythmicity white collar- 7 Arrhythmicity, low frq expression n.d. n.d.9 white collar-2 Arrhythmicity, low frq expression n.d. n.d.* Synechococcus kaiA BC Many alleles, including short period, Yes n.d l631 long period, arrhythmicity *‘Genes’ indicates known DNA and protein sequence. Eircadian rhythm of mRNA and/or protein expression. :n.d., not determined. BMutations originally identified as a blue light blind phenotype. f’eedback loop and co~dd be inhibited by a mammalian ver- [21”,22”.2~~,2-r’,2s l‘,26*]. Intcrcstingly. ;I light pulse sion of PER via its PAS domain. Furthermore, the GM administered during the subjective night results in rapid. mutation, an A+T transversion in the splice donor site of transient induction of mPPr2 expression (maximal induc- exon 19, results in a 51 amino acid deletion within the pro- tion within one hour) and delayed, transient inducrion of posed activation domain and is consistent with the rnl’er,’ (maximal induction within 1.5 to 2 hours) antimorphic nature of the (:lo& mutant phenotype [19”.20]. [23’,24*.2.5**,26*]. In contrast, mPd dots not acutcl? respond to a light pulse [2.5*‘]. Following the cloning of C/o&, the identification of per orthologs in human and in mouse by several independent Transcriptional activation by bHLI1 factors such as laboratories underscored the possibility of a D/aq&i/u-like CI,OCK require dimerization and binding to a IIN, pro- feedback loop in mammals. These genes, prefixed human motcr element called the E box (consensus (h) or mouse (m) Prr-l [21”,22”], Prr-2 [23’,24’], and f’f& .S’-CANNTG3’) [27]. The CLOCK dimerization partner. [25**], all encode proteins that contain a PAS domain. B?Jw/? (for brain and muscle ARX’Flike factor), \vas idcn- Circadian expression of mPr;rl, mPu-2, and mPuE? in the tified using a two-hybrid screen [28’,29”]: this gene of suprachiasmatic nuclei (SCN), the site of the mammalian previously unknown function [30] also encodes a b1 ILH- clock. suggests that these genes could be circadian clock PAS protein and is expressed in the SCN [29’*]. components [21”,22”.2~‘,2~‘,25”]. Confirmation of these Interestingly, an R box present in a 69 bp enhancer of the genes as circadian components requires functional evi- DT-O.SO@~U~PJ-promoter is required for 13~7mRNA cycling, dcnce: either ;I mutation within each gene or altered which suggests a role for bHLH transcription factors in the expression of each gene must result in an altered circadian circadian mechanism [31”]. ITsing a proximal fragment of phenotype in mice. In the SCN, mPer/ expression peaks at the mPerl promoter which contains three E boxes, Cl 6 whereas mPu-2 peaks at CT 9: mPer3 expression Gekakis et (11. [29”] find that CI,OCK and B~IXLl hct- reaches its maximum at CT 6 and remains at that level erodimers bind the mPe/-I promoter and activ;ltc until after Cl’ 9. In tissues throughout the body, such as transcription. Furthermore, CLOCK-HhlAI> heterodimers retina and skeletal muscle, expression of all mZ’Pr genes is can activate transcription from the three E boxcs alone. delayed -6 hours relative to their phase in the SCN whereas mutation of the E boxes abolishes DNA binding Circadian rhythms: molecular basis of the clock Wilsbacher and Takahashi 597 Figure 1 :LOCK EIMALI / Current molecular model of rhythm generation in Drosophila. The succession of events (a-f) occur over the course of -24 hours. (a) CLOCK-BMAL heterodimers bind the per and tim promoters and activate mRNA expression from each locus; peak expression occurs -CT 12. CLOCK-BMAL may also activate transcription other circadian-regulated genes (not shown). (b) per and tim mRNA are transported to the cytoplasm and translated into PER and TIM protein, respectively. (c) Regulation of protein levels occurs by two mechanisms: DBT protein phosphorylates and destabilizes PER, and light destroys TIM. Light during the early subjective night can phase-delay the clock. Small ‘blobs’ indicate degraded proteins. (d) PER and TIM levels slowly accumulate during the early subjective night; TIM stabilizes PER and promotes nuclear transport. Peak PER and TIM levels in the cytoplasm occur -CT 19. (e) PER and TIM dimers enter the nucleus and inhibit CLOCK-BMAL- activated transcription; peak nuclear PER/TIM levels occur -CT 21. (f) Protein turnover (combined with the lack of new PER and TIM synthesis) leads to derepression of per and tim mRNA expression; the cycle begins again (a) -CT 2. Light during the late subjective night can phase-advance the clock. and tmnscriptional activation [ZX’,W’]. Finally, the exon- the basic leucine zipper transcription factor albumin site 1%deleted mutant form of CLOCK failed to activate D-binding protein (DBP) oscillates under constant condi- transcription, a result fully consistent with the antimorphic tions in several tissues, including the SCN and the liver; Cl& mutant allele [29”]. These results identify CLOCK interestingly, the phase of the rhythm in the SCN is and BhlAL as positive elcmcnts in the transcription-trans- advanced four hours relative to that in peripheral tissues lation loop: whether mPER1 inhibits CI,OCK/BRIAL [32’]. Animals homozygous for a targeted DBP-null muta- activity awaits additional experimentation. tion are less active than wild-type littermates but still display free-running rhythms, although period is -30 minutes short- 1;urthei delineation of circadian output and input pathways er than wild-type [32’]. In addition, the G’&Jgene does not in mammals has also been achieved recently. Expression of require the DBP protein for its own expression [32’]. This 598 Differentiation and gene regulation information suggests that dt?p is an outpt,t gene rather than a (dh;4') results in pupal lethality [40°']. Remarkably, thc dht part of thc central oscillator mechanism. At the inpt,t level, gene encodes a kinase with extensivc honlol(~gy to human targeted disruption of the melatonin la (Mella) receptor casein kinase I~ [41"°]. dhlt' homozygous mutant embryos showed that this receptor mediates the melatonin-induced express high levcls of stable, unphosphorylatcd PEP, pro- inhibition of SCN neural activit% but that the phase-shifting tein independently of circadian timc whereas embryonic effects of melatonin may be mediated by the Melll, receptor t/m mRNA and protein rhythms arc abolished [40",42"1. or by an unidentified receptor [33]. In addition, a putative These findings support a model in which I)BT phospho- input gcne was identified in a cDNA subtraction screen for rylates and destabilizes PER, thereby contributing to the light-induced gene expression in the SCN; this gcne, the translational delay of PER accunlulation that is required zinc-finger transcription factor egr-3, is expressed in the ven- for rhythmicity [40",41 °'] (t:igure 1). Finally, a post-trans- tral SCN and is gated by the circadian clock [34]. Finall> the lational modification other than phosphorylation ~xithin mammalian blue-light photoreceptors cryptochrome l PER amino acids 637 and 848 appcars to regulate cyclical ((,';3,1) and cuptochrome 2 (6'0'2) are expressed in the retina PER degradation [43]. (CO, I and (,'0,2) and the SCN (CO,1 only), thereby providing a new class of photopigments as candidates for the photic At the level of circadian output, analysis of the /ad" genc entraimnent pathway in mammals [35°]. confirmed that it is under circadian clock control and thcrc- fore on the output pathway [44]. The /a/~' gcnc product The Drosophila clock bchaves like a reprcssor of cclosion, as thc lad" mutant Genetic and molccular approaches in Dros@/;i/a have lcd to allele results in early eclosion whereas additional copies of recent discoveries that maintain this species as the best wild-type/ar~" delay eclosion [45]. Despitc the absence of a understood in the circadian field. Mutagenesis screening lad' mRNA rhythm, I,ARK protein oscillates in abundance rcvealed the scmidominant mutation Jd" and the gene (peak and trough levels at CT 8 and (7I" 2(), rcspcctively) in dose-dependent mutation O,c/e to'c), both of which either thc presencc of a functional per gcnc [44]. Intcrcstingly, alter or abolish rhythms in behavior, per expression, and tim /ad" is exprcsscd both in lateral neurons, the proposcd site expression [36°',37"]. (](mcomitantly, dc/oc;~ was isolated in of the Drosop/;i/a master clock, as well as in eclosion-rcgu- a low-stringency screen with mClod', and a Drosopt;ila- lating cells in the ventral nervous system [44]. Thcsc exprcssed sequence ta R clone with homology to hl~ma/l rcsults suggest a specific output pathway for eclosion that is was identified [38"]: dc/ocx;- and dhmal were found to map controlled by the central circadian oscillator. to the .ld' and O'c loci, respectively. Darlington eta/. [38 °°] have shown that d(:LOCK (presumably with dBMAI~) The Neurospora clock binds E box sequences to activate transcription of per and (2ontinued analysis of the ~\.}'urospora./}¥ gcnc in the past year tim. ITurthcrmnre, co-exprcssion of PER and TIM inhibits has rcsulted in the identification of novel regulatory mecha- transcriptional actiwltion by dCLOCK [38°°]. "Fhesc results nisms. As in Dro.rop/;i/a, post-translational regulation plays a thus appear to closc the circadian loop: the positive ele- rolc in the :\'?uro.~7?o;~/ circadian clock: ./~¥ mRNA contains ments d(]I,O(]K and dBMAI, activate transcription of the alternative translation initiation sites, the choice of which is negative elements per and t/m, the products of which cven- mediated b\ environmental tempcrature [4~3°',47"]. At tually inhibit their own transcription via interaction ~xith moderate tcmperatures (25°C), t~vo forms arc cxprcssed, and d(]I,O(3K-dBMAI, (Figure 1). The precise molecular each can support rhythmicity; however, at high tcmpcraturcs interactions that mediate this inhibition are unknown; how- (30°C) the short form of FRQ (FRQl°(>';s% is unablc to ever, thc action of PER-TIM on d(]LOCK-dBMAL is maintain rhythmicity, whereas at low temperaturcs (18°(~) rclativcly direct, as the E box clement is necessary and suf- the full-length form cannot drive rhythnls [47"]. In addition, ficicnt for activation and inhibition of transcription [38"]. temperature-shifting experiments in iX>uro.spoJzl have demonstrated that a shift from low tcmperaturc (21 '(:) to Regulation of the Drosop/;ila circadian loop appears to high temperaturc (28°C) strongly rcscts the clock to (','1' 0. occur at both the post-transcriptional and the post-transla- whereas the oppositc shift resets the clock to ~(71" 12 I48"]. tional levcls. Using nuclear run-on experiments, So and Finalh:. FRQ protein was shown to translocate to the nucle- Rosbash [39 °] have demonstratcd that po'and tim arc tran- us and rcpress./~¥ mRNA cxpression within 4 hours of the./)¥ scribed at high levcls several hours before an RNasc mRNA peak, ~xith rccmery from this repression taking thc protection assay can detect their mRNA specics. In addi- remaindcr of the circadian day I49",501. Thus, thc details of tion, no rhythm in transcription rate was detected from a circadian regulation between Xe;;;os/~o;~'; and D;o.rophila con- promoterless per gene that weakly restores rhythms of per tinue to differ, as translational dela~ of PER and TIM in mRNA accumulation to pc~ ) mutants. "['hcsc rest, Its indi- Drosophila versus long recovery period in ,\~';;ro.~)0o;~/arc used cate that a post-transcriptional mechanism contributes to to maintain a 24-hour pcriod. the observed cycle in per and tim mRNA expression [39°]. An important post-translational rcgulatnry mechanism was The genes ~e~hite collar-I (~,'c-l) and ~e'hJt~ colla;~2 (~e'c-2), also discovered recently. The mutation double-time (dht) which encode zinc-finger proteins, appear to be in~ oiled in cither shortens (dbts) or lengthens (dbt I,) period, and a P circadian clock regulation [51"]. In particular, -~,;:/is neces- clement-induced null or strongly hypomorphic mutation sary for light-induced.l/q cxpression, whereas ~e'~:2 may bc Circadian rhythms: molecular basis of the clock Wilsbacher and Takahashi 599 required for circadian ,fiv expression [Sl’]. However, w-2 ‘I’his discovery provides dcfinitivc cvidcncc of brain-indc- and .a~-2 have not been shown to bind thefhq promoter or pendent mammalian clock cells. Furthermore, the relative directly activate j-q transcription. Sequence analysis indi- phases of r,%-2 and rPer-2 expression in cell culture match cates that FK(Z itself may be a transcription factor, as FRQ those found in the liver in civo [60**]. Finally the rPel-/ and shares moderate homology with known helix-turn-helix rl’w2 gcncs fulfill the criteria for immediate-early genes in transcription factors [.%I; but, again, no functional evidcncc that serum induction is rapid and independent of new pro- in support of this hypothesis is available. tcin synthesis [60”]. This finding is reminiscent of the immediate-early expression of cj& andJNt/-H in the SCN The cyanobacteria clock in response to light and suggests that immcdiatc-early sig- TIlc most recent circadian model system \vas established naling pathways may play a role in conveying photic for cynobactcrium S~~~&XVUUS strain XC 7932, which information to the circadian clock in the XX [61]. displays circadian rhythms in bioluminescence dcspitc a replication time of five to six hours [53’]. llsing gcnctic Comparison of mammalian per. expression data shows that complementation. a three-gene locus named kni;lHC’ was the phase of these circadian genes is advanced hctwccn shown to drive all circadian rhythms in this organism three and nine hours (depending on lighting conditions, [S-l”]. ‘I’hcsc genes share no homology \jith any known species, and laboratory) in the SCN relative to the rest of gcncs. ‘I’hc KaiC product represses acti\.ity of the cluster, the body [X*,23*,25** .32’,60”]. suggesting that the SCN and overexpression of this gcnc can rcsct the phase of the play a special role within the collection of cellular oscilla- clock. KaiA is required to drive ,&iC: expression. In cffcct, tors. Indeed, twenty-five years of physiological evidence a single gcnc cluster in cyanobacteria appears to contain has dcmonstratcd that the S(:N contains the required both ncgativc and positiw elements for a circadian nega- mammalian pacemaker - the oscillator that dribes period tivc feedback loop. These results demonstrate that and phase in other oscillating cells [62]. The method by circadian transcription-translation negative regulatory \vhich the SCX directs circadian rhythmicity throughout loops are conser\,ed among living systems but the undcrly- the body is unknown, hut two gcncrai mechanisms are ing genes differ among phyla. possible: the SCN could dri1.c rhythms in passive, non- oscillating cells, or, conversely, the S(:N could coordinate Circadian organization: central pacemakers cell-autonomous oscillators. The discovery of peripheral and peripheral oscillators oscillators strongly supports the latter model. Indeed, the Conventional wisdom has it that the circadian clock resides physiological organization of circadian rhythmicit); can be in the brain in higher animal organisms. The lateral neu- compared to the hierarchy of cardiac paccmakcrs: in the rons in Drosophih appear to be important for circadian heart. the sinoatrial (SA) node controls the period of car- regulation [42’.55], and the suprachiasmatic nuclei are the diac rate but, in the absence of the SA node, the site of the circadian clock in mammals [%I. In the past atrioventricular (A\‘) node regulates rhythm. In the year, holvever. brain-independent circadian oscillators absence of either node, indi\-idual cardiac cells are capable (cells capable of self-sustained rhythmic output) ha1.e been of rhythmic contraction. This analogy could be applied to detected in many peripheral tissues of Iho.wphi/u and with- the circadian system, where some unknow:n factor(s) place in cultured cell lines in mammals [57,58,59”,60”]. For the SCiY at the top of the circadian hierarchy to coordinate example, the hlalpighian tubules of both decapitated flies cells in the body as a precisely functioning unit. and non-decapitated control animals displayed identical circadian rhythms of PER-lacZ reporter expression and Conclusions nuclear localization [57]. Kay and colleagues have extend- The tremendous progress towards the molecular dissec- ed this observation using a real-time luciferase reporter tion of the circadian clock places the circadian field in an assay to show that the Drosoflhila body as a whole and in exciting era. The identification of new circadian gents cultured segments sustains circadian rhythms in pwdriven and the delineation of regulatory mechanisms in diverse expression [58,SY]. Furthermore, every cultured tissue model organisms have underscored the universal nature of could be entrained by light, indicating that non-neural the circadian clock yet also suggested phylogenetic differ- Drnsnphih cells are photoreceptive [SY]. ences in its assembly. Future studies will undoubtedly focus on each gene’s functional role and interactions with Significant new evidence has been found for the existence other circadian genes within the organism. Finally, the of oscillators throughout the mammalian body. As dis- discovery of brain-independent circadian clocks should cussed above, rhythms in mPerl, mPer,‘, and mPP/r? can be allow the elucidation of the molecular circadian mecha- found in many non-neural body tissues [22*~,23~,2S~*]. In nism and provide a better understanding of the cell culture. serum stimulation of rat-l fibroblasts and H3S physiological circadian hierarchy. hepatoma cells elicits expression of several genes, includ- ing rat (r) PerI, rPm2, dhp, and tef (thyroid embryonic factor). Remarkably, the expression patterns of these genes Acknowledgements then oscillate in a circadian manner in the presence of the cell cycle inhibitor cytosine P-D arabinofuranoside [60”]. 600 Differentiation and gene regulation References and recommended reading portion of the putative activation domain, which is consistent with the Papers of particular interest, published within the annual period of review, antimorphic nature of the mutation. have been highlighted as: 20. King DP, Vitaterna MH, Chang AM, Dove WF, Pinto LH, Turek FW, Takahashi JS: The mouse Clock mutation behaves as an l of special interest antimorph and maps within the wgH deletion, distal of Kit. l * of outstanding interest Genetics 1997, 146:1049-l 060. 1. Crews ST, Thomas JB, Goodman CS: The Drosophila sing/e- minded gene encodes a nuclear protein with sequence similarity 21. Tei H, Okamura H, Shigeyoshi Y, Fukuhara C, Ozawa R, Hirose M, to the per gene product. Cell 1988, 52:143-l 51. . . Sakaki Y: Circadian oscillation of a mammalian homologue of the Drosophila period gene. Nature 1997, 389:512-516. 2. Takahashi JS: Molecular neurobiology and genetics of circadian A novel PCR-based approach, which took advantage of relatively conserved rhythms in mammals. Annu Rev Neurosci 1995, l&531 -553. sequence within the functionally significant PAS domaln, is used here to identify a mammalian ortholog of the Drosophila period gene; significant 3. Rosato E, Piccin A, Kyriacou CP: Circadian rhythms: from behavior sequence homology occurs throughout the entire gene in- human,-mouse, to molecules. Bioessays 1997, 19:1075-l 082. and Drosophila. Mouse (m) Per expression cycles in a circadian manner in 4. Rosbash M: Molecular control of circadian rhythms. Curr Opin the SCN. Genet Dev 1995, 5:662-668. 22. Sun ZS, Albrecht U, Zhuchenko 0, Bailey J, Eichele G, Lee CC: 5. Hardin PE, Hail JC, Rosbash M: Feedback of the Drosophila period l * R/GUI, a putative mammalian ortholog of the Drosophilia period gene product on circadian cycling of its messenger RNA levels. gene. Cell 1997, 90:1003-l 01 1. Nature 1990, 343:536-540. This group cloned human R/GUI (now renamed hperl) in a chromosome 17 transcript mapping project. mPerl (called mRlGU/ here) expression 6. Edery I, Zwiebel LJ, Dembinska ME, Rosbash M: Temporal displays a circadian rhythm in the SCN as well as in the retina, the pars phosphorylation of the Drosophila period protein. Proc Nat/ Acad tuberalis, and the Purkinje neurons; interestingly, expression in the SCN is SC; USA 1994, 91:2260-2264. advanced 6-l 2 hours relative to the other tissues. 7. Sehgal A, Rothenfluh-Hilfiker M, Hunter-Ensor M, Chen Y, Myers MP, Young MW: Rhythmic expression of timeless: a basis for 23. Shearman LP, Zylka MJ, Weaver DR, Kolakowskl LFJ, Reppert SM: promoting circadian cycles in period gene autoregulation. Science . Two period homologs: circadian expression and photic regulation 1995, 270:808-810. in the suprachiasmatic nuclei. Neuron 1997, 19:1261-l 269. See annotation [25”]. 8. Hunter-Ensor M, Ousley A, Sehgal A: Regulation of the Drosophila protein timeless suggests a mechanim for resetting the circadian 24. Albrecht U, Sun ZS, Eichele G, Lee CC: A differential response of clock by light. Cell 1996, 84:677-685. . two putative mammalian circadian regulators, mperl and mper2, to light. Cell 1997, 91 :1055-l 064. 9. Myers MP, Wager-Smith K, Rothenfluh-Hilfiker A, Young MW: Light- See annotation [25”]. induced degradation of TIMELESS and entrainment of the Drosophila circadian clock. Science 1996, 271 :1736-l 740. 25. Zylka MJ, Shearman LP, Weaver DR, Reppert SM: Three period 10. Zeng H, Qian Z, Myers MP, Rosbash M: A light-entrainment l * homologs in mammals: differential light responses in the mechanism for the Drosophila circadian clock. Nature 1996, suprachiasmatic circadian clock and oscillating transcripts 380:129-l 35. outside of brain. Neuron 1998, 2O:l 103-I 1 10. These papers [23’,24’,25”,26’1 collectively Illustrate several new, 11. Zeng H, Hardin PE, Rosbash M: Constitutive overexpression of the fundamentally important results in mammalian circadian biology. First, at least Drosophila period protein inhibits period mRNA cycling. EM50 I three mammalian per homologs exist. Second, all three genes respond 1994, 13:3590-3598. differently to light admlnistered during the subjective night: mPerl expresston increases and decreases rapidly, much like the immediate-early gene 12. Gekakis N, Saez L, Delahaye-Brown A-M, Myers MP, Sehgal A, resoonse; mPer.2 exoression increases in a delaved fashion. then decreases Youna MW. Weitz CJ: Isolation of timeless bv PER Drotein rapidly; and mPer3 expression does not change’ln responsk to light. Finally, inter&ion’: defective interaction between t~me/e&p%ein and the phase of each mPer is advanced 3-9 hours in the SCN relative to other long-period mutant PERL. Science 1995, 270:81 5. I-81 body tissues including the retina, skeletal muscle, and testis. 13. Saez L, Young M: Regulation of nuclear entry of the Drosophila clock proteins period and timeless. Neuron 1996, 17:91 l-920. 26. Shigeyoshi Y, Taguchi K, Yamamoto S, Takekida S, Yan L, Tel H, . Moriya T, Shibata S, Loros JJ, Dunlap JC, Okamura H: Light-induced 14. Lee C, Parikh V, ltsukaichi T, Bat K, Edery I: Resetting the resetting of a mammalian circadian clock is associated with rapid Drosophila clock by photic regulation of PER and a PER-TIM induction of the mPer7 transcript. Cell 1997, 91 :1043-l 053. complex. Science 1996, 271 :1740-l 744. See annotation [25”] 15. Dunlap JC: Genetic and molecular analysis of circadian rhythms. 27. Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Annu Rev Genet 1996, 30:579-601. Blackwell TK, Turner D, Rupp R, Hollenberg S: The MyoD gene 16. Aronson BD, Johnson KA, Loros JJ, Dunlap JC: Negative feedback family: Nodal point during specification of the muscle cell lineage. defining a circadian clock: autoregulation of the clock gene Science 1991, 251:761-766 frequency. Science 1994, 263:1578-l 584. 28. Hogenesch JB, Gu Y-Z, Jain S, Bradfield CA: The basic helix-loop I 7. Crosthwaite SK, Loros JJ, Dunlap JC: Light-induced resetting of a . helix-PAS orphan MOP3 forms transcriptionally active complexes circadian clock is mediated by a rapid increase in frequency with circadian and hypoxia factors. Proc /Vat/ Acad Sci USA 1998, transcript. Cell 1995, 81 :I 003-l 012. 95:5474-5479. CLOCK is shown to be a dimerization partner of MOP3 (BMALl), and the 1 a. Antoch MP, Song E-J, Chang A-M, Vitaterna MH, Zhao Y, E-box promoter element is shown to direct reporter expression In the . . Wilsbacher LD, Sanaoram AM, King DP, Pinto LH, Takahashi JS: presence of CLOCK/MOP3 (BMALI). In addition, MOP3 forms Functional identification of the m&se circadian Clock gene by transcriptionally functional dimers with MOP4 (NPASP), HIFln, and HIF2n. transgenic BAC rescue. Cell 1997, 89:655-667. Complete rescue of the Clock mutant phenotypes is achieved using 29. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, bacterial artificial chromosome transgenes, which shows that functional . . King DP, Takahashi JS, Weitz CJ: Role of the CLOCK protein in the rescue by transgenesis is a viable method of gene cloning in mammals. mammalian circadian mechanism. Science 199!,,280:1564-1569. Another interesting observation: period length is shortened to values The authors of this paper describe the first definitive posltlve elements In the beyond wild-type in transgenic lines containing a high copy number of mammalian circadian clock mechanism: CLOCK and BMALI activate the transgene. mPer7 expression directly. The CLOCK/BMALl dimer can bind both the Drosophila per promoter E box and the mferl promoter E boxes to dnve 19. King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, expression of a reporter gene; furthermore, the mutant Clock product fails l * Antoch MP, Steeves TDL, Vitaterna MH, Kornhauser JM, Lowrey PL to transactivate the mf’erl promoter. et a/.: Positional cloning of the mouse circadian Clock gene. Cell 1997, 89:641-653. 30. lkeda M, Nomura M: cDNA cloning and tissue-specific expression Genetic and physical mapping is combined with a three-tiered molecular of a novel basic helix-loop-helix/PAS protein (BMALI) and approach to transcription unit analysis leading to the identification of the identification of alternatively spliced variants with alternative Clock gene sequence. Sequence analysis indicates that Clock encodes a translation initiation site usage. Biochem Siophys Res Commun bHLH-PAS transcription factor. The Clock mutation results in deletion of a 1997, 233:258-264. Circadian rhythms: molecular basis of the clock Wilsbacher and Takahashi 601 31, Hao H, Allen DL, Hardin PE: A circadian enhancer mediates PER phosphorylation in dbP mutants suggests that the dbt product . . dependent mRNA cycling in Drosophila melanogaster. MO/ Cell phosphorylates PER, which destabilizes PER and possibly contributes to its Biol 1997, 17:3687-3693. translational delay. The first indication that a bHLH transcription factor may be involved in the circadian mechanism came from this work. A 69 bp enhancer in the 42. Kaneko M, Helfrich-Forster C, Hall JC: Spatial and temporal Drosophila Per promoter that is necessary and sufficient for rhythmic . expression of the period and timeless genes in the developing expression of Per was isolated. This enhancer contains an E box, the DNA nervous system of Drosophila: newly identified pacemaker element that bHLH transcription factors bind. Mutation of the E box candidates and novel features of clock gene product cycling. abolished rhythmic per expression, which suggested that bHLH factors are J Neurosci 1997, 17:6745-6760. a part of the circadian loop. This elegant work demonstrates circadian expresslon of per and tim in Drosophila larvae. Expression of these genes was found In the lateral 32. Lopez-Molina L, Conquet F, Dubois-Dauphin M, Schibler U: The DBP neurons and a subset of the larval dorsal neurons; surprisingly, two larval . gene is expressed according to a circadian rhythm in the dorsal neurons express PER and TIM in antiphase lo the other neurons. suprachiasmatic nucleus and influences circadian behavior. EMf30 1997, 16:6762-677 1. 43. Dembinska ME, Stanewsky R, Hall JC, Rosbash M: Circadian cycling These authors made the first observation of a gene, dbp (albumin site D of a PERIOD-p-galactosidase fusion protein in bindmg protein), whose phase of expression is advanced In the SCN Drosophila:evidence for cyclical degradation. Jour Biol Rhyth 1997, relative to peripheral tissues. Targeted deletion of the dbp gene does not 12:157-172. abolish circadian rhythms in behavior but period length decreases by -30 minutes. 44. McNeil GP, Zhang X, Genova G, Jackson FR: A molecular rhythm mediating circadian clock output in Drosophila. Neuron 1998, 33. Liu C, Weaver DR, Jin X, Shearman LP, Pieschl RL, Gribkoff VK, 20:297-303. Reppert SM: Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock. Neuron 1997, 45. Newby LM, Jackson FR: Regulation of a specific circadian clock 19:91-102. output pathway by lark, a putative RNA-binding protein with repressor activity. J Neurobiol 1996, 31 :l 17-l 28. 34. Morris ME, Viswanathan N, Kuhlman S, Davis FC, Weitz CJ: A screen for genes induced in the suprachiasmatic nucleus by light. 46. Garceau NY, LIU Y, Lores JJ, Dunlap JC: Alternative initiation of Science 1998, 279:1544-l 547. . . translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 1997, 35. Miyamoto Y, Sancar A: Vitamin B2-based blue-light photoreceptors 89:469-476. . in the retinohypothalamic tract as the photoactive pigments for These authors provide the first demonstration of FRO. protein oscillations in setting the circadian clock in mammals. Proc Nat/ Acad Sci USA both abundance and moblllty. In addition, alternative initiation events leadlng 1998, 95:6087-6102. to two forms of FRQ are described. Expression of new photoplgments in the retina and SCN suggest the existence of an entrainment-specific photic response pathway. 47. Liu Y, Garceau NY, Loros JJ, Dunlap JC: Thermally regulated . . translational control of FRQ mediates aspects of temperature 36. Allada R, White NE, So WV, Hall JC, Rosbash M: A mutant responses in the Neorospora icircadian clock. Cell 1997, l * Drosophila homolog of mammalian Clock disrupts circadian 89:477-486. rhythms and transcription of period and timeless. Cell 1998, This paper introduced a new regulatory mechanism in Neurospora: alternative 93:791-804. translational lnltlatlon mediated by temperature. This work provides a The circadian mutation Irk severely disrupts rhythmicity in behavior, per molecular entry into the study of temperature compensation in Neurospora. expressIon and r/m expressIon In the heterozygous state and abolishes these rhythms in the homozygous state. Jrk is the Drosophila ortholog of 48. Liu Y, Merrow M, Joros JJ, Dunlap JC: How temperature changes mammalian Clock; the Jrk mutation results in truncation of a large portion of . . reset a circadian oscillator. Soence 1998, 281:825-829. the putative actlvatlon domaln. An elegant paper that details how temperature can reset a circadian oscillator by its effects on FRQ protein levels. 37. Rutlla JE, Surl V, Le M, So WV, Rosbash M, Hall JC: CYCLE is a . . second bHLH-PAS clock protein essential for circadian 49. Merrow MW, Garceau NY, Dunlap JC: Dissection of a circadian rhythmicity and transcription of Drosophila period and timeless. . oscillation into discrete domains. Proc Nat/ Acad Sci USA 1997, Cell 1998, 93:805-814. 94:3877-3882. The circadian mutation cycle lengthens behavioral period in the Using an inducible frq construct in a frq loss-of-function genetic heterozygous state and abolishes behavloral, per expression, and t/m background, these authors demonstrate that FRO protein rapidly inhibits frq expression rhythms in the homozygous state. cycle is the Drosophila transcription and that recovery from this inhibition requires the rest of the ortholog of Bmall; a nonsense mutation just downstream of the PAS-B circadian day. region corresponds well with the circadian phenotype. 50. Luo C, Lores JJ, Dunlap JC: Nuclear localization is required for 38. Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, function of the essential clock protein FRQ. EM50 1998, l 0 Steeves TDL, Wietz CJ, Takahashi JS, Kay SA: Closing the circadian 17:1228-l 235. loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 1998, 28O:i 599-l 603. 51. Crosthwaite SK, Dunlap JC, Loros JJ: Neorospora WC-I and WC-~: This group have independently identified the Drosophila orthologs of Clock . transcription, photoresponses, and the origins of circadian and bmall, then demonstrated the activities of the known circadian genes: rhythmicity. Science 1997, 276:763-769. first, dCLOCK activates expression of dper and dtim through the E box Evidence of zinc-finger proteins as positive elements in the circadian loop IS present In each gene’s promoter; and second, PER and TIM directly inhibit provided in this paper. The WC-~ gene is required for response to light, and dCLOCK to decrease their own expression. the WC-Z gene appears to be essential in circadian rhythm generation. 39. So WV, Rosbash M: Post-transcriptional regulation contributes to 52. Lewis MT, Morgan LW, Feldman JF: Analysis of frequency (frq) clock Drosophila clock gene mRNA cycling. EM60 1997, 16:7146-7155. gene homologs: evidence for a helix-turn-helix transcription ;sing the nuclear run-on assay, these authors confirm that per and tim factor. MO/ Gen Genet 1997, 253:401-414. mRNA transcription rates cycle in a circadian manner but that the amplitude 53. Kondo T, Mori T, Lebedeva NV. Aoki S. lshiura M. Golden SS: and phase of these rhythms differ unexpectedly from amplitude and phase . Circadian rhythms in rapidly diving cyanobacteria. Science 1997, in their mRNA abundance rhythms. This finding suggests that post- 275~224.227. transcriptional regulation, in addition to transcriptional activation, affects mRNA cycling of circadian genes. The unexpected presence of circadian rhythms in bioluminescence and mRNA expression In an organism with a generatlon time of five to six hours 40. Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW: suggests the existence of a universal circadian mechanism, but the details . . double-time is a new Drosophila clock gene that regulates of this mechanism in prokaryotes are expected to differ significantly from PERIOD protein accumulation. Cell 1998, 94:83-95. that in eukaryotes. See annotation [41”]. 54. lshtura M, Kutsuna S, Aoki S, lwasaki H, Andersson CR, Tanabe A, 41. Kloss B, Price JL, Saez L, Blau J. Rothenfluh A, Wesley C, Young MW: l * Golden SS, Johnson CH, Kondo T: Expression of a gene cluster . . The Drosophila clock gene double-time encodes a protein closely kaiA8C as a circadian feedback process in cyanobacteria. Science related to human casein kinase l-epsilon. Cell 1998, 94:97-l 07. 1998, 281:1519-1523. The isolation of a new clrcadtan mutant in Drosophila and the positional The cloning and characterization of the genes responsible for all circadian cloning of its gene introduce an important regulatory mechanism of the phenotypes in cyanobacteria indicate that the negative regulatory loop is circadian clock. double-time encodes a protein with high homology to indeed conserved throughout evolution; however, the nature of the gene human casein kinase IE; this homology combined with the lack of PER products differ among phyla. 602 Differentiation and gene regulation 55. Hall JC: Tripping along the trail to the molecular mechanisms of cells are photoreceptive, an observation that challenges the somewhat biological clocks. Trends Neurosci 1995, l&230-240. narrow view that only the brain can regulate rhythms In the body. 56. Ralph MR, Foster RG, Davis FC, Menaker M: Transplanted 60. Baisalobre A, Damlola F, Schlbler U: A serum shock induces suprachiasmatic nucleus determines circadian period. Soence . . circadian gene expression in mammalian tissue culture cells. Cell 1990, 247:975-978. 1996, 93:929-937. This paper demonstrates the existence of functional clrcadlan clocks in cell 57. Hege DM, Stanewsky R, Hall JC, Giebultowlcz JM: Rhythmic culture, a discovery that should allow the elucldatlon of the clrcadlan expression of a PER-reporter in the Malpighian tubules of mechanism sooner rather than later. In addttlon, the immediate-early kinetics decapitated Drosophila: evidence for a brain-independent of ctrcadian gene InductIon suggest a role of immediate-early gene circadian clock. J BKI/ Rhythms 1997, 12:300-308. expressjon In the ctrcadian pathway. 50. Plautz JD, Straume M, Stanewsky R, Jamlson CF, Brandes C, Dowse HB, Hall JC, Kay SF: Quantitative analysis of Drosophila 61. Kornhauser JM, Mayo KE, Takahashl JS: Light, immediate-early period gene transcription in living animals. J B/o/ Rhythms 1997, genes, and circadian rhythms. Behav Genef 1996, 26:221-240. 12:204-217. 62. Weaver DR: The suprachiasmatic nucleus: a 25-year retrospective. 59. Plautz JD, Kaneko M, Hall JC, Kay SA: Independent photoreceptive J B/o/ Rhythms 1998, 13:100-l 12. . . circadian clocks throughout Drosophila. Science 1997, 278:1632-l 635. 63. Kondo T, Tslnoremas NF, Golden SS, Johnson CH. Kutsuna S. Both whole bodies and cultured tissue segments of Drosophila are shown lshlura M: Circadian clock mutants of cyanobacteria. Soence to contain endogenous circadian oscillators. Remarkably, all per-expressing 1994, 266:1233-l 236.