The Genetics of Circadian Rhythms PDF
Preview The Genetics of Circadian Rhythms
Advances in Genetics, Volume 74 Serial Editors Stephen F. Goodwin University of Oxford, Oxford, UK Theodore Friedmann University of California at San Diego, School of Medicine, USA Jay C. Dunlap Dartmouth Medical School, Hanover, NH, USA AcademicPressisanimprintofElsevier 525BStreet,Suite1900,SanDiego,CA92101-4495,USA 225WymanStreet,Waltham,MA02451,USA 32JamestownRoad,London,NW17BY,UK Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands Firstedition2011 Copyright(cid:1)2011ElsevierInc.Allrightsreserved. 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ISBN:978-0-12-387690-4 ISSN:0065-2660 ForinformationonallAcademicPresspublications visitourwebsiteatelsevierdirect.com PrintedandboundinUSA 11 12 13 10 9 8 7 6 5 4 3 2 1 Contributors Numbersinparenthesesindicatethepagesonwhichtheauthors’contributionsbegin. Deborah Bell-Pedersen (55) Biology Department, 3258 TAMU, Texas A&M University,College Station, Texas, USA Stuart Brody (1, 55) Founder,Center for Chronobiology, DivisionofBiological Sciences,UniversityofCalifornia,SanDiego,LaJolla,California,USA JaynaL.Ditty(13) DepartmentofBiology,UniversityofSt.Thomas,St.Paul, Minnesota, USA Ying-Hui Fu (231) Department of Neurology, University of California, SanFrancisco, California, USA Susan S. Golden (13) Center for Chronobiology and Division of Biological Sciences,University ofCalifornia, SanDiego, California,USA Paul E. Hardin (141) Department of Biology and Center for Biological Clocks Research,Texas A&MUniversity, College Station, Texas,USA Chris R. Jones (231) Department of Neurology, University of Utah, Salt Lake City,Utah,USA Patricia L. Lakin-Thomas (55) Department of Biology, York University, TorontoOntario,Canada PhillipL.Lowrey(175) DepartmentofBiology,RiderUniversity,Lawrenceville, NewJersey,USA Shannon R. Mackey (13) Biology Department, St. Ambrose University, Davenport, Iowa, USA C. Robertson McClung (105) Department of Biological Sciences, Dartmouth College,Hanover, New Hampshire, USA LouisJ.Ptacek(231) DepartmentofNeurology,UniversityofCalifornia,San Francisco, California,USA Joseph S. Takahashi (175) Department of Neuroscience, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas,Texas, USA Luoying Zhang (231) Department of Neurology, University of California, SanFrancisco, California, USA ix 1 Introduction Stuart Brody Founder,CenterforChronobiology,DivisionofBiologicalSciences,University ofCalifornia,SanDiego,LaJolla,California,USA I. Introduction II. AnUnofficial,Abbreviated/AnnotatedVersionoftheHistory oftheGeneticsofCircadianRhythms III. CommonThemes IV. SomeReflections References I. INTRODUCTION Biological oscillations range from the rapid firing of neurons, on the order of milliseconds, to seasonal (oreven longer) oscillations that may be measured in monthsoryears.Inbetweenareoscillatorsthathaveperiodsofseveralminutes or hours (glycolysis), circatidal rhythms, daily rhythms (24h), and circalunar rhythms. This chapter and the subsequent chapters will focus just on the daily clocks,thatis,thecircadianrhythms. The generally agreed-on definition of a circadian rhythm has several parts: (1) anendogenousandself-sustainingoscillator(ESSO); (2) aperiodthatisapproximately,butnotexactly,24h(circadian); (3) capable of being shifted or entrained by environmental signals, primarily light; AdvancesinGenetics,Vol.74 0065-2660/11$35.00 Copyright2011,ElsevierInc.Allrightsreserved. DOI:10.1016/B978-0-12-387690-4.00001-5 2 Stuart Brody (4) a period that is temperature compensated, that is, when an organism, or a cell, is grown at a different temperature, the period remains roughly constant.Thispropertyisahallmarkofacircadianrhythm,asitisusually not found in other oscillators. This property is not called temperature independent, as the phase of the rhythm is still sensitive to abrupt temperaturepulses. The details about these general properties are given in the subsequent chapters. The study of circadian rhythms can be traced back prior to the rise of modern molecular biology. The application of genetic techniques to study the clockhadafairlyclearstartingpoint,asshowninSectionIIbelow.Thestudyof circadian rhythms was, to a large extent, descriptive in nature until recently. Now,ithasbecomeaveryvibrantexperimentalarea.Itmayverywellreturntoa moreholisticapproachoncethisexcitingreductionistphaseisover. The use of mutants in the “clockworks” has enabled us to sharpen the criteria for clock parameters, state variables, hands versus gears, etc. It has allowed us to distinguish major players, that is, key genes and proteins, from theancillaryonesasthisvolumepointsout.Ithasallowedusto(1)describethe circuitry of the clockworks in terms of positive and negative feedback loops; (2) attempt to provide the reasons for constructing clocks out of multiple oscillators; (3) understand the roles of input and output mechanisms, their connections to an oscillator, and the path from the oscillator; (4) understand the complexities of coupled oscillators and networks of cells containing inde- pendent oscillators; (5) understand the architecture of the clockworks, that is, howthefeedbackloopsareinterconnected;and(6)seewhatthecentralthemes andmotifsareinavarietyofclocksystems. The molecular circadian oscillator is now described in terms of many componentsinterlockedtoeachotherviapositiveandnegativefeedbackloops. Circuit diagrams for clocks in various species show many similarities, even though the key clock genes and proteins have very different sequences. It is interesting to point out that such a biological oscillator with positive and negative feedback loops was proposed many years ago (Monod and Jacob, 1961),butsomehowwasoverlookedbythe“clockcommunity.” II. AN UNOFFICIAL, ABBREVIATED/ANNOTATED VERSION OF THE HISTORY OF THE GENETICS OF CIRCADIAN RHYTHMS (1) The origins of these genetic studies may not be completely known, but a good place to start is Bu¨nning’s papers of 1932 and 1935. He analyzed different isolates of the plant, Phaseolus, based on rhythms of their leaf 1.Introduction 3 movement in constant darkness, employing QTL (quantitative trait loci) methods.Hecametotheconclusionthatthereweremanygenesthatmade smallcontributionstothefree-runningrhythm.Thischapterdiscouraged peoplefromlookingforsingle-gene“clock”mutantsformanyyears. (2) However, Konopka and Benzer (1971) isolated just such single-gene mutants in Drosophila, naming them pers (period-short), perl (period- long),andper0(arrhythmic).Theirsuccessdramaticallychangedthefield. (3) ClockmutantsinmicroorganismswerethenfoundinNeurosporacrassaby FeldmanandWaser(1971)andinChlamydomonasbyBruce(1972).The mutant approach allowed manipulative studies that could not be performedbyothermethods(FeldmanandHoyle,1973). (4) A parallel line of genetic studies was initiated by Brody and Martins (1973), summarized by Lakin-Thomas et al. (1990), employing a large number of existing Neurospora mutants. The aim of these studies was to determineifmutationsaffectingbiosyntheticpathwaysormitochondriaor developmentalprocessesalsohadclockeffects. (5) Molecular techniques then allowed cloning and sequencing of the Drosophila per gene (Bargiello and Young, 1984; Reddy et al., 1984) and theNeurosporafrqgene(McClungetal.,1989).Boththesegenescodedfor unusuallylongproteins,1200aminoacidsforper,andalmost1000forfrq. There was no sequence similarity between them, surprisingly enough, exceptforlongstretchesofGlyandSer/Threoninerepeats.Thefunction of these regions is still unclear although they may be domain “spacer” regionsorphosphorylationsites,etc.ThepresenceoftheGly-Serrepeats led to some good-natured teasing about cloning some homologue to the insect genes for silk! There was also some similarity to a mammalian proteoglycan,anextracellularcomponent. (6) Thefirstmammalianclockmutantdescribed,thetmutant,byRalphand Menaker (1988) ushered in another new chapter in clock genetics. Numerous studies employing this mutant have followed, and it encouragedpeopletolookformutantsinmice(seeChapter6). (7) More advances came when Young and his lab group employed a P-1 mediatedtransposoninsertiontoisolateanotherplayerintheclockfield, that is, mutants in the tim gene (Sehgal et al., 1994) of Drosophila. In Neurospora,thewhite-collar(wc-1andwc-2)mutants,whosephenotypes had been described previously, were found to have “clock” effects (Crosthwaite et al., 1997). In both cases, after these mutants were found, primitive circuit diagrams of feedback loops could then be constructed. (8) The mouse Clock mutant (Vitaterna et al., 1994) was the first circadian clock mutant found in mice and was found after a chemical mutagenesis screenthatlaidthegroundworkforsubsequentlarge-scalesearches. 4 Stuart Brody (9) Inplants,theisolationofmutantsinthetoc-1geneinArabidopsisbythe Kay lab (Millar et al., 1995) employing a luciferase-based screen was noteworthy for several reasons: (1) it was the first single-gene clock mutant in plants; (2) it employed a clever screen based on a new technology; and (3) it allowed for the unraveling of the circuitry of the clockinanotherspeciesforcomparativestudies. (10) Studiesonbacterialclocksweregivenasignificantimpetuswhenacluster encoding three clock genes was discovered (Ishiura et al., 1998) in cyanobacteria. These genes, kaiA, B, and C, were unlike the Drosophila orNeurosporaclockgenesandhaveledtoaseriesofinterestingdiscoveries andmodels(seeChapter2fordetails). (11) In further genetic analysis, additional mutants were found, double clock mutants were constructed, and dominant/recessive relationships were tested. An example of this for Neurospora was reported by Gardner and Feldman (1980). Most mutants were recessive or codominant, depending upon whether the organism was haploid or diploid or which part of the clocksystemthesemutantsaffected. (12) Clock gene interactions have been examined in many organisms. One of the first such studies was of the allelic frq series of N. crassa mutants and their interactions with the cel mutant and other mutants (Lakin-Thomas and Brody, 1985). The authors came to the conclusion that interactions could be classified in three ways: multiplicative in terms of periods; epistatic (one mutant negated the effects of the other); or interactive, where the resulting period suggested some type of gene product interaction. This general scheme could also be applied to inhibitor/ mutant interactions as well. Mutant interactions in Drosophila (Rothenfluhetal.,2000)followedthesamerules. (13) More clock mutants were later obtained in all of the clock systems. (see Chapters 2–5 for details). Many mutants had effects on light-resetting behavior, others thought to be in “output” processes, turned out to have some effects on period, and others were employed as critical tests of the transcription–translationloop(TTL)model. (14) Agrowingbodyofevidence,particularlyinNeurospora,pointedoutthat eveninclocknullmutants,suchasfrq10orwc-1KO,orwc-2KO,avisible rhythmcouldstillbeseen.Thisledtothepostulateofasecondoscillator, downstreamoftheFRQ/WCCoscillator(seeChapter3fordetails). (15) In a study identifying the first human clock mutant, the well-known familialadvancedsleep phasesyndrome(FASPS)wastraceddown(Toh, 2001)tomutationsinahumangenePER2,homologoustothepergeneof Drosophila andmice. This discovery hadan important effect on the field sinceitindicatedthatthecircuitryinhumansmightbesimilartothatin themodelorganisms(seeChapters5and6). 1.Introduction 5 (16) Moreclockgeneshavebeendescribedinanimalsasdetailedinathorough compilation of clock and clock-associated genes identified over the past decade(ZhangandKay,2010). Because I have been involved with the clock field since 1972 (Brody and Harris, 1973; Delmer and Brody, 1975), I have observed how the field has changedsincethen.Intheearlytomid-1970(s),thetechniquesemployedwere simple Mendelian genetics, some enzyme assays, some elementary modeling, somecytology,andlotsofdescriptivebiology.Now,onecanmakeapartiallist ofthegenetic/molecularfindingsortechniquesemployedintheclockfield:the selection for mutants, as opposed to just finding them; studies on alternative splicing;studiesthatemployantisense;genomics;modifiersandsuppressors;site- specific in vitro mutagenesis; RNAi; overexpression/gene dosage; mutants in promoter genes; dissection of regulatory regions (“clock boxes”) by deletion analysis; conditional promoters; yeast-2 hybrid “bait” methods; microarrays; QTLmethods;ChIPanalysis,proteomics,metabolomics,etc. III. COMMON THEMES A partial summary of the clock properties in many different organisms is compiled in Table 1.1. One can see many differences and similarities across the systems. But there are other themes common to many clock systems that emergefromthechaptersinthisvolumesuchas (1) thecentralityofTTL(ineukaryotes); (2) theinterlockingofpositiveandnegativefeedbackloops; (3) the hierarchal nature of these loops, ranging from a master/slave relationship to a parallel versus series relationship. The nature of the crosstalkbetweentheloopsstillremainstoelucidated; (4) feedbackfromthe“output”pathwaysbacktotheoscillator; (5) feedbackfromthecoreoscillatorbacktoinputprocesses,atypeofgating; (6) the property of temperature compensation, relatively unique to circadian oscillatorsasopposedtootherbiologicaloscillators; (7) thequalitiesofthesystemthatallowforrobustnessandforentrainment; (8) thewidespreadclockcontrolofgeneexpression,rangingfrom10%ofthe genome to almost 100%, depending upon species and sensitivity of the methodsemployed; (9) the wide variety of phase times where clock-controlled genes show their maximumexpression; (10) the incorporation of environmental sensors such as light-responsive elementsintotheclockloopsofsomeorganisms; Table1.1. APartialSummaryandComparisonsofImportantClockProperties Synechococcus Neurospora Drosophila Arabidopsis Hamsters/mice Humans Phosphorylationofclockproteins ✓ ✓ ✓ ✓ ✓ ✓ Dephosphorylationofclockproteins ✓ ✓ ✓ ✓ ✓ ✓ Nuclearlocalization – ✓ ✓ ✓ ✓ ? Clockproteinmultimerization ✓ ✓ ✓ ✓ ✓ ? Clockproteinheteromultimerization ✓ ✓ ✓ ✓ ✓ ? Lighteffects Onclockproteinlevelsorstates ViaSCN Multipleloops ? ✓ ✓ ✓ ✓ ? Positive/negativefeedbackloops ✓ ✓ ✓ ✓ ✓ ? Transcription/translationeffects ✓ ✓ ✓ ✓ ✓ ? Clockboxes(promoterregions) No ✓ ✓ ✓ ✓ ? Temperaturecompensation ✓ ✓ ✓ ✓ ✓ ? %Clock-controlledgenes(Est.) 30–100 10–20 10 30 10 Clock“fitness”experiments ✓ ✓ ✓ Citations/detailsaregivenintheindividualchapters. 1.Introduction 7 (11) thesimilarityofarchitectureoftheclockloops,eventhoughmanyofthe individualcomponentsarenotstructurallysimilar; (12) theestablishmentofthecriteriafordistinguishing“hands”versus“gears,”etc.; (13) theemployment ofgenetictechniques (forward,reverse, invitro,cloning, etc.)todissectclocksystems; (14) the emergence of some of the details behind clock-controlled chromatin remodeling; (15) the emergence of the role of “energy metabolism,” whether it be redox controlorenergychargeorotherkeyparametersinclockcontrol; (16) theroleofproteolysisinspecificregulationofclockcomponents. IV. SOME REFLECTIONS 1. Thenatureofclockmutations Mostclockmutationsappeartobehaplo-insufficientorincompletelydominant (also named as semidominant), while others are recessive, and a few are dominant. Based on classical biochemical–genetic studies, the explanation for dominanceisasfollows:Itcanbepostulatedthatifthelossof50%ofthedosage leadstoaphenotype,thentheoriginaldiploidstatewas“poised”ontheedgeofa rate-limiting step, and there was no excess capacity in that step. This would suggestthenthatmanyofthecomponentsoftheclockmechanismare“poised” in the linear range of a “rate-limiting” step with no excess capacity. Clearly, otherexplanationsfordominantmutationsarepossible,suchastheinterference ofdefectivemonomersinmultimericcomplexes. Themethodofscreeningformutantsclearlyplaysaroleindetermining the types of mutants found. In some instances, only dominant-negative or incomplete dominant mutants may be found. In other instances, lethals in essential metabolic steps would not show up in screens unless special techniquesareemployed. Oneofthesurprisingthingsaboutthegeneticsofcircadianrhythmsis the lack of a battery of temperature-sensitive (Ts) mutants, so elegantly employed for studying the yeast cell cycle, for instance. Such mutants are very useful for looking for mutations in genes that would otherwise be lethal to the cell or organism under one condition but could have a clock phenotype under anothercondition.Theyarealsomoreusefulthanstraighton/off“null”mutants, as one can vary the phenotype gradually with temperature. It should also be pointedoutthatcertainspecializedmethodssuchas“tilling,”targeting-induced locallesionsingenomes(Stemple,2004),havenotyetbeenwidelyemployed(or published)intheclockfield.
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