80 *Author ([email protected]) for correspondence Texas, North Sciences, Denton,TX76201,USA. University of Developmental Integrative Biological Biology Research Cluster, of Department in 1957(Nanney, 1957)andRiggsHolidayinthemid-seventies beginning withWaddington in1942(Waddington, 1942)toNanney 2014). Historically, theoriginaldescriptionsofepigenetics, underappreciated andpoorlyunderstood(Burggren andCrews, epigenetic effects incomparative biologyisstillbothgreatly identification oftheunderlyingmolecularmechanisms,scope of al., 1998;JablonkaandRaz,2009).Despitecontinuingadvances in Jablonka andLamb,1989;2002; et Chahwan etal.,2011; Hauseretal.,2011; HoandBurggren, 2010; effects (forreviewsseeBurggren, 2014;CantoneandFisher, 2013; decades haveseenaveritableexplosionofinterestinepigenetic Epigenetic phenomenahavelongbeenappreciated,butthelast few KEY WORDS:Acclimation,DNA methylation,Epigenetics epigenetic phenomena. course willrevealmuchaboutboththeimpactandmechanismsof even likely. Focusedinvestigationsthatconcentrateonthetime review representingepigeneticdynamicsarepossibleandsome sensitive, precisequantitativemethods.Allofthescenariosinthis persistence ofthephenomenonarebestconductedwithhighly effects (andintragenerationaleffects, forthatmatter) thatsearchfor wash outorin.Thus,studiesoftransgenerationalepigenetic the phenotypicmodificationofinterest,especiallywhenphenotypes dynamics isalsohighlydependentonthethresholdfordetectionof again acrossmultiplegenerations.Recognitionofepigenetic pressures ofenvironmentalstressorsthatwax,waneandthenwax and theremaybeunexploredadditiveeffects resultingfromthe an epigeneticeffect mightalso‘wash-in’ overmultiplegenerations, modified phenotypebothwithinandacrossgenerations.Conceivably, time-related changesthatcanpotentiallyinvolvethe‘washout’ of dynamic epigeneticphenomenamaybebetterdepictedbygraded, suddenly disappearsbetweenonegenerationandthenext.Rather, be regardedas‘digital’ (on–off), inwhichamodifiedtraitnecessarily dynamic perspectivesuggeststhatepigeneticphenomenashouldnot modifications changeinnon-linearpatternsovertime.Importantly, a transgenerational andintragenerationalepigeneticphenotypic in comparativebiology. Epigeneticdynamicsdescribeshowboth away whatmightbecalledthe‘dynamics’ of epigeneticsanditsrole prevails inmanyepigeneticstudiestodatehasdivertedattention decades. However, thestrongreductionistfocusonmechanismthat epigenetic phenotypicmodificationshaveproliferatedinthelastfew Epigenetic studiesofbothintragenerationalandtransgenerational Warren W. Burggren* intragenerational phenotype‘washout’ Dynamics ofepigeneticphenomena:intergenerationaland REVIEW © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,80-87doi:10.1242/jeb.107318 epigenetic studies contemporary thefocusIntroduction: andutilityof ABSTRACT unidentified variation incomparativebiological studies,withsuch phenomena mayadditionallybe asourceofconsiderablepreviously (Burggren andCrews,2014;Cropleyetal.,2012). provided forby‘conventional’ mechanisms ofevolutionselection responding toenvironmentalchange inaframeworkshorterthanis generations –canbeviewedas highlyresponsivemechanismsfor epigenetic phenotypicmodifications–eitherwithinoracross the environmentalstressorretreatsordisappears.Inthisrespect, environmental perturbation,butcanthenbe‘sunsetted’ whenandif phenotypic modificationscanbeinducedbyasignificant mutation ormoreslowlythroughnaturalselection,epigenetic phenotypic modificationsthatcanresultsuddenlyfrom gene environments subjecttoclimatechange.Unlikepermanent changing environmentalconditions,suchasthevariablenature of evolutionary studiesfocusingonmechanismsofsurvival in Epigenetics canalsobeofgreatrelevancetoecological and on themechanismsandimpactsoftransgenerationalinheritance. 2014). context-dependent epigeneticinheritance(Burggren andCrews, parental effects toincludepaternaleffects), asanexample of includes so-calledmaternaleffects (or, perhaps,moreexpansively, (i.e.theenvironmentalstressor).Thisframework causative agent consequently persistacrossgenerationsintheabsenceoforiginal organism isdirectlyaffected, andphenotypicmodifications ‘germline-dependent’ inheritanceresultswhenthegermlineofan present, thephenotyperemainsmodified.Bycontrast,so-called generations toanenvironmentalstressor. Aslongasthestressoris results fromdirectandcontinuingexposurewithinoracross ‘context-dependent’ epigeneticinheritancethataffects phenotype categories (forareview, seeBurggren andCrews,2014).So-called which itisinvestigated,epigeneticeffects canbeclassifiedintwo individual’s lifetime–e.g.‘theepigenetics ofcancer’. intragenerational phenomenonthatcreatespathologiesduringan common practiceinmedicinetoreferepigeneticsasan epigenetics hasbeenrenderedmorecomplexbytheincreasingly 2010). Noteworthyisthat,inrecentyears,terminologysurrounding Pishva etal.,2014;RuttenandMill,2009;SvrakicCloninger, psychological–behavioral field(Crews,2008;Peñaetal.,2014; Kuzawa andThayer, 2011; Varriale, 2014);andinthe Burggren andCrews,2014;Cropleyetal.,2012;Horowitz, or evolutionaryvantageratherthanasapathology(Burggren, 2014; 2013); inbiology, whereepigenetics isofinterestfromanadaptive Mazzio andSoliman,2014;MillHeijmans,2013;Oginoetal., epigenetics isviewedinapathologicallight(JonesandSung,2014; transfer. Anextensiveliteraturenowexistsinmedicine,where – thesephenotypicchangesinvolvedtransgenerationalphenotype without change(s)inthegenesequence.Sometimes–butnotalways (see Holliday, 2005),werein thecontextofphenotypealteration Epigenetics isalsoofgreat relevancebecauseepigenetic Epigenetics isofinteresttomorethancliniciansorthosefocused Irrespective ofhowepigeneticsisviewedorthefieldstudyin

The Journal of Experimental Biology generation, asoften assumed.Howsuchwashout phenomenamight in theF with themoretypicalviewofan epigeneticeffect thatisfully present generations, ultimatelyfallingbelow detectablelevels.Thiscontrasts 1 Fig. fashion and,ifso,howwould this bemanifested?Theleftsideof generations inmoreofagraded oranalogratherthanon–off digital Can epigeneticphenotypicmodificationsfadeor‘washout’ across transgenerational effects. epigenetic phenomena,aswewillnowexplore,beginning with may misinterpretormissaltogetherimportantimplications of effect. Yet, withoutunderstandingthedynamicsofepigenetics,we considered asan‘analog’ viewofatransgenerationalepigenetic graded insomeformwithinoracrossgenerations–whatmight be Less frequentlyhavestudiesconsideredwhetherthephenomenon is extent ordegree(hencethe‘digital’ descriptionofthisapproach). see whetheranepigeneticphenomenonispresentorabsent–not its generation. Putdifferently, investigatorshaveprimarilylookedto or moreoffspring generations,thenabruptly‘off’ inasubsequent modifications as‘digital’ –theyaretypicallyviewedas‘on’ forone typically treatedtransgenerationalepigeneticphenotypic when thetimecourseofepigeneticsisconsidered,investigatorshave ‘dynamics’ of epigenetics–havenotyetcometothefore.Indeed, ‘why’ andespecially the‘when’ –whatmightbereferredtoasthe ‘what’ and‘how’ ofepigeneticsarebeingintenselyinvestigated,the epigenetic effects achieved–i.e.whatisthemechanism?While phenomena producedbyepigeneticeffects? (2)howarethese epigenetic studies:(1)whatarethetransgenerationalphenotypic To date,therehavebeentwogeneralfocifortransgenerational changes, suchasalteredhypoxiaresistanceorbodymass. comparative physiology, thatconsiderscomplexphenotypic focusing onamoreorganismal levelperspective,befittingof the presenceorabsenceofasingleexpressedprotein,Iwillbe Thus, ratherthanrefertoamodifiedphenotypeas,atminimum, to variabledegreesbyDNA methylation, histonemodification,etc. major suitesofgeneswhosecollectedexpressionmaybesuppressed probably gobeyondevenpleiotropicgeneexpressiontoinclude biology, Iwillbeemphasizing‘complex’ phenotypic changesthat morphological, physiologicalorbehaviorallevels. absolutely noeffect ontheoverallphenotypeatcellular, environment (Noble,2015),theabsenceofthistranscriptmayhave ‘buffering’ oftheknockouteffect bythecellularandexternal biologist ashavingamodifiedphenotype.Yet, followingthe to expressthecorrespondingtranscriptisviewedbyamolecular Thus,ananimalwithasinglegeneknockoutthatfails the beholder’. epigenetic dynamics.Inmanyrespects,phenotypeis‘intheeyeof but itiscriticaltounderstandingthisperspectiveonpotential It mayseemcurioustohavediscussthemeaningof‘phenotype’, (Burggren andCrews,2014). to eithercontext-dependentorgermline-dependentexposure could resultfromepigenetictransgenerationaleffects thattrackback yet undeterminedbutpotentiallylarge componentofthisvariation regarded asinherent(andthusunavoidable)‘noise’.However, anas variation previouslyattributedto‘experimentalerror’ orsimply REVIEW Analog versusdigitalviewsoftransgenerationalepigenetics What is‘phenotype’ inacomparativebiologicalcontext? Epigenetic dynamics:aconceptualframework Transgenerational ‘washout’ In thefollowingdiscussionofepigeneticdynamicsincomparative shows ahypotheticalepigenetic effect thatslowlyfadesacross N generation beforethensuddenly disappearingintheF N +1 2014). Importantly, theseeffects graduallydeclinefromthe F vinclozolin) administeredduringgestation(Paoloni-Giacobino, generations arecausedbytwoendocrinedisruptors(methoxychlor, Decreased spermcountsthroughalteredmethylationpatternsacross entirely eliminated,suggestiveoftransgenerationalwashout. phenotypic traitsfadeovermultiplegenerationsorwithintheF (right) ‘washout’ ofepigeneticeffects. Fig. the overalltransgenerational phenotypicmodification isactually generations couldconceivably produce ‘additiveeffects’, in which environmental stressorintermittently presentduringalternating 3A illustrateshowan stressor levelsovermultiplegenerations. Fig. is changinginacomplexmanner withmultiplemodificationsto extent epigeneticphenomenacan beadditiveinanenvironmentthat blown effects totheircomplete absenceacrossasinglegeneration. generation, ratherthanshowinganabrupttransitionfromthe full- generation, andthensuddenlydisappearingintheF not simplyabsentintheF reveal themselvesisshownin broods. are reducedintheF abnormality, primordialfolliclelossandmaletumordevelopment Importantly, phenotypicmodificationincludingmalepubertal multiple phenotypicmodifications(Manikkametal.,2012). D) duringtheF stay constantforsometime(scenarioC)orslowlyincrease Fig. phenotypic traitcouldsimplywashout(scenarioA),asdescribedin of whichareoutlinedinFig. transgenerational phenotypetransfer, multiplescenariosemerge, some dynamic frameworkandhowsuchaperspectivemightaffect Once onebeginstorecognizetheimplicationsofanepigenetic could progressively‘washout’ oversubsequentgenerations(Fig. Rather, acontext-dependentorgermline-dependentepigeneticeffect F several examples,afewofwhichwillberecountedhere.Whenthe 0 Another poorlyexploredaspect ofepigeneticdynamicsistowhat Evidence fortransgenerationalwashoutislimited,butthereare Transgenerational is exposedtodioxininrats,theF

epigenetic effect .Hypothesizedtransgenerational(left) andintragenerational 1. 2A; alternatively, sucheffects couldslowlydecline(scenarioB), washout of The JournalofExperimentalBiology(2015)doi:10.1242/jeb.107318 1 generation. 3 compared withtheF F F F F F 0 4 3 2 1 0 , continuingunabatedthroughtheF i.2A. Fig.

2B: themagnitudeofamodified In thishypotheticalscheme, Brood/litter 2 Brood/litter 3 Brood/litter 1 1 to F In thisscenario,theeffect is 3 1 Intragenerational epigenetic effect generations experience generation, butarenot washout of N +1 1 over multiple generation. 1

to F 2A). 0+N 81 3

The Journal of Experimental Biology environmental hypoxia. portion ofitsphysiologicalrepertoire incasethereisareturnof parental exposuretohypoxia mightbenefitfromretainingthat phenotype thatincludesincreased hypoxiatolerancecreatedby Scenario B,themodifiedphenotypeslowlydeclinesinF variations ofthescenariopresentedinA (reproducedhereasScenarioA).In transgenerational phenotypetransferandsubsequentwashout,basedon across generations.(B)Additionalscenariosforpossibledynamicpatternsof begins towashoutordecayovertime,progressivelydecliningthroughand 82 REVIEW environmental stressor. Thus,forexample,anF represents aprotectionagainst thepossiblereturnof return topre-stressorphenotype) couldrepresenta‘bethedging’ that immediate sunsetting(i.e.notawashout,butratheranabrupt full conditions return(Burggren, 2014;Cropleyetal.,2012).However, environmental stressordisappearsandmorefavorable modification tothegenotype,canberapidly‘sunsetted’ ifthe Epigenetic effects, unlike phenotypic changesresultingfrom phenotype. because oftheelevatedbaselineexpressionmodified effect isthesame,butcompounded (overall)effect isincreased phenotype. Inthisscenario,themagnitudeofstressor-induced amplified bythelingeringpriorepigeneticmodification of parental exposuretoastressorappearsintheF the transgenerationalepigeneticmodificationofphenotypecausedby Fig. modification isstaticthroughouttheF begins towashoutinsubsequentgenerations.InScenarioC,thephenotypic be expressed. are butafewofthemanypossiblewaysinwhichepigeneticdynamicscan during thegrowthofF out. Finally, inScenarioD,thephenotypicmodificationactuallyincreases Is washoutofaphenotypictraitadaptiveormaladaptive? Generations Generations

phenotypic of modified .Examplesofepigeneticphenotype washout. 2. phenotypic Magnitude of modified Magnitude Stressor Stressor trait trait + + 0 0 A B F F Epigenetic 0 Epigenetic (Original parental) 0 (Original parental) →→→→ effect →→→→→ effect 1 (F generation, thenwashesout.Thesescenarios F (F F 1 0 1 0 generation only) generation only) A B C D 1 F F generation thensubsequentlywashes 2 2 Time Washout 1 F generation, butimmediately 3 F 3 (A) Inthisscenario 1 generation witha F 1 4 F generation then → 4 greater intheF the changesinsurvival,fecundity andhemocytenumberswere phenotypic modification, thoughwhether it wasprogressive, disappearing intheF phenyloxidase activity)not just intheF time, survival,fecundity, weight,hemocytenumbersand (TBT) causesnumerousphenotypicadjustments(e.g.development insect sparse, butdoesexist.Forexample,intheaquaticlarvaeof the generations. modified phenotypictraitthatwaspresentfromtheF dynamics indicatedinFig. epigenetically modifiedphenotypictrait,thecomplexepigenetic zenith. Ifonewassimplylookingforthepresenceorabsence of an generations, andthenactuallybegintowashoutafterreaching its modified phenotypictraitcouldincreaseacrossacouple of Fig. ‘wash in’ – that is,slowlybuildupoversuccessivegenerations. If epigenetictraitscanwashout,itisalsofeasiblethattheycould peak. successive generationsandthenbegintowashoutonceithasreachedits generation. (B)Anepigeneticallymodifiedphenotypictraitcouldbuildover Fig. et al.,2012).Interestingly, atthehighestF exposure toTBT, butallthewaythrough F Transgenerational ‘wash-in’ ‘overshoot’) whenastressorisexperiencedsecondtimeintheF epigenetic effect resultsinanevengreaterepigeneticmodification(an scenario portrayinganadditiveeffect, aslowlydecliningbutstilllingering Evidence fortransgenerationalwash-in,likethatwashout, is Generations

phenotypic of modified 3. Additionaltransgenerationalepigeneticdynamics. Magnitude Generations 3B showshowsuchaphenomenoncouldmanifestitself.A Stressor phenotypic of modified Magnitude Chironomus riparius,exposuretothepollutanttributylitin trait Stressor trait The JournalofExperimentalBiology(2015)doi:10.1242/jeb.107318 + 0 A + 0 F B Epigenetic 0 F (Original parental) 5 Epigenetic →→→→→ 0 (Original parental) effect →→→→ effect generation thanintheF (F 6 0 F and F (F generation. Thissuggestsawash-in of F 1 0 1 Wash-in

generations only) 3B wouldsimplybeinterpretedasa 2 generations) F F 2 2 Time 0 Washout exposure doseemployed, F F epigenetic 3 3 1 1 effect Peak Retained 5 following parental Washout generation, before Overshoot effect generation (Lilley F F 4 (A) Inthis 4 → 2 1 to F 4

The Journal of Experimental Biology magnitude ofknown causativeagentsforintragenerational epigenetic (Fig. generation acrosssuccessivebroods producedbytheF phenotypic effects couldfadeor‘washout’ withinasingleoffspring Little attentionhasbeenpaid towhetherinducedepigenetic in nodetectionofthephenotypicmodificationF effect isapparent.Evenworse, aless-sensitivethresholdcouldresult in anunderestimationofthenumbergenerationswhich an detection thresholdforamodifiedphenotypemaysimilarlyresults apparent inFig. phenotype isalsorelevanttothewash-inphenomenon, as is may benothresholdthatistoosensitivetorelevant. is tryingtodeterminemechanismandquantifyitspersistence, there they arefartoosmalltohaveanybiologicaleffect. However, ifone reached atwhichtracesofthemodifiedphenotypearepresent,but modification, thenonecouldargue thatapoint(ageneration)is assess thebiologicaleffects oftransgenerationalphenotype depends inpartuponthegoalsofexperiment.Ifgoalisto phenotype acrossgenerationsuntilithasfullywashedout?This threshold (browndashedlineinFig.4A). phenotype whencomparedwithevenaslightlymoresensitive might nonethelesscreateasignificantunderestimationofmodified (blue dashedlineinFig. phenotype slowlywashesout.Conventionaldetectionthresholds experimenter’s thresholdforphenotypicdetectionasthemodified epigenetic effect thatwouldbemadedependinguponthe 4A showsthedifferent interpretations ofatransgenerational Fig. or decay, ratherthansuddenlydisappearwithanewgeneration. have ratherlarge implicationswhenmodifiedphenotypeswashout as a‘threshold’ ornecessaryresolutionforphenotypedetectioncan to measure.Unfortunately, failuretorealizewhatmightberegarded observations totransgenerationalphenotypiceffects thataredifficult physiological studies(Burggren, 2014a),withoutextendingour uncontrolled sourcesofvariationin,forexample,comparative phenomena andmechanisms.Thereareenoughpotentially not? Manyofthesestudiesareexploringnewterritoryinepigenetic behaviors, anatomicalstructuresandmolecularmarkers.Andwhy straightforward andeasilymeasuredtraits,e.g.stereotypic Many studiesofepigeneticphenomenainvolverelatively F abnormality andpolycysticovarydiseasewerebothgreaterinthe exposure todioxininrats(Manikkametal.,2012).Femalepuberty F development andwasmoresevereacrossgenerationsasfollows, successive generations(Lietal.,2012).Obesityoccurredearlierin epigenetic inheritanceofobesityinmicefedhigh-fatdietsforthree Another exampleoftransgenerationalwash-inisfoundinthe exponential, stepwise,etc.couldnotbediscernedfromthestudy. REVIEW of thesepathologicalphenotypes. subsequent generationswhentheeffect wouldactuallyshowup! epigenetic effect atall,and toabandonanyobservationsof leading theexperimentertoerroneouslyconcludethattherewas no disease andspermphenotypicmodificationsresultingfromF final examplecomesfromtheepigeneticinheritanceofadult-onset a ‘transgenerationalaccumulationofepigeneticmodifications’.A phenotypic transfer Threshold/resolution effectsinassessmentoftransgenerational Intragenerational washout 2 2 >F Having highresolutionfordetectionofanepigeneticallymodified Is thegoaltoachieveabsolutedetectionofmodified generation thanintheF 1 1, right-handside).Certainly increases anddecreasesinthe >F 0 , suggestingaphenotypicwash-inor, astheauthorsputit, 4B. Inthisscenario,aconventional,less-sensitive

4A), evenarelativelysensitivethreshold, 1 generation, againsuggestingawash-in 0 generation, 1 offspring 0 exposure inducedareducedbodymassinthesubsequent F apparent germline-dependentepigeneticeffect, thisparentalhypoxic generation (Meyer etal.,2002). was evidentinthefirstandsecondbroodscomprisingF generation forthefirst4–6 female model forepigeneticstudies(Harrisetal.,2012).We exposedadult (Andrewartha andBurggren, 2012),whichisemerging asapromising evident inthebodymorphologyofwaterflea intragenerational washoutofepigeneticphenotypicmodifications below. Ourlaboratoryhascollectedpreliminaryevidencefor methylation withdevelopment,growthandsenescence,asdiscussed effects havebeenidentified,e.g.intragenerationalchangesinDNA text foradditionaldiscussion.) modified phenotype,sotoocanthewash-inofaphenotype.(See relatively lowsensitivitydetectionthresholdisinplaceforanepigenetically line). (B)Justasintergenerationalwashoutcanbeunder-detectedwhena particularly whenthedetectionthresholdisnothighlysensitive(bluedashed effect thatisprogressivelywashingoutcanbeconsiderablyunderestimated, the duration(measuredingenerations)ofatransgenerationalepigenetic (A) Dependinguponthethresholdfordetectionofmodifiedphenotype, Fig. 2002; MeyerandGiulio,2003) and fadeoutovertimewithintheF progressively lostduringlarval development(MeyerandDiGiulio, potentially acquiredthroughgermline-dependent epigeneticeffects are killifish ( intragenerational washoutatthe molecularlevelalsoexistsinthe whose parentshadnotbeenexposed tohypoxia(Fig. identical bodymasseswhencomparedwithcontrolF disappeared or‘washedout’ priortothethirdbrood,whichexhibited transgenerational transgenerational transgenerational transgenerational

4. Importanceofthresholdsfordetectioninepigeneticstudies. persistence persistence persistence persistence Reported Reported Actual phenotypic Actual of modified Magnitude D. magnatohypoxia(4%)for6 phenotypic of modified Magnitude Fundulus heteroclitus trait trait The JournalofExperimentalBiology(2015)doi:10.1242/jeb.107318 + 0 + 0 F B F A 1 Wash-in 1 →→ F →→ 1 Washout F toF 1 toatleastF F 3 generations 1

F toF days. Importantly, thisreductioninmass F F 2 2 threshold foridentifying Conventional detection 2 toatleastF ), wherearefractoryCYP1aphenotype modified phenotype 4 threshold foridentifying Conventional detection generations modified phenotype 4 generations Time F F 3 4 →→ 3 generations → i.5). days (Fig. Lowered detectionthreshold produces greaterestimates F of transgenerationaleffects Lowered detectionthreshold

produces greaterestimates of transgenerationaleffects 4 F 4 → Daphnia magna

2). Evidencefor 1 Through an generations 1 , buthad 83 1 1

The Journal of Experimental Biology Andrewartha (AndrewarthaandBurggren,2012). this semi-logplot. 84 REVIEW temporal issues integratedintophenotypicmodifications. Gender- To thispoint,wehaveconsidered‘epigeneticdynamics’ intermsof offspring ofthefirstandsecond–butnotthirdF parents createdasignificantlyreducedbodymass(shadedareas)inthe modifications in Fig. methylation causingepigenetic effects). mechanism isassociatedwitha particularphenomenon(e.g.DNA interesting semanticquestionofwhetherthepresence of a histone, nucleosomeposition,microRNAs,etc.Thisraises the the ‘traditional’ epigeneticmechanismsofDNA methylation, changes resultfrommechanismsthathavenotbeenassociated with 2012; Snell-Rood,West-Eberhard, 2003).Manyofthese developmental plasticity(deJongandLeyser, 2012;Kellyetal., environmental perturbations,ofcourse,aphenomenontermed maturation. Non-geneticmodificationsofphenotyperesult from trivialized. concept ofintragenerationalwash-inissimultaneouslyvalidated and modified (diseased)phenotypeis‘washingin’.Inthisrespect, the effect then,almostbydefinition,asthediseaseprogresses, through non-geneticmechanismsasanintragenerationalepigenetic intragenerational wash-in.Ifoneviewsadiseasethatdevelops consider themedicallyorientedviewofconcept Gender andepigeneticdynamics Epigenetic dynamics,gender andpopulations Phenotype normallychangesduringdevelopment,growthand Before leavingthetopicofintragenerationalepigeneticeffects,

5. Intra-generationalwashoutofmorphologicalepigenetic Development (days) 1000 1000 1000 100 100 100 10 10 10 (117,97) (78,94) (66,85) 02 * Brood 3 Brood 1 * Brood 2 Daphnia magna.Chronichypoxiaexposureofadult N values inparentheses.Graphsmodifiedfrom (99,90) (91,82) * * * (67,52) (62,77) (51,59) * * (42,54) * (126,108) (87,100) * * (84,109) Development (days) (15,15) (15,13) 8642 (15,15) (15,15) (15,15) (15,15) (15,15) (15,15) 1 (15,15) broods, asdepictedin Control to hypoxia Parents exposed (15,15) (15,15) (15,15) (15,15) (15,15) 1816141210 0 as aresultofthesegender-dependent effects. reproductive successandthusoverallfitnesscouldcomeintoplay could havequitedifferent profilesbasedongender. Again, the ‘washout’ (orpotentiallythe‘wash-in’)ofaphenotypictrait themselves ingender-dependent patterns(Dadaetal.,2012).Thus, female tissues(e.g.prostategland,mammaryglands)willmanifest epigenetic effects oneithermaleorfemalegametes outlasts thefemalesofthatsamepopulation.Obviously, specific phenotypic modificationthat,acrossgenerations(orbroods), a hypotheticalexample,malesofpopulationcouldhave other mechanismsofepigeneticmodificationphenotype.Thus,in differential resilienceofDNA methylation,histonemodificationor population beyondtheindividual(seebelow). trait suppressioncouldaltermatingsuccessandthusaffect the suppress malephenotypictraits.Intheextreme,suchgender-based CpG methylationofthepromoterregionsforthesecould expressed throughlifeandinmanytissues(Gaboryetal.,2015). (Basu andZhang,2011). Y chromosomegenesaretypicallywidely by methylation,andsuchgenesarethusmoreexpressedinfemales 2010). Significantly, someX-linked genescanescapeinactivation gender-based differences inDNA methylation(Gallou-Kabanietal., of epigeneticresponses.We know, forexample,thatthereare specific responsesmightbeconsideredanotherdynamiccomponent the dynamicsof knownepigeneticmodulators ofgeneexpression. dynamics’? To begintoanswerthisquestion,letusbriefly consider A key questionis‘whatarethepotentialmechanismsforepigenetic level. others, couldgenerateanexponential washoutatthepopulation individuals perpetuatingtheresponsethroughmoregenerations than in thepersistenceofresponsebetweenindividuals,with some phenotype disappearingcompletelywithanewgeneration,variation ‘digital’ epigeneticresponsetoastressor, withthe modified 6.Evenifallindividualsinapopulationactuallyshowed Fig. the populationlevelaswell.Considerscenarioportrayed in interest tocontemplatehowepigeneticdynamicsmightmanifest at population-level approachesinquantitativegenetics,itisalso of 2011). 2013) totheinterpretationoftwinstudies(OllikainenandCraig, in areasrangingfromepigeneticinheritanceathletes(Ehlert et al., individual outliers,althoughthesearejuststartingtobeconsidered genetic inheritance,hasnotsimilarlyembracedthesignificanceof individuals canbeaffected outsidethemechanismofconventional then, thatthefieldofepigenetics,withitsframeworkhow (e.g. Bennett,1987;Federetal.,2000;Williams, 2008).Itisironic celebrating outliersandwhattheseindividualresponsescantellus comparative biologistshaveincreasinglyrealizedtheimportanceof as representativeofconfounding(andannoying)variance, often interestingdatafromindividuals.Ratherthanviewingoutliers to expressacollectionofindividualresponsesde-emphasizesthe field ofepigenetics.Mostdirectly, thepracticeofusingmeanvalues honored statisticalapproachhassomesignificantimplicationstothe differences asmeansdrawnfromthesampledpopulation.Thistime- mechanisms inindividuals,andthenmostoftenexpressthese investigators trytoidentifyphenotypicchangesandunderlying – aretypicallyconsideredatthelevelofindividual.Thatis, Epigenetic dynamics–forthatmatter, mostepigeneticphenomena Mechanism(s) for epigeneticdynamics Individual versuspopulation-levelresponses Gender differences couldalsomanifestthemselvesthrough Paralleling theongoingdiscussionaboutindividualversus The JournalofExperimentalBiology(2015)doi:10.1242/jeb.107318

The Journal of Experimental Biology the situation,however, is that demethylationofpromoter regions of amodifiedphenotypewithin asinglegeneration.Confounding decline insuchmethylationcould underlieatime-dependentdecay DNA methylationisfarfromfixedinorganisms, aprogressive inter-organ changesinDNA methylation.However, giventhat not atallclearwhatthedrivers areforsuchintra-individualand within anorgan, suchastheplacenta(Gaboryetal.,2015). Itis DNA methylationofgenesresponsibleforspecifictissuetypes Smith andMeissner, 2013),whichleads tolarge differences in DNA aremoremethylatedthanothers(SchärandFritsch,2011; individual differences inDNA methylation,specificregions of Ooi andBestor, 2008;Wu andZhang,2010).Inadditiontointer- dictated inpartbytheenvironment(Ficz,2015;Horvath,2013; after theblastocyststageandbeyondinembryo,thought tobe ‘methylation erasure’ forDNA,followedby least inmammaliangermcellsandzygotesthereisaperiod of reported (Jaenisch,1997;Wu andZhang, 2010).Furthermore,at individual organism’s lifespanaswelldecreaseshave been increases inDNA methylationduringtheprogressionofan do notfollowuniversalpatternsorevengeneraltrends.Both 2013; Teschendorff etal.,2013).Yet, thesedevelopmentalchanges based on‘DNA methylationage’ (Grškovi tightly correlatedastoleadtheconceptofan‘epigeneticclock’ species, therelationshipbetweenCpGmethylationandageisso Meissner, 2013;vanOtterdijketal.,2013).Indeed,insome Gabory etal.,2015;Rodríguez-Rodero2010;Smithand development, maturationandaging/senescence(Ficz,2015; Within anindividual,DNA methylationchangesduringembryonic (Hunt etal.,2013;Schoofs2015;Weiner andToth, 2012). and mayevenbedifficult todetect,asinthefruitfly but inmanyinvertebratesthedegreeofmethylationfallsto<3% adulthood (Gallou-Kabanietal.,2010;SmithandMeissner, 2013), taxa. Inmammals,some60–80%oftheDNA ismethylatedin DNA methylation.DNA methylationalsovariesgreatlyamong within anorgan (e.g.placenta)showingquitedifferent levels of highly regionalandtissuespecific,withevendifferent tissues phenotype, ortheycanbeessential.Methylationpatternsarealso methylations canvariouslybesilentintermsofinfluenceon and itisnotmyintenttorevisitthisextensiveliterature.DNA papers inthisspecialissueonepigeneticscontributearea), DNA methylationhasbeenextensively reviewed(andseveral population mayshowananalogwashout. absent, ratherthananalogresponses.However, thestatisticalmeanof showing ‘digital’ responsesinwhichtheepigeneticeffect ispresentor as welltheindividuallevel. Fig. REVIEW The dynamicsofDNA methylationandotherepigeneticmodulators Generations

6. Epigeneticdynamicscanbeconsideredatthepopulationlevel phenotypic of modified Magnitude Stressor trait + 0 F 0 Individuals in parental) (Original →→→→→ population F (F 1 0 generation only) In thisscenario,individualsmayactuallybe F 2 Time washes out epigenetic effect Population-level F 3 ć etal.,2013;Horvath, de novo F 4 remethylation or absent effects present epigenetic Individual Drosophila are multiplepossibilities,twoofwhichwillnowbeexplored. ‘what arethepotentialmechanismsforepigeneticdynamics?’,there phenotypic traitmodifications.Returningtotheoriginalquestionof concert –ratherthanindividualmechanismsthatunderlie it is,wearelikelytofindthatissuitesofmechanismsactingin nucleosome repositioningormicroRNAs.Therealworldbeingwhat investigators focusonDNA methylation,histonemedication, considering thesemechanisms,weshouldrecognizethatmost modification lastingatleast10generations.Moreover, in 10–15% overthecourseoffourgenerationsaphenotypic data suggeststhattheextentoftransgenesilencingwasreducedby transgene silencingofGFP. However, acarefulexamination of their generational study, thefocus wasonthepresenceorabsenceof generations (Asheetal.,2012).Interestingly, inthismulti- workers notedstressor-induced changesinpiRNAsacross to beimplicatedwithadditionalstudies.Forexample,Asheandco- about thelife-cycledynamicsofthesemechanisms,buttheyaresure and Klose,2014;ZhangPradhan,2014).Farlessisknown positioning, non-codingRNAs,etc.(HughesandRando,2014;Rose molecular mechanisms,includinghistonemodification,nucleosome suppressed genes(Ficz,2015). does notautomaticallyleadtore-expressionofpreviously normal timing of thethirdbrood,anddetermining whetherthe Daphnia, bydelayingtheproductionof thefirstbrooduntil tested inananimalproducing multiple broodsovertime,suchas decline inDNA methylation(orothermechanism)canbereadily Gabory etal.,2015).Thishypothesis ofasimpletime-dependent (Alvarado etal.,2014a;Alvarado etal.,2014b;Bollati2009; squirrels, cichlidfishesandmany otherorganisms including humans DNA methylationhavebeenquantifiedinants,mice,ground each brood.Certainly, progressiveintragenerationalchangesin morphological modificationsasdevelopmentaltimeprogresses for the epigeneticeffect. This decaymayalsoexplainlossof gametes, andsopassageoftimeresultsintheobservedwashout of epigenetic mechanisms)simplymaynotpersistacrosstime in first orsecondbroods.Perhaps,then,DNA methylation(orother produced, butobviouslywasproducedseveralweekslaterthan the effect thatreducedbodymass, wasnotonlythethirdofthreebroods (Fig. et al.,2012).Consideragainourexperimentswith loss ofhistonemodificationovertimehasbeensuggested(Przybilla diminishment ofthecausalmechanismovertime.‘Spontaneous’ That is,justlikearadio-isotopedecaysovertime,theremaybe could involveatime-related‘passive’ decayofthecausalagent. modified phenotype. either disappearedorfallenbelowathresholdfordetectionofthe decline intheepigeneticallymodifiedphenotype,untileffect had epigenetic molecularagentcouldbethecausalbehinda 2013). Thus,patternsofdeclineinDNA methylationorother 2014a; Alvaradoetal.,2014b;Golbabapour2011; Ohnoetal., cycle andsubsequentinheritanceofaphenotype(Alvaradoetal., maintained, cleared(erased)andthenre-establishedduringthelife subsequent generation,DNA methylation patternsarecreated, extent ofwhichcanoccuracrossacontinuum.Infact,witheach are variableacrossgenerations,sotooisDNA methylation,the positioning, forexample.Justasepigeneticallymodifiedphenotypes DNA methylation,histonemodification, and/ornucleosome Across generations,therecouldbeanactive‘washout’ ordeclineof ‘Active’ and‘passive’ washoutofepigeneticmodulators Epigenetic effects are,ofcourse,alsomediatedbyother An alternativeoradditionalexplanationforepigeneticwashout 5). Thethirdbrood,whichhasrecoveredfromtheepigenetic The JournalofExperimentalBiology(2015)doi:10.1242/jeb.107318 Daphnia magna 85

The Journal of Experimental Biology molecular markersinthekillifish 86 REVIEW Biology throughitsannualworkshopseriesandtheUniversityofNorthTexas. Science Foundation[grantnumberIOS-1025823],theJournalofExperimental The authorisverygratefulforfinancialsupportthisstudyfromtheNational The authordeclaresnocompetingfinancialinterests. preparation ofthemanuscript. Dr BenjaminDubanskyandMariaRojasprovidedusefulcommentaryduring transgenerational epigeneticeffect laststhrough underlying mechanisms.Theaccuracyofassertionsthataspecific possible formsofepigeneticdynamicsandthenidentifyingthe studies, then,shouldbedirectedtowardsvalidatingthevarious even likely–arenotyetsupportedbyexperimentaldata.Future epigenetic dynamicspresentedinthispaper–whilepossible,and (respectively) epigeneticeffects. Yet, manyofthescenariosfor examples provideevidenceforgradedtrans-andintragenerational additive effects intheindividual,bothwithinandacrossgenerations. graded seriesofchangesthatcaninvolve‘washout’,‘wash-in’ and Rather, amorenuancedviewframesepigeneticdynamicsas epigenetic dynamicsisnotasuiteofdigital(on-off) phenomena. potentially acrossmanygenerations.Centraltothisviewisthat phenotypic modificationschangeinnon-linearfashionsovertime– to whichtransgenerationalandintergenerational epigenetic epigenetics (the‘when’).Epigeneticdynamicsdescribestheextent attention awayfromwhatmightbecalledthe‘dynamics’ of epigenetic studiestodate(the‘what’ andthe‘how’)hasdiverted The reductionistfocusonmechanismthatprevailsinmanyofthe time. phenotypic modificationispresent,orhasnotstoodthepassageof nrwrh,S .adBrge,W. W. Burggren, and S.J. Andrewartha, laao .S . aaua,R,Soe,K . buef .adFrad R. Fernald, and E. Abouheif, K.B., Storey, R., Rajakumar, S.M., Alvarado, M. Szyf, and K. Storey, R., Fernald, S., Alvarado, Conclusions directions andfuture References Funding Competing interests Acknowledgements occurs withthepassageofchronologicaltime? generations oracrossbroods,isitapassiveprocessthat‘simply’ DNA methylationisprogressively diminishedbydilutionacross Is washout,forexample,anactiveprocessinwhich, which epigeneticdynamicssuchasphenotypewashoutmightoccur. community. selection hasyettobeexploitedbythebroadepigenetic determining everythingfrommechanismtofitnessnatural examining outliers,thisincreasinglytime-honoredpracticefor comparative biologistshaveforyearsrecognizedthepowerof reported asaveragesfortheexperimentalpopulation.Although Almost allepigeneticeffects aremeasuredontheindividual,but best conductedwithhighlysensitivepreciseanalyticalmethods. for thatmatter)searchpersistenceofthephenomenonare transgenerational epigeneticeffects (andintragenerationaleffects, of thephenotypicmodificationinterest.Thus,studies then disappearsishighlydependentonthethresholdfordetection Physiol. 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