g epigenetic regulatory mechanisms in human disease · “genetic and epigenetic regulatory...
TRANSCRIPT
ARMENISE-HARVARDSYMPOSIUM2007
GENETICANDEPIGENETICREGULATORYMECHANISMS
INHUMANDISEASE11thAnnualSymposium
June8-11,2007,HyattRegencyNewportHotelandSpa,Newport,RhodeIsland
AbouttheSymposium
AlthoughmosteducatedpeopleviewDNAasthemoleculesolelyresponsibleforinheritance,amorecomplexandnuancedstoryunfoldedduringthe11thAnnualSymposiumoftheGiovanniArmenise-HarvardFoundation.HeldinNewport,RhodeIsland,onJune8-10,thisyear’sthemewas“GeneticandEpigeneticRegulatoryMechanismsinHumanDisease”.
Anyonetallenoughtopeerthroughamicroscopecanseethatfat,hemoglobin-richbloodcellsdon’tlookanythinglikeneuronswiththeirlong,spideryarms.YetthesameDNAispackedintothenucleiofthesewildlydifferentcells.SoDNA,scientistsnowsay,isnotallthereis.
Today’sconsensusisthatstructuralandfunctionaldifferencesamongcelltypesaredeterminedby“epigenetic”programsthatregulateDNA’sactivity,someofthemmarksfromenvironmentalassaults,someaformofmemory.WhatmakestheseproteinsandsmallmoleculessoimportantistheircapacitytochangegeneticexpressionwithoutalteringDNAsequenceitself.
Epigeneticchangesturnanembryonicstemcellintoaspecializedadultcellandtransformnormalcellsintotumors,accordingtoWhiteheadInstituteinvestigatorRudolphJaenisch.Inhiskeynoteaddress,Jaenischsaidunderstandinghowtoprogramandreprogramdifferenttypesofcellsisoneofthemostimportanttopicsinbiologytoday,becausesuchskillswouldholdenormouspromisefortreatingtheillsofhumankind.Epigenetic“reprogramming”isakeystepforcreatingstemcelltherapiesforcancerandprogressivedisorderssuchasParkinson’sdisease,yettheseeffortsareensnaredinethicalandpoliticalcomplications.Twodaysbeforethesymposiumbegan,theHouseofRepresentativesvotedtoexpandgovernment-supportedstemcellresearch,apieceoflegislationdestinedforPresidentialvetodespiteprotestsfrompatientsandfamilies.Onthesameday,NaturepublishedimportantstemcellfindingsfromJaenisch’slab.Elsewhere,cancerpatientswereparticipatinginclinicaltrialstestingdrugswithepigeneticactivity,hopingforcures.
Thesearethereal-worldcomplicationsofthebasicsciencepresentedbyJaenisch,18otherspeakersandnumerousposterpresentersatthe2007symposium.Participantsconvenedatthe
HyattRegencyNewportHotelandSpaonJune8-10,markingthesymposium’sreturntotheUnitedStatesafterfourconsecutivemeetingsinItaly.PowerfultiestoHarvardsciencemadeNewportanappropriatesettingforthisyear’smeeting.ItwasherethatAlexanderAgassiz,curatorofHarvard’sMuseumofComparativeZoology,constructedapioneeringmarinebiologylaboratoryin1875.Forthenext25years,hisresearchteamssetsailfromNewporttoprobethephysical,chemical,biologicalandgeologicalfeaturesofthegreatoceanbasins.
Agassiz’sengineeringtalentshadalreadymadehimwealthyintheminingindustry;inhissecondcareerheusedtheseskillstoinventnewtechnologiesforgatheringspecimensandtakingmeasurementsindeepwater.Longbeforeanyonedreamedofunmannedsubmersiblesorfiberoptics,hemadeobservationsaboutstarfishes,seaurchinsandothermarinecreaturesthatarestillcitedtoday.
Thecontemporaryquesttounderstandhowepigeneticfactorscontrolgeneticinformationisaschallengingasdeep-seaexplorationwasfortheVictorians.Yetprogressisbeingmade.Somepresentersdescribedhowepigeneticfactors,suchaschromatinpackagingandmethylation,establishacell’sidentityyetleaveitsusceptibletochange.Othersfocusedonepigeneticfunctionsasawayofrecordingwhathappenedtoonegenerationandpassingthatmemorytothenext.
Sixtyparticipantsacceptedtheinvitationtothisyear’ssymposium.ScientistsrepresentedHarvardMedicalSchoolanditsaffiliatedinstitutions,MassachusettsInstituteofTechnology,YaleUniversity,nineItalianuniversitiesandresearchinstitutesandonemultinationalpharmaceuticalcompany.FoundationPresidentJosephB.Martin,deanofHMS,presidedandPresidentEmeritusDanielC.Tosteson,formerdeanofthemedicalschool,attendedalongwithmembersoftheFoundation’sBoardofTrustees,ScientificAdvisoryboardandItalianScholarshipAdvisoryCommittee.Attheirannualmeeting,theBoardofTrusteesvotedtocreateanewArmeniseFoundationChairatHMS.CountGiovanniAulettaArmenise,whocouldnotbepresentinNewport,joinedthisdecisionviatelephonefromItaly.TheBoardnamedStephenC.Harrison,directoroftheArmenise-HarvardCenterforStructuralBiology,asthefirstholderofthisendowedchair.Harrison’sappointmentwasannouncedtosymposiumparticipantsbyDeanJosephMartin.
TheprogramfeaturedtalentedyoungscientistswhohavebenefitedfromFoundationprogramsatHMSandinItaly.FourwinnersofHMSJuniorFacultyGrantsparticipatedinthesymposium,includingtwoofthreeresearchershonoredin2007.TheywerejoinedbysevenrecipientsofCareerDevelopmentAwards,whichenableItalianstoreturnhomeandestablishtheirownlaboratoriesaftercompletingpost-doctoraltrainingabroad.TheDulbeccoTelethonInstitutealsomakesresearchgrantsforthispurpose,andtheFoundationinvitedTelethon-supportedepigeneticsresearcherstojointhisyear’ssymposiumandmeetpotentialcollaborators.
AlsoonhandweretwoItaliansciencejournalists,thelatestrecipientsoftheannualScienceWriterFellowshipsthatenableItalianreporterstoresearchstoriesoftheirchoosingatHMSandparticipateinthesymposium.
<palign=""left""><strong>Pluripotency,EpigeneticReprogrammingandEmbryonicStemCells</strong></p>
RudolfJaenisch
WhiteheadInstituteforBiomedicalResearchandDepartmentofBiology,MassachusettsInstituteofTechnology,Cambridge,MA
<ahref=""mailto:[email protected]"">[email protected]</a>
Thirtyyearsago,youngRudolphJaenischbecamefamousbeyondhisyearsbycreatingthefirsttransgeniclaboratoryanimalsinhistory.HeshowedthatwhenMoloneyleukemiaviruswasinjectedintoearlymouseembryos,viralgenesequencesnotonlyintegratedintotheDNAoftheseanimalsbutalsopassedtotheiroffspring.
OnthedaybeforetheArmenise-HarvardSymposiumbegan,Jaenischmadeheadlinesforadifferentreason:hisgroupandtwootherteamsrevealedthattheyhadtransformedadultskincellsintopluripotentembryonicstemcells.Thesecellswereincorporatedintodifferenttissuetypesandcouldbeinheritedbyoffspring.Importantly,thesegroupshadcreatedembryonicstemcellswithoutrunningafoulofcontroversiesaboutusingeggsordestroyingembryos.
<palign=""left"">IntheJaenischlab,thisaccomplishmentbuildsonmorethantwodecadesoftherapeuticcloningresearch.Usingmiceasamodel,theresearcherssubstituteDNAfromafibroblastfortheoriginalDNAinanunfertilizedegg,whichisthengrownintoablastocysttogeneratestemcellsforanimal“patients.”AlthoughJaenischbelievesthisapproach–knownassomaticcellnucleartransfer,orSCNT--couldbesuccessfulinhumanpatients,hesaiditis“notfinanciallyfeasible,itistoodifficulttoobtainenougheggs,andtherearemanyethicalbarriers.”</p>
<palign=""left"">Inthecourseofhisnuclearcloningexperiments,Jaenischwasintriguedbythepossibilityofresettingtheepigenetichallmarksofanadultdonornucleustothoseofanembryoniccell.Toaccomplishthis,Jaenischreckonedhewouldneedtounderstandhowembryonicstem(ES)cellsreplenishthemselveswhilestillbeingabletodifferentiateintonearlyanytypeofadultcell.</p>
<palign=""left"">Fortunately,histeamandothershavelearnedagreatdealaboutthemolecularcircuitryofpluripotencyandself-renewal,makingitpossibletodifferentiatepluripotentcellsintovariousspecificcelltypesandtotakesomaticcellsbackwardintime,restoringelasticitytheypossessedatanearlierdevelopmentalstage.</p>
<palign=""left"">JapaneseresearcherShinyaYamanakaandhiscolleaguesatKyotoUniversitywereengagedinasimilarquest,andinAugust2006theyreportedthatactivatinggenesforfourtranscriptionfactorsinskincellsfromadultmicehadreturnedthosecellstoapluripotentstate.Unfortunately,thesecellswerenotasflexibleasrealembryonicstemcellsandcouldnotbeusedtogeneratelivemice.</p>
<palign=""left"">Jaenisch’slabsetouttorefinetheseexperiments,andafterscreeningvarioustranscriptionfactorsforgenome-wideactivitytheresearchersdecidedtofocusonOct4andNanog.Thesefactorsareactive<em>only</em>infullypluripotentcells,wheretheystimulateanarmadaofgenesthatretainelasticityandrepressgenescontrollingdifferentiation.TheteamdevisedaclevermethodforcollectingcellssuccessfullyreprogrammedbyactiveOct4andNanog,thentestedtheircapacitytobehavelikeembryonicstemcells.</p>
Thereprogrammedcellscontributedtoeverytissuetypeafterbeinginjectedintoearly-stageembryosandtheseembryosdevelopedintolivemice.Whenthesemicewerebred,descendentsofthereprogrammedcellsweredetectedinthenextgeneration.Additionalexperimentswere
performedaswell,andthealteredcellswereindistinguishablefromembryonicstemcellsonallcounts.
<palign=""left"">Whenitcomestocell-basedtherapyforhumandisease,however,“wearenotthereyet,”Jaenischcautioned.Theretroviralvectorsusedtoreprogramthecellsareoncogenicandcouldnotbeethicallyusedinhumans,scientistsdon’tknowwhetherthetranscriptionfactorsthatreturnmousefibroblaststopluripotencywouldworkforhumans,andbetterwaysareneededtoseparatereprogrammedcellsfromadultcellsthatdidnotsuccumb.</p>
<palign=""left"">Ultimately,findingslikethesecouldpropelcell-basedtreatmentsforhumandiseaseoverscientificandethicalhurdlesassociatedwithusinghumanembryosfortherapeuticpurposes.Fornow,however,thesefindingsare“preliminaryandproofofprinciple,”Jaenischsaid.</p>
<strong>Presentations</strong>
<strong>SystemsAnalysisofHumanMitochondrialDisorders</strong>
VamsiK.Mootha,MD
DepartmentofSystemsBiology,HarvardMedicalSchool,
CenterforHumanGeneticResearch,MassachusettsGeneralHospital,andtheBroadInstituteofMITandHarvard
<ahref=""mailto:[email protected]"">[email protected]</a>
AssoonasmitochondrialDNAbecame“thefirsthumangenomeeversequenced,”backin1981,scientistsrecognizedthatitencodedonly13completeproteins.Sincethen,about50humandiseasesandsyndromeshavebeenattributedtomitochondrialabnormalities.Thesediseasesaredifficulttodiagnoseandsofartherearenocurativetherapiesforanyofthem.
What’sfascinating“isthatmostclinicalmitochondrialpatientsdonothavelesionsinthetinymitochondrialgenome–onlyabout15percentdo,”VamsiMoothasaid.“Theresthaveproblemswithnucleargenomeproteinsthatregulatemitochondrialactivity.”Itappearsthatmitochondriadrawnuclearproteinsintotheirorbit,andthenewrecruitscancauseproblems.
<palign=""left"">Mootha’slaboratoryuseshumangenomesequencedata,microarrays,andcomputeralgorithmstosystematicallylinkmitochondriatohumandiseases.Severalyearsago,MoothaandhiscollaboratorsusedthesetoolstoidentifythemutationresponsibleforLeigh
syndrome,FrenchCanadiantype.ThislethaldiseasewastragicallycommonamongchildreninaremotepopulationinQuebec,affectingoneinevery2,000.Mootha’sworkledtoaprenataltestnowbeingusedbyfamiliesatrisk.</p>
<palign=""left"">TheMoothateamhassincecreatedanewsystem,called“Maestro,”thatishelpingthemconstructacompleteproteinatlas,orproteome,formitochondria.Thissystemintegratesdatafromeightdifferentproteinscreeningmethods,cross-checkingoneagainsttheothers,topinpointproteinsactiveinmitochondria.Sofar,itappearsthatabout1,500proteinsfunctioninthistinybutindustriousorganelle.</p>
<palign=""left"">Theteamisusingthis“partslist”tolookforspecificgenedefectsassociatedwithraremitochondrialdisorders,andthesearchisgoingwell.MoothacollaboratedwithMassimoZeviani’slaboratoryattheNationalNeurologicalInstituteinMilantochangemisconceptionsabouthepaticmitochondrialDNAdepletionsyndrome,adisorderthathadbeenattributedtoperoxisomemalfunction.</p>
<palign=""left"">TherealculpritisadefectinMPV17,whichencodeskeycomponentsofthemitochondrialmembrane.InaposterpresentationattheSymposium,AntonellaSpinazzoladescribedtheimpactofthismutationinfamiliesstudiedinItalyandonaNavahoreservationintheUnitedStates,aswellasinamousemodelofdisease.</p>
<palign=""left"">OngoingworkintheMoothalabisaimedatachievingasystems-levelunderstandingofmitochondriathatwillimprovediagnosisandtreatmentformanyother,morecommonhumandiseases.</p>
<strong>TargetingtheB-cellAntigenReceptorinBLymphomaCells:LessonsfromMouseModels</strong>
<palign=""left"">StefanoCasola</p>
FoundationIFOM,theFIRCInstituteofMolecularOncology,Milano,andtheCBRInstituteofBiomedicalResearch,HarvardMedicalSchool
<ahref=""mailto:[email protected]"">[email protected]</a>
Theincidenceofnon-Hodgkin(NHL)lymphomashasdoubledoverthepasttwodecades,advancingittofifthplaceonthelistofcommoncancersamongmenandwomen.IntheUnitedStates,morethan63,000newNHLcasesarediagnosedeachyearandnearly20,000peopledie.Thisincreaseisunlikelytoleveloffbecausetheselymphomasareassociatedwithrisingratesofinfectiousdiseases,autoimmunedisorders,andtoxicenvironmentalexposures.
CliniciansneednewtherapiesforNHLpatients,80to90percentofwhomhaveB-celltumorsthatoriginateinlymphoidtissue.StefanoCasolausesmousemodelstopeerinsidetinyfactorieswheresomeBcellsareprogrammedtostandwatchagainstinvaders.Whilenormalsentriesdieattheendoftheirshift,otherslivetoolongandturntraitorous.AsurfacecomplexcalledtheBcellantigenreceptor(BCR)isimportantfortheirdifferentiation,proliferationandlifespan.MatureBcellsundergoapoptosiswhenBCRislostafterseveralweeks.
<palign=""left"">Incontrast,functionalBCRexpressionpersistsinmosttypesofNHLcells,includingBurkitt’s,follicularlymphomaanddiffuselargeB-celllymphoma.Casola’steamsetouttounderstandtheroleoftheBCRinthesecancercells,andtoseewhatwouldhappeniftheymanipulatedthiscomplexincellsfrommicewiththeequivalentofBurkitt’slymphoma.</p>
Ahumanc-MYConcogenewasusedtoinducetumorsthatlooklikehumanBurkitt’slymphomaunderthemicroscopeandhavethesamecellsurfacemarkers.Inaseriesof<em>invivo</em>and<em>invitro</em>experiments,theresearchersselectivelyblockedtheactivityofBCRandothergenesthoughttobeinvolvedinBcellsurvival,proliferationanddifferentiation.
WhentheresearchersdisabledBCR,B-lymphomacellsisolatedfromindependentprimarytumorsrapidlydied,both<em>invitro</em>and<em>invivo</em>.ComparedwithBCR+cells,theBCR-oneswerethreetimesmorelikelytoundergoapoptosis.Forreasonsthatarepoorlyunderstood,thecellssuddenlydeprivedofBCRdiedonlywhenBCR+cellswerepresent.It’spossiblethathavingBCRisanadvantageonlywhenBCR-cellsarealsopresentandcompetingfornutrients,asituationCasolacharacterizedas“restrainedgrowth.”HeseespreliminaryevidencethatBCR+cellsmaybebetterabletotakeupglucoseinthissetting,enablingthemtooutcompeteBCR-cells.
CasolaandhisteamarenowlookingforhighlyconservedstepsintheBCRsignalingcascaderegulatedbyepigeneticfactorssuchashistonemodifiers.TheymaybeabletohastenthedeathofmalignantBcellsiftheycanidentifysuchinterimsteps.
<strong></strong>
<strong>TumorSuppressorActivityoftheHistoneAcetyl-TransferaseTip60</strong>
BrunoAmati
DepartmentofExperimentalOncology,EuropeanInstituteofOncology(IEO),
IFOM-IEOCampus,Milano
<ahref=""mailto:[email protected]"">[email protected]</a>
Drunkenteenagerschallengeoneanothertojumpfromadangerouslyhighrailroadtrestle.Whenonemakesamovetowardtheedge,he’sunlikelytoleapifheisrestrainedbyafriendoneachside.Butifthereisonlyonefrienddragginghimback,thedangerincreasesthathe’llbreakfreeandtaketheplunge.
Apparentlythesameistrueforcancercells,whicharetornbetweenthedrivetoproliferatewildlyandthemoresoberimpulsetostopdividingandundergonormalapoptosis.Havingtwocopiesoftumorsuppressinggenesonhandreducestheriskofmalignanttransformation;butifonecopyislost–sothatthetumorcellsbecome“haplo-insufficient”–anythingcanhappen.
BrunoAmati’slabinvestigatesepigeneticregulationofhumordevelopment,andhissymposiumpresentationfocusedonTip60,anacetyl-transferasethatmodifieschromatinstructureandinfluencestranscriptionfactorsthateitherpromoteorsuppresstumorigenesis.Tip60hasbeen
characterizedasahaplo-insufficienttumorsuppressorinhumanbreastcancer,andAmatiusesmousemodelstoprobeitsactivitiesindetail.
Inthesemodels,theMyconcogenepromotesBcelllymphomawhileparadoxicallyactivatingDNA-DamageResponse(DDR)signaling,atypeoftumorsuppression.Pre-tumoralB-cellsofyoungEm-mycmiceshowedasustainedDDRasjudgedbyphosphorylationofATM,H2AX,Chk1,Bidandp53(Ser15).
WhenEm-myctransgenicmicewerecrossedwithheterozygousTip60knockoutanimals,DDRwasseverelyimpairedandtumorsgrewfaster,eventhoughtherewasnosignificantchangeinpre-tumoralB-cellexpansion,S-phaseorapoptosis.Theresearchersweresurprisedtoseethatthewild-typeTip60allelewasneverlostinlymphomas,butwasduplicatedinstead.TheyconcludedthatTip60actsasahaplo-insufficienttumorsuppressorduringearlytumordevelopment,butnotwhencancerismoreadvanced.
MycisastronginduceroftheARF-p53tumorsuppressorpathway.LymphomasarisinginEm-mycp53+/-micelosetheremainingp53allele,exceptwhenthislossofheterozygosity(LOH)isblockedbysecondarymutationsthatimpingeonp53activityand/orapoptosis.InlymphomasarisinginEm-mycp53+/-Tip60+/-mice,however,secondarymutationsdidnotpreventp53LOH.AsimilarobservationwasmadeforARFLOHinEm-mycARF+/-Tip60+/-mice.SelectivepressuretolosetheARF-p53pathwayinthesemiceimpliesthatTip60haplo-insufficiencydoesnotbypassARF-p53function,eventhoughp53phosphorylationisimpaired,Amatisaid.RedundantmechanismsforDDRmustbesuppressingtumorgrowthbyothermeans.
Finally,thehaplo-insufficienttumorsuppressoractivityofTip60appearstokickinonlywhenoncogenesareactiveinincipienttumorcells.Liketheboywhopullsafriendbackfromtheedge,restraintisneededonlywhendangerthreatens.
<strong>SonicHedgehog—ProteoglycanInteractionsinDevelopmentandDisease</strong>
RosalindSegal
DepartmentofNeurobiology,HarvardMedicalSchoolandDepartmentofPediatricOncology,Dana-FarberCancerInstitute
<ahref=""mailto:[email protected]"">[email protected]</a>
Sonichedgehogisoneofthebestknownmorphogensinbiology,andarguablythemostcolorfullynamedofthesignalingproteinsthattouchlargegroupsofembryoniccellsandguidethemtoformspinalcords,wingsorfingers.Sonichedgehog(Shh)promotessynchronizedgrowthandspecifiescellfateaspatternsdevelopincomplextissues.
Shhisanexceptionallyversatileproteinthatactsonmanyeukaryoticintracellularpathways.RosalindSegalexaminesShh’sinteractionswithproteoglycansfoundoutsidethecells,intheextracellularmatrix,andaskshowthesemightbeimportantforoncogenesis.ThisisaclinicallyimportantquestionbecauseShhoveractivityhasbeendescribedinsometumorsofthebrain,skin,breastandpancreas.
Inanimalmodels,asinhumancells,theCardin-WeintraubmotifinShhproteinbindstightlywithheparin-sulfateproteoglycans.WhenSegal’steamintroducedmutationsthatdisruptedthisbinding,themicehadfeaturesthatwerenormallypatternedbutabnormallysmall.Observationsinmiceand<em>Drosophila</em>indicatethatsuchdisruptionsinterferewithShh’snormalexpansionofstemcellpoolswithoutaffectingmorphogenesis.
Morerecently,theSegallabhasbeenexploringhowShh-proteoglycansinteractionsaffectwhathappensduringmitosis.Likemanyotherresearchers,she’sbecomefascinatedbyhowsignalsfromoutsidethecell,suchasproteoglycans,integratewithintrinsiccellfateprogramstoregulatecelldivision.Duringnormalstemcellmitosis,onedaughterisliketheoriginal–replenishingthestemcellpool–whiletheotherpreparestodifferentiate.This“asymmetric”divisionmaintainsnormalgrowthanddevelopment.
Inneuralstemcells,however,SegalandhercolleaguesintroducedmutationsthatdisturbedtypicalShh-proteoglycaninteractionsand–amongothereffects–induced<em>Gli3,Bmi1</em>and<em>cyclinD1/2.</em>Insteadofundergoingnormal,asymmetricdivision,thesecellsdividedmuchfasterthanusual.Heparin-sulfateproteoglycanslocalizesShhtomitogenicniches,andundercertainconditionsappearstoholdthepedaltothemetal,speedingupcelldivisiontoamalignantdegree.
Segalemphasizedthatthisisonlyapreviewofwhat’stocome.“Therearemoretypesofproteoglycansinamammalthantherearestarsinthesky,”shesaid,andshehopesthatgeneticapproachesto“glycomics”willbeabletofingermoreofthebadactors.Andthis,inturn,raisesthepossibilityofshrinkingtumors–withoutharmingnormaldevelopment–byblockingspecifictypesofproteoglycansactivity.
<strong>CellularandOrganismToxicityinaDrosophilaModelforthePolyglutamineDiseaseDRPLA</strong>
Manolis<strong></strong>Fanto
DulbeccoTelethonInstitute,DIBIT-SanRaffaeleScientificInstitute,Milano
<ahref=""mailto:[email protected]"">[email protected]</a>
AlthoughHuntington’sistheonlypolyglutamine(polyQ)diseasemostpeopleknowabout,itisonlyoneofaboutadozenprogressiveneurologicaldisordersinthisgroup.Whattheyshareisthegeneticequivalentofastutter–relentlessrepetitionofCAG,threenucleotidesthatencodetheaminoacidglutamine.Thishappensinaso-calledpolyglutaminetract,apartofthegenewheremultiplecopiesarenormal.Whenstutteringgeneratesexcesscopies,theresultingproteinnolongerplayswellwithothers,clogsnervecellswithdepositsthatstubbornlyresistdegradation,andultimatelykillsthecells.
Becauseeachdiseaseinthisgroupdamagesaspecificproteinanddestroyscellsinaspecificbrainregion,theirsymptomsaredifferent.Nevertheless,allthePolyQdiseasesworsenwithtimeandshareagrimprognosis.
Muchofwhat’sknownaboutthesedisorderscomesfromexperimentsusingthefruitfly<em>Drosophila</em><em>melanogaster</em>,andManolisFantousesnovelfliestoinvestigatedentatorubropallidoluysianatrophy(DRPLA),adisordercausedbyPolyQexpansioninthehumangeneforAtrophin-1.Healthypeoplehavesixto35copiesoftheCAGrepeatinthisgene;DRPLApatientshave49-88.
AlthoughsomeflymodelsforDRPLAusehumangenesorsyntheticconstructs,FantoandhiscolleagueshavegeneratedamodelbyexpandingendogenouspolyQin<em>atro.</em>
Theycombinegainoffunctionandclassiclossoffunctionanalysistounravelthecellularandorganismconsequencesofmutationsin<em>atro</em>anditspartner<em>fat.</em>Histeamanalyzescelldegenerationinneuronalphotoreceptorsanduseslifespanasameasureofoveralltoxicity.
WhenresearchersinducedtheflyequivalentofDRPLAbyexpandingapolyQtractinatrophin,theflyretinadevelopedabnormalvacuolesandtheanimalsdiedshortoftheirnormallifespan.NewfindingsindicatethatthelargerthepolyQexpansiontheworsetheretinaldamage,whichtheyattributetodisruptionofautophagyratherthaninductionofapoptosis,Fantoreported.Autophagynormallyprotectscellsbyclearingawayspentorganelles.
AbnormalfoldingmakesexcessPolyQproteinsclumsywhentheyinteractwithintracellularpartners.Atrophinandthegiganticcadherinandtumoursuppressor<em>fat</em>arepartofaneuroprotectivepathway,andFanto’sexperimentshaveshownthatcertainmutationsineitheronecancauseneurodegeneration,killingretinalneuronsandabbreviatinglifespan.Themostsurprisingfindingwasthattheorganismdiesfasterthanacelldegenerates,andthatthiseffectonviabilityisexertedbyglialcellsaswellasbyneurons,Fantosaid.
Althoughthesestudiesarefarfromcomplete,theyhintthatthecomplexityandvariationinpolyQdiseasesmayarisenotonlyfromthedeathofcellsinaspecificbrainarea,buttomorewidespreadtoxicityaffectingthewholenervoussystem.Thenextstepsaretodiscoverthemechanismsinvolved.
<strong>TargetingActiveGenesforHistoneModificationin<em>Drosophila</em></strong>
MitziI.Kuroda
HowardHughesMedicalInstitute,Harvard-PartnersCenterforGeneticsandGenomics,Brigham&Women’sHospitalandDepartmentofGenetics,HarvardMedicalSchool
<ahref=""mailto:[email protected]"">[email protected]</a>
MitziKuroda’slaboratoryfocusesonafundamentalquestioninbiology:howdoorganismsbalancegeneexpressioninmalesandfemales,whenmaleshaveonlyoneXchromosomeandfemaleshavetwo?
Theanswerliesin“dosagecompensation,”shesaid,adjustmentstochromatinstructurethatoperatedifferentlyindifferentorganisms.Inmammals,epigeneticmodificationsofhistoneinactivategenesononeoftheX’searlyinfemaledevelopment;infruitflies,modificationsincreaseexpressionofgenesonthesingleXsothattheymatchtheoutputofbothfemaleXchromosomesinmaleembryos.
In<em>Drosophila,</em>dosagecompensationiscarriedoutbyalargeribonucleoproteincomplexcontainingMSL(male-specificlethal)proteinsandnoncoding<em>roX</em>(RNAonX)RNAs.Thisribbon-likecomplexattachesitselfonlytothemaleXchromosome,whereitaltershistoneH4acetylationandencouragestranscriptionofX-linkedgenes.
The<em>RoX</em>RNAsdifferdramaticallyinsizeandsequence,yettheyarefunctionallyredundantandnecessaryforassemblingtheMSLcomplex.Oncethecomplextakesshape,itconsistsoffiveMSLproteins:ifoneismissingorinactivatedthemaleembryodies.
ResearchershavestruggledtounderstandhowtheMSLcomplextargetsspecificsitesontheXchromosomewherebindingwillincreasegeneexpression.Kuroda’steamuseda“ChIP-chip”strategy,whichcombinesmicroarraytechnologywithchromatinimmunoprecipitation,togeneratehigh-resolutionimagesofMSLbindingandhistonemodification.Theyobservedwild-typeMSLbindingto800sitesonXchromosome–97%ofthemovergenesandmostnearthe3’end,Kurodasaid.MSLisattachingitselftoH3K36me3,atargetingmarktypicallyconcentratednearthe3’endofgenesandabundantontheXchromosome.
Inanotherseriesofexperiments,KurodasetouttounderstandMSL’sstrongpreferenceforbindingtheXchromosomeandshunningautosomes.Whentheinvestigatorsinserteda<em>roX1</em>ora<em>roX2</em>genomictransgeneinanautosome,MSLwaseagertobindthesefamiliarpartners.TheyweresurprisedtofindthatonautosomestheMSLcomplexcouldalsospreadlongdistancesfromtheoriginal<em>roX</em>insertionsite,attachingitselftoactivegenesmarkedbyH3K36me3witha3’bias.
Together,thesedatasupportamodelinwhichMSLcomplexisinitiallyrestrictedtoachromatindomainbyhighaffinitysitessuchas<em>roX</em>genes,butthenscansthechromosomeforgeneralmarkssuchasH3K36me3.MSLcomplexandactivechromatinmarkssuchasH3K36me3areconservedinmammals,Kurodanoted,improvingtheoddsthatthisrecruitmentmechanismwillproveimportantformorethan<em>Drosophila.</em>
<strong></strong>
<strong>HistoneDemethylasesandHumanDiseases</strong>
YangShi
DepartmentofPathology,HarvardMedicalSchool
<ahref=""mailto:[email protected]"">[email protected]</a>
“Understandingbiologicalimportanceisthenextstep,”YangShisaidattheendofhis2006symposiumpresentation.Hislabpioneeredtheideathathistonemethylationisdynamicwhentheyclonedthefirstdemethylasein2004.Initiallytheycouldremoveonlyoneortwomethylgroupsfromalysineresidue,anditseemedthattrimethylgroupsmightbepermanentlyattached.Thiswasdiscouraging,becausethistypeofhistonemodificationappearstolockgenesassociatedwithvariouscancersandneurologicabnormalitiesinthe“on”position.
Then,shortlybeforethe10<sup>th</sup>Armenise-HarvardSymposiumlastyear,Shi’steamidentifiedanenzymefamilythatknockedtrimethylsofftheirhistoneperchesinculturedhumancellsandlivenematodes.Inthenematodes,addingandremovingmethylgroupsdeterminedwhethercellslivedordied.
Ahallmarkoftheseproteinsisthecruciform“Jumanji”domain,whichisfoundinabout30humanproteinfamilies.Theresearchersscreenedthesefordemethylatingenzymesanddiscoveredfourproteinsthatsnippedgroupsortwoorthreemethylsoffmedicallyinterestingsites.
OneoftheseisencodedbySMCX(orJARID1C),andcertainpointmutationsinthisgeneareassociatedwithX-linkedmentalretardation(XLMR).AlthoughpreviousresearchforgedlinksbetweenthesemutationsandXLMR,exactlyhowthesemutationsdamagedbraindevelopmentwasnotknown.
Shinowhasanidea.SMCXlostdemethylationcapacitywhenpointmutationsfoundinXLMRwereintroducedintoratcerebellargranuleneurons.Postsynapticneuronsnolongerdevelopednormaldendriticspines,anabnormalitypreviouslydescribedinthebrainsofmentallyretardedhumans.
SeparateexperimentsinzebrafishalsoraisethepossibilitythatincreasedneuronalcelldeathmaybepartlytoblameforXLMR.ResearchersfoundthatinzebrafishthehomologofSMCX,whichactsexclusivelyinthebrainduringdevelopment,isessentialfornormalneuronsurvival.
Althoughthere’sdefinitelymoretolearn,Shinotesthatstudieslikethesereinforceconnectionsbetweenbasicchromatinbiologyandhumandisease.
<strong></strong>
<strong>FunctionalInteractionbetweentheNucleosomeRemodelingFactorISWIandCovalentModifiersofChromatin</strong>
DavideF.V.Corona
IstitutoTelethonDulbeccoc/oDipartimentodiScienzeBiochimiche-Universita'degliStudidiPalermo
<ahref=""mailto:[email protected]"">[email protected]</a>
OnTVmakeovershows,swarmsofcarpenters,electriciansandpainterstransformcrampedtracthousesintoshowplaces,shovingpastoneanothertoknockoutwallsandimprovelighting.
Thenucleusofaeukaryoticcellisnotsodifferent.Busycrewsofepigeneticfactorsmodifyhistonesandremodelchromatin,pushingandpullingnucleosomessothatDNAisaccessible,inacontrolledmanner,fortranscription,repair,recombination,andotheressentialfunctions.
“Alltheseactivitiesintegrateinthenucleusandtheyworkatthesametime,”DavideCoronasaidatthesymposium,“butit’snotclearhowtheiractivitiesarecoordinatedtoregulatechromaticstructure,geneexpressionandothernuclearfunctions.”HislabusesgeneticandbiochemicaltoolstosearchforchromatinmodifyingfactorsthatactinconcertwithISWI,ahighlyconserved,ATP-dependentnucleosomeremodelingfactor.
In<em>Drosophilamelanogaster</em>,ISWIregulatesexpressionofabout5percentofthegenomeandisinvolvedinDNAreplication,RNAtranscriptionandchromosomeorganization,Coronasaid.Itsactivitycanbemodulatedbysite-specificacetylationofhistones,andCorona’steamusedflieswithaninheritedeyedefecttoscreenhundredsofmutantgenesthatmightbeconspiringwithISWItounderminenormalchromatincondensation.
Thescreenidentifiedtwomutantgenesofinterest:SIN3AandPARP.Botharehighlyconservedenzymesinvolvedinchromatinmodificationanddynamics.
SIN3Aanditspartner,RPD3,formahistonedeacetylasecomplexinvolvedintranscriptionalrepression.ImmunostainingassaysshowthatISWIandthiscomplexnormallysticktogetheronpolytenechromosomes,butsplitapartonISWImutantchromosomes.Aseriesofexperiments
confirmedinteractionsbetweenISWIandtheSIN3A-RPD3complexandrevealedwhatCoronacalled“afunctionalantagonismbetweenISWIandtheacetylationofhistoneH4onlysine16.”TheresearchershypothesizethatISWIrecruitstheSIN3A-RPD3complextoproductivelyremodelnucleosomes.
Thesecondinterestingmutationwasinthegenecodingforthepoly-ADP-ribosepolymerase,orPARP,achromatinenzymethatCoronasaidisimportantduringDNAdamageresponseandapoptosis.HeatshockproteinsrecruitPARP,whichloosenschromatinbyaddingpoly-ADP-ribose(PAR)andthuspromotestranscription(whichISWItendstorepress).ISWIispoly-ADP-ribosilated<em>invitro</em>and<em>invivo,</em>whichtheinvestigatorsweresurprisedtofindinhibitsitsATPaseactivitybymakingitlesslikelytosticktothenucleosome.ISWIandPARarelocalizedindifferentchromatindomainsonwildtypepolytenechromosome,butwhenISWIismutatedPARspreadsonthesechromosomes.Chromatin-boundISWIcanbejoltedoffpolytenechromosomesby<em>invivo</em>inductionofchromatinpolyADP-ribosylation,Coronareported.Thisindicatesthatpoly-ADP-ribosylationofISWIinhibitsitsATPaseactivity,causingISWItoloseitsgriponchromatin.
Thesedatashowdifferentepigeneticfactorsworkingtogetherasanetwork,withISWIandcovalentmodifiersofchromatinregulatingchromatinaccessibilityandothernuclearfunctions.Coronathankedthesymposiumorganizersforthe2004Armenise-HarvardFoundationCareerDevelopmentAward,whichmadepossiblethesefindingsandwhathecalled“mynewlifeinPalermo.”
<strong>PossibleRolesinSilencingforpiRNAs</strong>
BobKingston
MassachusettsGeneralHospital,Boston
<ahref=""mailto:[email protected]"">[email protected]</a>
Althoughmanygenesoscillatebetweentalkingandlistening,somegenesessentialformaintainingparticulartissuetypesmustspendthelifeoftheorganismturnedoninonecelltypeandoffinanother.BobKingston’slabisinterestedinepigeneticregulatorycomplexesthatareasdecisiveasTonySoprano:whentheysilenceagene,itstaysthatway.Thissilenceissoprofoundthatitisheritable,enshrinedinchromatinandpassedfromonegenerationtothenext.
In2006,theKingstonlabandthreeothergroupssimultaneouslydescribedanewclassofsmallregulatoryRNAsthatcanrendergenesmute.ThesehavebeendubbedPiwi-interactingRNA,orpiRNA,becausetheyglomontomembersofthePiwifamilyofproteins.Theseso-calledArgonaute
proteinsinteractwithpolycombproteins,whichsincethe1940shavebeenknownasheritablerepressorsof<em>Drosophila</em>genes.
OtherlaboratoriesworkingonpiRNAsbeganwiththeprotein-RNAcomplex,thenfiguredouthowtoremovetheRNAcomponentforanalysis.KnowingthatthesespecializedRNAsoccurinegg-andsperm-producingcellsinfruitflies,membersoftheKingstonlabtookadifferentapproach.
PostdoctoralfellowNelsonLaustartedwithrattestesandsetouttoextractRNAsthatmightfunctionastranscriptionalgenesilencers.HepulledoutacomplexcontainingsmallRNAsandRiwi,theratversionofhumanPiwi.TheresearchersdubbedthispiRC.ThesmallRNAsinpiRCarraythemselvesonchromosomesinapatterntypicalforregulatoryenzymes,Kingstonnoted.Approximately94percentofpiRNAsmapto100small(<100kb)genomicloci;atthesesitestheyadhereeithertoWatsonorCrickstrands,oriftheybindbothstrandstheydonotoverlap.Theselocations,Kingstonsaid,appeartobeconservedacrossspecies.
PreparationsofpiRCalsohookupwithrRecQ1,theratequivalentofahelicasethathelpssilencegenesin<em>Neurospora</em>.RecombinantRecQ1andPiwifamilyproteinsappeartointeractdirectly,andinfliesPiwihasbeengeneticallylinkedtotranscriptionalgenesilencing.ExperimentsshowthatthepurifiedcomplexhaspiRNA-directedsliceractivity,whichaddsweighttotheideathatpiRCisaheritablesilencerofmammaliangenes.Kingstonsaidmoreevidenceisneededtoconfirmthis.
<strong>TheRoleofChromosome3DOrganizationinPolycomb-DependentEpigeneticRegulation</strong>
ValerioOrlando
DulbeccoTelethonInstituteatIGBCNR,NaplesItaly
<ahref=""mailto:[email protected]"">[email protected]</a>
Profoundshiftsingeneexpressionenablecellstodifferentiateorchangemetabolicstate.MuchofthedramainvolvescovalentmodificationsofDNAandchromatinandthree-dimensionalreorganizationofchromosomesandgenesinthenucleus.Together,theseepigeneticfactorscollaboratetoensurethequality,stability,andheritabilityofcell-specifictranscriptionprograms.
ValerioOrlandowantstounraveltheepigenomicmechanismsthatcontrolcellidentityandcellfateplasticity.Inparticular,hislabstudiesPolycombgroupproteins(PcG),whichplayafundamentalroleindevelopment,stemcellrenewalandtumorprogressionbyshapingchromatinstructure.
In<em>Drosophila</em>,PcGsjoinwiththeirtargetsequences,PolycombResponseElements(PREs),toensureepigeneticheritabilityoftranscriptionprogramsandgenesilencing.Buthow?OnetheoryisthatPREsarecarpenters,buildingstructuresthatsequestercertaingenesandmaketheminaccessiblefortranscription.Likehouses,thesestructuresmaymaintainthestatusquobybeingpassedfromparenttooffspring.
Inaseriesof<em>invivo</em>experimentswith<em>Drosophila,</em>Orlando’steamexploredthedynamic,3-Dstructureofthehomeoticlocusbithoraxcomplex(BX-C).TheyusedChromosomeConformationCapture(3C)toidentifywherePcGbindstoBX-Cduringearlyembryogenesis,thencombinedthisinformationwithdatafromfluorescent<em>insitu</em>hybridization(FISH)andFISH-Immunostaining(FISH-I)analysis.
Theylearnedthatwhenhomeoticgenesarerepressed,PcGproteins,PREsandcorepromotershadinteractedatadistancetobuildtopologicallycomplexstructures.Incontrast,whenthehomeoticgenesareprogrammedtobeactive,PcG-controlledepigeneticDNAelementsdidnotinteract.Finally,Orlandoreportedthatmajorchangesinhigherorderstructuresareessentialtomaintainandstabilizealternativetranscriptionstates,whilehistonemodificationandreducedlevelsofPcGproteinsdefineanepigeneticswitchthatisonlypartiallyheritable.
FutureexperimentswillsearchforlargeandsmallRNAsinvolvedinhigherorderstructureswithinthenucleus,Orlandosaid,followingcluesthatimplicatetheDicerribonucleaseinchangesto3-Dchromatinstructures.
<strong>AChromosome-AssociatedSmallRNAAmplificationLoopRequiredforHeterochromatinFormation</strong>
DaneshMoazed
DepartmentofCellBiology,HarvardMedicalSchool
<ahref=""mailto:[email protected]"">[email protected]</a>
Heterochromatinwasfirstdescribedin1928,whenbiologistsrealizedthatcertaintightlycompactedregionsofchromatinstaineddifferentlyfromtherest.Andonlyafewyearslater,<em>Drosophila</em>researchersrecognizedthatgenesaresilencedintheseregions,whichareassociatedwithcentromeres,telomeres,repetitiveDNAelementsandcelldifferentiationgenes,DaneshMoazedsaidinhisopeningremarks.Oneofthemostfascinatingpropertiesofthesedomainsisthattheyareepigeneticallyinheritedeventhroughmanycelldivisions.
Moazedhasbeeninterestedinheterochromatinassemblyandepigeneticinheritanceformorethanadecade,andinitiallyheusedbuddingyeastasamodelsystem.Morerecentlyhislabbeganexperimentingwith<em>S.pombe,</em>orfissionyeast,becauseitsgenesilencingmechanismsmorecloselyresemblethoseofhigheranimals.
Threeyearsago,MoazedandhiscolleaguesdiscoveredsurprisingconnectionsbetweenRNAinterference,orRNAi,andtheassemblyofheterochromatinatfissionyeastcentromeres–aprocessalreadyknowntoinvolvehistonebindingproteinsandmethylationenzymes.TheyhavesinceidentifiedseveralmultiproteincomplexesthatphysicallylinktheRNAipathwaytoheterochromatinassembly,andhaveanalyzedtheiractivities<em>invitro</em>and<em>invivo</em>.
AstheMoazedlabfitspiecesintothiselaboratepuzzle,itappearsthatapositivefeedbackloopdeservescreditforkeepingheterochromatinstrongduringcelldivision.AcomplexdubbedRITSattachessmallRNAtagstospecificchromosomeregionsthatshouldbebundledintoheterochromatin.Anothercomplex,RDRC,workswithRITSandtheDicerribonucleasetogenerateasupplyofsmallinterferingRNA(siRNA).ThesesiRNAs,togetherwithhistoneH3lysine9(H3K9)methylation,createsapositivefeedbackloopthatgeneratesmoresiRNAandmaintainsH3K9methylation.Thesilencingmechanisminvolvesco-transcriptionaldegradationofRNAsthataretranscribedinheterochromaticdomainsbyRNAi-dependentand-independentpathways,aprocessthatMoazedcallsCTGS,orCo-TranscriptionalGeneSilencing.
ThediscoveryofadirectphysicalconnectionbetweenRNAiandheterochromatinraisesthepossibilityofdevelopingdrugsthatsilencegenesbeforetheyspeak,ratherthantryingtocounteractwhatthey’vealreadysaid.
<strong>HomologuePairing:RegulatingGenesandDrivingEvolution</strong>
TingWu
DivisionsofGeneticsandMolecularMedicine,HarvardMedicalSchool,
<ahref=""mailto:[email protected]"">[email protected]</a>
TingWuremembersatriptoSeattle,justafewyearsago,whereshefirstbecameinterestedinultraconservedelements,orUCEs.TheseDNAsequences,atleast200basepairslong,arevirtually100%identicalindistantlyrelatedorganisms.ThisstruckachordwithWu,whoselabhaslongbeeninterestedinhomologyandhomologouspairing.
“Homology–orthenumbertwo–playsahugeroleinthelivesofcells,”Wusaid.ShewonderediftheseUCEs,whichareenrichedinpartsofthegenomebelievedimportantforgeneregulationandexpression,mightbeinvolvedinpairing.Ifso,theymightalsohavesomethingtodowithcopynumbervariations,situationswhereDNAsegmentsdonotmarchdowntheaisleinneatpairs.Herlabwasalreadyinterestedinoddnumbersandthedynamiccapacityofthegenometoaccommodatemanydeletionsandduplicationsfromoneindividualtothenext.
Wususpectedtheremightbeaconnectionbetweenthesetwopuzzles,andthereportsheheardin2004providedcluesaboutwhatthiscouldbe.ResearchersfromtheUniversityofCalifornia-SantaCruzhadidentified481uniqueUCEsconservedamonghumans,rats,andmice.TheseUCEsoccureverywhere<em>except</em>chromosome21andtheYchromosome.
“What’sintriguingabout21andYistheyarethetwochromosomesmostlikelytobefoundinhumansinnumbersotherthantwo,”Wunoted.“Trisomy21(Downsyndrome)isthemostviablehumananeuploidandofcoursethereisasingleYinmales.”
“WethinkthatpossiblytheseUCEsarelikelytobecopy-counters,andtheirroleistomakesurethateachsegmentofthegenomeoccursexactlytwice,”Wusaid.SheandhercolleaguespredictedthatDNAsegmentspresentincopynumbersotherthantwowouldbedepletedofUCEs.
ComputationalgeneticistAdnanDertihelpedtestthismodelbydeterminingwhetherthreesetsofUCEs,totaling896elements,aredepletedamongtwosetsofhumansegmentalduplications(SDs)andsevensetsofCNVs.TheyfoundastrikingshortageofUCEsinthetwosetsofSDs(P<10<sup>-8</sup>)andsixofthesevensetsofCNVs(P<10<sup>-4</sup>).Theresultsareconsistentwith“astringentwatchingofcopynumbers,”Wusaid.
FutureexperimentswilllookforspecificmotifswithinUCEsandexplorewhethertheirduplicationordeletionisassociatedwithlossoffitnessordeath.
<strong>TheChicken-and-EggRelationshipbetweenTranscriptionandChromatinandImplicationsforEpigeneticInheritance</strong>
KevinStruhl
DepartmentofBiologicalChemistryandMolecularPharmacology,HarvardMedicalSchool
<ahref=""mailto:[email protected]"">[email protected]</a>
ExperimentswithyeasthaveenabledKevinStruhlandhiscolleaguestogainsurprisinginsightsintotherelationshipbetweenthe“beadsonastring”structureforDNAstorageandtheapparatusthattranscribesgenes.Theyhavelearnedthatsometranscriptionmechanismsdodoubleduty:notonlylooseningheterochromatinintoreadableeuchromatinbutalsorepackingitintheirwake.
Inlate2005,StruhlandhiscolleaguesmadewavesbydemonstratingthatRNApolymeraseII(PolII)doestwoseeminglyoppositethingsatonce.AsitmovesalongtheDNAtemplateitnotonlyexposesandreadsgenetictext,butalsoknocksoffacetylgroupsandcoversupwhatitjustread.DoingthiskeepsPolIIfrommistakenlybeginningatranscriptionpartwayalongthecodingregion.
Atthesymposium,Struhldescribedhowtheepigeneticreprogrammingofyeastillustratesthechicken-and-eggrelationshipbetweentranscriptionandchromatinstructure.ThesilencingproteinSir3isanestablishedtoolforblockingDNAtranscription,anditinducesheterochromatin
formationbycausingrapidlossofhistoneacetylation.Removalofeuchromatichistonemethylationwasmoregradual,andoverseveralcellgenerationstheresearchersobservedstructuresthatwereneithereuchromaticorheterochromatic.
Unexpectedly,Sir3bindingandthedegreeoftranscriptionalrepressionincreasedgraduallyforthreetofivecellgenerations,eventhoughtheintracellularlevelofSir3remainedconstant,Struhlreported.StrainslackingSas2histoneacetylaseorthehistonemethylasesthatmodifylysines4(Set1)or79(Dot1)ofH3displayacceleratedSir3accumulationat<em>HMR</em>oritsspreadingawayfromthetelomere,suggestingthatthesehistonemodificationsexertdistinctinhibitoryeffectsonheterochromatinformation.
MethylationofH3K4appearstorepresentwhatStruhlcalledaformofmemory,amarkthatsays“Iwasrecentlytranscribed.”Andthisisthesortofmarkthatmakesitpossibleforpatternsofgeneregulationtobeepigeneticallyinherited.Asfortherelevanceoftheseyeaststudiestohigherorganisms,hecitedthefamousdictumfromJacquesMonot:“What’struefor<em>E.coli</em>istruefortheelephantonlymoreso.”
<strong>EpigenomeandInflammation:InductionofahistoneH3Lys27me3demethylasebyNF-kB–alinkbetweeninflammationandPolycomb-mediatedgenesilencing</strong>
GioacchinoNatoli
EuropeanInstituteofOncology(IEO),CampusIFOM-IEO,Milan
<ahref=""mailto:gioacchino.natoli@ifom-ieo-campus"">gioacchino.natoli@ifom-ieo-campus</a>
Inflammationistheimmunesystem’sfirstresponsetoinfection,irritationordamage,anditsclinicalmanifestationsrangefromthefleetingpainofastubbedtoetochronicinflammatorydiseaseorlethalsepticshock.GioacchinoNatoliisintriguedbyconnectionsbetweeninflammatorystimuliandepigeneticcontrolofchromatinstructure,especiallytheroleoftranscriptionfactorsintheNF-kB/Relfamily.
In2002,heandhiscolleaguesreporteddynamicchangesinH3Lys9methylationinanexperimentalsystemthatusedlipopolysaccharide(LPS)stimulationofculturedhumandentriticcellstosimulatebacterialinfection.Demethylationandremethylationofthissitedeterminedwhatsortsofinflammatorygenesweretranscribedandwhen.
Morerecently,Natoliandhiscolleagueshavebeenexperimentingwithmouseproteinscontainingthe“Jumanji”domain,murineequivalentsofdemethylatingenzymesthatYangShi’slaboratorystudiesatHMS.ThenuclearproteinJmj8isinducedbyLPSstimulationonlywhenNF-kBispresent,Natolisaid,anditremovesthetrimethylgroupfromH3Lys27me3sites.Whenthatgroupisattached,Polycombrepressivecomplexes–whichsilencegenes–areinduced.
TheinvestigatorsnextusedmicroarraystoprobeconnectionsbetweenH3Lys27trimethylationandinflammation.DepletingJmj8didnotchangegloballevelsofH3K27me3,butdidincreasebasallevelsatHoxA11,Natolireported.
AdditionalexperimentsrevealedthatLPSstimulationhadeliciteddramaticallydifferentresponsesindifferentcelltypes.Inmyeloidprecursors,theresearcherswitnessedsustained,high-levelinductionofJmj8andglobalcontrolofHcLys27me3levels.Indifferentiatedmacrophages,Jmj8wastransientlyinducedatalowlevelandtherewasnoglobalimpactonmethylation,Natolisaid.
ThiscouldmeanthatinductionofthisH3Lys27demethylasebyinflammatorystimuligeneratesa“permissivechromatinstate”thatallowsonetypeofcelltoturnintoanother.Therehavebeensignsoftransdifferentiationinchronicallyinflamedcellsoftheeye,kidney,andintestinallining,hesaid.
Iffutureexperimentsconfirmthattransdifferentiationoccursduringinflammationinhismousemodels,duetoepigeneticreprogramming,thiscouldhavemanyimplicationsforhumandisease.
Histonedemethylasescouldturnouttobeadirectmolecularlinkbetweenchronicinflammationandperturbationofdifferentiationprograms(metaplasia)relevantforcancerdevelopment.
<strong>GrowthPropertiesofNormalandTransformedBreastStemCells</strong>
AngeloCicalese
DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan
<ahref=""mailto:[email protected]"">[email protected]</a>
Anti-canceragentswithepigeneticactionarealreadybeingtestedinpatientswithvariouslymphomas,andtheultimategoalofAngeloCicalese’sresearchistoidentifyepigenetictreatmentsforbreastcancer.Arecentseriesofexperimentswithmicesuggeststhatthismaybepossible,Cicalesesaidatthesymposium.
Likeothertissues,tumorsareorganizedhierarchically:theyoriginatefromandaremaintainedbyasmallsubsetofcancerstemcells,orCSCs.CicaleseandhiscolleaguesaskedwhetherepigeneticstrategiescoulddisableCSCsandshrinktumors.
Totestthisidea,theysetupprotocolsforpropagatingbreaststemcellsfromwild-typemousemammarygland(BSCs)andtransformedBSCsfromMMTV-ErbB2transgenicmice.BSCsnotonlysurviveinsuspension,butalsogeneratesphericalcellaggregatescalled“mammospheres.”Mammospheresareclonalinorigin,showthestemcellpropertiesofself-renewalanddifferentiationandarecomposedofBSCsandprogenitors,Cicalesesaid.Duringserialpassages,thenumberofmammospheresformedfromwild-typemousebreasttissuedecreasedatafixedratethrougheachpassage.TheErbB2mammospheres,incontrast,increasedatanexponentialrateanddisplayedanear-immortalphenotype.Mammosphereinitiatingcells,orMICs,accountedforthedisparity;thegrowthrateofprogenitorsdidnotchangeovertime.
TobetterstudythegrowthpropertiesofnormalandtransformedBSCs,theresearchersincorporatedalipophilicdye,PKH26(PKH),intostemcellplasmamembranes.Thisallowedthemtotrackawell-knownpropertyofstemcells:theirabilitytoremaingenerallyquiescentand/orundergofewdivisionswhilegivingrisetoanintermediatepopulationofactivelyproliferatingprogenitors.
Usingthisprocedurewithnormalbreasttissue,Cicalese’steamfoundthatonlyaminorityofPKH+cellscontinuedformingmammospheresafterbeingdissociatedandreplatedseveraltimes.Afewofthelabeledcellswereabletogeneratemammarytissuewhentransplantedintoanotheranimal’sfatpad,showingthattheyaretrulystemcells.
BSCsfromMMTV-Erb2micewereanentirelydifferentstory.BothPKH+andPKH-populationsformedmammospheres<em>invitro</em>andinitiatedtumorsaftertransplantation,Cicalesereported.TheseErbB2mammospheresandbreastcancertissuescontainedmoreBSCsthannormalmammospheresandtissue.TheresearchersattributethistoincreasedsymmetricdivisionsofcancerBSCs,atypeofgrowthpatternalsoseeninBSCsfrommicemissingthep53tumorsuppressorgene.
Knowingthatmutatedorfunctionallysuppressedp53isacommonfeatureofErbB2tumors,theinvestigatorsspeculatedthatp53malfunctionmightaccountforincreasedsymmetricdivisionsoftransformedBSCsintheirexperiments.TheyusedNutlin3,anMDM2antagonist,toblockp53degradationandrestoreitsfunction.Theresultwasacompletereversaloftheirearlierresults,hintingthatepigenetictreatmentsforbreastcancermaybepossibleinthefuture.
<strong>ExitfromMitosisinBuddingYeastSaccharomycescerevisiae</strong>
RosellaVisintin
DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan
<ahref=""mailto:[email protected]"">[email protected]</a>
Anyonewhohaseverbeateneggwhitesknowsthattimingiseverything:whiskafewsecondstoolongandyou’llhaveamess,notameringue.Mitosisisnodifferent:cellsmustexitatpreciselytherightmomentorruntheriskofaccumulatingchromosomesegregationerrorsandgeneticinstabilityassociatedwithcancer.
UnderstandinghowcellsexitmitosisisthefocusofRosellaVisintin’slaboratory,whichshefoundedin2005withsupportfromaCareerDevelopmentAward.Sheusesthebuddingyeast<em>Saccharomycescerevisiae</em>asamodelforstudyingcellcycleprogression,whichinalleukaryotesistriggeredandcoordinatedbyasetofproteinsincludingcyclins,cyclin-dependentkinases(CDKs)andtheirinhibitors.
PhosphorylationofproteinsbymitoticCDKsdrivesmitosis,andafterchromosomesareaccuratelysegregatedthesesameCDKsmustbeinactivatedsocellscanleavemitosisandentertheG1phase.Inyeast,thephosphataseCdc14emergesfromthenucleolus,whereithasbeenboundtobytheinhibitorysubunitCfi1,andspreadsintothenucleusandcytoplasm.Itzeroesinonitstargets,bringsmitosistoasuddenhalt,andretreatsswiftlytothenucleolus.
Visintinandhercolleagueshavebeenexploringhowtworegulatorynetworks,calledFEAR(CdcFourteenEarlyAnaphaseRelease)andMEN(MitoticExitNetwork)controltheassociationofCdc14withCfi1andpromoteCdc14’sreleasefromthenucleolus.Inhersymposiumpresentation,Visintinfocusedontheflipsideofthecoin:mechanismsthatrapidlyinactivateCdc14aftermitoticexit.
TheproteinkinaseCdc5,acomponentofboththeFEARandMENnetworks,helpsliberateCdc14fromCfi1soitcanleavethenucleolus.WorkintheVisintinlabindicatesthatinactivationofCdc5isnecessary–butprobablynotsufficient–toreturnCdc14toitsstorageplace.
“Cdc14plantstheseedsforitsowninactivation”viatheFEARandMENnetworks,Visintinsaid,butexactlyhowthisworksremainstobeseen.Answersfromyeastwillberelevantforhumans,becausethesameplayers–Cdc14,FEARandMEN–helpregulatecelldivisionandmaintaingenomicintegrityinhumans.
<palign=""left""><strong><brclear=""all""/></strong></p>
<strong>Dances-with-Retroviruses:The40MillionYearSagaofthe<em>TRIM5</em>Locus</strong>
WelkinJohnson
NewEnglandPrimateResearchCenter,DepartmentofMicrobiologyandMolecularGenetics,HarvardMedicalSchool
<ahref=""mailto:[email protected]"">[email protected]</a>
Despiteitsglobalprominenceformorethan20years,inthelongruntheHIV/AIDSsagamaybeasfleetingasaCNNheadlineaboutthelatestcarbombinBaghdad.Thehumangenomeisstuddedwiththefossilfootprintsofretrovirusencountersstretchingbacktensofmillionsofyears,WelkinJohnsonsaid,longbeforeAIDS.Thesebattlesbetweenhostandpathogenarememorializedinthegermlineandpassedfromonegenerationtothenextasfeaturesofinnateimmunity.
OneofthesefootprintsistheTRIM5locus.SincetheearlyyearsofHIV/AIDS,researchershaverealizedthatageneticbarrierkeptHIV-1fromthrivinginoldworldmonkeys–makingtheircellsunsuitableasmodelsforHIVreplicationandAIDS.Incontrast,humancellsarecatastrophically
susceptibletoHIV-1infection,butabletorepelsomeotherretrovirusinfections.Alandmark2004articleidentifiedtherhesusmonkeyTRIM5gene,andspecificallytheTRIM5 alphasplice-isoform,asadeal-breakerthatblocksHIVreplicationinthecytoplasm,preventingtheretrovirusfrompenetratingthehostnucleus.
TRIM5aisabroad-spectrumantiviralwhosespecificcapacitiesareshapedbyeachprimate’shistory.WhenJohnson’steamcompared<em>TRIM5</em>fromoldandnewworldprimatestheyfoundahighdegreeofinterspeciesdivergenceandevidence,fromdN/dScomparisons,thatstrongpositiveselectionwasimportant.Theseobservationsmotivatedthemtoexploreintraspeciesdiversity,orpolymorphism,intheprimate<em>TRIM5</em>locus.Theybeganbysurveyingindividualsrepresentingthreespecies,anAfricanmonkey(sootymangabeys)andtwoAsianmonkeys(rhesusmacaquesandpig-tailmacaques).Theyweresurprisedtofindahighdegreeofpolymorphism,includingmultipleinstancesofsharedpolymorphism,Johnsonreported.Bothnon-synonymousandsynonymousSNPswereclusteredintworegionsofthegene,correspondingtotwodistinctdomainsintheTRIM5a protein.
Thehuman<em>TRIM5</em>locus,incontrast,wasmoremonomorphicoverallanddisplayednovariationsinsomeregionsthatwerehighlypolymorphicinotherprimates.Forexample,contemporaryhumanshaveanargeninealleleatpositionaa334,whereprimatesarepolymorphic.ThehumanalleleisassociatedwithsusceptibilityofcellstoHIVreplication,whileprimateswithaprolineinthispositionresistHIV.FormillionsofyearstheTRIM5a locushasbeenthesceneofastruggle,Johnsonspeculated,betweenpositiveselectionpressureandotherforcesdeterminedtomaintainmultipleallelesatintermediatefrequenciesformillionsofyears,evenasspeciesdiverged.
Itispossiblethatonlyhumanswiththeargenineallelesurvivedaprehistoricretroviralepidemic,Johnsonsaid,onlytohavetheselectionofthatTRIM5sequenceopenthedoortoAIDSmillionsofyearslater.Ifthatistrue,theTRIM5alphastoryisbiology’sversionofaGreektragedy,wherethehero’sstrengthultimatelyprovestobehisundoing.
<strong></strong>
<strong>ANon-codingRNAPotentiallyRegulating4q35GenesExpressioninFSHD</strong>
<palign=""left"">DavideGabellini</p>
<palign=""left"">DulbeccoTelethonInstituteatStemCellResearchInstitute,DIBIT-HSR,Milan</p>
<ahref=""mailto:[email protected]"">[email protected]</a>
Thethirdmostcommonformofmusculardystrophyisnamedforthebizarrewaysymptomsemerge,withpatientsgraduallylosingtheuseofmusclesintheface,shouldergirdleandupperarms.Calledfacioscapulohumeralmusculardystrophy(FSHD),thisautosomaldominantdiseasestrikesapproximatelyonein20,000people.Thecourseoftheillnessvaries,butthehallmarkmusclegroupswastegradually,othermusclesweakenandmanypatientslosemobilitywithage.TherearecurrentlynotreatmentsforFSHD.
ThegoalofDavideGabellini’slaboratoryistocharacterizethemolecularpathogenesisofFSHDanddevelopnewtherapies.In2002,hewaspartofateamthattracedthediseasetoadeletioninthe4q35regionofchromosomefour.Thisregioncontainsthemolecularequivalentofastudy-hallmonitor:itkeepsbadboysquietandintheirseats,butremoveitandtheygowild.
Thelostsegmentof4q35isnotpartofaprotein-codinggene,butoccursinanon-codingrepetitiveelementcalledD4Z4.Healthypeoplehave11to150copiesofthisrepeat;FSHDpatientshaveoneto11copies.D4Z4isheavilymethylated,hasheterochromaticfeatures,andcontainsatranscriptionalsilencerwhosedeletionpermitsinappropriateover-expressionofnearbyupstreamgenes.
GabelliniandhiscolleagueslateridentifiedthreegenesthatbecameboisterousincellswiththeFSHDdeletion.Oneofthem,dubbedFRG1(forFSHDregiongene1)causedFSHDsymptomswhenover-expressedintransgenicmice,theyreportedin<em>Nature</em>inDecember2006.Theresearchersalsodiscoveredaberrantalternativesplicinginspecificpre-mRNAsinthemusclesofFRG1transgenicmiceandFSHDpatients.
Additionalstudiesinpatientsamplesindicatethatpeoplewithearly-onset,severeFSHDhaveveryfewcopiesofD4Z4.Oddlyenough,peopledevoidofD4Z4arehealthyandsymptomfree.
AlthoughdeletingD4Z4issufficienttocreateamousemodelforFSHD,Gabellenisaysthelossofregulationofneighboringgenes,suchasFRG1,probablyinvolvesotherfactorsaswell.AsforFRG1,itisprobablyoneofagroupofgenesthataffectRNAsplicing,whichinturndetermineswhat
proteinsaremadebycells.Whilehisteamcontinuestoinvestigatethemolecularpathogenesisofthisfascinatingdisease,theyalsohopetouseFRG1transgenicmicetoscreenforpossibletherapies.