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:jaenisch@wi.mit.edu"">jaenisch@wi.mit.edu</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>

&nbsp;

<strong>Presentations</strong>

<strong>SystemsAnalysisofHumanMitochondrialDisorders</strong>

VamsiK.Mootha,MD

DepartmentofSystemsBiology,HarvardMedicalSchool,

CenterforHumanGeneticResearch,MassachusettsGeneralHospital,andtheBroadInstituteofMITandHarvard

<ahref=""mailto:vmootha@broad.mit.edu"">vmootha@broad.mit.edu</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:stefano.casola@ifom-ieo-campus.it"">stefano.casola@ifom-ieo-campus.it</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:bruno.amati@ifom.ieo.campus.it"">bruno.amati@ifom.ieo.campus.it</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.

&nbsp;

<strong>SonicHedgehog—ProteoglycanInteractionsinDevelopmentandDisease</strong>

RosalindSegal

DepartmentofNeurobiology,HarvardMedicalSchoolandDepartmentofPediatricOncology,Dana-FarberCancerInstitute

<ahref=""mailto:rosalind_segal@dfci.harvard.edu"">rosalind_segal@dfci.harvard.edu</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:m.fanto@hsr.it"">m.fanto@hsr.it</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.

&nbsp;

<strong>TargetingActiveGenesforHistoneModificationin<em>Drosophila</em></strong>

MitziI.Kuroda

HowardHughesMedicalInstitute,Harvard-PartnersCenterforGeneticsandGenomics,Brigham&amp;Women’sHospitalandDepartmentofGenetics,HarvardMedicalSchool

<ahref=""mailto:mkuroda@genetics.med.harvard.edu"">mkuroda@genetics.med.harvard.edu</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:yshi@hms.harvard.edu"">yshi@hms.harvard.edu</a>

“Understandingbiologicalimportanceisthenextstep,”YangShisaidattheendofhis2006symposiumpresentation.Hislabpioneeredtheideathathistonemethylationisdynamicwhentheyclonedthefirstdemethylasein2004.Initiallytheycouldremoveonlyoneortwomethylgroupsfromalysineresidue,anditseemedthattrimethylgroupsmightbepermanentlyattached.Thiswasdiscouraging,becausethistypeofhistonemodificationappearstolockgenesassociatedwithvariouscancersandneurologicabnormalitiesinthe“on”position.

Then,shortlybeforethe10^thArmenise-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:dcorona@unipa.it"">dcorona@unipa.it</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.”

&nbsp;

<strong>PossibleRolesinSilencingforpiRNAs</strong>

BobKingston

MassachusettsGeneralHospital,Boston

<ahref=""mailto:kingston@molbio.mgh.harvard.edu"">kingston@molbio.mgh.harvard.edu</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(&lt;100kb)genomicloci;atthesesitestheyadhereeithertoWatsonorCrickstrands,oriftheybindbothstrandstheydonotoverlap.Theselocations,Kingstonsaid,appeartobeconservedacrossspecies.

PreparationsofpiRCalsohookupwithrRecQ1,theratequivalentofahelicasethathelpssilencegenesin<em>Neurospora</em>.RecombinantRecQ1andPiwifamilyproteinsappeartointeractdirectly,andinfliesPiwihasbeengeneticallylinkedtotranscriptionalgenesilencing.ExperimentsshowthatthepurifiedcomplexhaspiRNA-directedsliceractivity,whichaddsweighttotheideathatpiRCisaheritablesilencerofmammaliangenes.Kingstonsaidmoreevidenceisneededtoconfirmthis.

&nbsp;

<strong>TheRoleofChromosome3DOrganizationinPolycomb-DependentEpigeneticRegulation</strong>

ValerioOrlando

DulbeccoTelethonInstituteatIGBCNR,NaplesItaly

<ahref=""mailto:orlando@igb.cnr.it"">orlando@igb.cnr.it</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.

&nbsp;

<strong>AChromosome-AssociatedSmallRNAAmplificationLoopRequiredforHeterochromatinFormation</strong>

DaneshMoazed

DepartmentofCellBiology,HarvardMedicalSchool

<ahref=""mailto:danesh@hms.harvard.edu"">danesh@hms.harvard.edu</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.

&nbsp;

<strong>HomologuePairing:RegulatingGenesandDrivingEvolution</strong>

TingWu

DivisionsofGeneticsandMolecularMedicine,HarvardMedicalSchool,

<ahref=""mailto:twu@genetics.med.harvard.edu"">twu@genetics.med.harvard.edu</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&lt;10^-8)andsixofthesevensetsofCNVs(P&lt;10^-4).Theresultsareconsistentwith“astringentwatchingofcopynumbers,”Wusaid.

FutureexperimentswilllookforspecificmotifswithinUCEsandexplorewhethertheirduplicationordeletionisassociatedwithlossoffitnessordeath.

&nbsp;

<strong>TheChicken-and-EggRelationshipbetweenTranscriptionandChromatinandImplicationsforEpigeneticInheritance</strong>

KevinStruhl

DepartmentofBiologicalChemistryandMolecularPharmacology,HarvardMedicalSchool

<ahref=""mailto:kevin@hms.harvard.edu"">kevin@hms.harvard.edu</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.”

&nbsp;

<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.

&nbsp;

<strong>GrowthPropertiesofNormalandTransformedBreastStemCells</strong>

AngeloCicalese

DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan

<ahref=""mailto:angelo.cicalese@ifom-ieo-campus.it"">angelo.cicalese@ifom-ieo-campus.it</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.

&nbsp;

<strong>ExitfromMitosisinBuddingYeastSaccharomycescerevisiae</strong>

RosellaVisintin

DepartmentofExperimentalOncology,EuropeanInstituteofOncology,Milan

<ahref=""mailto:rosella.visintin@ifom-ieo-campus.it"">rosella.visintin@ifom-ieo-campus.it</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:wjohnson@hms.harvard.edu"">wjohnson@hms.harvard.edu</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:Gabellini.davide@hsr.it"">Gabellini.davide@hsr.it</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.