分子医学-表观遗传学

Epigenetics

ClassicalGenetics:TheCentralDogmaIntroductiontoEpigeneticsTheHumanGenomeProject(HGP)Keyfindings:approx.24,000genesinhumanbeingallhumanracesare99.99%alikeChallenges:about24,000genesvs.100,000proteinsnon-coding(98%)vs.codingDNA(2%)genotypevs.phenotypeIntroductiontoEpigeneticsTheymaylookthesame,butarethey?Position-effectvariegationParamutationatthemaizeEpigeneticbiologyIntroductiontoEpigeneticsEpigeneticdifferencesariseduringthelifetimeofmonozygotictwins5mCAcH4AcH3PNAS,July26,2005,Vol.102IntroductiontoEpigeneticsEpigeneticsDefinition:Thestudyofanypotentiallystableand,ideally,heritablechangeingeneexpressionorcellularphenotypethatoccurswithoutchangesinWatson-Crickbase-pairingofDNA.Oneexample:cellulardifferentiation/developmenttotipotentstemcellsbecomethevariouspluripotentcelllinesoftheembryowhichinturnbecomefullydifferentiatedcellsIntroductiontoEpigeneticsChange:Geneticsvs.EpigeneticsIntroductiontoEpigenetics1938,HermannMullerfirstdescribedthePosition-effectvariegation1942,ConradWaddington

definedepigeneticsas

“thebranch

ofbiologywhichstudiesthecausal

interactionsbetweengenesandtheir

products,whichbringthephenotype

intobeing”1950s,R.BrinkdiscoveredParamutationinmaize1961,MaryLyonproposedtherandominactivationofonefemaleXchromosometoexplainthemottledphenotypeoffemalemiceheterozygousforcoatcolorgenes1983,discoveredtheDNAmethylation……BriefHistoryofEpigeneticsIntroductiontoEpigeneticsEpigeneticsEventsparamutationbookmarkingimprintinggenesilencingXchromosomeinactivationpositioneffectreprogrammingtransvectionmaternaleffectsIntroductiontoEpigeneticsEpigeneticResearch:techniquesChIP;ChIP-on-chip;ChIP-seqfluorescentinsituhybridizationmethylation-sensitiverestrictionenzymes

HpaII(CCGG)andMspI(CCGG)DNAadeninemethyltransferaseidentification(DamID)NatProtoc,2007,2(6):1467–78.

bisulfitesequencing.computationalepigenetics……IntroductiontoEpigeneticsEpigeneticResearch:futureNoME-Seq:NucleosomeOccupancyandMethylomeSequencingoxBS-Seq:OxidativeBisulfiteSequencingBisChip-seq/ChIPBS-Seq:CombinedBisulfiteSequencing&ChromatinImmunoprecipitation

informmethylationstatusofhistone-modifiedDNANanofluidicEpigeneticAnalysis:SCAN(SingleChromatinAnalysisattheNanoscale)&NanofluidicDNAMethylationDetectionIntroductiontoEpigeneticsTheFocusesofEpigeneticReseachIntroductiontoEpigenetics

GenomicLevel

ChromatinRemodeling DNAmodificaition Histonemodification othermodifications

X-chromosomeinactivation:

DNAmethylation;ncRNA

genomicimprinting:

DNAmethylation;ncRNA

……RNAlevel

ncRNA RNAi(RNAInterference)&miRNA(microRNA) lncRNA RNA-interactingprotein ……TheLevelsofEpigeneticReseachIntroductiontoEpigeneticsMolecularinteractionDNAmodification

ProteinmodificationncRNAregulation

RNAmodification

ChromatinRemodelingThenucleosomeThespacingandpositioningofnucleosomesatcriticalcis-actingcontrolregions(i.e.,promoters)aretailoredfortheproperregulationofthelocus.Higher-OrderStructuringofChromatinChromatinRemodelingTheStructureofaNucleosomeEuchromatinvs.HeterochromatinChromatinRemodelingChromatinRemodelingChromatin-remodeling:Dynamicstructuralchangestothechromatinoccurringthroughoutthecelldivisioncycle.ThesechangesrangefromthelocalchangesnecessaryfortranscriptionalregulationtoglobalchangesnecessaryforchromosomesegregationSevendistinctmechanismsofchromatinremodelingChromatinRemodelingChromatinRemodelerNon-covalentmannerChromatinremodelingcomplexesHistonevariantsCovalentmannerHistonemodificationDNAmethylationNon-codingRNA

Fact:workcoordinatelyChromatinRemodeling:mechanismsChromatinremodeler:Non-covalentmannerareATP-hydrolyzingmachines

Functions: fullypackagethegenome restructure,mobilizeorejectnucleosomes specializechromatinregions provideregulatedDNAaccessibilityin packagedregions.ChromatinRemodelerChromatinremodelerFamiliesAllcontainaSWI2/SNF2-familyATPasesubunitcharacterizedbyanATPasedomainthatissplitintwoparts:DExx(red)andHELICc(orange).ChromatinRemodelerChromatinremodeler:acomplexChromatinRemodelerTheactionsofremodelersonnucleosomesChromatinRemodelerTheactionsofremodelersonnucleosomesChromatinRemodelerChromatinRemodelingChromatinRemodeling:covalentmannerDNAmethylation:isatypeofchemicalmodificationofDNAthatcanbeinheritedandsubsequentlyremovedwithoutchangingtheoriginalDNAsequenceHistoneModifications:changethechromatintemplatebycis-effectsthatalterinternucleosomalcontactsandspacing,orthetrans-effectscausedbyhistoneandnon-histoneproteinassociationswiththetemplate.ChromatinRemodelingMethylation(甲基化)

DNAmethylation:typicallyoccursatCpGsites

invertebrates catalyzedbytheenzymeDNAmethyltransferase.

DNAmethylationHypomethylationHypermethylation/reviews7/Taylor1.jpg

ImprintingXchromosomeinactivationHeterochromatinmaintenanceDevelopmentalcontrolsTissuespecificexpressioncontrolsTheRolesofDNAMethylationDNAmethylationDNAmethylationanddemethylation

inalifecycleDNAmethylationRegulationofGeneExpressionbyDNAmethylationInterferencewithtranscriptionfactorbindingAttractionofMethyl-CpG-bindingproteinsDNAmethylationDNAMethylationandCancerNatureReviewsGenetics,6:597DNAmethylationDNAMethylationandOtherHumanDiseases--ImprintingDisorder:Beckwith-Wiedemannsyndrom(BWS)Prader-Willisyndrome(PWS)Transientneonataldiabetesmellitus(TNDM)--Repeat-instabilitydiseasesFragileXsyndrome(FRAXA)Facioscapulohumeralmusculardystroph--DefectsofthemethylationmachinerySystemiclupuserythemtosus(SLE)Immunodeficiency,centromericinstabilityandfacialanomalies(ICF)syndromeDNAmethylationDNAMethylationAnalysis

BisulfiteGenomicSequencing(GoldStandard)PyrosequencingTheHELPassay(CCGGHpaII-CCGGMspI)ChIP-on-chipassaysMethylatedDNAimmunoprecipitation(MeDIP)MolecularbreaklightassayforDNAadeninemethyltransferaseactivity……Formoreinformation,see/wiki/DNA_methylationBisulfite-mediatedconversionofC

U

BisulfiteGenomicSequencingNucleotidesinblueareunmethylatedcytosinesconvertedtouracilsbybisulfite,whilerednucleotidesare5-methylcytosinesresistanttoconversion.

DNAmethylationanalysismethodsnotbasedonmethylation-specificPCR

Followingbisulfiteconversion,thegenomicDNAisamplifiedwithPCRthatdoesnotdiscriminatebetweenmethylatedandnon-methylatedsequences.Thenumerousmethodsavailablearethenusedtomakethediscriminationbasedonthechangeswithintheampliconasaresultofbisulfiteconversion.Formoreinformation,see/wiki/Bisulfite_sequencingBeCareful

Beaware:theprocessofdemethylationof5mC5mC 5-hydroxymethylcytosine(5hmC) 5-formylcytosine(5fC) 5-carboxylcytosine(5caC) C 5mCand5hmC,butnot5fCor5caC,arebothresistanttobisulfiteconversion

MethylatedDNAimmunoprecipitation(MeDIP)

TimelineofSequencing-BasedTechnologiesforMappingHumanEpigenomesCell,2013,155:39CoordinatedmodificationofchromatinThetransitionofanaïvechromatintemplatetoactiveeuchromatin(left)ortheestablishmentofrepressiveheterochromatin(right),involvingaseriesofcoordinatedchromatinmodification.Inthecaseoftranscriptionalactivation,thisisaccompaniedbytheactionofnucleosome-remodelingcomplexesandreplacementofcorehistoneswithhistonevariants(yellow)ChromatinRemodelingRNAEpigeneticsNatChemBiol,2011,7(12):885-7.N6-methyladenosineinnuclearRNAisamajorsubstrateoftheobesity-associatedFTO.fatmassandobesity-associatedprotein(FTO):oxidativedemethylationactivityDatademonstratethatmethylationofRNAhasacriticalroleinhumanenergyhomeostasisRNAModifica