基因组甲基化1

基因组甲基化LiuQingyou20080318主要内容基本概念基因组甲基化的原理基因组甲基化的研究方法基因组甲基化在胚胎发育中的模式与作用表观遗传学在基因组中除了DNA和RNA序列以外，还有许多调控基因的信息，它们虽然本身不改变基因的序列，但是可以通过基因修饰，蛋白质与蛋白质、DNA和其它分子的相互作用，而影响和调节遗传的基因的功能和特性，并且通过细胞分裂和增殖周期影响遗传。这就是表观遗传学（epigenetics），表观遗传学又称为实验遗传学、化学遗传学、特异性遗传学、后遗传学、表遗传学和基因外调节系统，它是生命科学中一个普遍而又十分重要的epigenetics是一种关于区别于DNA序列改变的基因表达可遗传变化基因组含有两类遗传信息,一类是传统意义上的遗传信息,即DNA序列所提供的遗传信息,另一类是表观遗传学信息,它提供了何时、何地、以何种方式去应用遗传信息的指令。组织细胞时空特异性的表达Nature441,143-145(11May2006)

CpGisolandCpG岛位于基因的启动子和第一个外显子区,富含CpG二核苷酸,故称为CpG岛,一般作为启动子和基因复制的起始区.在哺乳动物中,几乎所有的甲基化位点均在CpG二核苷酸,并通过对CpG的甲基化来调节基因的表达,因为DNA的甲基化可改变染色质的结构,从而引起不同DNA结合蛋白的结合。所有管家基因和4%组织特异性基因的5′端调控区存在CpG高频区CpG島（CpGislands）是指DNA上一個區域，此區域含有大量相聯的胞嘧啶（C）、鳥嘌呤（G），以及使兩者相連的磷酸酯鍵（p）。哺乳類基因中的啟動子上，含有約40%的CpG島（人類約70%）。一般CpG島的長度約300到3000個鹼基對（bp）。定義是指一個至少含有200bp的區域，其中GC所佔比例超過50%，且CpG的觀察值/預測值比例必須高於0.6。此部份的CpG島與基因相連，可用來作為限制酶的辨識位置。ZnFinger-DNAComplex

Helix-Turn-Helix

Motif

inDNABinding

DNAbindingThemodeofbindingismadesolelythroughinteractionswiththeDNAbackbone.Theplatinum-basedmolecules,“track”thebackboneofDNAthrougharepetitivemotif.ImageprovidedbyJ.Moniodis/VCU-UniversityofWesternAustralia.Farrell’sgroupmadethefindingwhileworkingtoimprovetheactionofcisplatin,oneofthemosteffectiveanti-cancerdrugsintheclinic.DiscoveryofthenewmodeofDNAbindingisthefirstsincetwoscientistsexaminingdruginteractionswithDNAmadeseparatediscoveriesinthe1960sand1970sthatsetthestandardfordrugdesignefforts.

鉑複合物需要電荷和順式位置的平衡才能表現其抗腫瘤效果前已知鉑複合物可以廣泛地抑制動物和人類的腫瘤DNA甲基化DNA甲基化是指真核生物以S-腺苷甲硫氨酸(SAM)作为甲基供体,在DNA甲基转移酶(DNAmethyltransferases,DNMTs)的作用下,将甲基转移到特定碱基C上,形成5甲基胞嘧啶(5mC)。FunctionsofDNAmethylationinmammals

TranscriptionalgenesilencingChromatincompactionGenomestabilitySuppressionofhomologousrecombinationbetweenrepeatsGenomedefense（抵御）Xchromosomeinactivation(females)Methylgroups

(red)inDNA

(mapby

Dr.CraigCooney)

DNAMethylationReactionCatalyzedbyDNAMethyltransferase(DNMT)

S-腺苷甲硫氨酸(SAM)MoleculargraphicsofmethylatedDNA

BacterialDNAmethyltransferaseintheactofcatalysis.

DNMT与DNA相互作用investigatingCalculationsofbindingaffinitiesforMGMT/inhibitorcomplexeswithcomputersimulationtechniquesDNA甲基化转移酶今后可能象限制性内切酶一样成为实验室常用的工具酶。来自立陶宛的HHMI研究人员Saulius

Klimasauskas领导的小组利用DNA甲基化转移酶把一些化学分子转移到DNA上，为基因诊断和纳米生物技术的应用奠定了基础，有关结果发表在《Nature

Chemical

Biology》。DNMT1themostabundantandcatalyticallyactiveenzymeinmostcelltypes,whichassociateswithS-phasereplicationfociviainteractionwithPCNA(Yokochietal.2002).

Itsprimaryroleisbelievedtobethatofamaintenancemethyltransferase(Bestor2000),copyingDNAmethylationpatternsfollowingDNAreplication.

MurineknockoutsofDnmt1areembryoniclethalatdayE8.5.

DNMT2functionremainsunclearsinceitpossessesverylowenzymaticactivity

invitroandknockoutofthegeneinmiceproducesnodiscernablephenotype(Okanoetal.1998;Yoderetal.1998;Hermannetal.2003).

DNMT3AandDNMT3Bdenovomethyltransferasessincetheyarehighlyexpressedatthestageofmurineembryonicdevelopmentwhenwavesofdenovomethylationareoccurringinthegenome(Okanoetal.1999).

MurineDnmt3aknockoutmicearebornlivebutdiebeforereachingfourweeksofage.

Dnmt3bknockoutmiceareembryoniclethalby~dayE14.5.

Dnmt3aknockoutmiceexhibitsubtleDNAmethylationdefectsinmaternallyimprintedregions(Hataetal.2002),whileDnmt3bknockoutmiceshowmarkeddemethylationofpericentromericsatelliterepeats(Okanoetal.1999).

knockoutofDnmt3L,whichisnotafunctionalenzymeduetolackofcriticalcatalyticsitemotifs,resultsinmaternalDNAmethylationimprintfailureandmalesterilityinmice(Hataetal.2002).TherearecurrentlyfivemembersoftheDNAmethyltransferasefamilyinmammaliancells.

AlloftheseproteinshavetheircatalyticdomainintheC-terminalregion,and(withtheexceptionofDNMT2)aregulatorydomainintheN-terminalregion.

Mostoftheprotein-proteininteractionsaremediatedbytheN-terminalregion.DNAmethyltransferasesinteractwithmanyotherproteins.

ProteinsinvolvedinmodulatingchromatinstructureincludepRb,HDACs,HP1,SIN3A,hSNF2H,SUV39H1,andhCAP-C/E.

Transcriptionallyactivechromatinregionstendtobehyperacetylatedandhypomethylated.

IfaregionofDNAorageneisdestinedforsilencing,chromatinremodelingenzymessuchashistonedeacetylasesandATP-dependentchromatinremodelerslikelybeginthegenesilencingprocess.

OneormoreoftheseactivitiesmayrecruitDNAmethyltransferaseresultinginDNAmethylation,followedfinallybyrecruitmentofthemethyl-CpGbindingproteins.

TheregionofDNAwillthenbeheritablymaintainedinaninactivestate.

DNM与染色质remodelDNAMethylationasaBiomarkerforCancerLossofnormalDNAmethylationpatternsinsomaticcellsresultsinlossofcellgrowthcontrol.

Innormalcells,DNAmethylationisfoundpredominantlyintherepetitivefractionofthegenome,includingsatelliteDNAandparasiticelements(LINES,SINES,endogenousretroviruses)(Yoderetal.1997).

Tumorcellsexhibitglobalhypomethylationofthegenomeaccompaniedbyregion-specifichypermethylationevents(Baylinetal.2001).

MostofthehypomethylationoccursinrepetitiveDNAthatisnormallyheavilymethylated(Yoderetal.1997).

Innormalcells(top)DNAmethylationisconcentratedinrepetitiveregionsofthegenomeandmostCpGislandpromotersareunmethylated.

Intumorcells,thecompartmentalizationbreaksdownandrepetitiveDNAlosesmethylationwhileCpGislandpromotersacquireit,resultinginsilencingoftheassociatedgene.

TheDNMTsarelikelytargetedtoparticularregionsviaprotein-proteininteractionswithinchromatin.DNAMethylationandChromatinRemodelingModificationstothechromatinandthecorehistone‘tails’impactgeneexpressionandtheabilityofproteinsthatactonDNAtoaccesstheirtargetsequences.

Recentstudiesindicatethattheseepigeneticprocesses,aswellaschromatinremodeling,cooperatetocontrolchromatinstructureandultimatelygeneexpressionandDNAmethylationitself.

ChromatinremodelingproteinsoftheSNF2family,whichutilizeATPtoalterthestructureofchromatinthroughdisruptionofhistone/DNAcontacts(Havasetal.2001),alsoinfluenceDNAmethylationpatterns.

mammalianLsh,SNF2helicasefamilymemberexertsprofoundeffectsonDNAmethylationpatterns.

Lsh

knockoutmiceexhibitdrasticlossesofDNAmethylation,bothinrepetitiveelementsand