Gene regulation in eukaryotes 2.docx
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Gene regulation in eukaryotes 2.docx
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Generegulationineukaryotes2
VI.GenesilencingbymodificationofhistonesandDNA
Activatorsandrepressorsbindnearageneandswitchitonoroff.Theeffectsarelocal,andtheiractionsareoftencontrolledbyspecificextracellularsignals.
Genesilencingisapositioneffect—ageneissilencedbecauseofwhereitislocated,notinresponsetoaspecificenvironmentsignal.Also,silencingcanspreadoverlargestretchesofDNA,switchingoffmultiplegenes.
Themostcommonformofsilencingisassociatedwithheterochromatin.Inmammalincells,-50%ofgenomeisestimatedtobeinheterochromatin,includingtelomeres,centromeres.
真核生物转录调控以正调控为主,负调控为辅。
原核以负调控为主。
原核的负调控作用多是局部的效应。
并且是对特殊的环境作出应答,以适应环境。
而真核细胞所特有的的负调控方式-genesilencing(特点:
)1、是一种位置效应,因为某个基因在染色质中处于一定的位置(常染区或者异染区)因而被抑制,而不是对环境信号作出的应答。
2、可以沿染色质扩散到一个较大的区域,使大面积基因被抑制。
如异染色质区占大半。
Bothactivationandrepressionoftranscriptionofteninvolvemodificationofnucleosomestoalteraccessibilityofgene.
HeterochromaticsilencingcanbeinducedbyDNAmethylation.Thiskindofsilencingisn’tfoundinyeastbutiscommoninmammals.
DNAmethylationcaninhibitbindingofproteins,suchastranscriptionmachinery,orcanberecognizedbyrepressorsrecruitinghistinedeacetylase.
核小体的修饰一方面使组蛋白被共价修饰,另一方面与DNA的甲基化有关(都不涉及DNA序列的改变,但是都可以抑制基因表达)。
DNA甲基化可以抑制基因转录的原因是:
1、DNA甲基化可以提供一种识别信号,被特异的蛋白质结合将启动子封闭,使转录因子不能结合。
2、被repressor识别并结合后,repressor募集组蛋白去乙酰化酶,使组蛋白去乙酰化结合得更紧密。
Silencingatyeasttelomere:
deacetylationandhistonemethylation
Rap1recognizeandbindtelomererepeats,recruitSir(silentinformationregulator)complex.Sir2ishistonedeacetylase.
Deacetylationspreadsalongchromatin,producingextendedregionofheterochromatin.
Howisspreadslimitedtotelomereandothersilentregions?
HistonemethylationtransferasesattachmethylgroupstoLysintailsofhistonesH3andH4.H3methylationblocksSir2binding,andstopsspreading.
酵母端粒区高凝集状态的形成:
在端粒区有大量得重复序列,处于高度凝集得状态,成因:
有识别端粒区重复序列得蛋白质叫做Rap1,Rap1与端粒重复序列结合后可以募集Sir2(3,4)蛋白(silentinformationregulator)Sir2是一个组蛋白去乙酰化酶将附近低甲基化状态的组蛋白去乙酰化。
去乙酰化得组蛋白结合得更紧密,使端粒处于高凝集状态,另一方面,去乙酰化的组蛋白可以被Sir2直接识别,从而直接募集Sir2,这使得去乙酰化作用可以沿DNA扩散,使异染色质区扩大。
但是扩散的长度是受限制的,组蛋白甲基转移酶对此起作用,甲基转移酶可以使H3,H4的Ntail甲基化,甲基化的H3可以阻止Sir2的扩散。
Silencingmechanismofyeasttelomere
MultiplecopiesofRAP1bindtoasimplerepeatedsequenceateachtelomereregion,whichlacksnucleosomes(top).Thisnucleatestheassemblyofamultiproteincomplex(bottom)throughprotein-proteininteractionsbetweenRAP1,SIR2,SIR3,SIR4,andthehypoacetylatedN-terminaltailsofhistonesH3andH4ofnearbynucleosomes.SIR2deacetylatesthehistonetails.Theheterochromatinstructureateachtelomereencompasses≈4kbofDNAneighboringtheRAP1-bindingsites,irrespectiveofitssequence.Associationofseveralcondensedtelomeresformshigher-orderheterochromatincomplexes.
AbandDNAprobescolocalizeSIR3inyeastheterochromatin
(a)Confocalmicrograph0.3umthickthroughthreediploidyeastcells,eachcontaining68telomeres.Telomereswerelabeledbyhybridizationtoafluorescenttelomere-specificprobe(yellow).DNAwasstainedredtorevealthenuclei.The68telomerescoalesceintoamuchsmallernumberofregionsnearthenuclearperiphery.(b,c)Confocalmicrographsofyeastcellslabeledwithatelomerespecifichybridizationprobe(b)andafluorescent-labeledantibodyspecificforSIR3(c).NotethatSIR3islocalizedintherepressedtelomericheterochromatin.SimilarexperimentswithRAP1,SIR2,andSIR4haveshownthattheseproteinsalsocolocalizewiththerepressedtelomericheterochromatin.
组蛋白修饰酶可以被repressor和activator募集
Repressor-directedhistonedeacetylationofN-terminaltailsinyeast
TheDNAbindingdomain(DBD)oftherepressorUME6interactswithaspecificupstreamcontrolelement(URS1)ofthegenesitregulates.TheUME6repressiondomain(RD)bindsSIN3,asubunitofamultiproteincomplexthatincludesRPD3,ahistonedeacetylase.DeacetylationofhistoneN-terminaltailsonnucleosomesintheregionoftheUME6-bindingsiteinhibitsbindingofgeneraltranscriptionfactorsattheTATAbox,therebyrepressinggeneexpression.Activator-directedhyperacetylationofhistoneN-terminaltailsinyeast
TheDNA-bindingdomainoftheactivatorGCN4interactswithspecificupstreamactivatingsequences(UAS)ofthegenesitregulates.TheGCN4activationdomain(AD)theninteractswithamultiproteinhistoneacetylasecomplexthatincludestheGCN5catalyticsubunit.SubsequenthyperacetylationofhistoneNterminaltailsonnucleosomesinthevicinityoftheGCN4-bindingsitefacilitatesaccessofthegeneraltranscriptionfactorsrequiredforinitiation.Repressionandactivationofmanygenesinhighereukaryotesoccursbysimilarmechanisms.
组蛋白修饰酶可以被repressor和activator募集
Repressor或activator一般具有两个结构域,DBD或AD/RD,DBD结合特异的上游序列(UAS上游激活序列/URS上游抑制序列)AD/RD募集组蛋白修饰复合物,含有组蛋白修饰酶作为亚基,改变附近基因启动子去的甲基化状态。
使TATAbox暴露或封闭,使其更容易或不易接近TFIID。
Thechromatinimmunoprecipitationmethodcanrevealacetylationstateofhistonesinchromatin
Histonesarelightlycross-linkedtoDNAinvivousingacell-permeable,reversible,chemicalcrosslinkingagent.Nucleosomeswithacetylatedhistonetailsareshowningreen.Step1:
Cross-linkedchromatinisthenisolatedandshearedtoanaveragelengthoftwotothreenucleosomes.Step2:
Anantibodyagainstaparticularacetylatedhistonetailsequenceisadded,and(step3)boundnucleosomesareimmunoprecipitated.Step4:
DNAintheimmunoprecipitatedchromatinfragmentsisreleasedbyreversingthecross-linkandthenisquantitatedusingasensitivePCRmethod.ThemethodcanbeusedtoanalyzetheinvivoassociationofanyproteinwithaspecificsequenceofDNAbyusinganantibodyagainsttheproteinofinterestinstep2.
乙酰化的组蛋白有专门的抗体。
Histonecodehypithesis
组蛋白密码:
4种组蛋白的N端都存在多个特殊的修饰位点,可以转录后水平进行乙酰化,甲基化,磷酸化等修饰,这些修饰方式又存在多种组合方式,使组蛋白存在多种修饰状态,每种组蛋白的修饰状态不同意味着不同的含义,可以被不同的含有特殊结构域的蛋白质(或复合物)(bromodomain-acetyl,chromodomain-methyl)识别起到不同的调节基因表达的作用。
H3,H4的修饰比H2,H1重要。
Differentpatternofmodificationsonthetailscanbe“read”tomeandifferentthings:
resultsofdirecteffectsofmodification.
Specificpost-translationalmodificationsoftheN-terminaltailsinhistonesH3andH4arefoundineuchromatin,whichisaccessibletoproteinsandtranscriptionallyactive.Differentmodificationsarefoundinheterochromatin,whichiscondensedandthuslargelyinaccessibletoproteinsandtranscriptionallyinactive.
(不背)Lys9inH3tail:
Methylationheterochromatin
Acetylationsilencedregion
Unmodifiedsilencedregion
Lys4inH3tail:
Methylationincreasedtranscription
Insomeorganisms,thesamemethylatedLysbindsproteinstoestablishandmaintainheterochromaticstate,strongerthanthatassociatedwithdeacetylatedhistone.(组蛋白的甲基化对基因表达的负调控作用比组蛋白去乙酰化更强烈)
Chromodomain
克罗莫结构域,既染色质结构修饰结构域(chromatinorganizationmodifierdomain)真核生物蛋白质模体之一,高度保守,含30-50各氨基酸,其作用是结合甲基化的氨基酸残基,存在于动物、植物细胞和内参与调节染色质结构的若干蛋白质。
例:
果蝇HP1蛋白,特异识别H3N端tail中甲基化的Lys残基。
DNAmethylationisassociatedwithsilencedgenesinmammals
LargeregionofmammaliangenomearemarkedbyDNAmethylationandheterochromatic.
ThemethylatedsequencesareoftenrecognizedbyDNA-bindingproteins,suchasMeCP2,recruitinghistonedeacetylasesandhistonemethylasestomodifychromatin.
DNA甲基化后提供了DNA结合蛋白质的识别位点,如MeCP2,这些蛋白质与DNA结合后可以募集组蛋白修饰酶如组蛋白去乙酰化酶或组蛋白甲基化酶。
DNA甲基化对形成异染色质区和基因表达抑制中扮演重要角色。
人类基因组转座子占了45%,其中大部分是逆转座子,可以编码逆转座酶,如果任凭这些逆转座子编码逆转座酶,频繁转座基因组将遭破坏。
DNA甲基化使占基因组一半的转座子失活,不能表达转座酶,不能转座。
GeneoffbyDNAandhistonemodification
Unmodifiedgenereadilyon,butneverfirmlyshutoff.Thisisnotgoodenough:
sometimesithastobeoffpermanently.
ThisisachievedthroughmethylationoftheDNAandmodificationofthelocalnucleosomes.Thus,whenthegenesisnotbeingexpressed,aDNAmetyltransferase(amethylase)cangainaccessandmethylatecytosineswithinthepromotersequence,thegeneitself,andtheupstreamactivatorbindingsites.Themethylgroupisaddedtothe5’positioninthecytosinering,generating5-methylcytosine.Thismodificationalonecandisruptbindingofthetranscriptionmachineryandactivatorsinsomecases.Butitalsobindsotherproteins(forexample,MeCP2)thatrecognizeDNAsequencescontainingmethylcytosine.Theseproteins,inturn,recruitcomplexesthatremodelandmodifylocalnucelsomes,switchingoffexpressionofthegenecompletely.
只在组蛋白修饰的水平上抑制基因表达是不可靠的,因为是可逆的,有一些基因需要永远处于关闭状态,这时DNA甲基化是最可靠的方式。
通过DNA甲基化通过覆盖基因的启动子,上游调节序列以及基因本身,与特殊识别甲基化胞嘧啶的蛋白质(MeCP1)再募集组蛋白修饰酶,改变局部核小体结构,这样可以使有些基因永远关闭(如一些逆转座子编码逆转座酶的基因)
Metylationpatternsofglobingenes(血红蛋白基因)inhumanembryos
通过甲基化调节可以确保一些成簇的基因按一定的顺序表达。
如人胚胎发育过程中,6个月时,ε-globin启动子未甲基化ε-globin表达,而其下游的γ-globin启动子甲基化γ-globin被抑制不表达。
12个月时,ε-globin启动子甲基化ε-globin不再表达,而其下游的γ-globin启动子去甲基化γ-globin开始表达。
ε-globin是胚胎型血红蛋白,γ-globin是胎儿型血红蛋白。
Imprinting
印记的核心是DNA甲基化,印记是表观遗传调控的一个例子。
Imprintingdescribesachangeinagenethatoccursduringpassagethroughthespermoreggwiththeresultthatthepaternalandmaternalalleleshavedifferentpropertiesintheveryearlyembryo.MaybecausedbymethylationofDNA.Insituhybridization(cytologicalhybridization)
DNAmethylationliesattheheartofimprinting.
Indiploid,therearetwocopiesofmostgenes,oneinheritedfromfather,anotherfrommother,inmostcases,twoalletesareexpressedatcomparablelevelsbecausebothhavethedamesequencesandregulators.
Butthereareafewcaseswhereonecopyofageneisexpressedwhiletheotherissilent.
Imprintingisaexampleofepigeneticregulation.
表观遗传:
是指在生物减数分裂和有丝分裂过程中,基因表达发生了可遗传的变化,但是与孟德尔遗传学不同的是这种基因表达的变化不涉及基因编码序列本身的改变,而是除DNA编码序列之外的因素如:
组蛋白的修饰状态发生了可稳定遗传的改变,导致表型的改变。
表观遗传调控的主要机制包括:
DNA甲基化,组蛋白修饰(如甲基化,乙酰化,磷酸化)和非编码RNA的调控。
Imprintingdescribesachangeinagenethatoccursduringpassagethroughthespermoreggwiththeresul
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