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General Mechanisms of Transcriptional Regulation General Mechanisms of Transcriptional Regulation in Eukaryotes 真核基因表达概况：时空多样性真核基因表达概况：时空多样性真核基因表达概况：时空多样性真核基因表达概况：时空多样性 研究基因表达的手段：基因芯片、蛋白电泳、二维电泳、研究基因表达的手段：基因芯片、蛋白电泳、二维电泳、研究基因表达的手段：基因芯片、蛋白电泳、二维电泳、研究基因表达的手段：基因芯片、蛋白电泳、二维电泳、WESTERN BLOTWESTERN BLOT 真原核转录调控的异同真原核转录调控的异同真原核转录调控的异同真原核转录调控的异同 真核染色质结构对基因表达的影响（组蛋白修饰、核小体动态结构）真核染色质结构对基因表达的影响（组蛋白修饰、核小体动态结构）真核染色质结构对基因表达的影响（组蛋白修饰、核小体动态结构）真核染色质结构对基因表达的影响（组蛋白修饰、核小体动态结构）真核基因的调控序列：增强子、绝缘子真核基因的调控序列：增强子、绝缘子真核基因的调控序列：增强子、绝缘子真核基因的调控序列：增强子、绝缘子 Eukaryotic Eukaryotic Transcriptional Transcriptional ActivatorsActivators Eukaryotic Eukaryotic Transcriptional RepressorsTranscriptional Repressors Signal Integration and Combinatorial ControlSignal Integration and Combinatorial Control Signal Transduction and the Control of Transcriptional RegulatorsSignal Transduction and the Control of Transcriptional Regulators Gene Silencing by Modification of Histones and DNAGene Silencing by Modification of Histones and DNA Epigenetic Gene RegulationEpigenetic Gene Regulation Gene Regulation in EukaryotesCell Types are highly diversifiedCell Types are highly diversified and Different Cell Different Cell Types Types Synthesize Different Sets of RNAsSynthesize Different Sets of RNAsmRNA levels of 1800 genes(top to bottom)in 142 different human tumor cell lines(Ieft to right),each from a different patientEach type of tumor has a characteristic gene expression pattern.Gene expression Gene expression profilingprofiling:RNA level detection RNA level detection using DNA microarraysusing DNA microarraysDifferent Cell Types Synthesize Different Sets of ProteinsDifferent Sets of ProteinsAt any given time in a cells life history,only a fraction of the only a fraction of the RNAs and proteinsRNAs and proteins encoded in its genome are expressedProtein 2-D electrophorysis4PAGE -Polyacrylamide Gel Electrophoresis2-D electrophorysis 2-D electrophorysis=SDS-PAGE+Isoelectric FocusingSDSProteinSDS-Sodium Dodecyl Sulfate:an ionic detergent 1.4 g of SDS binds 1 g of proteinSDS and ProteinsIn the presence of SDS,all proteins have uniform shape and charge per unit lengthSDS nonpolar chains arrange themselves on proteins destroy secondary,tertiary and quarternary structrureThe protein SDS complex becomes rod shapedBecause of a constant mass-to-charge ratio and the presence of a sieving polymer matrix,the molecules are separated by size巯基乙醇8Western blot10Comparison of Southern,Northern and Western bolt hybridizationIsoelectric FocusingEach protein has its pIEach protein has its pIContinuous and stable pH gradientContinuous and stable pH gradient2D-Electrophoresis2D-electrophoresis allows separation of proteins by bothboth sizesize and isoelectric pointisoelectric point.Each spot represents a different proteinEach spot represents a different protein.13Proteomics:IdentificationIdentification of the full setfull set of proteins produced by a cell or a tissue under a particular set of conditions1.2-D gel electrophoresis for protein separationprotein separation 2.MMassass spectrometry spectrometry for precise determinationprecise determination of the molecular weight and identity of a protein 3.Bioinformatics for assigning assigning proteins and peptides to the predicted products of protein coding sequence in the genome Features of the eukaryotic genomes and their expression1.1.Much largerMuch larger and and highly packagedhighly packaged genomes;genomes;2.2.mRNAs are mono-cistrons mRNAs are mono-cistrons;Split genes Split genes andand postposttranscriptional modificationtranscriptional modification3.3.UnUncoupled transcription coupled transcription andand translation translation4.4.RNA polymerase II(RNAPII)is much larger RNA polymerase II(RNAPII)is much larger andand more complex more complex-it must synthesize RNA-it must synthesize RNA and coordinate the special processing events and coordinate the special processing events 5.5.Patterns of gene expression Patterns of gene expression can be can be extraextraordinarily complex:ordinarily complex:基因表达时空特异性基因表达时空特异性基因表达时空特异性基因表达时空特异性6.6.Totipotency Totipotency:细胞全能性细胞全能性细胞全能性细胞全能性细胞的全能性细胞的全能性细胞的全能性细胞的全能性(totipotency)(totipotency)指细胞经分裂和指细胞经分裂和指细胞经分裂和指细胞经分裂和分化后仍具有产生完整有机体的潜能分化后仍具有产生完整有机体的潜能分化后仍具有产生完整有机体的潜能分化后仍具有产生完整有机体的潜能 Binding of RNAP II to promoter does notdoes not produce efficient transcription by itselfTranscription requires binding of GTFs,GTFs,usually constitutively expressed usually constitutively expressed,to promoter-proximal elementspromoter-proximal elementsEukaryotic Eukaryotic transcriptional machinery is more elaboratetranscriptional machinery is more elaborate:17SimilaritiesSimilarities1.Principles are the same:signalssignals /activators and repressorsrecruitment and allostery,cooperative binding 2.Transcription initiation is the most pervasively(普遍地普遍地)regulated step.General MechanismsGeneral Mechanisms in in transcription transcription regulationregulation:SimilaritiesSimilarities/differences differences signalssignalsRNAPRNAPRegulatorRegulatorDifferenceDifferences:s:In bacteria:naked DNAnaked DNA/single activator single activator oror repressor protein/repressor protein/operonsoperons In eukaryotes:accessaccess to promoters is restricted by nucleosomes and their modifiers more numerous proteins more numerous proteins andand DNA sequences DNA sequences participating in gene regulation few operonto to expose promoter sequences expose promoter sequences or to or to recruit RNAP II directly recruit RNAP II directly through bindingthrough bindingIn bacteria,RNAP can usually In bacteria,RNAP can usually begin transcription begin transcription unless a unless a repressor protein blocks itrepressor protein blocks itActivator proteins increase the Activator proteins increase the binding of RNAP to promoters binding of RNAP to promoters where a little help is neededwhere a little help is needed.The packaging of DNA with The packaging of DNA with nucleosomes nucleosomes preventsprevents the the RNAP IIRNAP II-GTFs bindingGTFs binding,unless unless other regulatory other regulatory proteins are presentproteins are present.General MechanismsGeneral Mechanisms in transcription regulation:in transcription regulation:SimilaritiesSimilarities/differences differences The eukaryotic The eukaryotic chromatin Packaging chromatin Packaging play important play important role inrole in transcription regulationtranscription regulationHistone modificationsHistone modifications affect chromatin packaging chromatin packaging and gene expressiongene expression PhosphorylationPhosphorylation,MMethylation,ethylation,Acetylation Acetylation LYSINE LYSINE ACETYLATIONACETYLATION ANDAND METHYLATIONMETHYLATION ARE ARE COMPETING REACTIONSCOMPETING REACTIONS.and their meanings are differentMeanings of histones modificationsof histones modifications-the histone codethe histone codeWWriting riting and R Readingeading histone code histone code:modifications of the tails are recognized by various proteins recognized by various proteins R Readingeading and wwritingriting the histone code the histone code during transcription initiationtranscription initiationThe nucleosome positioning has some rules:and DNA structure preferencethrough protein interactionNucleosomes have dynamic structuresSliding Nucleosome position can be changed:remodelingTransfering Eukaryotic genes haveEukaryotic genes have more more regulatory binding sitesregulatory binding sites and are and are controlledcontrolled by more by more regulatory proteinsregulatory proteins Enhancers Enhancers are bound by are bound by Specific TFsSpecific TFs that regulate that regulate smaller subsets of genes,smaller subsets of genes,throughthrough controlling access of GTFs and RNAP II.controlling access of GTFs and RNAP II.Often,an enhancer will act in Often,an enhancer will act in only one only one oror a few cell types a few cell types.Promoters Promoters andand promoter-promoter-proximal elementsproximal elements are are bound by bound by GTFsGTFs that affect that affect expression of expression of many genesmany genes.Multicellular organismsMulticellular organisms have moremore regulatory sequences,usually further further awayaway from promoter,than yeast30vvEnhancerEnhancer:a short(50-1500 bp)region of a short(50-1500 bp)region of DNADNA that can be bound with specific specific TFTFs s to activate gene transcription.vThere are hundreds of thousandshundreds of thousands of enhancers in the human genome.Forming loops:Forming loops:1.Some proteins(TFs)bind to DNA sites between the enhancer and the promoter to form multiple miniloopsmultiple miniloops,bringing the promoter and enhancer into closer bringing the promoter and enhancer into closer proximityproximity.2.2.SMC(Structural Maintenance of Chromosomes)proteins can stabilizstabilize e enhancer enhancer promoter loopspromoter loops Enhancers can actEnhancers can act at great distances at great distances(1Mb),(1Mb),can be can be upup-/-/downstream downstream,and in,and in eithereither directiondirection,when modulating activity of the transcriptional apparatus.when modulating activity of the transcriptional apparatus.Enhancers may containcontain binding sites binding sites for many transcription factorsmany transcription factors,which bind and interact cooperativelycooperatively.CohesinCohesin glues sister chromatids together,glues sister chromatids together,condensincondensin reorganizes chromatin into higher reorganizes chromatin into higher structure.structure.SMC proteins:SMC proteins:Structural Maintenance of Chromosomes Structural Maintenance of ChromosomesEnhanceosome:Enhanceosome:large protein complex,acting s synergisticallyynergistically to activate transcription;formed by formed by binding ofbinding of multiple regulatory proteins multiple regulatory proteins to the to the multiple binding sites inmultiple binding sites in an enhanceran enhancerInsulators:Regulatory sequencesRegulatory sequences between enhancersenhancers and promoterspromoters,ensuring activatorsactivators to work discriminatelydiscriminatelyHow to avoid the long-distance crosstalk?How to avoid the long-distance crosstalk?Insulators are recognized by specific DNA-binding proteinsspecific DNA-binding proteins which bind cohesincohesin to form chromatin loop chromatin loop containing only enhancer containing only enhancer andand the specific the specific promoterpromoter,preventing enhancers from forming loops with other promotersp经常不断地学习,你就什么都知道。你知道得越多,你就越有力量pStudyConstantly,AndYouWillKnowEverything.TheMoreYouKnow,TheMorePowerfulYouWillBe写在最后谢谢你的到来学习并没有结束，希望大家继续努力Learning Is Not Over.I Hope You Will Continue To Work Hard演讲人：XXXXXX 时 间：XX年XX月XX日
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