講義分子課件2013bio2000liqing

1、2013.9.25 Bio2000生命科學學院: li.qingChromatin Assembly & Epigenetic InheritanceEpigenetics 表觀遺傳學Chromatin染色質Chromatin Assembly 染色質組裝Histone組蛋白Histone modifications 組蛋白修飾Histone Variants 組蛋白變體Histone chaperone 組蛋白Nucleosome 核小體Euchromatin 常染色質Heterochromatin 異染色質伴侶Chromatin reming complex 染色質重塑因子DNAm

2、odificationsRNA-based mechanismsGenomeEpigenomeHistone modifications and variantsModified from Baylin and Schuebel, Nature (2007)Epigenetic information is stored in chromatinDNAGenomeHistone chaperoneNucleosomeHistones: H3, H4, H2A and H2B+147bp DNAChromatinEpigenomeH2A-H2BH3-H4Nucleosomethe buildin

3、g block of chromatinThe NucleosomeAcetylation of H3 lysine 56 (H3K56Ac)Phosphorylation AcetylationMethylation UbiquitinationPost translational modifications on histones are one type of epigenetic marksLugar et al., Nature 1997DNAreplicationDaughter chromosomeReassembled chromatinGene TranscriptionCh

4、romatin AssemblyRepaired chromosomeRegenerated or specialized chromatinDNA repairHistone turnover/ ExchangeChromatin assembly is important for chromatin structureF Histone chaperones-Depositing new histones and transferring old histonesF Histone modifications-Regulating nucleosome assemblyF Chromati

5、n rem nucelsomesing factorsPositioningKey players in nucleosome assembly:A group of Histone interacting proteins which can stimulate reactions involving histone transfer without being part of the final product.Possible role of Histone chaperones:1.2.3.StorageHistone exchangeHistone deposition (Speci

6、fic variants/Specialized regions)Nucleosome assembly4.Nucleoplasmin, N1/N2, NAP-1, NAP-2, Spt6, Asf1, HIRA, CAF-1, Rtt106, DaxxHistone chaperonesEpigenetics 表觀遺傳學Chromatin染色質Euchromatin 常染色質Heterochromatin 異染色質Transcriptional silencing 轉錄沉默(2)Chromosome foldingNLevel 2:30 nm fiberLevel 3: Nuclear sc

7、affoldingLevel 4: Mitotic (metaphase)chromosomeLevel 1: ucleosome formation?An amazing architectural feat: multiples levels of DNA packagCinhgroma/n packaging heirarchyNucleosome positioning1. ATP dependent rem complexesing2.DNA binding factors3.DNA sequenceWhat positions nucleosomes?MonoDualMonoMon

8、oBasic repeating structure can be probed (protect and seq method)- Digestion enzyme cuts accessible regions of DNA- DNA protected by nucleosome is not cutNucleosome positioning/ OccupancyPartial micrococcal nuclease digestion generates mono, di, trinucleosomes(Furuyama and Biggins ,PNAS)Micrococal n

9、uclease digestionUnderlying DNA sequence has apredominant influence on occupancylevels in vivo.Segal, E. Kaplan, N. Nature 442, 772778 (2006). Nature 458, 362366 (2009)Nucleosome positioning & Nucleosome occupancyHow nucleosomes become uniformly spaced and precisely positioned in vivo?B Franklin

10、 Pugh NSMB 17,923 2010At the transcription start sites. Distinct nucleosome organization at the 5 end of genes. Whitehouse., 2007 Nature 450, 1031-1035(Toshio Tsukiyama Lab)ATP-dependent chromatin-reming complexes regulategene expression by either moving, ejecting or restructuring nucleosomes.Possib

11、le roles:By hydrolysis of ATP allowing these reming complexes toreposition (slide, twist or loop) nucleosomes along the DNA, expel histones away from DNA or facilitate exchange of histone variants, and thus creating nucleosome-free regions of DNA for gene activationFive major families:SWI/SNF, ISWI,

12、 NuRD/Mi-2/CHD, INO80 and SWR1ATP-dependent chromatin reming complexISW2-ATPdependent DNA translocase involved in chromatinremingTsukiyama T, Whitehouse. (1999), Genes Dev 13(6):686-97;., Nature 450, 1031-1035Loss of ISW2 leads to directional nucleosome repositioning at the 5 end and 3 end of genes.

13、At the DNA replication initiation sites.Replication origin-a paticuliar sequence in genome where DNA replication is initiated;ACS (ARS consensus sequence)- a short (11 bp) essential DNA sequence that recruits replication proteins to form pre-RC in budding yeastEaton., 2010 G&D(David MacAlpine La

14、b)Replication origins are associated with an asymmetric NFR flanked by well-positioned nucleosomes.ORC- origin recognition complex; a highly conserved six subunits protein complex essential for the initiation of the DNA replication in eukaryotic cells.Adapted from: Sivaprasad, U., Dutta, A., and Bel

15、l, S.P. (2006). Assembly of pre- replication complexes. In DNA Replication and Human Disease, M.L. Depamphilis, ed. (Cold Spring Harbor, NY, Cold Spring Harbor Press)A mfor pre-replicative complex (pre- RC) formation. ORC is necessary for precise nucleosome positioningEaton., 2010 G&D(David MacA

16、lpine Lab)Rhee and Pugh, 2011 CellChIP-Exo: Protein-DNA Interactions Detected at Single-Nucleotide ResolutionTATA-less: PICs and TSSs were positionally constrained at TFIID-engaged downstream +1 nucleosomesTATA-Box: +1 nucleosome was positioned to be in competition with the PICRhee and Pugh, 2012 Na

17、tureChIP-Exo: Protein-DNA Interactions Detected at Single-Nucleotide ResolutionNLevel 2:30 nm fiberLevel 3: Nuclear scaffoldingLevel 4: Mitotic (metaphase)chromosomeLevel 1: ucleosome formationAn amazing architectural feat: multiples levels of DNA packagCinhgroma/n packaging heirarchyHigher order ch

18、romatinstructure:Ø Mapping the 3D organization of genomes;Ø Mapping the long-range chromatin interactionStained, diploid spread of chicken metaphase chromosomesCremer, T. & Cremer, C. Nature Reviews Genetics 2, 295.What do chromosomes look like in the nucleus of a cell between cell div

19、isions?Chromosome territory mCarl Rabl in 1885"spaghetti" mCremer, T. & Cremer, C. Nature Reviews Genetics 2, 295.Ms of Chromosome OrganizationCremer, T. & Cremer, C. Nature Reviews Genetics 2, 295.Testing the ms Introduce the term CT (Rabl 1885 Boveri 1909) Experiment in 1980s def

20、ined CT: use laser to first induce genome damage CT m: predict damage onlylocalized to a small subset of chromosomes Random m: predict damageonly distributed on many chromosomesDamages mostly localized to chromosome 1 & 2(Heard & Bickmore, 2007)Chromosome territoriesA Functional nuclear land

21、scapeHow can we visualize CT in the cell?(Lindsay Shopland ,Ins9tute for Molecular Biophysics)dsDNA infixed cellFluorescence imagingdenatureLabeled DNAprobe* hybridize*Probing chromosome structure in the nucleus by Fluorescence in situ hybridization (FISH)Chromosome Territory MforOrganization of Chr

22、omatin in the Interphase Cell NucleusChromosome 1 (red),Chromosome 9 (green)Are chromosome territories randomly arranged in the nucleus, or are they organized into patterns?Mouse lymphocytesLiver cellsVisualization of chromosometerritories by FISHCremer, T. & Cremer, C. Nature Reviews Genetics 2

23、, 295.Chromosome territories in the chicken Chromosomes have preferred position with respect to the center or periphery of the nucleus Variability between cell-types Non-random neighbors: purpose is to facilitate proper gene expression!Chromosome paintingChromosome Territories: a unit of nuclear org

24、anizationGiant loop with active genes extending from chromosomal territoryActive genes (white) congregate where transcription factories (polymerases, splicing factors etc) are enrichedActive genes (white) move away from het (yellow), and back again when silent (black)Gene poor, late replicating doma

25、ins within chromosome territories localize closer to the nuclear peripheryDifferent chromatin densitieswithin a chromosome territoryCremer & Cremer (2001) Nat. Rev. Genet. 2(292)Overlay transcriptional and chromosomal territoriesHow is chromatin organized with a CT? Chromosome conformation captu

26、re (3C) MethodFix CellsLigate Bio/n LabelingCan hybridize to microarray/large scale sequenceing to get systems wide info (4C)5C, Hi-CDekker Dostie., Science 2002 3C., Genome Research 2006; 5CLieberman-Aiden.,Science 2009 Hi-CHiC vs. FISHMouse ES cells (from 433 Million Reads)Dixon., Nature, 2012Hi-C

27、 for genome-wide analysis of higher order chromatin structure Tome is composed of megabasesized topological domainsTopological domains in the mouse ES cell genome.Dixon., Nature 2012 (Bing Ren Lab)Topological Association Domains (TADs)- New building Blocks of chromosomeHi-C analysis reveals that the

28、 mammalian genome consists of mega-base sized topological domains (also known as TADs).Topological domains are stable across cell typesand largely pduring evolution,suggesting that they are a basic property of the chromosome architecture.TADs boundaries are enriched for the insulator binding protein

29、 CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposonsDixon., Nature 2012 (Bing Ren Lab)Topological Association Domains (TADs)- New building Blocks of chromosomeBayesian 3D constructor for Hi-C dataHu. PLoS Comp. Biol. 2013Spatial organization of genomic and

30、epigenetic featuresZJ Duan. Nature (2010)Three-dimensional mof the yeast genome.How are distinct chromatin domainsestablished?From A. J. Courey, Mechanisms of Transcription Regulation (2008).Genome architecture: chromatin domainsTelomereReplication origin: Initiation of DNA replicationReplication or

31、iginTelomere: Protection of chromosome endCentromereCentromere: chromosome segregationTelomereIn higher eukaryotic cells, DNA replication origin and Centromeres are specified epigenetically or by chromatin structureThe basic elements that constitute an eukaryotic chromatin.EuchromatinHeterochromatin

32、 Regions “rich” in genes Regions active in transcription Hyperacetylated histones Replicate early in S phase of the cell cycle Regions “poor” in genes Regions that silence transcription Hypoacetylated histones Replicate late S phaseEuchromatin and heterochromatinAllis, Science(2001)Histone Codes at

33、“Euchromatin” and “Heterochromatin”1949 Murray L. Barr and E.G.Bertram(Nature. 163(4148): 676-7).Dark, drumstick-shaped masses in nuclei of nerve cellsPattern only seen in cats 1961 Mary LyonThe Lyon Hypothesis (Lyonization) (Nature. 190 (4773): 372-3)Random inactivation of onefemale X chromosomBarr

34、 body2011 Lyon Law at the EMBO 50-years of X-inactivation conferenceh=p://wiki/Barr_bodyBarr body: X chromosome inactivationHeterochromatin formation & Epigenetic silencingHow are Silent Chromatin Domains established and Maintained?1> Decision making process: Target specific s

35、ilencing complex to the DNA sequence to be inactivated;2> Chromatin structuring process: Efficient inhibition of RNA polymerases or other nuclear enzymes3>Propagation the silent chromatin through DNA replication and mitosisChromatin based silencing:Heterochromatin in Saccharomyces cerevisiaeye

36、ast transcriptional silencingChromosome IIIHMLHMRChromosome XIIrDNA (150copies) Other chromosomesChromosome IIIMATHMRHML24 224224441ORC3ARsilencer E1-4: Sir1-4; R: Rap1; A: Abf1EuchromatinHeterochromatinsilencer Ea2a1silencer IHeterochromatin silencing in S. cerevisiaeSir1RAsilencer E1-4: Sir1-4; R:

37、 Rap1; A: Abf1Heterochromatin EuchromatinORC1Heterochromatin silencing in S. cerevisiaeSir4Sir1ORCsilencer E1-4: Sir1-4; R: Rap1; A: Abf1Heterochromatin Euchromatin41RAHeterochromatin silencing in S. cerevisiaeSir1Sir4Sir2ORCRAsilencer E1-4: Sir1-4; R: Rap1; A: Abf1Heterochromatin Euchromatin4 21Het

38、erochromatin silencing in S. cerevisiaeSir2Sir3Sir4Sir1ORCsilencer E1-4: Sir1-4; R: Rap1; A: Abf1Heterochromatin Euchromatin4 213RAHeterochromatin silencing in S. cerevisiaeSir4 Sir2Sir3Sir124 224224441ORC3ARsilencer E1-4: Sir1-4; R: Rap1; A: Abf1Heterochromatin EuchromatinHeterochromatin silencing

39、in S. cerevisiaeSir2Sir3Sir4Sir124 224224441ORC3ARsilencer E1-4: Sir1-4; R: Rap1; A: Abf1Heterochromatin EuchromatinHeterochromatin silencing in S. cerevisiaeSir2, a histone deacetylaseSir3Sir4Sir1S2ir244 22422441ORC3ARsilencer E1-4: Sir1-4; R: Rap1; A: Abf1Heterochromatin Euchromatin Assembly of Si

40、lent Chromatin in Budding YeastDanesh Moazed Cell 2011 Assembly of Silent Chromatin in Fission YeastClr1DNA bindingClr3HDAC(Hda1)Clr4HMT(Suv39)Clr6HDAC(Rpd3)Swi6HP1Rik1DNA binding(WD40 repeats) Assembly of Silent Chromatin in Fission YeastSilent mating type loci (S. pombe)DNA: centromere-like region

41、 (cenH) boundary elements ( IR-L, IR-R)Order of events:1. Deacetylation (Class I (Hda1 Clr3), Class II (Clr6) key residue H3K92. Methylation H3K9 Clr43. Binding HP1 Swi6, Chp24. Establishment of HeterochromatinRITSCLRC: Clr4 methyltransferase complexRITS: RNA induced transcriptional silencing comple

42、x RDRC: RNA-directed RNA polymeraseShiv Grewal Danesh MoazedRNAi-mediated heterochromatin assembly in fission yeast1> Polycomb complexes are multiprotein complexes that may organize and modify the structure of chromatin and reduce the transcriptional potential of genes in an epigenetic fashion2&g

43、t; Proteins of the Polycomb Group (PcG) were first discovered in Drosophila for their role in the regulation of homeotic genesPolycomb mechanismPolycomb-mediated gene silencingPolycomb repressive complex 1 (PRC1) and 2 (PRC2)1.In Drosophila, DNA sequences known as Polycomb response elements (PRE) ar

44、e targets for PcG protein recruitment;Long ncRNAs are becoming recognized as important participants inPRC2 function;PRC2 also contributes to chromatin compaction, and catalyses the methylation of histone H3 at lysine 27 through its enzymatic subunits EZH1 and EZH2. (Transcription silencing initiatio

45、n)PRC1 compacts chromatin and catalyses the monoubiquitylation ofhistone H2A Lys 119 via the ubiquitin ligase RING1A and RING1b; .2.3.4.(Transcription silencing Maintenance)Danesh Moazed Cell 2011Polycomb-mediated gene silencingPolycomb repressive complex 1 (PRC1) and 2 (PRC2)Li and Zhang ABBS 2012L

46、inking DNA replication to heterochromatin silencing and epigenetic inheritanceDanesh Moazed Cell 2011Epigenetic memory during cell divisions after the switchInheritance of chromatin statesExperiment: replicate mixed chromatin and nucleosome-free templates in vitroResults:Replisome copied through nuc

47、leosomes!Nucleosomes were found only on the daughters of the chromatin plates No nucleosomes moved to the daughters of the nucleosome-free templateReplication distributesnucleosomes to the daughtersH2A-H2B H3-H4AcetylationParental nucleosome New nucleosomeReplisomeDNA replication coupled (RC) nucleo

48、some assembly is important for epigenetic inheritanceHow is DNA methylation maintained?How is histone modifications inherited?How is the initial epigenetic state restored?Chromatin replicationMaintenance of DNA methylation Newly replicated DNA is hemi-methylated Methylation is restored by DNMT1-UHRF

49、1 (Bestor, Jacobsen, koseki labs.)Histone modifiers Parental histones are diluted by new histone incorporationHow is the initial epigenetic state restored? Establishment of H3K27Me3 and H3K9me3 onnew histones is a slow process (Scharf.,2009; Lanzuolo., 2011; Xu., 2011)Chromatin replicationReplisomeC

50、hromatin factors*Proposed functionOrganism components*ORCHP1Replication initiation: ORC recruitment; heterochromatin organizationHs, Xl, DmSIR1SilencingScHBO1Replication initiationSc, HsORCA (also known as LRWD1)Replication initiationHsEBNA1Initiation of viral replicationEBV-infected human cellsHMGA

51、1aReplication initiationHsORC1TRF2Replication initiation: ORC recruitment at telomeresHsCDC6HP1Replication initiationSpCDT1HBO1Replication initiationHsMCM27H3H4Fork progression; histone dynamicsHs, MmASF1Fork progression; histone dynamicsHs, MmHBO1Replication initiation; histone acetylationHsFACTRep

52、lication initiation and elongation; histone dynamicsHs, ScMCM7SMC1Sister chromatid cohesionHsRFC15ASF1Replisome integrity; nucleosome assemblyScRPAFACTFork progression; histone dynamicsScPol FACTFork progression; histone dynamicsScPol Silencing complex DOS2RIK1 and MMS19Heterochromatin maintenance (

53、H3K9me2)SpPCNACAF1Chromatin assemblyHs, Mm, Dm, Sc, SpDNMT1DNA methylation maintenanceHs, MmSMARCAD1Heterochromatin maintenanceHsLamin A, lamin B and lamin CDNA replicationHs, XlWSTFSNF2Preventing unwarranted heterochromatin formationHsHDAC1Histone deacetylationHsHDAC2Histone deacetylationHsESCO1/2Sister chromatid cohesionHs, ScInteractions between replisome components and chromatin regulatorsPolycomb Proteins Remain Boun