分子生物学(双语)10-Chromatin课件.ppt

Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title styleClick to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Chapter 10,Chromatin,Individual nucleosomes are released by digestion of chromatin with micrococcal nuclease.The bar is 100 nm,Reprinted from Cell,vol.4,P.Oudet,M.Gross-Bellard,and P.Chambon,Electron microscopic and biochemical evidence.,pp.281-300.Copyright 1975,with permission from Elsevier.Photo courtesy of Pierre Chambon,College of France.,Basic Structures,nucleosome,The basic structural subunit of chromatin,consisting of 200 bp of DNA and an octamer of,histone,proteins.,histone tails,Flexible amino-or carboxy-terminal regions of the core histones that extend beyond the surface of the nucleosome.,Histone tails are sites of extensive posttranslational modification.,The 10 nm fiber in partially unwound state can be seen to consist of a string of nucleosomes,Photo courtesy of Barbara Hamkalo,University of California,Irvine.,The 10 nm fiber is a continuous string of nucleosomes,30 nm fiber,A coil of nucleosomes.,It is the basic level of organization of nucleosomes in chromatin.,nonhistone,Any structural protein found in a chromosome except one of the histones.,Basic Structures,The 30 nm fiber is a two start helix consisting of two rows of nucleosomes coiled into a solenoid,Reprinted from Cell,vol.128,D.J.Tremethick,Higher-order structure of chromatin.,pp.651-654.Copyright 2007,with permission from Elsevier.,DNA Is Organized in Arrays of Nucleosomes,MNase(micrococcal nuclease),cleaves linker DNA and releases individual nucleosomes from chromatin.,95%of the DNA is recovered in nucleosomes or multimers when,MNase,cleaves DNA in chromatin.,The length of DNA per nucleosome varies for individual tissues or species in a range from,154,to,260 bp,.,Nucleosomal DNA is divided into the core DNA and linker DNA depending on its susceptibility to MNase.,Footprinting:Characterizing RNA PolymerasePromoter and DNAProtein Interactions,footprinting,A technique for identifying the site on DNA bound by some protein by virtue of the protection of bonds in this region against attack by nucleases.,A protein protects a series of bonds against nuclease attack,The nucleosome is a cylinder with DNA organized into 1 2/3 turns around the surface,DNA occupies most of the outer surface of the nucleosome,The nucleosome consists of approximately equal masses of DNA and histones(including H1),Possible model for the interaction of histone H1 with the nucleosome,20,21,22,The crystal structure of the histone core octamer is represented in a space-filling model,Photos courtesy of E.N.Moudrianakis,Johns Hopkins University.,Linker DNA is the region of 8 to 114 bp,and this is the region that is susceptible to early cleavage by the enzyme.,Changes in the length of linker DNA account for the variation in total length of nucleosomal DNA.,H1 is associated with linker DNA and may lie at the point where DNA enters and leaves the nucleosome.,25,The histone fold consists of two short a-helices flanking a longer a-helix,Structures from Protein Data Bank 1HIO.G.Arents,et al.,Proc.Natl.Acad.Sci.USA 88(1991):10145-10152.,The crystal structure of the histone core octamer is represented in a ribbon model,Structures from Protein Data Bank 1AOI.K.Luger,et al.,Nature 389(1997):251260.,The crystal structure of the histone core octamer is represented in a ribbon model,Structures from Protein Data Bank 1AOI.Luger,K.,et al.,Nature 389(1997):251-260.,FIGURE CO:Chromatin,Structure from Protein Data Base 1ZBB.T.Schalch,et al.,Nature 436(2005):138-141.Photo courtesy of Chris NelsonUniversity of Victoria.,The 30 nm fiber is a two start helix consisting of two rows of nucleosomes coiled into a solenoid,Reprinted from Cell,vol.128,D.J.Tremethick,Higher-order structure of chromatin.,pp.651-654.Copyright 2007,with permission from Elsevier.,The histone tails are disordered and exit from both faces of the nucleosome and between turns of the DNA,Structure from Protein Data Bank 1AOI.K.Luger,et al.,Nature 389(1997):251-260.,FIGURE 13:The histone fold domains of the histones are located in the core of the nucleosome,Figure 29.20:The structures of histone tails are not defined.,Figure 29.21:Histone tails emerge between DNA turns.,37,38,Histone tails have many sites of modification,Adapted from The Scientist 17(2003):p.27.,The positive charge on lysine is neutralized upon acetylation,while methylated lysine and arginine retain their positive charges,41,FIGURE 20:Most modified sites in histones have a single,specific type of modification,but some sites can have more than one type of modification,43,FIGURE 18:Acetylation during replication occurs on specific sites on histones before they are incorporated into nucleosomes,FIGURE 19:Acetylation associated with gene activation occurs by directly modifying specific sites on histones that are already incorporated into nucleosomes,Figure 2.Proposed mechanism by which DNA methylation leads to transcriptional repression.,(a)Transcriptionally active chromatin is predominantly unmethylated and has high levels of acetylated histone tails(short black squiggles).(b)Methylation at CpG dinucleotides can be carried out by one of the three known human DNA methyltransferases(DNMT1,3a and 3b),resulting in DNA with high levels of CpG methylation(purple circles),but still containing predominantly acetylated histone tails.DNA in this form would still be expected to be transcriptionally competent.(c)Methylated DNA is targeted by methyl-binding domain(MBD)proteins such as MBD2 and MeCP2,which are found associated with large protein complexes such as the NuRD complex(MBD2)and the Sin3a complex(MeCP2).Histone deacetylase(HDAC1 and 2)and chromatin-remodelling activities(Mi-2 and Sin3a)within these complexes result in alterations in chromatin structure,producing chromatin that is refractory to transcriptional activation(pink streaks represent deacetylated histone tails).The functional roles of other components in these complexes are not yet known.Abbreviations:MTA2,metastasis-associated protein 2;RbAp46/48,retinoblastoma-associated protein 46/48;RNA pol II,RNA polymerase II;SAP18/30,Sin3-associated polypeptides 18/30(,fig002gsb).,Proposed mechanism by which DNA methylation leads to transcriptional repression,G.Strathdee and R.Brown,46,47,Covalent Modification of Histones,Photo courtesy of Sean D.Taverna,Johns Hopkins University School of Medicine,and Haitao Li,Memorial Sloan-Kettering Cancer Center.Additional information at S.D.Taverna,et al.,Nat.Struct.Mol.Biol.14(2007):1025-1040.,Numerous protein motifs recognize methylated lysines,Bromodomains are protein motifs that bind acetyl lysines,Structure from Protein Data Bank 1E6I.D.J.Owen,et al.,EMBO J.19(2000):6141-6149.,Numerous protein motifs recognize methylated lysines,Structure from Protein Data Bank 1KNE.S.A.Jacobs and S.Khorasanizadeh,Science 295(2002)2080-2083.,Numerous protein motifs recognize methylated lysines,Structure from Protein Data Bank 2GFA.Y.Huang,et al.,Science 12(2006):748-751.,Histone Variants Produce Alternative Nucleosomes,and potentially Alternative Nucleosomal Organization,All core histones except H4 are members of families of related variants.,Histone variants,can be closely related or highly divergent from canonical histones.,Different variants serve different functions in the cell.,The major core histones contain a conserved histone-fold domain,Adapted from K.Sarma and D.Reinberg,Nat.Rev.Mol.Cell Biol.6(2005):139-149.54,55,-H2AX is detected by an antibody(yellow)and appears along the path traced by a laser that produces double-strand breaks.H2AX has SEQL/Y in C-term which can be phosphorylated.,Rogakou et al.,1999.Originally published in The Journal of Cell Biology,146:905-915.Photo courtesy of William M.BonnerNational Cancer Institute,NIH.,The nucleosome is a cylinder with DNA organized into 1 2/3 turns around the surface,Organized bending of chains around a spherical,or circular object.,nucleosomes,and,bicycle chains.,58,Nicks in double-stranded DNA are revealed by fragments when the DNA is denatured to give single strands,Sites for nicking lie at regular intervals along core DNA,as seen in a DNase I digest of nuclei The most exposed positions on DNA recur with a periodicity that reflects the structure of the double helix,Rotational positioning describes the exposure of DNA on the surface of the nucleosome,DNA Structure Varies on the Nucleosomal Surface,DNA is wrapped 1.65 times around the histone octamer.,The structure of the DNA is altered so that it has:,an increased number of base pairs/turn in the middle,a decreased number at the ends,62,The Periodicity of DNA Changes on the Nucleosome0.6 negative turns of DNA are absorbed by the change in bp/turn:,from 10.5 in solution,to an average of 10.2 on the nucleosomal surface,This explains the linking-number paradox.,63,DNA is wrapped 1.67 times around the histone octamer.,DNA on the nucleosome shows regions of smooth curvature and regions of abrupt kinks.,The structure of the DNA is altered so that it has an increased number of base pairs/turn in the middle,but a decreased number at the ends.,DNA structure in nucleosomal DNA,Structures from Protein Data Bank:1P34.U.M.Muthurajan,et al.,EMBO J.23(2004):260-271.,0.6 negative turns of DNA are absorbed by the change in bp/turn from 10.5 in solution to an average of 10.2 on the nucleosomal surface,which explains the linking-number paradox.,DNA structure in nucleosomal DNA,Adapted from T.J.Richmond and C.A.Davey,Nature 423(2003):145-150.,The supercoils of the SV40 minichromosome can be relaxed to generate a circular structure,whose loss of histones then generates supercoils in the free DNA,Chromatin Is Fundamentally Divided into Euchromatin and Heterochromatin,Individual chromosomes can be seen only during mitosis.,During interphase,the general mass of chromatin is in the form of,euchromatin,which is slightly less tightly packed than mitotic chromosomes.,Regions of compact heterochromatin are clustered near the nucleolus and nuclear membrane,Photo courtesy of Edmund Puvion,Centre National de la Recherche Scientifique,Nucleosome positioning places restriction sites at unique positions relative to the linker sites cleaved by micrococcal nuclease,Nucleosomes may form at specific positions as the result of either:,the local structure of DNA,proteins that interact with specific sequences,The most common cause of nucleosome positioning is when proteins binding to DNA establish a boundary.,In the absence of nucleosome positioning,a restriction site can lies at any possible location in different copies of the genome,Translational positioning describes the linear position of DNA relative to the histone octamer,Figure 29.33:Phasing controls exposure of linker DNA.,71If nucleosomes are not randomly deposited on the DNA,(a)Are they are located by site specific interaction between specific DNA sequences and nucleosome proteins(INTRINSIC).,(b)Is the location of certain histones preferentially assembled giving rise to clear boundary patterns(e.g.at a promoter)at the 5 end of the gene and subsequent nucleosomes are spaced out evenly from that point,(EXTRINSIC).,72,73,Are Transcribed Genes Organized in Nucleosomes?,Nucleosomes are found at the same frequency when one digests with micrococcal nuclease:,transcribed genes,nontranscribed genes,Some heavily transcribed genes appear to be exceptional cases that are devoid of nucleosomes.,Figure 29.36,74,Individual rDNA transcription units alternate with nontranscribed DNA segments,Reproduced from O.L.Miller and B.R.Beatty,Science 164(1969):955-957.Photo courtesy of Oscar Miller.,RNA polymerase is comparable in