Chromatin Structure andFunctionHistone Octomers and NucleosomesTransactions between Chromatinand the Transcription MachineryChromatin Remodeling, HistoneModification and Gene RegulationEukaryoticgenomes arepackaged withchromatinproteinsNucleosomes as seen in the electron microscopeThese electron micrographs show chromatin strands before and after treatments that unpack,or "decondense," the native structure to produce the "beads-on-a-string" form. The nativestructure, known as the 30-nm fiber), is shown in (A).The decondensed, "beads-on-a-string" form of chromatin is shown at the same magnificationin (B). (A, courtesy of Barbara Hamkalo; B, courtesy of Victoria Foe.)Schematic of MNase DigestionAssayMicrococcal nuclease digestion of chromatin1234Crystal Structure of NucleosomeA Variety of Biological Phenomena Involve ChromatinVariety of Phenomena InvolveChromatinNucleosomesinhibittranscription atmultiple stagesWorkman and KingstonAnn. Rev. Biochem. 67: 545 (1998)Dnase HS site (indirect end-labelling)Chromatin Immunoprecipitation(ChIP) AnalysisAntirepression Model for GeneActivation(or, “unrepressed state”;= Naked DNA in vitro)How to activate (repress) chromatin fortranscription1. Covalentlymodifyhistonetermini: H3lysine 9acetylation /methylation2. Use histonemodificationsto recruit otherproteins3. Movenucleosomesout of or intothe way ofthe promoterSWI/SNF is anATPaseenzyme thatreduces thekinetic barrierto mobilizingDNA withinnucleosomesModels for Transcriptional Regulation by Protein AcetylationModel 1: Acetylation induces a conformational change in the corehistones.HAT (histone acetyl transferase) mechanismRoth SY, Denu JM, Allis CD. Annu Rev Biochem. 2001;70:81-120.Note the charge neutralization that accompanies acetylation!Activator-directed histone acetylation.Activator-directed hyperacetylation of histone N-terminal tails. The DNA-binding domain of Gcn4interacts with specific upstream-activating sequences (UAS) of the genes it regulates. The Gcn4activation domain (AD) then interacts with a multiprotein histone acetylase complex that includesthe Gcn5 catalytic subunit. Subsequent hyperacetylation of histone N-terminal tails on nucleosomesin the vicinity of the Gcn4-binding site facilitates access of the general transcription factors requiredfor initiation.Repressor-directed histone deacetylation.Repressor-directed deacetylation of histone N-terminal tails. The DNA-binding domain (DBD) ofthe repressor Ume6 interacts with a specific upstream control element (URS1) of the genes itregulates. The Ume6 repression domain (RD) binds Sin3, a subunit of a multiprotein complexthat includes Rpd3, a histone deacetylase. Deacetylation of histone N-terminal tails onnucleosomes in the region of the Ume6-binding site inhibits binding of general transcriptionfactors at the TATA box, thereby repressing gene expression.Model 2: Acetylation is a transducing signal (histone code)Model 3: Acetylation affects the activity of a nonhistone protein.Covalent modification of histone terminiModified residues are protein-binding sitesDescribed protein modules of histone-modifying enzymes that have been shown to interact with site-specific methylation (chromodomain) or acetylation (bromodomain) marks in histone NH2-termini. Aprotein module that would selectively recognize phosphorylated positions is currently not known.Abbreviations: HMT, histone methyltransferase; HAT, histone acetyltransferase; HDM, histonedemethylase; PPTase, protein phosphatase; HDAC, histone deacetylaseJenuwein T, Allis CD. Science 293:1074-80 (2001)Modifying enzymeReversing enzymeBindingproteinMajor Points1. DNA is packaged into nucleosomes to form chromatin: DNA wrapped around histone octomers - inhibition of transcription2. Assays to probe nucleosome structure and binding of transcription factors to chromatin (Micrococcal DNAse,ChIP)3. Gene activation by chromatin remodeling and chemical modification of histones4. Anti-repression versus activation: role of His acetylation and de-acetylation by HATs and HDACs5. ATP-dependent nucleosome mobilization by SWI/SNF chromatin remodeling co-activators6. Multiple covalent modifications (acetyl-, phospho-,methyl-) of histones and non-histone proteins help regulate
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