Bài giảng Molecular Biology - Chapter 13 Chromatin Structure and Its Effects on Transcription

Molecular BiologyFifth EditionChapter 13Chromatin Structure and Its Effects on TranscriptionLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1Chromatin StructureEukaryotic genes do not exist naturally as naked DNA, or even as DNA molecules bound only to transcription factorsThey are complexed with an equal mass of other proteins to form chromatinChromatin is variable and the variations play an enormous role in chromatin structure and in the control of gene expression213.1 HistonesEukaryotic cells contain 5 kinds of histonesH1H2AH2BH3H4Histone proteins are not homogenous due to:Gene reiterationPosttranslational modification3Properties of HistonesAbundant proteins whose mass in nuclei nearly equals that of DNAPronounced positive charge at neutral pHMost are well-conserved from one species to anotherNot single copy genes, repeated many timesSome copies are identicalOthers are quite differentH4 has only had 2 variants ever reported413.2 NucleosomesChromosomes are long, thin molecules that will tangle if not carefully foldedFolding occurs in several waysFirst order of folding is the nucleosome, which have a core of histones, around which DNA windsX-ray diffraction has shown strong repeats of structure at 100Å intervalsThis spacing approximates the nucleosome spaced at 110Å intervals5Histones in the NucleosomeChemical cross-linking in solution:H3 to H4H2A to H2BH3 and H4 exist as a tetramer (H3-H4)2Chromatin is composed of roughly equal masses of DNA and histonesCorresponds to 1 histone octamer per 200 bp of DNAOctamer composed of:2 each H2A, H2B, H3, H41 each H16H1 and ChromatinTreatment of chromatin with trypsin or high salt buffer removes histone H1This treatment leaves chromatin looking like “beads-on-a-string”The beads named nucleosomesCore histones form a ball with DNA wrapped around the outsideDNA on outside minimizes amount of DNA bendingH1 also lies on the outside of the nucleosome7Nucleosome StructureCentral (H3-H4)2 core attached to H2A-H2B dimersGrooves on surface define a left-hand helical ramp – a path for DNA windingDNA winds almost twice around the histone core condensing DNA length by 6- to 7-XCore histones contain a histone fold:3 a-helices linked by 2 loopsExtended tail of abut 28% of core histone massTails are unstructured8Crystal Structure of a Nucleosomal Core Particle9The 30-nm FiberSecond order of chromatin folding produces a fiber 30 nm in diameterThe string of nucleosomes condenses to form the 30-nm fiber in a solution of increasing ionic strengthThis condensation results in another six- to seven-fold condensation of the nucleosome itselfFour nucleosomes condensing into the 30-nm fiber form a zig-zag structure10Models for the 30-nm FiberThe solenoid and the two-start double helix model each have experimental supportA technique called single-molecule force spectroscopy was employed to answer the question, ‘which model is correct?’Results suggested that most of the chromatin in a cell (presumably inactive) adopts a solenoid shape while a minor fraction (potentially active) forms a 30-nm fiber according to the two-start double helix11Higher Order Chromatin Folding30-nm fibers account for most of chromatin in a typical interphase nucleusFurther folding is required in structures such as the mitotic chromosomesModel favored for such higher order folding is a series of radial loopsSource: Adapted from Marsden, M.P.F. and U.K. Laemmli, Metaphase chromosome structure: Evidence of a radial loop model. Cell 17:856, 1979.12Relaxing Supercoiling in Chromatin LoopsWhen histones are removed, 30-nm fibers and nucleosomes disappearLeaves supercoiled DNA duplexHelical turns are superhelices, not ordinary double helixDNA is nicked to relax1313.3 Chromatin Structure and Gene ActivityHistones, especially H1, have a repressive effect on gene activity in vitroHistones play a predominant role as regulators of genetic activity and are not just purely structuralThe regulatory functions of histones have recently been elucidated14Effects of Histones on Transcription of Class II GenesCore histones assemble nucleosome cores on naked DNATranscription of reconstituted chromatin with an average of 1 nucleosome / 200 bp DNA exhibits 75% repression relative to naked DNARemaining 25% is due to promoter sites not covered by nucleosome cores15Histone H1 and TranscriptionHistone H1 causes further repression of template activity, in addition to that of core histonesH1 repression can be counteracted by transcription factorsSp1 and GAL4 act as both:Antirepressors preventing histone repressionsTranscription activatorsGAGA factor: Binds to GA-rich sequences in the Krüppel promoterAn antirepressor – preventing repression by histones16A Model of Transcriptional Activation17Nucleosome PositioningModel of activation and antirepression asserts that transcription factors can cause antirepression by: Removing nucleosomes that obscure the promoterPreventing initial nucleosome binding to the promoterBoth actions are forms of nucleosome positioning – activators force nucleosomes to take up positions around, not within, promoters18Nucleosome-Free ZonesNucleosome positioning would result in nucleosome-free zones in the control regions of active genesAssessment in SV40 DNA, a circular minichromosome, was performed to determine the existence of nucleosome-free zones - with the use of restriction sites it was found that the late control region is nucleosome free19Detecting DNase-Hypersensitive RegionsActive genes tend to have DNase-hypersensitive control regionsPart of this hypersensitivity is due to absence of nucleosomes20Histone AcetylationHistone acetylation occurs in both cytoplasm and nucleusCytoplasmic acetylation carried out by HAT B (histone acetyltransferase, HAT) Prepares histones for incorporation into nucleosomesAcetyl groups later removed in nucleusNuclear acetylation of core histone N-terminal tailsCatalyzed by HAT ACorrelates with transcription activationCoactivators of HAT A found which may allow loosening of association between nucleosomes and gene’s control regionAttracts bromodomain proteins, essential for transcription21Histone DeacetylationTranscription repressors bind to DNA sites and interact with corepressors which in turn bind to histone deacetylasesRepressorsMad-MaxCorepressorsNCoR/SMRTSIN3Histone deacetylases - HDAC1 and 222Model for participation of HDAC in transcription repressionAssembly of complex brings histone deacetylases close to nucleosomesDeacetylation of core histones allows Histone basic tails to bind strongly to DNA, histones in neighboring nucleosomesThis inhibits transcription23Model for Activation and Repression24Chromatin RemodelingActivation of many eukaryotic genes requires chromatin remodelingSeveral protein complexes carry this outAll have ATPase harvesting energy from ATP hydrolysis for use in remodelingRemodeling complexes are distinguished by ATPase component25Remodeling ComplexesSWI/SNF In mammals, has BRG1 as ATPase9-12 BRG1-associated factors (BAFs)A highly conserved BAF is called BAF 155 or 170Has a SANT domain responsible for histone bindingThis helps SWI/SNF bind nucleosomesISWIHave a SANT domainAlso have SLIDE domain involved in DNA binding26Models for SWI/SNF Chromatin Remodeling27Mechanism of Chromatin RemodelingMechanism of chromatin remodeling involves: Mobilization of nucleosomesLoosening of association between DNA and core histonesCatalyzed remodeling of nucleosomes involves formation of distinct conformations of nucleosomal DNA/core histones when contrasted with: Uncatalyzed DNA exposure in nucleosomesSimple nucleosome sliding along a DNA stretch28Remodeling in Yeast HO Gene ActivationChromatin immunoprecipitation (ChIP) can reveal the order of binding of factors to a gene during activationAs HO gene is activated:First factor to bind is Swi5Followed by SWI/SNF and SAGA containing HAT Gcn5pNext general transcription factors and other proteins bindChromatin remodeling is among the first steps in activation of this geneOrder could be different in other genes29Remodeling in the Human IFN-b Gene: The Histone CodeThe Histone Code: The combination of histone modifications on a given nucleosome near a gene’s control region affects efficiency of that gene’s transcription This code is epigenetic, not affecting the base sequence of DNA itselfActivators in the IFN-b enhanceosome can recruit a HAT (GCN5) HAT acetylates some Lys on H3 and H4 in a nucleosome at the promoterProtein kinase phosphorylates Ser on H3This permits acetylation of another Lys on H330Remodeling in the Human IFN-b Gene: TF BindingRemodeling allows TFIID to bind 2 acetylated lysines in the nucleosome through the dual bromodomain in TAF1TFIID bindingBends the DNAMoves remodeled nucleosome asidePaves the way for transcription to begin31HeterochromatinEuchromatin: relatively extended and open chromatin that is potentially activeHeterochromatin: very condensed with its DNA inaccessibleMicroscopically appears as clumps in higher eukaryotesRepressive character able to silence genes as much as 3 kb away32Formation at the tips of yeast chromosomes (telomeres) with silencing of the genes is the telomere position effect (TPE)Depends on binding of proteinsRAP1 to telomeric DNARecruitment of proteins in this order: SIR3SIR4SIR2Heterochromatin and Silencing33SIR ProteinsHeterochromatin at other locations in chromosome also depends on the SIR proteinsSIR3 and SIR4 interact directly with histones H3 and H4 in nucleosomesAcetylation of Lys 16 on H4 in nucleosomes prevents interaction with SIR3Blocks heterochromatin formationHistone acetylation also works in this way to promote gene activity34Histone MethylationMethylation of Lys 9 in N-terminal tail of H3 attracts HP1This recruits a histone methyltransferaseMethylates Lys 9 on a neighboring nucleosomePropagates the repressed, heterochromatic stateMethylation of Lys and Arg side chains in core histones can have either repressive or activating effects35Histone MethylationMethylation of Lys 4 in N-terminal tail of H3 is generally tri-methylated (H3K4Me3) and is usually associated with the 5’-end of an active geneThis modification appears to be a sign of transcription initiationGenome-wide ChIP analysis suggests that this may also play a role in controlling gene expression by controlling the re-starting of paused RNA polymerases36Histone modifications can affect gene activity by two mechanisms: 1. By altering the way histone tails interact with DNA and with histone tails in neighboring nucleosomes, and thereby altering nucleosome cross-linking2. By attracting proteins that can affect chromatin structure and activitySummary37Modification CombinationsMethylations occur in a given nucleosome in combination with other histone modifications:AcetylationsPhosphorylationsUbiquitylationsEach particular combination can send a different message to the cell about activation or repression of transcriptionOne histone modification can also influence other, nearby modifications38Nucleosomes and Transcription ElongationAn important transcription elongation facilitator is FACT (facilitates chromatin transcription) Composed of 2 subunits:Spt16 Binds to H2A-H2B dimersHas acid-rich C-terminus essential for these nucleosome remodeling activitiesSSRP1 binds to H3-H4 tetramers39Nucleosomes and Transcription ElongationFACT facilitates transcription through a nucleosome by promoting loss of at least one H2A-H2B dimer from the nucleosomeAlso acts as a histone chaperone promoting re-addition of H2A-H2B dimer to a nucleosome that has lost such a dimer40