4Chromatin-structure - reminder Chromatin - a nucleoprotein complexCauses condensation and organization of DNAprotein:DNA = 2:1 mass ratiohistone:DNA = 1:1 mass ratioSeveral levels of condensation10 nm extended fiber with nucleosomes like beads-on-a-string (7x compaction)30 nm condensed fiber (40-50x compaction)And higherStructural unitsstrings of nucleosomes compose the primary structural unit.Formation of 30-nm fibers through histone tail–mediated nucleosome-nucleosome interactions provides a secondary level of compaction,tail-mediated association of individual fibers produces tertiary structures (such as chromonema fibers).
6Chromatin compaction influences activity of DNA in transcription Heterochromatin -transcriptionally silentEuchromatin - transcriptionally active
7The nucleosome - repeat unit of chromatin Nucleosomesrepeat unit in chromatinRepeat length: bpNuclease digestionsTrace of nuclease: oligo-nucleosomesMore nuclease: chromatosome with/ 166bp DNA + octamer + H1Even more nuclease: core particle 146 bp DNA + octamerDNA lefthanded superhelix 2 turns aroundDNA bended and kinkedDNA is wrapped so that it forms 1.7 turns of a left-handed superhelix within the nucleosome core particle
9The histones The histone fold Tails Octamer = (H2A, H2B, H3 and H4)2 Three-helix core domainForms a handshake-arrangementTailsDisordered N-terminal and or C-terminal tails that protrude from the nucleosome (through the minor-groove channels)Ideal located for covalent modificationsOctamer = (H2A, H2B, H3 and H4)2Assembly: H3-H4 tetramer +2 H2A-H2B dimers
10Nucleosome 3D structure Crytals structure of the nucleosome core particlelow resolution 1984, high resolution 19972.8 Å resolution.146 bp of DNA wrapped around a histone octamer coreNote outside position of histone tailsDNA is wrapped aroundso that it forms 1.7 turns of a left-handed superhelix within the nucleosome core particleLuger et al., 1997.
12Histone H1 - linker histone associated with linker DNA between nucleosomes (about one H1 per nucleosome)Binds DNA at entry/exitstimulates folding 10 nm 30 nm fiberrepressive effect on transcriptionH1 binds weaker to acetylated nucleosomesOrdinary nuclear extracts contain high levels of H1
13Chromatin - a general repressor of transcription
14Different logic of gene regulation - prokaryotes versus eukaryotes Prokaryotic gene regulation through three elementsPromoters recognized by RNA polymeraseOperators recognized by repressorsPositive control elements recognized by activators”Transcriptional ground state” a useful conceptThe inherent activity of promoters in vivo in the absence of specific regulatory sequences (and hence activators and repressors).Prokaryotes: ground state = non-restrictiveNo inherent restriction on RNA pol to gain access to promoterRepressors block accessActivators required only on some inherently weak promotersEukaryotes: ground state = restrictivedue to chromatin templateA strong core promoter is essentially inactive in eukaryotic cellsEssentially all eukaryotic genes require activatorsGROUND STATE1. Step2. Step
15Two mechanisms for gene activation Activation = Anti-repression of inhibitory chromatin structure which indirectly increases recruitment of pol II machineryRemodelling by cooperation of three actorsTranscription factorsHistones/nucleosomerRemodelling factorsActivation = Recruitment by the activator of components of the pol II machinery - many targets both in TFIID and in holoenzymeI1. StepII2. Step
16Chromatin maintains the transcriptionally restrictive ground state Chromatin - a general inhibitor of protein access to DNA, but with interesting differences:TBP unable to bind nucleosomal DNANucleosomes prevent binding of TBP to TATA-elements in vitroTBP does not associate with most core promoters in vivo in the absence of activatorsMany activators bind their targets in nucleosomal DNAChromatin maintains the restrictive ground state by blocking access of basic pol II machinery to core promoters, while permitting many activators to bind their targetsTFIIDTBPActivator
17Changing chromatin to allow transcription Opening and closing the chromatin template
18Changing the chromatin-template to help transcription factors Swi/SnfRSC1. Opening of chromatin through directed modication of histone tails (acetylation and methylation)Histone-modification by HAT and HMT activityBasis for the histone code hypothesis2. Opening of chromatin through directed nucleosome mobilizationATP-dependent process3. Positioning of nucleosomes creates promoters with different requirement for remodelingNURFCHRACHDACsACFHATs
201. Acetylation of core histones N-term tails reversible acetylated in Lys, particularly in H3+H4While the globular core is involved in histone-histone packing and DNA-contact, the N-terminal tails point outwards and is available for interactionThe consequences of acetylationAcetylation of -amino groups in lysines leads to reduced positive charge, believed to weaken the DNA-interaction of the tails (controversial)Changes the conformation of nucleosomes and destabilizes internucleosomal contactsremoval of the histone tails results in nucleosomal arrays that cannot condense past the 10 nm fiber.Improves the availability of chromosomal DNAMay change the interaction with other regulatory proteins+++
22A link histone acetylation - transcription Acetylated histone-tails enriched in transcribed chromatinImmunofractionated chromatin: acetylated fraction enriched in active chromatinNeutralizing mutations in histone-tails affect trxChange in level of acetylation using inhibitors of deacetylation enhance trxChromatin immunoprecipiations (ChIP) show enrichment of acetylated histones in active promotersAcetylation makes nucleosomal DNA more accessible for TF-binding - a role in “potentiation”
23GCN5-related HATs the first link to transcription The first cloning of HAT-A (p55) from Tetrahymena revealed large similarity with yeast GCN5Gcn5p was a know trx regulator in yeastPCAF and hGCN5 identified from its homology with yGCN5Human GCN5 in short and long formTrx forskning - studier av Gcn5pHiston forskning - kloning av HATlink
24General themes of histone modifications XBinding proteinMod enzFunction?Specificity?Demod enzFunction?
26The enzymes: the HATs Histone acetyltransferases (HATs) Catalyze the transfer of acetyl groups from acetyl-CoA onto histone tailsTwo main groups: A-type and B-type HATsA-Type HATs catalyze trx-related acetylationsB-Type HATs are cytoplasmic and catalyze acetylations linked to transport of newly synthesized histones from the cytoplasm to the nucleusHistone deacetylases (HDACs)
27HAT families GNAT family MYST CBP/p300 GTF-HATs Gcn5-related N-acetyltransferaseMany contains bromodomainsMYSTNamed for its founding members: MOZ, Ybf2/Sas3, Sas2, Tip60Several contain chromodomainsCBP/p300GTF-HATsTAFII250Nuclear receptor linked HATsSRC1, ACTR
28GNAT-family of HATs In larger complexes like ySAGA and hSTAGA and hTFTCSAGA (Spt-Ada-Gcn5-Acetyltransferase)2 MDa complex with 14 subunits incl TAFsInteracts with both TBP and acidic activatorsHuman GCN5 found in a similar complex STAGATFTC (TBP-free TAF-containing complex)
29HAT-enzymes found in larger complexes Activator contactGNAT-related HAT complexes
30HAT structure The structure of the HAT domain (GNAT-type) a central conserved core unit important for acetyl-CoA cofactor bindingA cleft used for substrate recognition that lies directly over the cofactor pocketPeptide binding pocketthe H3 peptide bound to Gcn5HAT adopts a random coil structureExtended backbone contactsphosphorylation of Ser 10 in the H3 peptide generates additional interactionsExplains how one histone modification can influence the creation of another modification.Phosphorylation at Ser10 can enhance Lys14 acetylation and together promote transcription
31MYST-type HAT complexes Involved in a wide range of cell functionsTrx activation and silencingFive complexes isolatedyNuA4 complex with essential Esa1yNuA3 complexhTip60 complexSome contain chromodomains (recognizing methylated histones)
33Unexpected large number of HAT-complexes Several coactivators have HAT activityhTAF250, dTAF230, yTAF130p300/CBPACTR and SRC-1P/CAFGcn5Why so many?Evidence for gene-specific HATsPit-1 krever CBP-HAT by cAMP-aktivering, men PCAF-HAT by vekstfaktor-aktiveringMice with p300, CBP, PCAF or GCN5 knocked-out exhibit distinct developmental defects
34Histone modification - specificity SAGAMethylationUbiquitinationPhosphorylationAcetylationADP-ribosylationUbGcn5Gcn5NuA4Esa1pDistinct HAT specificitieswhich Lys are modified?whether histones or nucleosomes are substrates?Free Gcn5 can only acetylate free histones, but as part of the SAGA-complex it modifies also nucleosomes
35So far ... X Function? Function? Binding protein Specificity? Mod enz Demod enzFunction?
36Trx-link: Activation/silencing determined by the equilibrium between HATs and HDACs Acetylation chromatin activationMany coactivators = HATsTetrahymena HAT = homolog of yeast GCN5, a known trx-coactivatorThe coactivator p300/CBPP/CAF = “p300/CBP associated factor”hTAF250, dTAF230, yTAF130Several coactivators for nuclear receptors (SRC-1, ACTR)Point to a coupling between histone- histon-acetylation and gene activationImplies histone acetylation as a directed phenomenonPresent model: HAT-activity becomes recruited to promoters through specfic interactions with activators, leading to local acetylation of nucleosomes around promoters.
37Recruitment was the missing link HATs were for a long time not considered interesting for trx because they were assumed to act globallyRecruitment changed this misconceptionHAT-activity in coactivator-complexes recruited to promoters by TF-interaction
38Global versus targeted acetylation/deacetylation Global acetylationBulk acetylation levels surprisingly highOn average, 13 of the 30 tail Lys residues in a histone octamer are acetylated.this steady-state level of acetylation is maintained by the opposing actions of HAT and HDAC complexes.Targetingof HAT and HDAC complexes to promoter regions then creates inspecific patterns of hyper- and hypoacetylation in a background of global acetylation that correlate with transcription activation and repression, respectively.
39HATs and coactivators in the same complex HAT: local acetylationcoactivator: bridge
40So far ... X Function? Function? Binding protein Specificity? Mod enz Demod enzFunction?
41Bromodomains - binding modules for acetylated histones Bromo-domains recognize acetylated lysines in histone tails100 aa motif in many chromatin-ass proteins, including HATsFour-helix bundle with a hydrophobic pocket able to interact with acetyl-lysSingle bromo = weak affinity, but affinity dramatically increases with tandem bromodomains such as in TAFII250Bromo in HAT logic - ordered recruitment of bromodomain-containing trx complexes
42Recruitment followed by further associations and modifications Once targeted, SAGA acetylates histone H3 in the vicinity of the promoter.Targeted acetylation by SAGA stabilizes its binding and that of a targeted SWI/SNF chromatin-remodeling complex.This requires the bromodomains of Gcn5 and Swi2, respectively, which bind to acetyl-lysine.Due to bromodomains in other proteins, acetylation might stabilize interactions of several complexes
43Summary X Function? Function? Several HATs In large coactivator complexesXBromo-domain proteinsBinding proteinMod enzFunction?Trx activationSpecificity?Yes,complex patternsDemod enzHDACsFunction?
44Other histone modifications PhosphorylationH3 Ser10 - mitosis associatedEGF-stimul - Rsk2 mediated phosphorylation of S10 in H3Phospho-S10 may enhance acetylation of surrounding residuesMethylationUbiquitinationPhosphorylationAcetylationADP-ribosylationUbMethylation
45Histone modifications Histones are among the most conserved proteins known in evolution, but..…are also among the most variable in post-translational modification.MethylationUbiquitinationPhosphorylationAcetylationADP-ribosylationUb
46A role in both activation and repression Histone methylationA role in both activation and repression
47Histone methylation multiple variants on Lys and Arg Both Lys (K) and Arg (R) can be methylated at more than one methyl-groupHistone methylation is a relatively stable modification with a slow turnover rate.No histone demethylases are found.An ideal epigenetic mark for more long-term maintenance of chromatin states.Methylated residues are present both in eu- and heterochromatin.
48The substrates: histone tails - arginine and lysine methylation asymmetricArgsymmetricLysIncrease in the basicity of lys
50The enzymes: Histone lysine methyltransferases (HKMTs) The first HKMT, SUV39H1was identified in 2000.It specifically methylates Lys 9 of histone H3.Later several other HMTs have been identified (see table below)A SET-domain is required for HKMT activity5 lysines within H3 (K4, K9, K27, K36 and K79) and 1 lysine within H4 (K20) are methylated by specific HKMTs
51SET-domains Most HKMTs characterized by a conserved SET-domain Humans: >30 SET-domainsYeast genome: 6 SET-domains>300 foundThe structure of DIM5, a homolog ofSUV39H1, which methylates lysine 9 (orange) in H3, is shown in complex with H3 tail (blue), cofactor (purple), and four zinc ions (pink).
52Function in long-term epigenetic maintenance Epigenetics = heritable changes in gene expression that do not result from alterations in DNA sequencesHMTs involved in a complex process that controls aspects of short- and long-term transcriptional regulation
53Function - Activation or repression? 2 sites - 2 HKMTs - 2 effects Histone methylation was traditionally linked to repression, but turns out to be linked also to activationH3 K9 methylation correlates with heterochromatin formation.Met-K4 in H3 correlates with trx activationSet1p associated with HAT complexSUV39H1Set1pMeMe[H3] ARTKQTARKSTGGKAPRKQL49Activationof trxRepressionof trx
54Function of histone methylation Repression (H3-K9, H3-K27, H4-K20) - more next lectureNumerous reports on silencing effects linked to H3-K9 methylationHeterochromatin formation linked to H3-K9 methylation and HP1 bindingHighly condensed centromeric regions related to methyl addition to H3-K9.H3-K9 and H3-K27 methylation might be important for the X chromosome inactivation process.Activation (H3-K4, H3-K36, H3-K79)H3-K4 methylation is generally associated with trx active chromatin.di-methyl H3-K4 appears to be a global epigenetic mark in euchromatic regions and tri-methylation of H3-K4 correlates with active transcriptionboth H3-K4 and H3-K79 participate in establishing euchromatic regions by preventing the spreading of heterochromatic regions.Role of histone lysine methylation in trx elongation
55Activating effects of methylation - two levels Chromosome level - preventing spread of heterochromatinThe methylation of H3-K4 specifically impairs methylation at H3-K9, thereby blocking a major pathway of heterochromatin formationbinding of the histone deacetylase NuRD repression complex to the H3 N-terminal tail is precluded by methylation at K4, but not K9Gene leveldi-methyl H3-K4 appears to be a global epigenetic mark in euchromatic regions and tri-methylation of H3-K4 correlates with active transcriptionElongation - more later
56Recognition - the chromodomains Methylated lysines are recognized by the conserved chromodomain modules found in several chromatin-associated proteins.The chromodomain binds to histone tails bearing methyllysine in a highly specific manner, with the affinity being highest for trimethyllysine and lowest for monomethyllysine.
57Recognition: Methylated histones recognized by spec chromodomains Chromo-domains as recognition modulesHP1 binds to histones methylated by SUV39H1H3 K9 methylation correlates with heterochromatin formation.Heterochromatin protein 1 (HP1) is a methyl-lysine binding protein.HP1 recruits SUV39H1 leading to propargation of methylation
58Modification of H3 Lys9 - a molecular ”switch” between eu- and heterochromatin? PAMHATHMTTranscriptionSilencingJenuwein, 2001.
60The PRMT domainThe catalytic module that methylates specific arginines is known as the PRMT (protein R methyltransferase) domain.The PRMT domain transfers the methyl group from SAM to the guanidino group of arginines to produce monomethylarginine or dimethylarginine.Some PRMT modules specifically prepare symmetric dimethylarginine and others produce asymmetric dimethylarginine.
61FunctionMethylation of specific arginines in histones H3 and H4 correlate with the active state of transcription.Ex: Methylation of Arg 3 of histone H4 facilitates H4 acetylation and enhances transcription activation by nuclear hormone receptors.
62Removing histone methylation? histone methylation is an extremely stable modificationconsidered an irreversible epigenetic markhistone demethylases have not been discoveredlong-term consequences of histone methylationStill possible to remove?processes exist that remove stable histone methylation modifications, perhaps through histone exchange
63Summary X Function? Function? Several HKMTs And HRMTs Chromo-domain proteinsBinding proteinMod enzFunction?Trx repression and activationEpigeneticsSpecificity?Not identifiedYes,complex patternsDemod enzFunction?
65PARP PARP recruitment Modification = Poly-ADP-ribosylation Stays open During activation, PARP is recruited by TFs to condensed chromatin (green).Modification = Poly-ADP-ribosylationPARP induces dissociation of nucleosomes (beige) by adding long ADP-ribose tails to their histone proteins.Stays openPARP prevents rebinding of transcription factors to the DNA by adding ADP-ribose tails to them
69The histone code Histone encoded info DNA encoded info Modifications of histone tails in specific patterns represent an encoded information extending the information content of the genome beyond the DNA sequenceThis hypothesis predicts thatDistinct modifications of the histone tails will induce interaction affinities for chromatin-associated proteinsModifications may be interdependent generate various combinationsLocal concentrations and combinations of differently modified nucleosomes determine qualities of higher order chromatin
70Combinatorial possibilities large - a language? Acetylated = acitve, deacetylated = inactive True but probably too simpleMany combinations - link to active/inactive chromatin complex
71Synergistic or antigonistic modifications of histones One modification can enhance or inhibit another modification
72Cross-talk between histone modifications Science 2001, 293,Cross-talk between histone modifications
73Cross talk between histone and DNA modifications Methylated DNA is correlated with condensed chromatin.MBD (Methyl CpG Binding Proteins) binds methylated DNA.Zhang & Reinberg, 2001.
74Reading / translating the histone code Modifications = binding sites for protein modulesBromodomains bind acetylated lysines75 bromo-containing proteins in humansChromodomains bind methylated sitesDomains recognizing phosphorylated tails unknownHow to read?
75Writing & Reading the histone code The authors: HMTs, kinases, HATs, PPTases, HDACs, HDM?The readers: Chromo- and bromo domain proteins.Genome Biology 2001, 2(4): reviews
76Mendel’s gene is more than just a DNA moiety!! HAT/ HDACHMT/ HDM?Kinase/ phosphataseHistone modificationsShort term changesLong term changesLocal structural changesBinding sites for proteinsEpigenetic mark for eu- and heterochromatinBiological events
78Changing the chromatin-template to help transcription factors Swi/SnfRSC1. Opening of chromatin through directed modication of histone tails (acetylation and methylation)Histon-modification by HAT and HMT activityBasis for the histone code hypothesis2. Opening of chromatin through directed nucleosome mobilizationATP-dependent process3. Positioning of nucleosomes creates promoters with different requirement for remodelingNURFCHRACHDACsACFHATs
79First discovered complex that remodels chromatin: the SWI/SNF complex SWI/SNF complex originally identified genetically as a positive regulator of HO and SUC2 genesSWI - HO-genet inolvert in mating type switchingSNF - Sucrose non-fermentor: SUC2 involvert in sucrose-fermenterngSWI/SNF complex needed for full activity of some TFsIn its absence : GAL4 tenfold reducedbasal transcription not affectedExists as a complex of about 2 MDaSeveral subunits: SWI1, SWI2, SWI3, SNF5,SNF6, SNF11, TFG3/ANC1 and SWP73, +some othersFew molecules per cell ≈ 100Chromatin-link: Supressors of defect SWI/SNF complex = chromatin componentsseveral histone mutatants restore activator responseSuggested a function in counteracting chromatin-dep repression
80Associates with nucleosomal DNA DNA-interactionSwi/Snf binds DNA and nucleosomes with high affinityKd = nM rangeBinding to DNA is ATP-dependent and targets minor grooveHMG-likeAfter binding to DNA starts an ATP-dependent remodelling of nucleosomes1000 ATP molecules consumed pr minThe Nucleosome is not removed during this process
81SWI2 - the motor in the machinery - running on ATP SWI2-subunit harbours a DNA-stimulated ATPase activitymutated ATPase defect SWI-SNF functionFunction: Use ATP hydrolysis to increase the accessibility of nucleosomal DNAInactive ground stateATPDerepressed stateMore accessibleClosedHow?
82Three possible activities of Swi/Snf TFTFNucleosomeremodelingTFTFNucleosomeslidingTFTFOctamertransfer
83Nucleosome disruption by Swi/Snf Unclear exactly what”disruption” meansStructurally changed nucleosomes or removal of the octamer?Or some sort of altered DNA conformationRemodeled chromatin is more accessible for TFs and other factorsPurified complex stimulates binding of GAL4-AH to nucleosmal DNA foldModel: partial ”unwrapping” of DNA from the surface of the nucleosomeReduced DNA-mass relative to protein-mass pr nucleosomeReduced DNA-supercoiling pr nucleosomeTF
85Remodeled chromatin also through ”sliding” or ”transfer” Removal of nucleosomes can happen by local displacement along the same strandin cis (sliding)by ”spooling” or ”twisting”Or by transferi trans (transfer)Swi/Snf+ATPcistrans
86Two main mechanisms Sliding Conformational change Octamer DNA path May itself cause slidingHigh-enery stategenerated by ATP?
87updateATPase motors that drive these enzymes seem to function as ATP-dependent DNA translocases with limited processivityCan also be regarded as ‘nucleosome isomerases’
88update Mechanisms 1. Twist defect diffusion 2. Bulge diffusion In this model, small local alterations from mean DNA twist (‘defects’) propagate around the nucleosome2. Bulge diffusionIn the bulge diffusion model, unpeeling of DNA from the histone octamer at the entry/exit of the nucleosome and subsequent rebinding of more distal sequences to the same histone contact points would establish nucleosomal particles harbouring excess DNA.Subsequent migration of the bulge around the nucleosomal superhelix would result in the nucleosome apparently stepping bp
89updateHistone dimers can be removed or exchanged between nucleosomes during the course of remodelling reactionsFurthermore, remodelling may cause alteration of chromatin composition
90Recruitment Recruitment model Swi/Snf acts probably only on a subset of genesGenome-analysis in yeast shows that <5% of all gener require Swi/SnfLimited number of molecules pr cell ( Swi/Snf pr celle)Swi/Snf must be recruited to those promotere requiring Swi/Snf functionRecruitment modelGene-specific activators recruit Swi/Snf-complex directlySeveral activators shown to have direct interaction with the Swi/Snf-complexGCN4, SWI5, GAL4-VP16, GR, C/EBPß
91The order of action: Swi/Snf acting before or after TF? Helping the TF bindHelping subsequent steps
92What comes first - the hen or the egg? An apparent paradox:TF binds chromatin only after the promoter is remodeled.Remodeling requires prior binding of a TF that then recruits the remodeling complex.Both statements could be correct, but for different TFsPromoter activation probably occurs through a specific chain of events including both TF binding and chromatin remodeling.Example Swi5-Swi/Snf-SAGA-SBF pathway on the HO promoterSpecific chain of events!Høna eller egget?
93Implications of ordered action: new principles of gene regulation Promoter modification through steps separated in timeChromatin modification can occur in steps - temporally separated and independently regulatedThe generation of an active chromatin structure (poised state) can be separated from the process of transcriptional activation per seRemodelling complexes (like Swi/Snf) can be recruited by some activators (acting after the activator) and be required for other activators to bind (acting before the activator)A wave - acting over large distancesChromatin modifying activities can progress from upstream sites to core promoter - wave of chromatin opening - alternative to loopingHøna eller egget?
94Implications of ordered action: new principles of gene regulation TF action after it has leftA TF can activate at gene without being associated when the gene is expressedBinding of an activator to its target site can be required for transcription even though the activator is not associated with the promoter when transcription actually occursEpigenetic memoryChromatin modifying activities recruited can remain after the TF has left - constitute an epigenetic memoryHøna eller egget?
95Each gene a specific pathway It appears that there is no obligate order for function of ATP-dependent remodelers and covalent modifiers that is general for all promoters.Rather, it seems that each individual promoter will work using a set order of action by these complexes that differs from promoter to promoter.
96Resolution of the paradox requires that some TFs can invade chromatin alone Some cis-elements available for TFs without disruption of the nucleosomeOthers hidden. These elements require nucleosome displacement before TF bindingMultiple TFs bound in a cooperative fashion necessary for invastion
97Three families of ATP-driven remodeling factors Swi/Snf-familienYeast: Original Swi/Snf + more abundant RSCDrosophila dSWI/SNF with brahma (brm)Humant: hSWI/SNFMed BRG1 and hBRMMi-2/CHD-familienMi-2 complexNuRDISWI-familienyeast: ISW1 and ISW2Drosophila NURF(Nucleosome remodelling factor) and ACF and CHRACHuman RSF and hACF and hCHRAC
102Changing the chromatin-template to help transcription factors Swi/SnfRSC1. Opening of chromatin through directed acetylationHiston-modification by HAT activity2. Opening of chromatin through directed nucleosome mobilizationATP-dependent process3. Positioning of nucleosomes creates promoters with different requirement for remodelingNURFCHRACHDACsACFHATs
1033. Different promoters differ in their dependence on remodeling Promoters vary w.r.t. dependence on remodelingAT-rich regions can be relative nuclesome-free and hence ”poised” for activationNucleosome position may be decisive
104Effect of nucleosomes - dependent of position Translational versus rotational positionpå langsinnover/utover
105Nucleosome positioning - translational Def: Translational positioning= position along the DNA-strandNucleosomes in vivo: - having a specific translational positioning?position determined partially by DNA-sequence, partially by bound TFsDNA seq varies mht flexibility, bendability bending around octamer in preferred positionstermodynamisk pref for AT-rik minor groove “inn” and GC-rik “ut”TFs kan virke som “bokstøtter” for en serie nucleosomer
106Nucleosome positioning - translational Repressed state aktivated state : often changed translational positiontranslational position may be part of a repressive mechanismeks. 2 repressor in yeast posisjonert nucleosom over TATAmutert H4 posisjon endret delvis derepresjonrepressormekanisme trolig by transl.pos. + direkte PIC-effectmay also be part of an activation mechanism by its effect on promoter architecture /3D-position of associated TFs
107Positive position effects of nucleosomes TFPIC(a) Packed nucleosomes generally act as a block to transcription,(b) Nucleosomes may serve a positive role by positioning two distinct DNA segments to create a binding site.(c) Nucleosomes may position independent activator binding sites to permit synergy(d) Nucleosomes may stimulate interactions required for transcription.
108Rotational positioning of nucleosomes Def: orientation of DNA relative to octamer surface (“in-out”)Determine availability of sitesA role in presentation of cis-elements5 bp sliding of a response element will change rot.pos. from “in” to “out”Represents a “third language” in DNAThe classical MMTV LTR example:+hormone GR goes to nucleus binds rotationally exposed GRE recruits SWI-SNF complex H1 dissociates NF1 + OCT site become exposed for binding TFIID recruited activationin/out
109Other news: Histone variant H2A.Z Only 60% conserved relative to H2AAssociated with transcribed regionspossibly involved in establishing active chromatin at promotersEssentialThe H2A.Z nucleosome has distinct featuresAltered contactsMetal binding site
111A new role for Z-DNA?Z-DNA = energetically unfavourable DNA conformation, stabilized by neg supercoiling, detected in active promotersMinor effect in transient transfections, major effect in chromatinized templates
112Model Z-DNA not compatible with nucleosome formation Z-DNA = ”open conformation”The mammalian SWI/SNF-like BAF complex cooperates with Z-DNA binding protein to Induce Z-DNA