Chromatin - Essential Player In Trx - UiO

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Chromatin- essential player in trxTTBTATPromote

MBV4230Packing of DNA into chromatin2 m of DNA in anucleus with adiameter of 5-10 µmChromatin not only packages DNA,but also regulates DNA accessibility throughmodifications in chromatin structure.Odd S. Gabrielsen

Histones andNucleosomes

MBV4230Chromatin-structure - reminder Chromatin - a nucleoprotein complex Causes condensation and organization of DNA Several levels of condensation protein:DNA 2:1 mass ratiohistone:DNA 1:1 mass ratio10 nm extended fiber with nucleosomes like beads-on-a-string (7x compaction)30 nm condensed fiber (40-50x compaction)And higherStructural units strings of nucleosomes compose the primary structural unit.Formation of 30-nm fibers through histone tail–mediated nucleosomenucleosome interactions provides a secondary level of compaction,tail-mediated association of individual fibers produces tertiarystructures (such as chromonema fibers).Odd S. Gabrielsen

MBV4230Multiple levels of chromatin foldingOdd S. Gabrielsen

MBV4230Chromatin compaction influencesactivity of DNA in transcription Heterochromatin transcriptionally silentEuchromatin transcriptionallyactiveOdd S. Gabrielsen

MBV4230The nucleosome- repeat unit of chromatin Nucleosomes repeat unit in chromatin Nuclease digestions Trace of nuclease: oligo-nucleosomesMore nuclease: chromatosome with/ 166bp DNA octamer H1Even more nuclease: core particle 147 bp DNA octamerDNA lefthanded superhelix 2 turns around Repeat length: 180-210 bpDNA bended and kinkedDNA wrapped 1.65 turns around the histone octameras a left-handed superhelixOdd S. Gabrielsen

MBV4230The nucleosome- repeat unit of chromatinOdd S. Gabrielsen

MBV4230The histones The histone fold Tails Three-helix core domainForms a handshake-arrangementDisordered N-terminal and or Cterminal tails that protrude fromthe nucleosome (through theminor-groove channels)Ideal located for covalentmodificationsOctamer (H2A, H2B,H3 and H4)2 Assembly: H3-H4 tetramer 2 H2A-H2B dimersOdd S. Gabrielsen

MBV4230Nucleosome 3D structure Crytals structure of thenucleosome coreparticle low resolution 1984, high resolution 1997 2.8 Å resolution.146 bp of DNA wrapped arounda histone octamer coreNote outside position of histonetails DNA is wrapped around DNA wrapped 1.65 turns aroundthe histone octamer as a lefthanded superhelix14 contact points betweenhistones and DNA, making thenucleosome one of the moststable protein-DNA complexesunder physiological conditionsLuger et al., 1997.Odd S. Gabrielsen

MBV4230Nucleosome 3D structureOdd S. Gabrielsen

MBV4230Histone H1 - linker histone H1 linker histone associated with linker DNA between nucleosomes (about one H1 pernucleosome) Binds DNA at entry/exitstimulates folding 10 nm 30 nm fiberrepressive effect on transcription H1 binds weaker to acetylated nucleosomes Ordinary nuclear extracts contain high levels of H1Odd S. Gabrielsen

Chromatin - a generalrepressor oftranscription- unwrapping needed

MBV4230Chromatin restricts transcription Chromatin - a general inhibitor of protein access to DNA Packed chromatin will block access of basic pol IImachinery to core promoters Chromatin needs to be unpacked, unwrapped or openedbefore transcription can take placeRNAPIITFIIDActivatorTBPOdd S. Gabrielsen

MBV4230Different logic of gene regulation- prokaryotes versus eukaryotes Prokaryotic gene regulation - three elements The inherent activity of promoters in vivo in the absence of specificregulatory sequences (and hence activators and repressors).Prokaryotes: ground state non-restrictive GROUND STATE”Transcriptional ground state” a useful concept Promoters recognized by RNA polymeraseOperators recognized by repressorsPositive control elements recognized by activatorsNo inherent restriction on RNA pol to gain access to promoterRepressors block accessActivators required only on some inherently weak promotersEukaryotes: ground state restrictive 1. Step2. Stepdue to chromatin templateA strong core promoter is essentially inactive in eukaryotic cellsEssentially all eukaryotic genes require activatorsOdd S. Gabrielsen

MBV4230Two steps to activate a gene 1. Activation step Anti-repressionof inhibitory chromatin structureallowing recruitment of the pol IImachinery 2. Activation step Recruitment bythe activator of components of thepol II machineryI1. StepII2. StepOdd S. Gabrielsen

MBV4230Chromatin maintains the transcriptionallyOFF state Chromatin - a general inhibitor of protein access to DNA,but with interesting differences:TBP unable to bind nucleosomal DNA Nucleosomes prevent binding of TBP to TATA-elements in vitroTBP does not associate with most core promoters in vivo in the absence ofactivatorsMany activators bind their targets in nucleosomal DNAChromatin maintains the restrictive ground state byblocking access of basic pol II machinery to corepromoters, while permitting many activators to bind theirTFIIDtargetsActivatorTBPOdd S. Gabrielsen

Changing chromatin toallow transcriptionOpening and closing thechromatin template

MBV4230Changing the chromatin-templateto help transcription factors 1. Opening of chromatin throughdirected modication of histone tails(acetylation and methylation) NURFHDACsRSCCHRACACFHATs2. Opening of chromatin throughdirected nucleosome mobilization Histone-modification by HAT and HMT activityBasis for the histone code hypothesisSwi/SnfATP-dependent process3. Positioning of nucleosomes createspromoters with different requirementfor remodelingOdd S. Gabrielsen

MBV4230Histone modificationsHistones are among the most conservedproteins known in evolution, but. are also among the most variable inpost-translational modification.Nomenclature:H3K4me3histone H3, lysine no 4,tri-methylatedH4K16Ac20Odd S. Gabrielsen

Histone acetylation

MBV42301. Acetylation of core histones N-term tails reversible acetylated inLys, particularly in H3 H4 The N-terminal tails point outwards and is availablefor interactionThe consequences of acetylation Acetylation of ε-amino groups in lysines leads toreduced positive charge, believed to weaken theDNA-interaction of the tails.Improves the availability of chromosomal DNAChanges the conformation of nucleosomes anddestabilizes internucleosomal contactsMay change the interaction with otherregulatory proteins Odd S. Gabrielsen

MBV4230Change of charge 00000Odd S. Gabrielsen

MBV4230A linkhistone acetylation - transcription Acetylated histone-tails enriched in transcribedchromatin Change in level of acetylation using inhibitors of deacetylation (TSA) enhancetrxChromatin immunoprecipiations (ChIP) show enrichment of acetylatedhistones in active promotersAcetylation makes nucleosomal DNA more accessiblefor TF-binding - a role in “potentiation”Odd S. Gabrielsen

MBV4230History - GCN5-related HATsthe first link to transcription The first cloning of HATA (p55) from Tetrahymenarevealed large similaritywith yeast GCN5 Gcn5p a known trx regulator inyeastPCAF and hGCN5identified from itshomology with yGCN5 Human GCN5 in short and longformTrx research - studies of Gcn5plinkHistone research - cloning of HATOdd S. Gabrielsen

MBV4230General themesof histone modificationsXBinding proteinMod enzFunction?Specificity?Demod enzFunction?Odd S. Gabrielsen

MBV4230The subtrates:Histone tails - multiple modficationsOdd S. Gabrielsen

MBV4230The enzymes: the HATs Histone acetyltransferases (HATs) Catalyze the transfer of acetyl groups from acetyl-CoA onto histone tails Two main groups: A-type and B-type HATs A-Type HATs catalyze trx-related acetylationsB-Type HATs are cytoplasmic and catalyze acetylations linked to transportof newly synthesized histones from the cytoplasm to the nucleusHistone deacetylases (HDACs)Odd S. Gabrielsen

MBV4230HAT families GNAT family MYST Named for its founding members:MOZ, Ybf2/Sas3, Sas2, Tip60Several contain chromodomainsCBP/p300GTF-HATs Gcn5-related N-acetyltransferaseMany contains bromodomainsTAFII250Nuclear receptor linkedHATs SRC1, ACTROdd S. Gabrielsen

MBV4230GNAT-family of HATs In larger complexes likeySAGA and hSTAGA and hTFTC SAGA (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)Odd S. Gabrielsen

MBV4230HAT-enzymes found inlarger complexesActivator contactGNAT-related HAT complexesOdd S. Gabrielsen

MBV4230HAT structure The structure of the HATdomain (GNAT-type) a central conserved core unit important foracetyl-CoA cofactor bindingA cleft used for substrate recognition thatlies directly over the cofactor pocketPeptide binding pocket the H3 peptide bound to Gcn5HAT adopts arandom coil structurephosphorylation of S10 in the H3 peptidegenerates additional interactions. Explainshow one histone modification can influencethe creation of another modification.Phosphorylation at Ser10 can enhance K14acetylation and together promotetranscriptionOdd S. Gabrielsen

MBV4230MYST-type HAT complexes Involved in a widerange of cellfunctions Trx activation andsilencingFive complexesisolated yNuA4 complex withessential Esa1yNuA3 complexhTip60 complexSome containchromodomains(recognizing methylatedhistones)Odd S. Gabrielsen

MBV4230MYST-type HATsOdd S. Gabrielsen

MBV4230Unexpected large numberof HAT-complexes Several coactivators have HATactivity hTAF250, dTAF230, yTAF130 p300/CBP ACTR and SRC-1 P/CAF Gcn5 Why so many?Evidence for gene-specific HATs Mice with p300, CBP, PCAF or GCN5 knocked-outexhibit distinct developmental defectsOdd S. Gabrielsen

MBV4230Histone modification - specificitySAGAGcn5Gcn5NuA4Esa1pDistinct HATspecificitieswhich Lys are modified?whether histones ornucleosomes are substrates?Free Gcn5 can only acetylatefree histones, but as part of theSAGA-complex it modifiesalso UbiquitinationADP-ribosylationOdd S. Gabrielsen

MBV4230So far .XBinding proteinMod enzFunction?Specificity?Demod enzFunction?Odd S. Gabrielsen

MBV4230Trx-link: Activation/silencing determined by theequilibrium between HATs and HDACs Acetylation chromatin activationMany coactivators HATs Tetrahymena HAT homolog of yeast GCN5, a known trx-coactivatorThe coactivator p300/CBPP/CAF “p300/CBP associated factor”TAF1: 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 topromoters through specfic interactions with activators,leading to local acetylation of nucleosomes aroundpromoters.Odd S. Gabrielsen

MBV4230Recruitment - the missing link HATs were for a long timenot considered interestingfor trx because they wereassumed to act globally Recruitment changed thismisconceptionHAT-activity in coactivatorcomplexes recruited topromoters by TF-interaction Odd S. Gabrielsen

MBV4230Global versus targeted acetylation/deacetylation Global acetylation Targeting Bulk acetylation levels surprisingly highOn average, 13 of the 30 K residues in a histone octamer are acetylated.this steady-state level of acetylation is maintained by the opposing actions ofHAT and HDAC complexes.of HAT and HDAC complexes to promoter regions then creates inspecificpatterns of hyper- and hypoacetylation in a background of global acetylation thatcorrelate with transcription activation and repression, respectively.Specific pattern or cumulative charge effect? Distinct patterns of lysine acetylation on histones have been proposed to specifydistinct downstream functions.Another view posits that the functions of histone acetylation rely primarily on thenumber of lysines modified (a cumulative effect). This because of the greater lossof positives charges making acetylated histones easier to displace from DNA.Odd S. Gabrielsen

MBV4230So far .XBinding proteinMod enzFunction?SpecificityDemod enzFunction?Odd S. Gabrielsen

MBV4230Bromodomains - binding modulesfor acetylated histones Bromo-domains recognize acetylated lysinesin histone tails Four-helix bundle with a hydrophobic pocketable to interact with acetyl-lys 100 aa motif in many chromatin-ass proteins, including HATsSingle bromo weak affinity, but affinity dramatically increaseswith tandem bromodomains such as in TAFII250Bromo in HAT logic - ordered recruitment ofbromodomain-containing trx complexesOdd S. Gabrielsen

MBV4230SummarySeveral HATsIn largecoactivator complexesXBromo-domain proteinsBinding proteinMod enzFunction?Trx activationSpecificityYes,complex patternsDemod enzHDACsFunction?Odd S. Gabrielsen

MBV4230Other histone modificationsPhosphorylationH3 Ser10 - mitosis associatedEGF-stimul - Rsk2 mediatedphosphorylation of S10 in H3Phospho-S10 may enhanceacetylation of surrounding ylationUbUbiquitinationADP-ribosylationOdd S. Gabrielsen

Histone methylationA role in both activation andrepression

MBV4230Histone modificationsHistones are among the most conservedproteins known in evolution, but. are also among the most variable inpost-translational modification.Nomenclature:H3K4me3histone H3, lysine no 4,tri-methylatedH4K16Ac46Odd S. Gabrielsen

MBV4230Histone methylationmultiple variants on Lys and Arg Both Lys (K) and Arg (R) can be methylated at morethan one methyl-groupHistone demethylases was for a long time thought tonot exist, but have just recently been identifiedMethylated residues are present both in eu- andheterochromatinOdd S. Gabrielsen

MBV4230The substrates: histone tails- arginine and lysine methylationasymmetricArgsymmetricLysIncrease in the basicity of lysOdd S. Gabrielsen

MBV4230The subtrates:Histone tails - multiple methylations Black Arginine (R)methylationRed Lysine (K)methylationAbove activationBelow repressionOdd S. Gabrielsen

MBV4230The enzymes: Histone lysinemethyltransferases (HKMTs) The first HKMT, SUV39H1 was 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 lysinewithin H4 (K20) are methylated by specific HKMTsOdd S. Gabrielsen

MBV4230SET-domains Most HKMTs characterized by a conservedSET-domain Humans: 30 SET-domains Yeast genome: 6 SET-domains 300 foundThe structure of DIM5, a homolog ofSUV39H1, which methylates lysine 9(orange) in H3, is shown in complex withH3 tail (blue), cofactor (purple), and fourzinc ions (pink).Odd S. Gabrielsen

MBV4230Many HKMTs identified since yeast, redworm, yellowfly, pinkmammalian, purple Odd S. Gabrielsen

MBV4230Summary 200753Odd S. Gabrielsen

MBV4230Function inlong-term epigenetic maintenance? Epigenetics heritable changes in geneexpression that do not result from alterations inDNA sequencesHMTs proposed to be involved in a complexprocess that controls aspects of short- andlong-term transcriptional regulationDogma currently being challengedOdd S. Gabrielsen

MBV4230Function - Activation or repression?2 sites - 2 HKMTs - 2 effects Histone methylation was traditionally linked torepression, but turns out to be linked also to activation H3 K9 methylation correlates with heterochromatin formation.Met-K4 in H3 correlates with trx activationSet1p associated with HAT ctivationof trxRepressionof trxOdd S. Gabrielsen

MBV4230Function of histone methylation Repression (H3-K9, H3-K27, H4-K20) - more next lecture Activation (H3-K4, H3-K36, H3-K79) Numerous 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 chromosomeinactivation process.H3-K4 methylation is generally associated with trx active chromatin.di-methyl H3-K4 appears to be a global epigenetic mark in euchromatic regions andtri-methylation of H3-K4 correlates with active transcriptionboth H3-K4 and H3-K79 participate in establishing euchromatic regions bypreventing the spreading of heterochromatic regions.Role of histone lysine methylation in trx elongationOdd S. Gabrielsen

MBV4230Activating effects of methylation- two levels Chromosome level - preventing spread of heterochromatin The methylation of H3-K4 specifically impairs methylation at H3-K9, thereby blocking a majorpathway of heterochromatin formation binding of the histone deacetylase NuRD repression complex to the H3 N-terminal tail is precludedby methylation at K4, but not K9 Gene level di-methyl H3-K4 appears to be a global epigenetic mark in euchromatic regions and tri-methylationof H3-K4 correlates with active transcription Elongation - more laterOdd S. Gabrielsen

MBV4230Recognition - the chromodomains Methylated lysines are recognized by the conservedchromodomain modules found in several chromatin-associatedproteins.The chromodomain binds to histone tails bearing methyllysine ina highly specific manner, with the affinity being highest fortrimethyllysine and lowest for monomethyllysine.Odd S. Gabrielsen

MBV4230Recognition: Methylated histonesrecognized by spec chromodomains Chromo-domains asrecognition modules HP1 binds to histonesmethylated by SUV39H1 H3 K9 methylation correlates withheterochromatin formation.Heterochromatin protein 1 (HP1)is a methyl-lysine binding protein.HP1 recruits SUV39H1 leading topropargation of methylationOdd S. Gabrielsen

MBV4230An increasing list of recognitionmodules The repressive proteins heterochromatin protein 1 (HP1) and theDrosophila Polycomb (PC) protein contain a chromodomain thatallows them to specifically recognize the repressive methylationmarks (H3K9 and H3K27), whereas the chromodomain helicaseDNA-binding protein 1 (CHD1) activator protein from yeast uses itschromodomain to bind the activating methylated H3K4.Odd S. Gabrielsen

MBV4230Histone arginine methyltransferases(HRMTs) – PRMTs Several Arg HMTases identified: Carm1 (Coactivator-associated arginine methyltransferase). PRMT1 (protein arginine methyltransferase).Odd S. Gabrielsen

MBV4230The PRMT domain The catalytic module thatmethylates specific argininesis known as the PRMT(protein R methyltransferase)domain.The PRMT domain transfersthe methyl group from SAMto the guanidino group ofarginines to producemonomethylarginine ordimethylarginine.Some PRMT modulesspecifically preparesymmetric dimethylarginineand others produceasymmetric dimethylarginine.Odd S. Gabrielsen

MBV4230Function Methylation of specific arginines in histones H3and H4 correlate with the active state oftranscription. Ex: Methylation of Arg 3 of histone H4 facilitates H4 acetylation andenhances transcription activation by nuclear hormone receptors.Odd S. Gabrielsen

A dogma beingchallenged

MBV4230Removing histone methylation? histone methylation has for long been believedto be an extremely stable modification,evenconsidered an irreversible epigenetic markhistone demethylases was for long notdiscoveredHypothesis - histone methylation a long-termepigenetic markStill possible to remove?Odd S. Gabrielsen

MBV4230Enzymatic mechanisms Amine oxidationCH3 cleaved off afteroxidation asformaldehydeOdd S. Gabrielsen

MBV4230Recently discovered enzymesdemethylating Lys: LSD1 LSD1 LSD1 (lysine-specificdemethylase) is able todemethylate H3K4 using anamine oxidase reactiondependent on FAD (flavinadenine dinucleotide). Demethylation by LSD1 islimited to mono- or dimethylated H3K4: it cannotdemethylate tri-methylatedH3K4. The androgen receptor alterthe specificity of LSD1 fromH3K4 to H3K9, and therebyconverts the demethylasefrom a repressor to anactivator of transcription.LSD1Odd S. Gabrielsen

MBV4230Recently discovered enzymesdemethylating Lys - JmjC-domain enzymes JHDM1 The JmjC domain is a signaturemotif for a new family of histonedemethylases JHDM1 (JmjC domain-containinghistone demethylase 1) specificallydemethylates histone H3 at lysine 36(H3-K36). Demethylation generatesformaldehyde succinate andutilizes the same oxidativedemethylation mechanism as theAlkB DNA demethylases. JHDM1JMJD2AJMJD2A The JmjC domain-containing proteinJMJD2A is a lysine trimethylspecific demethylase able to reversetrimethylated H3-K9/K36 to di- butnot mono- or unmethylated products.Odd S. Gabrielsen

MBV4230Current list of histone demethylates69Odd S. Gabrielsen

MBV4230Recently discovered enzymesdemethylating Arg: deimination PADI4 Reversal of arginine methylationin histones can occur through theconversion of the methyl-arginineresidue into citrulline. This process is termeddeimination, since the methylgroup is removed along with theimine group of arginine. The enzyme that mediates thisreaction, peptidyl argininedeiminase 4 (PADI4), convertsunmodified arginine and monomethylated (but not dimethylated) arginine to citrullineat specific sites on the tail of H3and H4.Odd S. Gabrielsen

MBV4230SummarySeveral HKMTsAnd HRMTsXChromo-domain proteinsBinding proteinMod enzFunction?SpecificityYes,complex patternsTrx repressionand activationEpigeneticsSeveral new enzymes areDemod enznow being discoveredFunction?Trx repressionand activationEpigeneticsOdd S. Gabrielsen

The histone code

MBV4230Odd S. Gabrielsen

MBV4230The histone codeHistone encoded infoDNA encoded info Modifications of histone tails in specific patternsrepresent an encoded information extending theinformation content of the genome beyond the DNAsequenceThis hypothesis predicts that Distinct modifications of the histone tails will induce interaction affinitiesfor chromatin-associated proteins Modifications may be interdependent generate various combinations Local concentrations and combinations of differently modifiednucleosomes determine qualities of higher order chromatinOdd S. Gabrielsen

MBV4230Combinatorial possibilities large- a language? Acetylated acitve, deacetylated inactiveTrue but probably too simpleMany combinations - link to active/inactivechromatin complexNumber of combinations very highOdd S. Gabrielsen

MBV4230Synergistic or antigonisticmodifications of histones One modificationcan enhance orinhibit anothermodificationOdd S. Gabrielsen

MBV4230Cross-talkbetween histonemodificationsScience 2001, 293, 1074-1079.Odd S. Gabrielsen

MBV4230Reading / translatingthe histone code Modifications binding sites for proteinmodules Bromodomains bind acetylated lysines How to read?75 bromo-containing proteins in humans Chromodomains bind methylated sites Domains recognizing phosphorylated tails unknownOdd S. Gabrielsen

MBV4230Reading the code (A) Domains used for the recognition of methylated lysines, acetylatedlysines, or phosphorylated serines. (B) Proteins found that associate preferentially with modified versions ofhistone H3 and histone H4.79Odd S. Gabrielsen

MBV4230Writing & Reading the histone code The authors: HMTs, kinases, HATs, PPTases, HDACs,HDM? The readers: Chromo- and bromo domain proteins.Genome Biology 2001, 2(4): reviews003.1.6Odd S. Gabrielsen

MBV4230Mendel’s gene is more than just aDNA moiety!!HAT/HDACShort termchangesLocal structuralchangesHMT/HDM?Kinase/phosphataseHistone modificationsBinding sites forproteinsLong termchangesEpigenetic mark foreu- andheterochromatinBiological eventsOdd S. Gabrielsen

MBV4230ORF patterns of histone modifications Distribution of histonemodifications along ageneral ORF (Open reading frame)H3K4 monomethylation is enrichedtoward the 3 - end, dimethylationpeaks in the middle, whereastrimethylation occurs around theTSS and the 5 -end of the ORF The importance of H3K4methylation might be due torecruitment. Chromatin-remodelingfactors(NURF) and histonemodification complexes (hTip60,yNuA3, etc.) contain PHD domainsthat recognize H3K4 methylation,recruiting complexes to activate/repress transcription.82Odd S. Gabrielsen

ATP-dependentremodelling

MBV4230Changing the chromatin-templateto help transcription factors 1. Opening of chromatin throughdirected modication of histone tails(acetylation and methylation) NURFHDACsRSCCHRACACFHATs2. Opening of chromatin throughdirected nucleosome mobilization Histone-modification by HAT and HMT activityBasis for the histone code hypothesisSwi/SnfATP-dependent process3. Positioning of nucleosomes createspromoters with different requirementfor remodelingOdd S. Gabrielsen

MBV4230First discovered complex that remodelschromatin: the SWI/SNF complex SWI/SNF complex originally identified genetically as apositive regulator of HO and SUC2 genes SWI/SNF complex needed for full activity of some TFs In its absence : GAL4 tenfold reducedbasal transcription not affectedExists as a complex of about 2 MDa SWI - HO-genet inolvert in mating type switchingSNF - Sucrose non-fermentor: SUC2 involvert in sucrose-fermenterngSeveral subunits: SWI1, SWI2, SWI3, SNF5,SNF6, SNF11, TFG3/ANC1 andSWP73, some othersFew molecules per cell 100Chromatin-link: Supressors of defect SWI/SNF complex chromatin components several histone mutatants restore activator responseSuggested a function in counteracting chromatin-dep repressionOdd S. Gabrielsen

MBV4230Associates with nucleosomal DNA DNA-interaction Swi/Snf binds DNA and nucleosomes with high affinity Kd nM range Binding to DNA is ATP-dependent and targets minor groove After binding to DNA starts an ATP-dependent remodelling ofnucleosomes 1000 ATP molecules consumed pr minThe Nucleosome is not removed during this processOdd S. Gabrielsen

MBV4230SWI2 - the motor in the machinery- running on ATP SWI2-subunit harbors a DNA-stimulatedATPase activity mutated ATPase defect SWI-SNF functionFunction: Use ATP hydrolysis to increase theaccessibility of nucleosomal DNAMore accessibleClosedATPDerepressed stateInactive ground stateHow?Odd S. Gabrielsen

MBV4230Recruitment Swi/Snf acts probably only on a subset ofgenes Genome-analysis in yeast shows that 5% of all gener require Swi/Snf Limited number of molecules pr cell (100-500 Swi/Snf pr celle) Swi/Snf must be recruited to those promotere requiring Swi/Snffunction Recruitment model Gene-specific activators recruit Swi/Snf-complex directly Several activators shown to have direct interaction with theSwi/Snf-complex GCN4, SWI5, GAL4-VP16, GR, C/EBPßOdd S. Gabrielsen

MBV4230Mechanims of nucleosome disruptionunwrapping / unpeeling Unclear exactly what ”disruption”means Remodeled chromatin is moreaccessible for TFs and other factors Structurally changed nucleosomes or removal of theoctamer?Or some sort of altered DNA conformationPurified complex stimulates binding of GAL4-AH tonucleosmal DNA 10-30-foldModel: partial ”unwrapping” of DNAfrom the surface of the nucleosomeprovides DNA access Reduced DNA-mass relative to protein-mass prnucleosomeReduced DNA-supercoiling pr nucleosomeTFOdd S. Gabrielsen

MBV4230Remodeled chromatinalso through ”sliding” or ”transfer” Removal of nucleosomes can happen by localdisplacement along the same strand in cis (sliding) by ”spooling” or ”twisting”Or by transfer i trans (transfer)Swi/Snf ATPcistransOdd S. Gabrielsen

MBV4230Two main mechanismsHigh-enery stategenerated by ATP?Odd S. Gabrielsen

MBV4230More mechanisms ATPase motors thatdrive these enzymesseem to function as ATPdependent DNAtranslocases with limitedprocessivity Can also be regarded as‘nucleosomeisomerases’Odd S. Gabrielsen

MBV4230Mechanisms 1. Twist defect diffusion In this model, small local alterationsfrom mean DNA twist (‘defects’)propagate around the nucleosome2. Bulge diffusion In the bulge diffusion model,unpeeling of DNA from the histoneoctamer at the entry/exit of thenucleosome and subsequent rebindingof more distal sequences to the samehistone contact points would establishnucleosomal particles harbouringexcess DNA. Subsequent migration of the bulgearound the nucleosomal superhelixwould result in the nucleosomeapparently stepping - 40-60 bpOdd S. Gabrielsen

MBV4230Mechanisms Histone dimers can beremoved or exchangedbetween nucleosomesduring the course ofremodelling reactionsFurthermore,remodelling may causealteration of chromatincompositionOdd S. Gabrielsen

MBV4230Histone eviction and variantincorporation Eviction - displacement of histones Histone dimers of H2A and H2B can be rather easily exchanged in andout of nucleosome Entire histone octamers, including H3 and H4, can also be displaced(evicted) or exchanged under certain circumstances TF binding, chromatin-remodeling complexes and actively transcribingPol II can all mediate histone displacement. Variant histone incorporation Many variant forms of histones exist, distinguished from canonical corehistones mainly by being expressed outside of S phase and incorporatedinto chromatin in a DNA replication-independent manner.95Odd S. Gabrielsen

MBV4230Example: Histone variant H2A.Z Only 60% conserved relativeto H2A Associated with transcribedregions EssentialThe H2A.Z nucleosome hasdistinct features Facilitates TBP bindingIs evicted upon activationAltered contactsMetal binding siteH3.3 also assocated with trx Different from canonical H3 in onlyfour amino acidsOdd S. Gabrielsen

MBV4230Three families of ATP-drivenremodeling factors Swi/Snf-familien Yeast: Original Swi/Snf more abundant RSCDrosophila dSWI/SNF with brahma (brm)Humant: hSWI/SNF Mi-2/CHD-familien Med BRG1 and hBRMMi-2 complexNuRDISWI-familien yeast: ISW1 and ISW2Drosophila NURF(Nucleosome remodelling factor) and ACF and CHRACHuman RSF and hACF and hCHRACOdd S. Gabrielsen

MBV

MBV4230 Odd S. Gabrielsen Nucleosome 3D structure Luger et al., 1997. Crytals structure of the nucleosome core particle low resolution 1984, high resolution 1997 146 bp of DNA wrapped around a histone octamer core Note outside position of histone tails DNA is wrapped around DNA wrapped 1.65 turns around the histone octamer as a left-

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TRX Skater Squat.30. TRX Training Manual www.FighterAbs.com 3 Disclaimer The information provided in this workout program is for educational purposes only. The author is not a doctor and this information shouldn’t be taken as medical advice. You should get File Size: 2MBPage Count: 30Explore furtherTRX MMA Workout - TRX Training Program for Fighters [PDF]www.coretrainingtips.comPrintable TRX Bodyweight Combo Workout Routine - 4 Day Splitsuspensionrev.comTRX Workouts – 30 minute home workout plan [PDF]www.coretrainingtips.comTRX Workout: 44 Effective Exercises for Full-Body Strengthgreatist.comRecommended to you b

point for your TRX Suspension Trainer and a wall hanger for your favorite photos or artwork. The TRX Rip Group Training Station supports groups up to 10. Made of high-grade steel, this collapsible station includes a cable and lock. Wheels allow for easy portability inside or out. TRX S-FRAME TRX MULTIMOUNT KIT TRX RIP GROUP STATION

of the TRX 2.5 Racing Engine. Old tech tips and tricks that may have boosted the power of other engines could seriously diminish the performance of the TRX 2.5. There's more advanced thinking, development and testing in the stock parts of your TRX 2.5 than in many aftermarket manufacturer's so-called performance parts. The TRX 2.5 is .