Nucleosomes: what, why and where?rpdata.caltech.edu/.../lectures/NucleosomeClass3.pdf ·...

Nucleosomes:what,whyandwhere?

RobBrewster

OutlineWhatisanucleosome?

‐howisDNApackaged/organizedinEukaryotes?

Whydonucleosomesform?

‐DNAissEff,howdo~100bploopsformsoreadily?

Wheredonucleosomesform?

‐ Whatcontrolsthespacingandstructureofnucleosomesonthechromosome?

AccessibilityofDNAinthenucleosomes

‐HowisDNAinsideanucleosomesaccessed?

DNAOrganizaEoninEukaryotesDNAispackagedandcondensedintochromosome

‐ Humangenomeisbig,nucleusissmall~2billionbasepairs≈2mnucleusradius~6µm

‐ ManydifferentlevelsoforganizaEon‐ Compactschromosome‐ RegulatestranscripEonbymakingporEonsofthechromosomemore/lessaccessible(upto80%isinaccessibletoproteinbinding)

‐ WewillfocusonnucleosomeformaEon

(Alberts,EssenEalCellBiology1998)

(LeeWetal.,Nat.GeneEcs2007)

ElectronmicrographofchromaEnatlowionicstrength

‐Nucleosomesappearas“beadsonastring

BasicrepeaEngstructurecanbeprobed(protectandseqmethod)

‐DigesEonenzymecutsaccessibleregionsofDNA

‐DNAprotectedbynucleosomeisnotcut

Whatisanucleosome?

(FuruyamaandBiggins,PNAS)

(ElectronMicrographfromOlinsandOlins)

Mono Dual Mono Mono

EnzymaEcdigestsParEcularenzymescancutdoublestrandedDNAbetweenbasepairs

‐MosthavespecificrecogniEonsites

‐Micrococcalnucleasecleaveseverythingitcan(nospecificseq.)DiluEonofnuclease1:3

(picturesfromNEB)

(PeterJ.Russell,iGene%cs)

RepeaEngstructurecontains5differentproteins

‐Mainbodyisanoctomerformedfrom:twocopieseachofH2A,H2B,H3,H4

‐DNAwraps147bp(1.75turns)aroundthecore

‐14nonspecificadhesivecontactswith histone(majorgroove–histone)

‐H1agachestothelinkerregionandchangestheconformaEon;requiredforchromaEnformaEon

‐H1covers30‐50bp

Structureofindividualnucleosome

DNArigidityDNAissEff…

‐~150bppersistencelength,λ,forlysedbacterialgenome

‐loopssmallerthanλshouldberare

…butnotthatsEff‐singleturnaroundhistonecore~100bp

BackofthepowerpointcalculaEonEnergypaidtobendloopof147bpDNAin16.5bpcircle

Assumeλ=150bpthen:

Thiscostmustbebalancedout(andthensome)bythe14contactssoeachcontactmustcontribute~3KT

However,nucleosomecontactsare~1.5KT(PolachandWidom,2005;Schiessel,2003)

ReversingthisandsolvingformaximumsEffnessforstablenucleosomes,λ<~80bp

Whatgives?

(ClouEerandWidom,MolecularCell2004)

Loopingprobabilityforsmallfragmentsislargerthanexpected

‐~100bpfragmentsformloopsmorereadilythanpredicted

‐certainsequencesaremoreflexiblethanothers

‐sequenceswhichloopmorereadilyalsomorereadilyformnucleosomes(asmuchas10^3‐folddifference)

DoesthissequencedependencecontrolwherenucleosomesareposiEoned?

BendingofshortDNAfragments

TheEukaryoEcgenome!‐FormaEononYeastgenomeismoreprobablethanfore.coligenome

‐ImpliesEuk.GenomehassequencepreferenEalfornucleosomes

EukaryoEcgenomeshowsspecificpagerns‐AA/AT/TTdinucleoEdefrequency~10bp(oneDNAtwist)

WhereareNucleosomes?

(Zhangetal.,Naturestruct.&mol.bio.,2009)

PowerSpectrumofAA/AT/TTrepeats

Nucleosomecode

10bpPeriodicityisevidentbothinvivoandinvitro‐GCis5bpoutofphasewithATdinucleoEdes

‐duetobendingdifferencesinGCandAT?

(Kaplanetal,nature2009)

Majorgroove

MajorgrooveWrap5bp

Moreexamples

(Segaletal.,Nature,2006)

Nucleosomevsrandomsequence

Periodicityismissingfromarbitrarysequences

(Segaletal.,Nature,2006)

Method:Frominvivooccupancydata,calculatetheprobabilityofanydinucleoEdepairatagivennuc.posiEon.

Probabilitythat147bpsequenceSiswrapped:

ThestaEsEcalweightofalongersequencewithmulEplenucleosomesisjusttheproductoftheprobabilityforeverybasepairtobeinthatstate,

TheprobabilitymustnowbecomputedcomputaEonallyduetotheenormousnumberofpossibleconfiguraEons(C)

(Segaletal.,Nature,2006)

ProbabilisEcmodelofnucleosomeoccupancy

ProbabilisEclandscapeforoccupancy

AtaparEcularnucleosomalcoveragecanpredictwherestablenucleosomeswillbelocated

(Kaplanetal,nature2009)

PredicEonsfromthermodynamicmodel

Prob.ofcoveragebynuc

Predictedstablenuc

Exp.Determinednuc

Successratewithin32bpofpredictedposiEon

StaEsEcalPosiEoning–A(semi)compeEngview

(Zhangetal.,Naturestruct.&mol.bio.,2009)

Variou

sTranscribe

dGen

es

Nucleosomespagernemergesfromstericexclusiononaline‐Promoterregionisalways“nucleosomefree”‐DNAsequenceappearstoplayonlyasmallrole

Less

more

InVitro

Barrier HardSpheres

Random1Dhardspheregas?Occurrence

DistancefromTSS

PhasinginvivoresemblesRDFof(forinstance)LJGas

(Zhangetal.,Naturestruct.&mol.bio.,2009)

Canasimplemodelofa1DhardspheregaspredictnucleosomeposiEoningpagern?

ConclusionsonposiEoning

DNAsequencemagersfornucleosomesposiEons…

‐preferenEallybindtoparEcularlyrepeaEngsequences

‐avoidlongtracksofA/T

…however,thiseffectseemstobeminimizedinvivo

‐Simple1DgasmodelfitsinvivonucleosomeposiEonswell (datanotshown)

Howdoesoneresolvetheapparentconflictintheseviews?

AccessingnucleosomalDNAHowisprotectedDNAaccessed?

‐DNAreplicaEon,repairandtranscripEonallrequireaccesstooccludedDNA

‐Specializedmotorscalled“remodelingfactors”disassembleandperturbnucleosomestoallowaccess

‐However,evenwithoutthesemotors,wrappedDNAhassomeaccessibility

‐DigesEonassaysprobeequilibriumaccessibility

‐Fretprobesdynamicaccessibility

Invitroaccessibilitymechanism

Whatisthemechanismtoaccessasiteburieddeepinthenucleosome?

TranslaEonalongdna

?? ?

Transientunwrapping

Bulgedstructures

Invitroaccessibilitymechanism

Whatisthemechanismtoaccessasiteburieddeepinthenucleosome?

TranslaEonalongdna

? ?

Transientunwrapping

Bulgedstructures

Invitroaccessibilitydoesnotshowlengthdependencebeyond~100bp (Andersonetal,2002)

FretonnucleosomalcomplexOnefretdyeisputonthenucleosome,oneisputontheendoftheDNAstrand

Exciteatlowwavelengths:

Dyesareclose–signal~680nmishigh

Dyesarefar–signal~680nmislow

(LiandWidom,2004)

Invitroaccessibilitymechanism

Whatisthemechanismtoaccessasiteburieddeepinthenucleosome?

TranslaEonalongdna

!

Transientunwrapping

Bulgedstructures

Invitroaccessabilitydoesnotshowlengthdependencebeyond~100bp

FRETexperimentsbyLiandWidom

Probingaccessibility

SpecificrestricEonenzymebindingsite

DNAiscleavedwhensitebecomesaccessiblefromunwrapping

MeasureprobabilityforbeingcutasafuncEonofburialdepth

Accessibilityisreduced(comparedtonakeddna)sitesburiedshallow10‐100x,deep~105x

(PolachandWidom,2000)

Measuringunwrapping

Themoreflexiblesequenceislessaccessible!

WhereΔGisthechangeinfreeenergyassociatedwithopeningthebindingsite

TheoryInterlude

NucleosomepreferenceisenErelybending?!

γ=‐0.24kT/bp

Moreflexiblesequence(601TA)Morerigidsequence(5S)

γ=‐0.16kT/bp

Fizngbendingenergy

b

WithR=13.5bp,a=10.5bp,L=147bp

However,forClouEerandWidomdata

bp

RNAPtranscripEonthroughthenucleosome

(Hodgesetal,2009)

BeadagachedtoendofDNAandRNAP

‐RNAPmovementthroughthenucleosomecanbemeasured

‐DistancebetweenbeadsisareadoutforRNAPdepthinnucleosome

ModelforRNAPtranscripEonthroughnucleosome

Conclusions

TheEnd

DNAorganizaEon

E.Coligenome~4millionbp≈1.2mmE.Colidimensions~2µm^3

Humangenome~6billionbp≈2000mmNucleusdimensions~750µm^3

(Maghews,DNA Structure Prerequisite Informa%on.1997)

Sequencepreferenceofnucleosomes

Nucleosomecode‐AA/TT/TAdinucleoEderepeats appearwith10bpperiodicity

‐GCrepeats5bpoutofphase

DuetodifferenceinbendingofdinucleoEdes?

‐HelicityofDNAmeansbendingatmajorandminorgrooveis opposite

‐NucleosomesmayposiEonAA/TA/TTatminor/major,GCatmajor/minorgrooveduetodifferenceinbending

(Segaletal.,Nature,2006)

WhatisaNucleosome(howdoweknow)?DigesEonwith

‐ Humangenomeisbig,nucleusissmall~2billionbasepairs≈2mnucleusradius~6µm

‐ ManydifferentlevelsoforganizaEon

‐ WeareinteresEnginNucleosomeformaEon

DNAorganizaEoninEukaryotes

OlinsandOlins

DNAorganizaEoninEukaryotes

Topics:‐GeneralintroducEontoNucleosomes ‐compacEoncomputaEon?‐ProtectandSeq:Widomhgp://www.wisdom.weizmann.ac.il/~eran/NucleosomeModel.pdf

weissmanhgp://www.nature.com/nmeth/journal/v6/n4/full/nmeth0409‐244b.html

‐Lacrastuff,pullingexp‐WidomAccessibility