Nucleosomes:*what,*why*and*where?*Rob*Brewster*Outline*What*is*a*nucleosome?**‐*how*is*DNA*packaged/organized*in*Eukaryotes?*Why*do*nucleosomes*form?**‐*DNA*is*sEff,*how*do*~100*bp*loops*form*so*readily?*Where*do*nucleosomes*form?*‐ *What*controls*the*spacing*and*structure*of*nucleosomes*on*the*chromosome?*Accessibility*of*DNA*in*the*nucleosomes**‐*How*is*DNA*inside*a*nucleosomes*accessed?*DNA*OrganizaEon*in*Eukaryotes*DNA*is*packaged*and*condensed*into*chromosome*‐ Human*genome*is*big,*nucleus*is*small*~*2*billion*basepairs*≈*2m*nucleus*radius*~*6*µm*‐ *Many*different*levels*of*organizaEon*‐ *Compacts*chromosome*‐ *Regulates*transcripEon*by*making****porEons*of*the*chromosome****more/less*accessible*(up*to*80%****is*inaccessible*to*protein*binding)*‐ *We*will*focus*on*nucleosome****formaEon*(Alberts,*EssenEal*Cell*Biology**1998)*(Lee*W*et*al.,*Nat.*GeneEcs**2007)*Electron*micrograph*of*chromaEn*at*low*ionic*strength**‐*Nucleosomes*appear*as*“beads*on*a*****string*Basic*repeaEng*structure**can*be*probed*(protect*and*seq*method)**‐*DigesEon*enzyme*cuts*accessible*regions*of*DNA**‐*DNA*protected*by*nucleosome*is*not*cut*What*is*a*nucleosome?*(Furuyama*and*Biggins*,PNAS)*(Electron*Micrograph*from*Olins*and*Olins)*Mono*Dual*Mono*Mono*EnzymaEc*digests*ParEcular*enzymes*can*cut*double*stranded*DNA*between*basepairs**‐*Most*have*specific*recogniEon*sites**‐*Micrococcal*nuclease*cleaves*everything*it*can*(no*specific*seq.)*DiluEon*of*nuclease*1:3*(pictures*from*NEB)*(Peter*J.*Russell,*iGene%cs)*RepeaEng*structure*contains*5*different*proteins******** *‐*Main*body*is*an*octomer*formed*from:****two*copies*each*of*H2A,*H2B,*H3,*H4**‐*DNA*wraps*147*bp*(1.75*turns)*around****the*core**‐14*non*specific*adhesive*contacts*with* ******histone*(major*groove*–*histone)**‐*H1*agaches*to*the*linker*region*and*****changes*the*conformaEon;*required****for*chromaEn*formaEon**‐*H1*covers*30‐50bp*Structure*of*individual*nucleosome*DNA*rigidity*DNA*is*sEff…**‐*~150bp*persistence*length,*λ,*for*lysed****bacterial*genome**‐*loops*smaller*than*λ*should*be*rare*…*but*not*that*sEff**‐*single*turn*around*histone*core*~100*bp*Back*of*the*powerpoint*calculaEon*Energy*paid*to*bend*loop*of*147*bp*DNA*in*16.5*bp*circle*Assume*λ=150*bp*then:**This*cost*must*be*balanced*out*(and*then*some)*by*the*14*contacts*so*each*contact*must*contribute*~3*KT*However,*nucleosome*contacts*are*~*1.5*KT*(Polach*and*Widom,*2005;*Schiessel,*2003)*Reversing*this*and*solving*for*maximum*sEffness*for*stable*nucleosomes,*λ*<*~*80*bp**What*gives?*(ClouEer*and*Widom,*Molecular*Cell**2004)*Looping*probability*for*small*fragments*is*larger*than*expected**‐*~100*bp*fragments*form*loops*more****readily*than*predicted**‐*certain*sequences*are*more*flexible*****than*others**‐*sequences*which*loop*more*readily****also*more*readily*form*nucleosomes****(as*much*as*10^3‐fold*difference)*Does*this*sequence*dependence*control*where*nucleosomes*are*posiEoned?*Bending*of*short*DNA*fragments*The*EukaryoEc*genome!**‐*FormaEon*on*Yeast*genome*is*more***probable*than*for*e.*coli*genome**‐*Implies*Euk.*Genome*has*sequence****preferenEal*for*nucleosomes*EukaryoEc*genome*shows*specific*pagerns**‐*AA/AT/TT*dinucleoEde*frequency**~10*bp*(one*DNA*twist)*Where*are*Nucleosomes?*(Zhang*et*al.,*Nature*struct.*&*mol.*bio.,*2009)*Power*Spectrum*of*AA/AT/TT*repeats*Nucleosome*code*10bp*Periodicity*is*evident*both*in*vivo*and*in*vitro**‐*GC*is*5bp*out*of*phase*with*AT*dinucleoEdes**‐*due*to*bending*differences*in*GC*and*AT?*(Kaplan*et*al,*nature*2009)*Major*groove*Major*groove*Wrap*5bp*More*examples*(Segal*et*al.,*Nature*,*2006)*Nucleosome*vs*random*sequence*Periodicity*is*missing*from*arbitrar y*sequences*(Segal*et*al.,*Nature*,*2006)*Method:*From*in*vivo*occupancy*data,*calculate*the*probability*of*any*dinucleoEde*pair*at*a*given*nuc.*posiEon.*Probability*that*147bp*sequence*S*is*wrapped:*The*staEsEcal*weight*of*a*longer*sequence*with*mulEple*nucleosomes*is*just*the*product*of*the*probability*for*every*basepair*to*be*in*that*state,*The*probability*must*now*be*computed*computaEonally*due*to*the*enormous*number*of*possible*configuraEons*(C)*(Segal*et*al.,*Nature*,*2006)*ProbabilisEc*model*of* nucleosome *occupancy*ProbabilisEc*landscape*for*occupancy*At*a*parEcular*nucleosomal*coverage*can*predict*where*stable*nucleosomes*will*be*located*(Kaplan*et*al,*nature*2009)*PredicEons*from*thermodynamic*model*Prob.*of*coverage*by*nuc*Predicted*stable*nuc*Exp.*Determined*nuc*Success*rate*within*32bp*of*predicted*posiEon*StaEsEcal*PosiEoning*–*A*(semi)compeEng*view*(Zhang*et*al.,*Nature*struct.*&*mol.*bio.,*2009)*Various*Transcribed*Genes*Nucleosomes*pagern*emerges*from*steric*exclusion*on*a*line**‐*Promoter*region*is*always*“nucleosome*free”**‐*DNA*sequence*appears*to*play*only*a*small*role*Less*more*In*Vitro*Barrier*Hard*Spheres*Random*1D*hard*sphere*gas?*Occurrence*Distance*from*TSS*Phasing*in*vivo*resembles*RDF*of*(for*instance)*LJ*Gas*(Zhang*et*al.,*Nature*struct.*&*mol.*bio.,*2009)*Can*a*simple*model*of*a*1D*hard*sphere*gas*predict*nucleosome*posiEoning*pagern?*Conclusions*on*posiEoning*DNA*sequence*magers*for*nucleosomes*posiEons…**‐*preferenEally*bind*to*parEcularly*repeaEng*sequences**‐*avoid*long*tracks*of*A/T*…*however,*this*effect*seems*to*be*minimized*in*vivo**‐*Simple*1D*gas*model*fits*in*vivo*nucleosome*posiEons*well***(data*not*shown)*How*does*one*resolve*the*apparent*conflict*in*these*views?*Accessing*nucleosomal*DNA*How*is*protected*DNA*accessed?**‐*DNA*replicaEon,*repair*and*transcripEon*all*require*access*to*****occluded*DNA**‐*Specialized*motors*called*“remodeling*factors”*disassemble*and****perturb*nucleosomes*to*allow*access
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