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Moreover, many of these reader-writer complexes also contain anATP-dependent chromatin remodeling protein, and the reader, writer, andremodeling proteins work in concert to either decondense or condense longstretchesof chromatin as the reader moves progressivelyalong the nucleosomepackaged DNA (Figure 4-46).Some idea of the complexity of the processesjust described can be derivedfrom the results of genetic screensfor mutant genesthat either enhance or suppress the spreading and stability of heterochromatin in tests for position effectvariegation in Drosophila (see Figure 4-37). As pointed out previously, morethan 50 such genes are knonrn, and most of them are likely to function as subunits in one or more reader-writer-remodeling protein complexes.Blockthe Spreadof Reader-WriterBarrierDNASequencesDomainsChromatinNeighboringandtherebySeparateComplexesThe above mechanism for spreading chromatin structures raises a potentialproblem.
Inasmuch as each chromosome consists of one continuous, very longDNA molecule, what prevents a cacophony of confusing cross-talk betweenadjacent chromatin domains of different structure and function? Early studies ofposition effect variegation had suggested an answer: the existence of specificDNA sequencesthat separate one chromatin domain from another (seeFigure4-37).
Severalsuch barrier sequenceshave now been identified and characterized through the use of genetic engineering techniques that allow specificregions of DNA sequenceto be deleted or added to chromosomes.For example, a sequence called HS4 normally separates the active chromatin domain that contains the B-globin locus from an adjacent region ofsilenced, condensed chromatin in erythrocytes (see Figure 7-61)' If thissequenceis deleted, the B-globin locus is invaded by condensed chromatin.
Thischromatin silencesthe genesit covers,and it spreadsto a different extent in different cells, causing a pattern of position effect variegation similar to that227Figure4-45 How the recruitmentof acode-reader-writercomplex can spreadchromatinchangesalong aThecode-writeris anchromosome.enzymethat createsa specificon one or moreof the fourmodificationAfteritshistones.nucleosomalto a specificsiteon arecruitmentchromosomeby a generegulatorYwith aprotein,the writercollaboratesproteinto spreadits markcode-readerbYto nucleosomefrom nucleosomemeansof the indicatedreader-writerto work,complex.Forthis mechanismthe samethe readermust recognizehistonemodificationmarkthat the writerproduces(seealsoFigure4-43).228Chapter4: DNA,Chromosomes,and Genomes" r e a d e r - w r i t e r "c o m p l e x\D,'1onm-ill;*'".1,",11"Ti0",,"nl.- @com'rexf--oo'l.@f-(A)oo'S P R E A D I NWGA V EO FCHROMATICNONDENSATIONobserved in Drosophila.
As described in chapter 7, this invasion has dire consequences:the globin genes are poorly expressed,and individuals who carry sucha deletion have a severeform of anemia.matin modifications are knor,tmthat can also protect genesfrom silencing.Thechromatinin centromeresRevealsHowHistoneVariantscanCreateSpecialStructuresThe presence of nucleosomes carrying histone variants is thought to producemarks in chromatin that are unusually long lasting.
consider, for example, theformation and inheritance of the chromatin that forms on centromeres, theDNA region of each chromosome required for the orderly segregation of thechromosomes into daughter cells each time a cell divides (see Figure 4-21). rnmany complex organisms, including humans, each centromere is embedded ina stretch of special centric heterochromatinthatpersists throughout interphase,even though the centromere-mediated movement of DNA occurs only duringFigure4-46 How a complexcontainingreader-writer and ATP-dependentchromatinremodelingproteinscanspreadchromatinchangesalong achromosome.(A)A spreadingwaveofchromatincondensation.Thismechanismis identicalto that in Figure4-45,exceptthat the reader-writercomplexcollaborateswith an ATP-deoendentchromatinremodelingprotein(seeFigure4-29)to repositionnucleosomesandpackthem into highlycondensedarrays.Thisis a highlysimplifiedview of themechanismknownto be ableto spreadamajorform of heterochromatinfor long(seedistancesalongchromosomesFigure4-36).The heterochromatinspecificproteinHPI playsa majorroleinthat process.HP1bindsto trimethyllysine9 on histoneH3,and it remainsassociatedwith the condensedchromatinasone of the readersin areader-writer-remodelingcomplexthat,while incompletelyunderstood,isconsiderablymore intricatethan thatshownhere.(B)Theactualstructureof achromatinreader-remodelingcomplex,showinghow it is thoughtto interactwith a nucleosome.Modeledin grayisthe yeastRSCcomplex,whichcontains1 5s u b u n i t s - i n c l u d i nagn A T P dependentchromatinremodelingproteinand at least4 subunitswith codereaderdomains.(8,from A.E.Leschzineret al.,Proc.Natl Acad.Sci.U.S.A.104:4913-4918, 2007.With permissionfrom NationalAcademyof Sciences.).THEREGULATIONOFCHROMATINSTRUCTURE(A)229Figure 4-47 Some mechanismsof barrieraction.Thesemodelsare derivedfromof barrieraction,and adifferentanalysescombinationof severalof them mayfunction at any one site.(A)The tetheringof a regionof chromatinto a largefixedsite,suchasthe nuclearporecomplexillustratedhere,canform a banierthatstopsthe spreadof heterochromatin.(B)Thetight bindingof barrierproteinstocancompetewitha groupof nucleosomes(C)Byspreading.heterochromatinrecruitinga groupof highlyactivehistonebarrierscan erasethemodifyingenzymes,histonemarksthat arerequiredforto spread.Forexample,aheterochromatinpotentacetylationof lysine9 on histoneH3 will competewith lysine9 methylation,therebypreventingthe HP1proteinbindingneededto form someformsof(seeFigure4-46).(Basedheterochromatinon A.G.Westand P Fraser,Hum.Mol.Genet.from11, 2005.With permission14:R101-R1OxfordUniversityPress.)n u c t e a rp o r eb a r r i e rp r o t e i nb a r r i e rp r o t e i nb a r r i e rp r o t e i nmitosis.
This chromatin contains a centromere-specific variant H3 histone,known as CENP-A (see Figure 4-41), plus additional proteins that pack thenucleosomes into particularly dense arrangements and form the kinetechore,the special structure required for attachment of the mitotic spindle.A specific DNA sequence of approximately 125 nucleotide pairs is sufficientto serve as a centromere in the yeast S.
cereuisiae.Despite its small size, morethan a dozen different proteins assemble on this DNA sequence; the proteinsinclude the CENP-A histone H3 variant, which, along with the three other corehistones, forms a centromere-specific nucleosome. The additional proteins atthe yeast centromere attach this nucleosome to a single microtubule from theyeast mitotic spindle (Figure 4-48).The centromeres in more complex organisms are considerably larger thanthose in budding yeasts.For example, fly and human centromeres extend overhundreds of thousands of nucleotide pairs and do not seem to contain a centromere-specific DNA sequence.
These centromeres largely consist of short,repeated DNA sequences, knor,tn as alpha satellite Dl/A in humans. But thesame repeat sequencesare also found at other (non-centromeric) positions on./normalucleosomenucleosomwe ithcentromere-specifi ch i s t o n eH 3(A)s e qu e n c e - s p e c i f i cD N A b i n d i n gp r o t e i ny e a s tc e n t r o m e r i cD N Am t c r ot u b ul eyeastkinetochore(B)Figure4-48 A model for the structureofa simplecentromere.In the YeastSaccharomycescerevisiae, a speciaIaDNAsequenceassemblescentromericin whichtwo copiesofsinglenucleosomeinan H3 varianthistone(calledCENP-Athe normalH3.replacesmostorganisms)uniqueto thisvariantPeptidesequenceshistone(seeFigure4-41)then helptoadditionalproteins,someofassembleThiswhichform a kinetochore.is unusualin capturingonly akinetochorehumanshavemuchsinglemicrotubule;and form kinetochoreslargercentromeresthat can capture20 or more microtubules(seeFigure4-50).The kinetochoreisin detailin Chapter17.discussed(Adaptedfrom A.
Joglekaret al.,Nat Cel/2006.With permissionBiol.8:381-383,Ltd.)from MacmillanPublishers230Chapter4: DNA,Chromosomes,and Genomesh i g h e r - o r d er e p e a t:+it+a l p h as a t e l l i t eD N A m o n o m e( 1 7 1n u c l e o t i d peairs)actlve centromere(A)tlankingi n a c t i v ec e n t r o m e r eheterochromatin withnonfunctionala l p h as a t e l l i t eD N A(B)n e o c e n t r o m e r feo r m e dw i t h o u t a l p h as a t e l l i t eD N AFigure4-49Evidencefor the plasticity(A)A seriesof humancentromereformation,of A-T-richalphasatelliteDNAsequencesarerepeatedmanythousands(red),of timesat eachhumancentromeresurroundedby pericentric(brown).heterochromatinHowever,dueto anancientchromosomebreakageandrejoiningevent,somehumanchromosomescontaintwo blocksof alphasatelliteDNA'eachof whichpresumablyfunctionedasa centromerein itsoriginalchromosome.Usually,thesedicentricchromosomesarenotstablypropagatedbecausetheyattachimproperlytothespindleandarebrokenapartduringmitosis.Inchromosomesthatdo survive,however,oneof thecentromereshassomehowinactivated,eventhoughit containsallthenecessaryDNAsequences.Thisallowsthechromosometo bestablypropagated.(B)Ina smallfraction(1/2000)of humanbirths,extrachromosomesareobservedin cellsof theoffspring.Someof theseextrachromosomes,whichhaveformedfroma breakageevent,lackalphasatelliteyetnewDNAaltogether,(neocentromeres)centromereshavearisenfromwhatwasoriginallyeuchromaticDNA.chromosomes, indicating that they are not sufficient to direct centromere formation.
Most strikingly, in some unusual cases,new human centromeres (calledneocentromeres) have been observed to form spontaneously on fragmentedchromosomes. some of these new positions were originally euchromatic andIack alpha satellite DNA altogether (Figure 4-45).It therefore seemsthat centromeres in complex organisms are defined by anassembly of proteins, instead of by a specific DNA sequence.when antibodiesthat stain specific modified nucleosomes are used to examine the stretchedchromosome fibers from centromeres, one observesstriking alternation of twomodified forms of chromatin (Figure 4-50). It appears that this arrangementallows the centric heterochromatin to fold so as to position the cENp-A-containing nucleosomes on the outside of the mitotic chromosome, where theybind the set of proteins that form the kinetechore plates.
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