HumanChromosomeStructure
andAberrationMechanisms
LyndaJCampbell
lynda.campbell@svhm.org.au
Tjio&Levan,Thechromosomenumbersof
man.Hereditas 42,16(1956)
Nucleosomes
• Thebasicunitofchromatinisthenucleosome.
• Nucleosomes contain9histones anda 166bpsofDNA:
• Histones areafamilyofsmall,positivelychargedproteins
termedH1,H2A,H2B,H3,andH4.
• DNAisnegativelycharged,duetothephosphategroupsin
itsphosphatesugarbackbone,sohistones bindtightlyto
DNA.
• Twoeachofthehistones H2A,H2B,H3,andH4forma
histone octamer,whichbindsandwrapsa 1.7turnsof
DNA,ora 146bp
• TheadditionofoneH1proteinwrapsanother20base
pairs,resultingintwofullturnsaroundtheoctamer.
• Everychromosomecontainshundredsofthousandsof
nucleosomes,whicharejoinedbythelinkerDNAthatruns
betweenthem(anaverageofabout20basepairs).
• Eachchromosomeisthusalongchainofnucleosomes.
Chromatinhashighlycomplexstructurewithseverallevelsoforganization.
ThesimplestlevelisthedoublehelicalstructureofDNA.
© 2005 Pierce,Benjamin.Genetics:AConceptualApproach,2nded.(NewYork:W.H.FreemanandCompany),292
DNAPackaging:Nucleosomes andChromatin
• Thehaploidhumangenomecontainsapproximately3
billionbasepairsofDNApackagedinto23chromosomes.
• Mostcellsinthebody(exceptfemaleovaandmalesperm)
arediploid,with23pairsofchromosomes(total:6billion
basepairsofDNApercell).
• Eachbasepairisaround0.34nanometerslong
• Eachdiploidcellthereforecontainsabout2metersofDNA
(0.34× 109)× (6× 109).
• Itisestimatedthatthehumanbodycontainsabout50
trillioncells— i.e.100trillionmetersofDNAperhuman.
• TheSunis150billionmetersfromEarth.Thismeansthat
eachofushasenoughDNAtogofromheretotheSunand
backmorethan300times,oraroundEarth'sequator2.5
milliontimes!
Annunziato, A. (2008) DNApackaging:Nucleosomes andchromatin. NatureEducation 1(1)
Chromosomecompactionduringmetaphase
• Chromosomesaregenerally
decondensed duringinterphase
• Thus,inthenucleusofacell
preparingforcelldivision,the
chromatinstrandsarelong,thin,
fragilehelices.
• Toseparatethechromatinstrands
intotwoidenticalportionswithout
breakage,DNAmustbecondensed
intocompactform.
• Thecondensationofinterphase
chromosomesoccursduringM
phaseandisintimatelyconnected
withprogressionofthecellcycle.
• Itrequiresaclassofproteinscalled
condensins whichdrivethecoiling
ofeachchromosome.
HeterochromatinandEuchromatin
• Twotypesofchromatin,heterochromatinand
euchromatin,arefunctionallyandstructurallydistinct
regionsofthegenome.
– Heterochromatinisdenselypackedandinaccessibleto
transcriptionfactorssoitisrenderedtranscriptionally silent
(RichardsandElgin2002).
– Euchromatin islesscondensed,moreaccessible,andtherefore
transcriptionally active(Hennig 1999).
• Euchromatin makesupmostofinterphase chromosomes
andprobablycorrespondstoloopeddomainsof30nm
fibres.
• Heterochromatiniscommonlyfoundaroundcentromeres
andneartelomeres,butalsointerruptseuchromatin at
otherpositionsonchromosomeswherethe30nmfibres
aresubjectedtoadditionallevelsofpacking,rendering
themresistanttogeneexpression.
Centromeres
• Theword"centromere"isderivedfromtheGreek
wordscentro ("central")andmere ("part"),butonly
metacentric chromosomeshavecentromeres attheir
middle;
• Inotherchromosomes,centromeres arelocatedata
varietyofpositionsthatarecharacteristicforeach
particularchromosome.
RoleofCentromeres
• Thecentromere wasfirstdescribedbyGermanbiologist
WalterFlemming inthe1880sasthe"primaryconstriction"
ofthechromosome.
• Thecentromere playsacriticalroleinmitosisbyensuring
thattheparentcell’schromosomesreachtheircorrect
destinationi.e.thattwodaughtercellsareproducedthat
eachcontainthesamenumberofchromosomesasthe
parentcell.
• Thecentromere providesthefoundationforkinetochore
assemblyandservesasasiteforsisterchromatid
attachment.
• Errorsincentromere orkinetochore functionare
catastrophicforcells:canleadtoaberrantdivisionand
chromosomalinstability,bothofwhichareoftenobserved
incancercells.
O'Connor, C. (2008) Chromosomesegregationinmitosis:Theroleofcentromeres. NatureEducation 1(1)
Centromere Function
• Sisterchromatids arejoinedtogetheratcentromeres
• Foraccuratemitoses,sisterchromatids mustremain
attacheduntilthespindlecheckpointhasbeenpassed.
• Theattachmentofsisterchromatids ismediatedbythe
cohesin complexofproteins
• Chromatid attachmentoccursonadifferentfaceofthe
chromosomefromthekinetochore.
• Asacellentersanaphase,cohesin isprecipitously
degraded,andthecell'ssisterchromatids separateto
oppositepolesofthespindle.
TheCentromere Kinetochore Region
Attheheartofthekinetochore
isthespecializednucleosome
thatcontainscentromere
protein(CENP)A,ahistone H3
homologue.
TheNdc80/HEC1complex
seemstobedirectlyinvolved
inmicrotubulebinding.
Severalmicrotubuleplusend
bindingproteins(+TIPs)are
importantformicrotubuleto
kinetochore attachment.
Musacchio,A.etal. Thespindleassemblycheckpointinspaceandtime.NatureReviews
MolecularCellBiology 8, 385,2007.
Telomeresofhumanchromosomes
• Inthe1930s,HermannMullernotedthatthe
endsofchromosomeshaduniqueproperties.
• Mullernamedtheseendstelomeres(fromthe
Greektelo,meaning"end,"andmere,meaning
"part"),basedontheirpositiononchromosomes.
• Hefoundthattheendsofchromosomeswere
resistanttotheeffectsofmutagenicXrays.
• Mullerthereforehypothesizedthat"theterminal
genemusthaveaspecialfunction,thatofsealing
theendofthechromosome"andthat"forsome
reason,achromosomecannotpersistindefinitely
withouthavingitsendsthussealed"
O'Connor, C. (2008) Telomeresofhumanchromosomes. NatureEducation 1(1)
Telomeres
• ElizabethBlackburnfoundthatthetelomeresofthe
protozoanTetrahymena contained2070tandemcopies
ofahexanucleotide:5'CCCCAA3'ononestrandand5'
TTGGGGontheother(Blackburn&Gall,1978).
• Allvertebratetelomeres,includinghumans,consistof
repeatsofthesame6basesequence:CCCTAA/TTAGGG.
• Humantelomeresareapproximately0.5– 15kbin
lengthandconsistofa3008,000preciserepeatsofthe
sequence.
• Inalltelomeres,theGrichstrandmakesupthe3'end
ofthechromosomeandtheterminalportionoftheG
richstrandissinglestranded,generatingasocalled“G
tail”
• ThelengthoftheGtailisvariable,averagingbetween
75300nucleotidesinhumans.
Telomeresandtelomerase
• WhenthemechanismofcellularDNAreplicationwas
clarified,itwasrealizedthatthiswouldresultintheendsof
chromosomesprogressivelyshorteningwitheachroundof
DNAreplication.
• DNApolymeraserequiresshortRNAprimerstoinitiate
replication,anditthenextendstheprimersina5'to3'
direction.
• Asthereplicationforkmovesalongthechromosome,one
ofthetwodaughterstrandsissynthesizedcontinuously.
Theotherstrand,orlaggingstrand,issynthesized
discontinuouslyinshortfragmentsknownasOkazaki
fragments,eachofwhichhasitsownRNAprimer.
• TheRNAprimersaresubsequentlydegraded,andthegaps
betweentheOkazakifragmentsarethenfilledinbythe
DNArepairmachinery.
Telomeresandtelomerase
• Aproblemarisesattheendofthechromosome,
becausetheDNArepairmachineryisunableto
repairthegapleftbytheterminalRNAprimer.
• Thus,thenewDNAmoleculeisshorterthanthe
parentDNAmoleculebyatleastthelengthof
oneRNAprimer.
• Withoutasolutiontothisendreplication
problem,chromosomeswouldprogressively
shortenovermanycelldivisions,aprocessthat
wouldbringaboutcatastrophicconsequences.
Telomerase
• CarolGreider,agraduatestudentinElizabethBlackburn's
laboratory,purifiedanenzymethatcouldlengthen
telomeres(Greider &Blackburn,1985).
• Thisenzyme,telomerase,isahighlyspecializedreverse
transcriptase anenzymethatsynthesizesDNAfroman
RNAtemplate.
• TelomerasebindstotheGtailofthetelomerethroughthe
RNAtemplateandthencatalyzestheextensionoftheG
tail.
• Telomeraseisabletorepeatthiscyclemultipletimesby
movingtonewbindingsitesalongthenewlysynthesizedG
tail.Then,duringthenextroundofDNAreplication,DNA
polymeraseandtheDNArepairenzymesfillintheother
strand.Thus,telomeraseisabletobothmaintainand
extendthelengthoftelomeres.
3’
5’
3’
Newstrands
RemovaloftheRNAprimerleadstothe
shorteningofthechromosomeaftereachround
ofreplication,eventuallyleadingtocelldeath
5’
Telomere
3’
AnRNAsequencein
telomeraseactsasa
templateforDNA.
Telomerase
Thisenzymeaddsthe
3’
telomeric sequenceto
the3’endofthe
RNAtemplate chromosome.
Telomerase
3’
3’
Gap
Originallengthof
chromosomeis
restored(leavinggap
wheretheprimerfor
DNAreplicationhas
beenremoved.
Tloop
• Mosteukaryoticchromosomesbendback
onthemselvestoformaphysicalloopat
thetelomere.
• Tloopssizecorrelateswithtelomere
length.
• Formationoftloopsrequiresboththe
telomeric repeatsandtheGtail.
• AnarrayoftelomerespecificDNAbinding
proteinsarerecruitedthatcatalyzethe
formationofatloop.
• TheGtailatthe3'endofthechromosome
invadesdoublestrandedtelomeric DNA,
locallydisplacingonestrandofthehelix.
• Thedisplacedstrandisthenboundbyan
essentialsinglestrandedbindingprotein
designatedPOT1,for"protectionof
telomeres."
• Othermultisubunitproteincomplexes
bind,somecatalyzingDNAbending,
regulatingtelomerelength,orplayinga
primarilyprotectiverole.
Telomerase
• Telomeraseplaysakeyroleintheregulationof
telomerelength.
• Telomerestendtoshortenincellswithouttelomerase
overtime,andcellsmaystopdividingandbecome
senescentafterthetelomeresshortenbelowacritical
length.
• Inmostanimals,embryoniccellsandgermcells
possesstelomeraseactivity,butmanysomaticcellsdo
not.Theexceptionsincludehighlyproliferativecells:
skin,haematopoietic tissuesandgutepithelium.
• Atthecellularlevel,telomerasedeficientmiceshow
chromosomalabnormalitiesseveralgenerationsbefore
defectsbecomeapparentatthetissuelevel,withsome
showingendtoendfusionsaspredictedbyMullerand
McClintock.
Structureofanacrocentric chromosome
• Shortarmheterochromatinappearstobe
anextensionofthecentromere regionand
containsshortsequencerepetitiveDNA
stalks
• Stalkscontainmultiplecopiesofthegenes
parm
codingfor18Sand28SribosomalRNA.
Theystainwithsilverstainingandarealso
centromere
calledNucleolar OrganizerRegions(NORs).
• Humanribosomalgenerepeatsare
distributedamongfiveNORsontheparms
qarm
ofacrocentric chromosomes.
• Onexitfrommitosis,nucleoliformaround
individualactiveNORs.Ascellsprogress
throughthecycle,thesemininucleolifuse
toformlargenucleoliincorporating
multipleNORs.
satellites
Chromosomebanding
• Eachchromosomehasauniquebandingpatternwhentreatedwitha
weaktrypsin solutionandstainedwithaRomanovsky dyesuchas
Giemsa.
• Eventhethinnestbandsprobablycontain>millionnucleotidepairs.
• Thehumangenomecontainslargenonrandomblocksofsequence
thataresignificantlyhigherorlowerinGCcontentthatthegenome
averageof41%.
• Theblockscorrelatewiththebandingpatternofmetaphase
chromosomes.
• BandsstaineddarklybyGiemsa (Gbands)correlatewithDNAthatis
lowinGCcontent;lightbandscorrespondtoDNAofhigherthan
averageGCcontent.
• GCrichregionsofthegenomehaveahigherdensityofgenes,
especiallyof“housekeeping”genesthatareexpressedinvirtuallyall
celltypes.
• ThebandingpatternmayberelatedtogeneexpressionandtheGband
lightanddarkbandsmayreflectthewayinwhichchromatinloopsare
packaged,withlightbandedareascontainingconcentrationsofthe
cellularcomponentsinvolvedingeneexpression.
Chromosometerritories
• Chromosomesininterphase resembleabowlof
spaghettibutistheirlocationtrulyrandom?
• Cremeretaldevelopedalaserthatcouldbeinduce
DNAdamageintheilluminatedregionsofasinglecell.
• Theythenaddedradioactivelylabelled nucleotides,
whichthecellincorporatedintoitsDNAduringthe
repairprocess.
• Whenthecellenteredthenextmitosis,themarked
regionsofeachchromosomewereanalyzedby
radiographyandshowedthatonlyafewchromosomes
percellweredamaged,aresultthatstrongly
supportedachromosometerritorymodel.
Misteli, T. (2008) Chromosometerritories:Thearrangementofchromosomesinthenucleus. NatureEducation 1(1)
Doeschromosomelocationaffectgene
transcription?
• Thespatiallocalizationofterritoriesisthoughttobe
importantforgeneexpression thepositionof
chromosomesrelativetooneanotherdiffersfromcell
tocellandsuchdifferencesreflectvariationingene
expressionpatterns.
• Invertebrates,chromosomalregionswithlowgene
densityarelocatedclosetothenuclearperiphery.
• Finlan etalrelocatedspecifichumanchromosomesto
thenuclearperipherybytetheringthemtoaproteinof
theinnernuclearmembrane
• Theirdatashowedthattheradialpositionwithinthe
nucleuscouldinfluencetheexpressionofsome,but
notall,genes.
Finlan etal,PLoS Genet2008
TheodorBoveri (18621915)
• TheodorBoveri wasthefirsttosuggest
thatmalignanttumourscouldbedueto
anabnormalchromosomeconstitution
• Healsoforeshadowedthediscoveryof
oncogenes andtumoursuppressorgenes
ashepostulatedtheexistenceof
enhancingorsuppressingchromosomes
withmalignantgrowthresultingfromloss
ofsuppressingorpredominanceof
enhancingchromosomes.
Ph
Nowelland
Hungerford,
1960
MechanismsofChromosomeAberrations
• Commonchromosomeaberrations:
–
–
–
–
Deletion
Translocation
Inversion
Amplification
• Mechanismsbywhichchromosomesare
rearranged:
– DoublestrandedDNAbreaksinducedbychemical
agentsorirradiation
– Aberrationsofnormalchromosomerearrangements
suchasoccursinIg genesduringBcelldevelopment
– Telomerefusion
In1973,JanetRowleyshowedthePhchromosometo
resultfromabalancedrearrangement:t(9;22)(q34;q11.2)
9
22
Whydogenefusionsarise?
• 3Dchromosomalarchitecturewithintheinterphase
nucleusprobablyplaysasignificantrole
• Lociinvolvedinthefollowingtranslocationsareclose
toeachotherinthecorrespondingnormalcelltype:
–
–
–
–
BCRABL1 inCML
PMLRARA inAPL
RETCCDC6 inthyroidcancer
IGH@MYC,IGH@CCND1,IGH@BCL2 inlymphoid
malignancies
• Otherfactorssuchassharedsequencemotifsat
chromosomalbreakpointsprobablyplayarole
Mitelman etal,NatureReviewsCancer2007
Causativeagents
• Whilstmostareunknown,anumberofgenotoxic
and/orenvironmentalexposureshavebeen
identifiedthatincreasetheriskofspecific
translocations,deletionsornumerical
aberrations:
–
–
–
–
–
Topoisomerase IIinhibitors
Alkylating agents
Agriculturalpesticides
Benzene
Radiation
11q23abnormalities
MLL generearrangements
• TopoisomeraseIIinhibitorsareknowntoinduce
therapyrelatedAMLassociatedwithMLL
translocationsandtheassumptionhasbeenthat
doublestrandedDNAbreaksareinducedwithin
MLL bytheTopoIIinhibitors
• tAMLafterTopoisomeraseIIinhibitorsand
infantacuteleukaemia(thathasbeenrelatedto
maternalingestionofoestrogens)showMLL
breakpointsthatclustertowardsthe3’endofan
8.3kbBCRincludingexons 814
• However,theexactmechanismbywhichTopoII
inhibitorsproduceDNAcleavageisunclear.
Leetal,GenesChromosomesandCancer2009
Hostfactors:Chromosomeinstability
• Cancerpredisposingdisorderssuchas
chromosomeand/orDNAbreakage
syndromes
e.g.AtaxiaTelangiectasia iscausedbymutationof
theATMgene(11q22)thatplaysanimportant
roleintherecognitionandrepairofDNAdouble
strandedbreaks.Affectedindividualsareproneto
developtranslocationsinvolvingTCRorIGgenes.
Immunoglobulingenetranslocations
DoublestrandedDNAbreaksmaybecausedby
differentmechanisms:
– VDJtypeinwhichtheRAGendonuclease createsasite
specificDSB5’ofarecombinationsignalsequenceinearly
BorTcells
– Classswitchrecombinationtypeinwhichactivation
inducedcytidine deaminase (AID)deaminates cytidines at
theIg switchregionsleadingtoDSBinactivatedBcells
– Randomtypebreakageduetoreactiveoxygenspeciesor
ionizingradiationgeneratingDSBrandomlythroughout
thegenome
– CpG type– DSBatCpG islandsinearlyBcells(IGH@BCL2)
Tsaietal,Cell2008
Telomereloss
• Themagnitudeoftelomerelosswitheachcelldivision
isgreaterthancanbeexplainedbythe“endreplication
problem”
• Inhumans,theobservedlossis50200bpsperdivision
• Oxidativestressisconsideredoneofthemainfactors
astelomeric DNAismoresusceptibletooxidative
stressthannontelomeric DNA
• Onceacriticallengthisreachedandthechromosome
capceasestofunctionatsomeends,genome
instabilitywithendtoendjoiningofchromsomes may
occur.
Monaghan,AnnN.Y.Acad Sci,2010