Eukaryotic chromosome

4 LEVELS OF DNA PACKAGING

1. THE ORGANIZATION OF EUKARYOTIC
GENOMES
Dr. ESTHER SHOBA R
ASSISTANT PROFESSOR
KRISTU JAYANTI COLLEGE

2. •Geneexpressionineukaryoteshastwomain
differencesfromthesameprocessinprokaryotes.
•First,thetypicalmulticellulareukaryoticgenome
ismuchlargerthanthatofabacterium.
•Second,cellspecializationlimitstheexpression
ofmanygenestospecificcells.
Introduction

3. •Theestimated35,000genesinthehumangenome
includesanenormousamountofDNAthatdoesnot
programthesynthesisofRNAorprotein.
•ThisDNAiselaboratelyorganized.
–NotonlyistheDNAassociatedwithproteintoform
chromatin,butthechromatinisorganizedintohigher
organizationallevels.
•Levelofpackingisonewaythatgeneexpressionis
regulated.
–Denselypackedareasareinactivated.
–Looselypackedareasarebeingactivelytranscribed.

4. •Whilethesinglecircularchromosomeofbacteriais
coiledandloopedinacomplex,butorderlymanner,
eukaryoticchromatinisfarmorecomplex.
•EukaryoticDNAispreciselycombinedwithlarge
amountsofprotein.
•Duringinterphaseofthecellcycle,chromatinfibersare
usuallyhighlyextendedwithinthenucleus.
•Duringmitosis,thechromatincoilsandcondensesto
formshort,thickchromosomes.
1. Chromatin structure is based on successive levels of
DNA packing

6. •Eukaryoticchromosomescontainanenormousamount
ofDNArelativetotheircondensedlength.
–Eachhumanchromosomeaveragesabout2x10
8
nucleotidepairs(20,00,00,000)
–Ifextended,eachDNAmoleculewouldbeabout6
cmlong,thousandsoftimeslongerthanthecell
diameter
–Onanaveragehumancellcontain6.4billionbase
pairsofDNAin46chromosomes

12. I LEVEL OF PACKAGING
•2nm ofDNAconvertsto11nmof
structure
•Byformationofnucleosome
•ReportedbyRogerKorenbergin1974

13. I LEVEL OF DNA PACKAGING

14. I LEVEL OF DNA PACKAGING

15. I LEVEL OF DNA PACKAGING

16. I LEVEL OF DNA PACKAGING

18. I LEVEL OF DNA PACKAGING

20. Formation of Histone Octamer

27. •Thebeadedstringseemstoremainessentially
intactthroughoutthecellcycle.
•HistonesleavetheDNAonlytransientlyduring
DNAreplication.
•TheystaywiththeDNAduringtranscription.
–Bychangingshapeandposition,nucleosomesallow
RNA-synthesizingpolymerasestomovealongthe
DNA.

32. •Aschromosomesentermitosisthebeadedstring
undergoeshigher-orderpacking.
•Thebeadedstringcoilstoformthe30-nm
chromatinfiber.
•Thisissecondlevelofpackaging

33. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 19.1

34. 11nm to 30nm –II level of packaging

36. 2 models –zig zag and solenoid

38. Solenoid model

40. ZIG -ZAG MODEL

41. ZIG -ZAG MODEL

42. Inthetwo-startzigzagmodel,straightlinkerDNAconnects
twoopposingnucleosomecores,creatingtheopposingrows
ofnucleosomesthatformsocalled“two-start”helix.
Inzigzagmodel,alternatenucleosomes(forexample,N1and
N3)becomeinteractingpartners.
Interestingly,somestudiesofferamodel,whereintermediate
30nmfiberscontainboththesolenoidandzigzag
conformations,suggestinginsteadthatobservationsmadein
invitroexperimentsmightbeanisolationartifactdueto
strictlycationiclow-saltenvironmentorchemicalcross-
linking(e.g.,glutaraldehydefixation).

44. II LEVEL OF PACKAGING

47. III LEVEL OF PACKAGING

48. III LEVEL OF PACKAGING

51. •Folding of the 30-nm fiber form looped
domains that attached to a scaffold of non-
histone proteins
•Numerous loops of DNA (30 -90 loops)
attached to a protein scaffold. Each loop is a
30nm fiber with 180 -300 nucleosomes.

52. Loops, Domains and Scaffold
•The30nmfibresintheformofasolenoidisorganizedinstructuresata
higherleveloforganization.
•Thesehigherlevelsoforganizationshavebeenexaminedusing
metaphaseandinterphasechromosomes.
•IthasbeenshownbyDNAasedigestionthatDNAintheformof
nucleosomesisorganizedinloops,eachwith85kbofDNAanda
lengthof10-30μm
•Theseloopshaveactuallybeenobservedtoemanatefromacentral
scaffold
•Scaffold-TheeukaryoticchromosomestructureremainingwhenDNA
andhistoneshavebeenremoved;madefromnonhistoneproteins.The
centralframeworkofachromosometowhichtheDNAsolenoidis
attachedasloops;composedlargelyoftopoisomerase.
•

54. SARS/MARS
•Ininterphasenuclei,anuclearmatrixisfoundasa
filamentousstructureontheinteriorofnuclearmembrane.
•
•Chromatinremainsattachedtothismatrixthroughmatrix
attachmentregions(MARs).
•SeveralMARsequenceshaveactuallybeenidentified.Itis
believedthatthesamesequencesofDNAworkasMARin
interphasenucleiandasSAR(scaffoldattachmentregions)
inmetaphasechromosomes.
•Bothmatrixandscaffoldareproteinaceousinnature.

55. •Matrixattachmentregions(MARS)arespecificelementsof
DNAofthegenomethatassociatewithorattachtothe
nuclearmatrixduringtheinterphase.
•
•MARSarerich(about70%)inadenine(A)andthymidine
(T)bases,butnoconsensussequencehasyetbeendefined.
•
•ThelengthsofMARSrangefromaminimumof200to350
basepairs(bp)toseveralthousand.
•Higherorderchromatinstructureinvolvesdiscrete,and
topologicallyindependent,loopeddomainsof5to200kbp
withinbothmitoticchromosomesanddispersedinterphase
chromatin.

56. •MARSformthebasesoftheseloopeddomainsand
anchortheloopstothemitoticchromosomescaffoldsor
totheinterphasenuclearmatrix.
•
•Inmitoticchromosomes,theseelementshave
traditionallybeenreferredtoasSARSforscaffold
attachmentregions.
•Inthecaseoftheinterphasematrix,theseAT-rich
elementsareusuallycalledMARS.

57. SCAFFOLD PROTEINS
•Thechromatinloopdomainsareanchoredtothematrixby
proteinsthatpreferentiallybindAT-richsequences
•Amongtheproteinscharacterizedare
(1)thespecialAT-richbindingprotein1,SATB1,thatisexpressed
predominantlyinthymocytes;
(2)the95-kDaattachmentregionbindingprotein,(ARBP)
(3)theAandBlaminsofthenuclearenvelope,
(4)thenuclearintermediatefilament-typeproteincalledNuMA,and
(5)thehnRNPUprotein.

58. •Mostinterestingly,topoisomeraseIIassociateswithboth
interphaseMARSandmitoticSARS.
•TopoisomeraseIImayrelievetorsionalstressgeneratedby
transcriptionwithinindividualloopeddomainsduringthe
interphase(asitbindstoMARS),andtheenzymemay
serveasastructuralproteinwithinthechromosome
scaffoldduringmitosis(asitbindsSARS).

60. Chromatid (700nm diameter)
•Theloopeddomains(300nmchromatin
fiber)coilfurtherduringmitosistoforma
700-nmchromatid(oneofthelongitudinal
subunitsofthemetaphasechromosome).
•Suchtightpackingmakesthegenesonthe
DNAinactive.
•Thisstructuregivesapackagingratioof
~700

61. Metaphase chromosome (1400nm diameter)
•Apairofsisterchromatidscomprisingachromosome
measuresabout1400nm.
•Fullycondensedchromosomeis10,000foldshorterand
400–foldthickerthanDNAalone.
•Metaphasechromosomesarethemostcondensedof
normaleukaryoticchromosomes.
•Thisstructuregivesapackagingratioof~10,000
•Theroleofthesehighlycondensedchromosomesisto
organizeandpackagethegiantDNAmoleculesof
eukaryoticchromosomesintostructuresthatwillfacilitate
theirsegregationtodaughternucleiwithouttheDNA
moleculesofdifferentchromosomesbecomingentangled
and,asaresult,beingbrokenduringtheanaphase
separationofthedaughterchromosomes.

64. •Wheneukaryoticcellsdivide,genomicDNAmustbe
equallypartitionedintobothdaughtercells.To
accomplishthis,theDNAbecomeshighlycompacted
intotheclassicmetaphasechromosomesthatcanbeseen
withalightmicroscope.Onceacellhasdivided,its
chromosomesuncoilagain
•Comparingthelengthofmetaphasechromosomestothat
ofnakedDNA,thepackingratioofDNAinmetaphase
chromosomesisapproximately10,000:1(dependingon
thechromosome).Thiscanbethoughtofasakinto
takingaropeaslongasafootballfieldandcompactingit
downtolessthanhalfaninch

67. •Interphase chromatin is generally much less
condensed than the chromatin of mitosis.
–While the 30-nm fibers and looped domains remain,
the discrete scaffold is not present.
–The looped domains appear to be attached to the
nuclear lamina and perhaps the nuclear matrix.
•The chromatin of each chromosome occupies a
restricted area within the interphase nucleus.
•Interphase chromosomes have areas that remain
highly condensed, heterochromatin, and less
compacted areas, euchromatin.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

71. Difference between heterochromatin and euchromatin

73. Telomeres

80. Significance of telomeres

81. Telomeres and cell division

85. Telomerase
•Somecellshavetheabilitytoreversetelomereshorteningbyexpressing
telomerase,anenzymethatextendsthetelomeresofchromosomes.
•TelomeraseisanRNA-dependentDNApolymerase,meaninganenzyme
thatcanmakeDNAusingRNAasatemplate.
•TheenzymebindstoaspecialRNAmoleculethatcontainsasequence
complementarytothetelomericrepeat.
•Itextends(addsnucleotidesto)theoverhangingstrandofthetelomere
DNAusingthiscomplementaryRNAasatemplate.
•Whentheoverhangislongenough,amatchingstrandcanbemadebythe
normalDNAreplicationmachinery(thatis,usinganRNAprimerand
DNApolymerase),producingdouble-strandedDNA.
•Theprimermaynotbepositionedrightatthechromosomeendandcannot
bereplacedwithDNA,soanoverhangwillstillbepresent.However,the
overalllengthofthetelomerewillbegreater.

89. Histone modifications
•Histonesareprimaryproteincomponentsofeukaryotic
chromatinandplayaroleingeneregulation.
•
•H3andH4histoneshavetailsprotrudingfromthe
nucleosomethatcanbemodifiedpost-translationallytoalter
thehistone'sinteractionswithDNAandnuclearproteins,
leadingtoepigeneticchangesforregulatingmanynormal
anddisease-relatedprocesses.
•Post-translationalmodificationstohistones–referredtoas
marks–regulategeneexpressionbyorganizingthegenome
intoactiveregionsofeuchromatin,whereDNAis
accessiblefortranscription,orinactiveheterochromatin
regions,whereDNAismorecompactandlessaccessible
fortranscription.

90. •HistoneH3isthemostmodifiedhistone.Modificationsto
histoneH3canpredictthetypeofchromatin
(heterochromatinvseuchromatin),distinguishbetween
functionalelementsofthegenome(promoters,enhancers,
genebodies),anddeterminewhethertheseelementsareinan
activeorrepressedstate.

91. Histone Methylation
•Themechanismknownashistonemethylationisapost-
translationalepigeneticmodificationthatinvolvesthe
transferofmethylgroupstohistoneproteinsviahistone
methyltransferases(HMTs).
•Methylgroupsareaddedtothe“tails”thatprotrudefromthe
histoneproteins,whichisthemostcommonlocationforpost-
translationalmodifications,especiallyN-terminaltails.
•FromS-adenosylmethionione

92. •Alternatively,histonedemethylationistheremovalof
methylgroupsfromthehistonetailscatalyzedbyhistone
demethylases(HDMs).
•Histonemethylationandhistonedemethylationare
epigeneticmodificationsthathavethepowertoreduceor
bolstergeneexpression,especiallyasaresultofaltering
chromatinstructure.
•Transcriptionalrepressionoractivationcanoccurasaresult
ofhistonemethylationordemethylationduetothe
looseningorrestrictionofthechromatinstructure.

93. •Whereandhowmanymethylgroupsareaddedtothehistones
largelydetermineswhetherthechromatinisavailableto
transcriptionornot.
•Tailresidues—lysine(K)andarginine(R)—maybe
methylatedatvaryingdegreeswithdifferingoutcomes.
•Forinstance,whenhistoneH4ismonomethylatedonlysine20
(H4K20me1),thiscommonhistonemodificationresultsinthe
contractionofchromatin.Restrictingthechromatinstructure
preventstranscriptionfromoccurringandreducesgene
expression.
•Alternatively,monomethylationofhistoneH3onarginine17
(H3R17me1)leadstotranscriptionalactivation.

94. •Lysinemethylationhasbeeninvolvedinbothtranscriptional
activation(H3K4,K36,K79)aswellassilencing(H3K9,
K27,H4K20)
•(H3K4me1orH3K4me3)areactivemarks,butH3K4me1
isfoundattranscriptionalenhancers,whileH3K4me3is
foundatgenepromoters.
•Tri-methylationofK36(H3K36me3)isassociatedwith
transcribedregionsingenebodies.
•Tri-methylation
ofK9andK27onhistoneH3(H3K27me3andH3K9me3)
arebothrepressivesignals

95. •ArgininemethylationofhistonesH3andH4promotes
transcriptionalactivationandismediatedbyafamilyof
proteinargininemethyltransferases(PRMTs).
•Thereare9typesofPRMTsfoundinhumansbutonly7
membersarereportedtomethylatehistones.
•Theycanmediatemonoordimethylationofarginine
residues.
•Basedonthepositionofthemethylgroupaddition,PRMTs
canbeclassifiedintotypeI(CARM1,PRMT1,PRMT2,
PRMT3,PRMT6,andPRMT8)andtypeII(PRMT5and
PRMT7).

96. •TypeIIPRMTsarefoundtobestronglyimplicatedin
diseaseslikecancer.1
•Forexample,PRMT5playsaroleintherepressionofcertain
tumorsuppressorgenessuchasRBtumorsuppressorswhile
PRMT7overexpressionisobservedinbreastcancer.
•DetectionofactivityandinhibitionoftypeIIPRMTsaswell
asotherHMTswouldbeimportantinelucidating
mechanismsofepigeneticregulationofgeneactivationand
silencing,aswellasbenefitingcancerdiagnosticsand
therapeutics.

97. Histone Acetylation/ deacetylation
•Histoneacetylationoccursbytheenzymaticadditionofan
acetylgroup(COCH3)fromacetylcoenzymeA.
•Theprocessofhistoneacetylationistightlyinvolvedinthe
regulationofmanycellularprocessesincludingchromatin
dynamicsandtranscription,genesilencing,cellcycle
progression,apoptosis,differentiation,DNAreplication,DNA
repair,nuclearimport,andneuronalrepression.
•Themodifyingenzymesinvolvedinhistoneacetylationare
calledhistoneacetyltransferases(HATs)andtheyplayacritical
roleincontrollinghistoneH3andH4acetylation.
•
•Morethan20HATshavebeenidentifiedwhichcanbe
classifiedintofivefamilies:

98. •GNAT1,MYST,TAFII250,P300/CBP,andnuclearreceptor
coactivatorssuchasACTR.1HistoneH3acetylationmaybe
increasedbyinhibitionofhistonedeacetylases(HDACs)and
decreasedbyHATinhibition.
•Histonedeacetylaces(HDACs)catalyzethehydrolytic
removalofacetylgroupsfromhistonelysineresidues.
•Animbalanceintheequilibriumofhistoneacetylationhas
beenassociatedwithtumorigenesisandcancerprogression.
•DetectingwhetherhistoneH3isacetylatedatitslysine
residueswouldprovideusefulinformationforfurther
characterizationofacetylationpatternsorsites,thereby
leadingtoabetterunderstandingofepigeneticregulationof
geneactivationaswellasthedevelopmentofHAT-targeted
drugs.
•SimilartoHATs,HDACsplayacriticalroleinvarious
cellularprocessesinvolvinghistoneH3andH4.

99. •Histoneacetylationislargelytargetedtopromoterregions.
Forexample,acetylationofK9andK27onhistoneH3
(H3K9acandH3K27ac)isnormallyassociatedwith
enhancersandpromotersofactivegenes.
•Lowlevelsofacetylationarealsofoundthroughout
transcribedgenes,althoughthefunctionofthisisstill
unclear.
•Histoneacetyltransferases(HAT)anddeacetylases
(HDACs)aretheenzymesresponsibleforwritingand
erasingtheacetylationofhistonetails.Lysineresidues
withinhistoneH3andH4arepreferentialtargetsforHAT
complexes.

100. Histone phosphorylation
•Phosphorylationofcorehistonesisacriticalintermediate
stepinchromosomecondensationduringcelldivision,
transcriptionalregulation,andDNAdamagerepair.
•Unlikeacetylationandmethylation,histone
phosphorylationseemstofunctionbyestablishing
interactionsbetweenotherhistonemodificationsand
servingasaplatformforeffectorproteins.
•Thisleadstoadownstreamcascadeofevents.

101. •PhosphorylationofhistoneH3atS10(H3phosphoS10)and
histoneH2AonT120aremitoticmarkers:these
modificationsareinvolvedinchromatincompactionandthe
regulationofchromatinstructureandfunctionduringmitosis.
•PhosphorylationofH2AXatS139(resultinginγH2AX)has
beenidentifiedasoneoftheearliesteventsoccurringafter
DNAdouble-strandbreaksandservesasarecruitingpoint
forDNAdamagerepairproteins.
•Histonephosphorylationalsohasabroaderroletoplay:H2B
phosphorylation,forexample,facilitatesapoptosis-related
chromatincondensation,DNAfragmentation,andcelldeath

102. Histone ubiquitylation
•Ubiquitylation,alsoreferredtoasubiquitination*,isthe
processofattachingubiquitin,asmallproteinfoundin
almostalltissuesofeukaryoticorganisms,toanother
targetedprotein.
•HistoneH2AandH2Baretwoofthemosthighly
ubiquitylatedproteinsfoundinthenucleus
10
.Themost
abundantformsaremonoubiquitylatedH2AonK119and
monoubiquitylatedH2BonK123(yeast)/K120
(vertebrates).

103. •MonoubiquitylationofH2Aiscatalyzedbypolycombgroup
proteins,anditismostlyassociatedwithgenesilencing.
•ThemainenzymeresponsibleformonoubiquitylatedH2Bis
Bre1inyeastanditshomologsRNF20/RNF40inmammals.
•UnlikeH2A,monoubiquitylatedH2Bisassociatedwith
transcriptionactivation.