Genomic Structure and Evolution of the Ancestral Chromosome Fusion Site in 2q13–2q14.1 and ...

13 Pages·2002·0.53 MB·English

Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
Letter
Genomic Structure and Evolution of the Ancestral
Chromosome Fusion Site in 2q13–2q14.1
and Paralogous Regions on Other
Human Chromosomes
Yuxin Fan, Elena Linardopoulou, Cynthia Friedman, Eleanor Williams,
and Barbara J. Trask1
DivisionofHumanBiology,FredHutchinsonCancerResearchCenter,Seattle,Washington98109,USA
Human chromosome 2 was formed by the head-to-head fusion of two ancestral chromosomes that remained
separate in other primates. Sequences that once resided near the ends of the ancestral chromosomes are now
interstitially located in 2q13–2q14.1. Portions of these sequences had duplicated to other locations prior to the
fusion. Here we present analyses of the genomic structure and evolutionary history of >600 kb surrounding the
fusion site and closely related sequences on other human chromosomes. Sequence blocks that closely flank the
inverted arrays of degenerate telomere repeats marking the fusion site are duplicated at many, primarily
subtelomeric, locations. In addition, large portions of a 168-kb centromere-proximal block are duplicated at
9pter, 9p11.2, and 9q13, with 98%–99% average sequence identity. A 67-kb block on the distal side of the fusion
site is highly homologous to sequences at 22qter. A third ∼100-kb segment is 96% identical to a region in
2q11.2. By integrating data on the extent and similarity of these paralogous blocks, including the presence of
phylogenetically informative repetitive elements, with observations of their chromosomal distribution in
nonhuman primates, we infer the order of the duplications that led to their current arrangement. Several of
these duplicated blocks may be associated with breakpoints of inversions that occurred during primate evolution
andofrecurrentchromosomerearrangementsinhumans.
[Supplemental material is available online at http://www.genome.org. The following individuals kindly provided
reagents,samples,orunpublishedinformationasindicatedinthepaper:T.Newman,C.Harris,andJ.Young.]
Humanshave46chromosomes,whereaschimpanzee,gorilla, The subtelomeric regions of human chromosomes are
andorangutanhave48.Thismajorkaryotypicdifferencewas particularlydynamicrelativetomostofthehumangenome.
causedbythefusionoftwoancestralchromosomestoform Sequenceshaverecurrentlyexchanged,recombined,anddu-
humanchromosome2andsubsequentinactivationofoneof plicated among the ends of nonhomologous chromosomes
thetwooriginalcentromeres(YunisandPrakash1982).Asa (for review, see Mefford and Trask 2002). Thus, the entrap-
resultofthisfusion,sequencesthatonceresidedneartheends mentofsubtelomericregionsatthemoresequesteredinter-
oftheancestralchromosomesarenowlocatedinthemiddle stitialfusionsiteprovidesapotentialopportunitytocompare
ofchromosome2,nearthebordersofbands2q13and2q14.1. thecompositionoftwoancestralsubtelomerestotheircoun-
Forbrevity,wereferhenceforthtotheregionsurroundingthe terparts that have persisted at, and propagated among, sub-
fusionas2qFus.Twohead-to-headarraysofdegeneratetelo- telomericlocations.
mererepeatsarefoundatthissite;theirhead-to-headorien- Martin et al. (2002) recently presented a clone contig
tationindicatesthatchromosome2resultedfromatelomere- encompassingthefusionandshowedhomologywithseveral
to-telomere fusion (Ijdo et al. 1991). Furthermore, cross- interstitial sites, in addition to subtelomeric sites. Here, we
hybridization between 2qFus and various subtelomeric providemoredetailonthestructureoftheDNAsurrounding
regionshasbeenobservedbyfluorescenceinsituhybridiza- thefusionsiteandtheseparalogousrelationships.Wequan-
tion(FISH)(Ijdoetal.1991;Trasketal.1993;Hoglundetal. tifytheextentanddegreeofhomologybetweenthisregion
1995;Martin-Gallardoetal.1995;Ningetal.1996;Leseetal. and paralogous segments elsewhere in the human genome,
1999; Ciccodicola et al. 2000; Park et al. 2000; Bailey et al. includingsitesnotdescribedpreviously.Usingthesedataand
2002;Martinetal.2002).Thus,thefusionmusthaveoccurred observationsofthechromosomallocationofthesesequences
aftersubtelomericsequencespresentattheendsoftheances- in nonhuman primates, we infer the history of some of the
tral fusion partners had already duplicated to/from at least duplications and rearrangements that have occurred during
oneotherchromosomeend. recent primate evolution. The extensive homology among
2qFus-relatedregionsofthegenomemayhavemediated—or
1Correspondingauthor.
been the result of—some of the rearrangements that distin-
[email protected];FAX[phone].
Articleandpublicationareathttp://www.genome.org/cgi/doi/10.1101/ guish the karyotypes of higher primates and that may now
gr.337602. interacttocausechromosomerearrangementsinhumans.
12:1651–1662 ©2002 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/02 $5.00; www.genome.org Genome Research 1651
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
Fan et al.
RESULTS National Center for Biotechnology Information (NCBI)
(http://genome.ucsc.edu/andhttp://www.ncbi.nlm.nih.gov)
Chromosomal Distribution of Sequences andrecentanalysesbyMartinetal.(2002).
from the 2q13–2q14.1 FusionRegion Weusedacombinationofthreeapproachesinorderto
Bacterial Artificial Chromosome (BAC) RP11–395L14 confirmtheassignmentofBACsformingthe614-kbcontigto
(AL078621) contains 789 bp of degenerate telomere repeats chromosome2qFusandtoinvestigatethechromosomaldis-
organizedintwohead-to-headarraysandoverlapstheances- tributionofparalogoussequences.
tralfusionsite(Fig.1A)(Martinetal.2002).UsingthisBACas
PCRAnalysesofMonochromosomalHybridPanel
the seed, we independently assembled a 614-kb contig sur-
rounding the fusion site using publicly available BAC se- First,wedesigned48PCRprimerpairs,whichamplifyDNA
quences(Fig.1A).ThefinishedBACsinthecontigare99.9%– freeofknownrepeats,acrossthecontigandperformedPCR
100%identicalintheirregionsofoverlap.Our2qFuscontigis assays on DNA from a panel of hybrid cell lines, each con-
consistent with the automated assembly of this region per- tainingadifferenthumanchromosomeagainstarodentback-
formedbyUniversityofCaliforniainSantaCruz(UCSC)and ground(Fig.1C).Asexpected,allprimerpairsamplifiedprod-
Figure1 Chromosomaldistribution,GCcontent,andrepeatcontentof614-kbDNAsequencesurroundingthe2q13–2q14.1fusionsite(2qFus).
(A)EachblacklineindicatesthesequencecoverageoffinishedBacterialArtificialChromosome(BAC)clonesassembledintothe2qFuscontigon
thebasisoftheir99.9%–100%identityinregionsofoverlap.BACRP11–559H14isunfinished(grayline);itoverlapsRP11–480C16and–395L15
with99.7%identity,therebyconfirmingtheiroverlap.Clonenamesarefollowedbychromosomallocationsdeterminedbyfluorescenceinsitu
hybridization;accessionnumbersaregivenbelowthelines.RP11–432G15extends35kbofftherightsideofthemap.NotethatfinalGenBank
entriesforsomeoftheBACshavebeentrimmedtoremoveoverlapamongclonesexceeding2kb.Thegreenverticallinemarksthelocationof
theinverteddegeneratetelomererepeatsatthefusionsite.Thefusionsiteisimmediatelyflankedbyatelomere-associatedrepeat,TAR1,arepeat
thatiscommonlyfoundincloseassociationwithterminaltelomerearraysandsometimesfoundnearinterstitialdegeneratetelomererepeats.(B)
TheG+Ctraceshowsagraphof%GCcontentwithwindowsizesof500bpforlocalcontent.Red,blue,andgreenregionsrepresentH3,H1–H2,
andLisochores,respectively,asdefinedbyGESTALTusinga30-kbslidingwindow.Thelocation,strand,age,andtypeofinterspersedrepetitive
elementsareshowninthethirdtrace,alsogeneratedwithGESTALT(LongInterspersedElements[LINEs],green;Alus,red;Mammalian-wide
Interspersed Repeats [MIRs], purple; all other repeats including retroviruses, Long Terminal Repeats [LTRs], Mammalian Apparent LTR-
retrotransposons[MaLRs],etc.,brown).Theageofeachelementisindicatedbytheheightofthefeature;tallerfeaturesrepresentevolutionarily
morerecentinsertions.(C)Chromosomaldistributionsofsequencehomologoustothe2qFusregionasdeterminedbyPCRassaysonamono-
chromosomalhybridpanel.FilledandopencirclesdenotepositiveandnegativePCRassays,respectively.ThepreciselocationsofthePCRassay
areindicatedbytheverticaltickmarks.Thecoloredbardelineatesregionswithdifferentchromosomaldistributions.Thelightgrayblockwithin
grayblockindicatessequenceduplicatedwithinchromosome2(seeFig.3).
1652 Genome Research
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
2q Fusion: Structure, Paralogy, and Evolution
ucts from chromosome 2. However, only a portion of the fivetestedindividuals,despitethefactthatthePCRanalyses
assembled sequence—a total of ∼350 kb on the ends of the oftheY-containinghybridindicatedthepresenceof∼100kb
contig—isuniquetochromosome2. ofparalogytothetwoclonesusedasprobes.FISHalsofailed
Sequencescloselyflankingthe
telomere-repeat arrays (red zone,
H C G O H C G O
Fig. 1C) amplified from seven or
morechromosomes,withoneassay
amplifying a product from 13 dif-
ferent chromosomes, including
chromosome 22. A 40-kb block
common to only chromosomes 2
and22anddefinedbyfourPCRas-
says(bluezone)adjoinstheregion
of multichromosomal segments.
9
Oneassaywithinthisblockisalso
positiveforchromosome15,dueto H C G O
the retrotransposition of a pro-
cessedpseudogeneofSNRPA1from
the intron-containing copy on
chromosome 15 prior to the seg- 2 22
mentalduplicationthatgaveriseto cen tel e
tt2wh0e0ee2ln)a.r2gOqenrFutbshloeacnokdpop2fo2sqhittoeemrs(ioFdlaeongoyefttbhaele-. F humanchimpanze gorillaorangutan
telomere-repeat arrays is a 150-kb
block (green zone) defined by 21
PCR assays that are common to
chromosomes2,9,andYinthehy-
bridpanel.
FluorescenceInSitu
Hybridization(FISH)
Second, in order to more precisely *
definethechromosomallocationof 1 3 4 5 6 7 X
sequences homologous to this re-
gion, we performed FISH analyses
usingthefiveBACclonescompris-
ingthe2qFuscontig.Theresultsare
summarized in Figure 1A and
shown schematically for three of
theBACsinFigure2.Sequencesin
RP11–395L14, which contains the
fusionsite,hybridizetofivepromi-
nent sites, 2qFus, 9q13, 9p11.2, 8 10 11 12 13 14 15
9pter(9p24),and22qter(22q13.3),
aswellastoseveralotherchromo-
somal ends with lower intensity.
RP11–480C16 produces FISH sig-
nalsat2qFus,2q11.2,9p11.2,9q13,
and 9pter, as observed recently by
Martin et al. (Martin et al. 2002).
16 17 18 19 20 21 Y
RP11–65I12 produces signals at
2qFusand2q11.2(notshown).On Figure2 Summaryoffluorescenceinsituhybridization(FISH)analysesofhominidchromosomes
the other side of the fusion site, usingBACsRP11–480C16(green),RP11–395L14(blue),andRP11–432G15(red)derivedfromthe
ancestralfusionsiteinhuman2q13–q14.1.Dataforchromosomes2,9,and22,whichcarrythemajor
RP11–432G15 produces FISH sig-
blocks of paralogy in human, are shown in the top panels so that the banding patterns are not
nalsat2qFusand22qter.Although
obscuredbytheFISHsignals.Otherchromosomesareshowninthebottompanel.Atotalofatleast
the hybrid-panel analyses had im- sixmetaphasespreadswereexaminedforeachprobeineachspecies(oneindividualeach).Hybrid-
plicated these chromosomes, FISH izationsignalsseenateachlocationwerescoredfromdigitizedimagesonanintensityscaleof1–4on
demonstratesthatthereareatleast eachprobeineachspecies.Thecumulativescoreswerenormalizedtothatofthelocationwithhighest
threesitesofhomologyonchromo- cumulativescoreineachexperiment.Theareaofeachdotisproportionaltothisnormalizedscore.
DotsarealignedwiththemidpointofobservedFISHsignalsforeachlocation.Theasteriskindicates
some 9 and two sites on chromo-
wherehybridizationwasseenononehomologonly.IdeogramsareredrawnfromYunisandPrakash
some2. (1982).AccessionnumbersforthethreeclonesaregiveninFig.1.Human2p-and2q-specificclones,
Surprisingly,FISHsignalswere RP11–90H11andRP11–47E6,respectively,wereusedtoverifytheidentityofhominidchromosomes
notobservedonchromosomeYin orthologousto2pand2q(i.e.,chimpanzee12and13,respectively)(notshown).
Genome Research 1653
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
Fan et al.
to detect this homologous sequence in metaphase chromo- weaksignalat2qFus(notshown).Althoughtheyare99.0%
somesprepareddirectlyfromthehybridline.Avisiblyintact identicalover∼16kb,clonesRP11–34G16and(cid:1)468G5rep-
Y was the only human material detected in this hybrid cell resentdistinctparalogousblocksin2q11.2.Sequencesneigh-
line when 20 cells were analyzed by FISH with a human- boringtheparalogyareverydifferent(Fig.3),andadissimi-
specificrepetitive-sequenceprobeorbyreversepainting(not larityof1%isgreaterthanexpectedfromthecombinationof
shown). However, these techniques could miss a small frag- allelicvariationandsequencingerrors.Thetwo2q11.2blocks
ment of non-Y human material, especially if present in a arenotresolvablebyFISHinmetaphasechromosomes,however.
small subpopulation of cells. Because the sequences of PCR The paralogy between 9pter (9p24) and 2qFus was re-
productsgeneratedfromtheYhybridare99.8%identicalto portedtoresideinRP11–174M15andRP11–143M1byMartin
sequencesderivedfromthe9q13paralog(seefollowing),over et al. (2002), but was not analyzed in detail. Our analyses
atotalof3.7kbsampled(notshown),weconcludethatthe show that the paralogy extends at least 168 kb with 98.9%
“Y”homologyisactuallya9q13contaminantinthehybrid overallidentity.Theclonesoverlapby49kbwith100%iden-
line. tity, and FISH to 9pter (most intensely), 9p11.2, 9q13, and
2qFus. In addition, RP11–143M1, the more distal clone, hy-
DatabaseMiningandSequenceAlignment
bridizes to multiple chromosomal ends (Supplementary Fig.
Third,weconductedaBlastNsearchofallfinishedanddraft IB,availableonlineathttp://www.genome.org).
sequencespubliclyavailableasofFebruary28,2002inorder Paralogy in 9q13 can be found in overlapping finished
to identify sequences paralogous to the 614-kb region of sequences from RP11–561O23, RP11–88I18, and RP11–
2qFus.ResultsaresummarizedinTable1andFigure3.Some, 274B18. The first two clones overlap by ∼35kb with 99.7%
but not all, of these paralogous segments were detected in identity, and the latter two by ∼25 kb with 99.9% identity
earlier whole-genome scans for duplications (Bailey et al. (beforeoverlapsweretrimmedto2kbinthelatestGenBank
2001;Martinetal.2002). entries),andthusareverylikelytoderivefromthesamelo-
Atleasttwoparalogoussegmentsresidein2q11.2.One, cus. Our FISH analyses of RP11–561O23 confirm its assign-
called 2q11.2-A, is found in the finished sequence of RP11– mentto9q13byNCBIandUCSC.ItgeneratesFISHsignalson
34G16;itencompasses(cid:1)98kbandis95.8%identicaltothe 9q13,9p11.2,2qFus,and9pter,and9q13isthebrightestsite
centromere-proximalportionof2qFus.OurFISHanalysesof (Supplementary Fig. IC, available online at http:/www.
this clone confirm its 2q11.2 location as indicated by the genome.org).This9q13paralogyto2qFusspansatleast149
NCBI and UCSC assemblies: Signals are observed at both kbwithoverallidentityof98.2%.
2q11.2and2qFus(SupplementaryFig.IA,availableonlineat Weidentifytwoadditionalblocksof2qFusparalogythat
http://www.genome.org).Thisintrachromosomalidentityex- derive from 9p11.2 or 9q13. One segment, which we call
plainswhyclonesRP11–65I12and(cid:1)480C16from2qFusgive (9p11.2)-A, spans >42 kb within RP11–15J10 and is 98.5%
FISHsignalson2q11.2:over55kband45kboftheirinserts, identical to 2qFus within the region that is paralogous to
respectively, match this paralogous segment in 2q11.2 at 9q13.Asecondblockof2qFusparalogy,called(9p11.2)-B,is
>95% identity. A second 20.5-kb block of 2qFus homology in RP11–403A15. This clone contains ∼63 kb homology at
(96.0%identity),called2q11.2-B,isinthefinishedsequence 98.1% identity to 2qFus, and ∼110 kb homology at 98.8%
of RP11–468G5. FISH confirms the 2q11.2 location of this identity to the 9q13 sequence (red and blue lines in Fig. 3).
paralogy:ThisBACgivesastrongFISHsignalin2q11.2anda RP11–15J10and(cid:1)403A15hybridizebyFISHmostintensely
Table1. ExtentandDegreeofSequenceSimilarityAmongParalogousBlocksRelatedto2qFus
2q11.2-A 2q11.2-B 9pter 9q13 (9p11.2)-A (9p11.2)-B (19pter) 22qter
34G16 468G5 174M15/143M1 561O23/88I18/274B18 15J10 403A15 34P13 n1g3/n94h12
AC008268 AC009238 AL356244/ AL353608/AL161457/ AL512605 AL445925 AL627309 AC002055/
AL449043 AL353616 AC002056
2qFus (cid:1)97.5kb 20.5kb (cid:1)167.8kb (cid:1)149.4kb (cid:1)42.3kb (cid:1)62.5kb (cid:1)29.5kb (cid:1)67.3kb
95.8% 96.0% 98.9% 98.2% 98.5% 98.1% 98.8% 98.6%
[355,2740] [84,278] [257,6822] [270,12821] [63,541] [240,12524] [41,104] [83,3715]
2q11.2-A (cid:1)16.1kb
99.0%
[22,76]
9pter (cid:1)150.5kb (cid:1)42.1kb (cid:1)64.2kb (cid:1)8.5kb
98.2% 98.4% 98.1% 99.6%
[321,12908] [71,413] [224,12100] [6,11]
9q13 (cid:1)42.4kb (cid:1)109.8kb
99.0% 98.8%
[75,380] [193,1286]
9p11.2-A ?
Extentofhomologyandpercentidentityofparalogousblocksrelatedto2q13–q14.1fusionregion.Onlybasesubstitutions,notinsertionsor
deletions,areconsideredinthecalculationsofpercentidentity(seeMethodssection).Thenumberofinsertionsordeletionsandthetotal
numberofbasesinthesegapsaregiveninbrackets.Theextentofhomologyisdefinedbythesequenceindicatedatthebeginningofeach
rowandisaminimalestimateinallbutonecase,sinceavailablesequenceterminateswithintheregionsofhomology.Allclonesarefromthe
RP11BAClibraryexceptthechromosome-22cosmids,n1g3andn94h12.
1654 Genome Research
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
2q Fusion: Structure, Paralogy, and Evolution
Figure 3 Summary of regions of homology with portions of the 614-kb sequence surrounding the fusion site on 2q13–2q14.1 that were
identifiedbyBlastNsearchesoffinishedgenomicsequencepubliclyavailableasofFebruary28,2002.Table1givestheclonenamesandaccession
numbers for the paralogous segments. For simplicity, only one of the sequences with homology with only the 68-kb region immediately
surroundingthefusionsiteisshown.Redsolidlinesindicatetheregionswith>95%averageidentitytothe2qFussequence.Theselinesaredrawn
withreferencetothe2qFussequence;theactuallengthsoftheparalogoussegmentsmaybeslightlylongerorshorterthanthosedrawnbecause
ofdistributedinsertionsanddeletions.Reddottedlinesindicateadjoiningregionswithnoavailablesequence,butthatwereshownbyPCRtobe
homologousto2qFus(seeFig.1).Differentcolorsareusedtoindicatedivergentsequence,withsolidlinesindicatingtheextentofcontiguous
sequencecoverage,anddottedlinesindicatingeitherunavailablesequenceorneighboringsequencethatlackshomologywithanyoftheother
segmentsshown.Blocks9p11.2-Aand-Bmaptothepericentromericregionof9byfluorescenceinsituhybridizationandhybridpanelanalyses
and are tentatively assigned to 9p11.2. Orientation indicated in parentheses is tentative; the presence of alpha-satellite-like repeats in the
right-mostportionofthe9p11.2-Asequenceindicatesthatitrunstelomeretocentromereasdrawn.(<?)Indicatesthat2qFushomologycould
extendfartherindirectionofarrowhead.ThesmallredsymbolsonstalksindicatethemostrecentAluinsertionsinthe2qFusregionand/orits
paralogs.Themostrecentlyactivefamilies,AluYb8andAluYa5,areindicatedbysquaresandtriangles,respectively,andcirclesindicatetheolder
classofAluYelements.Filledandopensymbolsindicatethattheparticularelementispresentorabsent,respectively.Thepartiallyfilledsymbol
in9pterindicatesthatthisAluYb8elementisnotpresentinall9pteralleles(seetext).TheasterisksmarkthepositionofahighlyvariableSATR1
repeatcommontoatleastfouroftheparalogoussegments;itis15,109bplongin9q13,15,021bpin9p11.2-B,4919bpin9pter,and3881
bpin2qFus.
to 9p11.2 and 9q13, and less intensely to 2qFus, 9pter, and some 19 in 22 individuals sampled from different ethnic
manypericentromericsites(SupplementaryFig.IDforRP11– groups(180chromosomes)(Meffordetal.2001).
15J10,availableonlineathttp://www.genome.org).Although Over67kbofsequencedistaltothefusionsiteis98.6%
these clones are assigned to 9q13 in the NCBI and UCSC identicaltotheq-terminusofchromosome22.Thishomol-
maps,thereisnostrongjustificationforthisassignmentover ogyendsjust∼1.4kbfromthearrayofdegeneratetelomere
9p11.2. These sequences are not connected to other 9q13 repeatsandwasdescribedpreviously(Ningetal.1996;Eichler
BACs by overlapping sequence or end-sequenced BACs, and etal.1997;Martinetal.2002).Theextentofthishomology
theycontainnoradiation-hybridorlinkagemarkersthatare explains why the 2qFus-clone RP11–395L14 was initially
unambiguously assigned to one side of the chromosome-9 given a GenBank assignment of chromosome 22 when the
centromereortheother.RP11–403A15and(cid:1)15J10haveno SangerCentre(http://www.sanger.ac.uk/HGP)sequencedit.
sequenceincommon,butmustliesufficientlyclosetoeach Theputative150-kbregionofparalogyonchromosome
otherthattheyarenotresolvablebymetaphaseFISH. YisnotpresentinthesequenceavailableforchromosomeY
AsexpectedfromourhybridpaneldataandFISHobser- inGenBankorCelera’shumangenomeassembly,consistent
vationsmadebyusandothers(referencesearlier),themulti- withtheideathattheY-hybridresultsareduetoa9q13con-
copy regions immediately flanking the fusion site match taminant.
many publicly available BAC and cosmid sequences. Clones Insummary,nomorethan254kbofthe614-kb2qFus
with homology with these multicopy regions belong to at contigissinglecopyinthegenome((cid:2)22kband104kbon
least15differentcontigsrepresentingdifferentchromosomal the two sides and 28 kb between the regions of homology
ends(notshown).Weshowonlyoneofthelongestavailable with2q11.2-Aand9pter).Theremainderofthesequenceis
homologies, that in RP11–34P13 (AC073186/AL627309), duplicatedinatleastoneotherlocation.Sofar,wehavede-
whichis98.8%identicalto2qFusover(cid:1)29.5kband99.6% tected16locationswithatleast5kbofhomologywith2qFus
identicaltothe9ptersequence.Thisclonecontainsnochro- byatleasttwoofthreemethods(areproducibleFISHsignal,
mosome-specific DNA and has been variously assigned to morethanonepositivePCRassay,or>5kbof>95%sequence
chromosome 1, 7, 18, and 21 in GenBank entries and draft match in a chromosomally assigned genomic sequence). Of
assembliesoverthelast2years.Itdoesnotderivefromchro- these locations, 11 are subtelomeric, and 3 are pericentro-
mosome1,18,or21,sinceitdoesnotcross-hybridizebyFISH meric. An additional 14 sites of homology (of which 11 are
tothesechromosomesinanyofseveralindividualsanalyzed subtelomeric)weredetectedwithonlyasinglemethod.The
(notshown).Itislikelytobeavariantchromosomealleleof failuretodetectthese14siteswithmorethanonemethodis
19, as it shares extensive homology with the 19pter allele likely due to incomplete sequence coverage, insensitivity of
sequencedbytheDepartmentofEnergyJointGenomeInsti- FISH, low density of PCR assays, mismatches to primer se-
tute(http://www.jgi.doe.gov)andcontainssequencevariants quences,and/ornormalpolymorphismamongthechromo-
oftheolfactoryreceptorgenemostoftenfoundonchromo- somesanalyzedinthethreemethods.
Genome Research 1655
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
Fan et al.
FISH Analyses of NonhumanPrimates andtherestarecommontodifferentcombinationsoftwoor
threespecies.
We confirmed the centromere–telomere orientation of the Almost all gorilla chromosome ends and half of chim-
2qFuscontigbyFISHanalysesofconstituentclonesonchim- panzee ends are capped with AT-rich, DAPI-bright bands.
panzeechromosomes(Fig.2).Chimpanzeechromosomes12 Thesecapsarenotpresentonhumanororangutanchromo-
and 13 are homologous to human 2p and 2q, respectively somes. 2qFus homologous sequences are invariably found
(Yunis and Prakash 1982; Wienberg et al. 1994). RP11– centromereproximalofthesecapswhenbotharepresent.
480C16, from one end of the 2qFus contig, hybridizes to
chimpanzee 12, indicating that it maps to the centromere- Genomic Structure
proximalsideofthefusionsite.RP11–432G15,fromtheother
endofthe2qFuscontig,hybridizestochimpanzee13,indi- Base-PairComposition
catingthatitliesonthecentromere-distalsideofthefusion TheGCcontentofthe2qfusionregionaverages44%,butit
siteinhuman.Asexpected,RP11–395L14,whichcontainsthe fluctuatesmarkedlyacrossthe614-kbsequence(Fig.1B).The
fusion site, generates signals on both chimpanzee chromo- GESTALT program (Glusman and Lancet 2000) divides the
somes12and13. regionintofiveisochores,fromcentromeretotelomere:H3,
FISH analyses also reveal changes in location and copy H1–2,L,H1–2,andL,asdefinedbyBernardiandcolleagues
number of paralogous segments that have occurred during (Bernardi1995).Eachoftheisochoreboundariesexceptone
hominidevolution.Inbothhumanandchimpanzee,RP11– correspondstoaboundarybetweenblocksduplicatedondif-
432G15(redsymbolsinFig.2)hybridizesonlytotheregions ferentsetsofchromosomes(compareFig.1Bwith1C),con-
corresponding to 2qFus and 22qter. These two sites are also sistentwiththeevolutionofthe2qFusregionasapatchwork
detectedingorillaandorangutan,indicatingthatthetransfer ofpiecescopiedfromothergenomiclocations.Forexample,
ofmaterialbetweentheselocationspredatedhominiddiver- the breakpoint in homology between 9q13 and 2qFus (and
gence.However,sequenceshomologoustothisclonearedis- 9pter) is marked in 2qFus/9pter by an L-to-H1–2 isochore
tributedonatleast38additionaltelomeresandtwointersti- transition, whereas it lies in the middle of an L isochore in
tialsitesingorilla.Hybridizationisdetectedat14ofthesame 9q13(Fig.4).Similarly,thebreakpointbetween2q11.2-Aand
locations in orangutan. Given the generally accepted homi- 2qFus creates an isochore transition in 2qFus (H1–2 to L),
nidlineage(ChenandLi2001),eitherorangutanandgorilla whereas it lies amid an H1–2 isochore in 2q11.2-A (not
independently acquired copies of portions of the RP11– shown).
432G15sequenceattheselocations,orhomologoussequence
was deposited at these sites before hominids diverged and InterspersedRepeats
thenwaslostintheancestorofhumanandchimpanzee.One Thedensityandnatureofrepetitiveelementsalsovaryacross
interstitialand26subtelomericintegrationsitesareuniqueto the614-kb2qFussequence(Fig.1B).Overall,interspersedre-
gorilla, indicating that a burst of duplications also occurred peats occupy 40% of the sequence, with Short Interspersed
along the gorilla-specific branch. One subtelomeric and five Elements(SINEs)andLongInterspersedElements(LINEs)ac-
interstitialsitesareuniquetoorangutan. countingfor12%and15%ofthesequence,respectively.Re-
SequencesinRP11–480C16,whichhybridizebyFISHto cent repeat activity helps to date some of the duplication
fivesitesinhuman(twoon2,threeon9),arepresentinfour events involving the 2qFus sequence. The full-length AluY,
of the orthologous sites in chimpanzee and three in gorilla AluYa5, and AluYb8 insertions into the 2qFus-paralogous
andorangutan(greensymbolsinFig.2).Chimpanzee,gorilla, blocksareindicatedinFigure3.Thesearetheyoungestclasses
and orangutan all lack cross-hybridizing sequences at the ofAluelementsfoundintheregion.TheAluYa5andAluYb8
9p11.2-equivalent location, and gorilla and orangutan are subfamilieshavebeentranspositionallyactiveveryrecently:
missinganadditionalsignalcorrespondingto9pteror9q13. 99%oftheinsertionsoftheseelementsarehumanspecific,
Becauseofaninversionwithbreakpointsinthesebandsthat and ∼25% exhibit presence/absence polymorphism in hu-
differentiateshumanchromosome9fromitscounterpartin
gorillaandorangutan,itisnotclearwhethertheremaining
conserved location corresponds to 9pter or 9q13, but other
evidence(seebelow)indicatesthat9q13holdstheancestral
copy.Fiveadditionalsubtelomericlocationshavedetectable
homologywithRP11–480C16inchimpanzee.
Asinhuman,blocksimmediatelyflankingthefusionsite
and contained in RP11–395L14 are multicopy in the chim-
panzee, gorilla, and orangutan genomes, and the copies are
primarilysubtelomericallylocated(bluesymbols,Fig.2).Be-
causethisBACencompassesblockswhosechromosomalpo-
sitionswereassayedbythetwoBACsdiscussedinthepreced-
Figure4 Disruptionofisochore,L1repetitiveelement,andPGM5
ingtwoparagraphs,weexpectedtoseemarkedspeciesdiffer-
gene by the breakpoint of duplication between 9q13 and 2qFus.
ences in the distribution of its FISH signals. Indeed, of 30 (Green)Lisochore;(blue),H1–2isochore.Theregionsshowncorre-
subtelomericlocationsdetectedineitherhumanorchimpan- spondtonucleotides381651to423067inthe2qFuscontigand,for
zee with reasonable efficiency, 15 are common to both spe- 9q13,fromnt139050inRP11–561023tont9713inRP11–88I18.
cies,and15areseeninonlyoneofthetwospecies.Signals Thebreakpointofhomology(dottedline)wasdeterminedbycross_
match (http://www.genome.washington.edu/phrap_documenta-
werealsoobservedintwoadditionalinterstitiallocationsin
tion.html)analysis,theL1PBarepeatwasidentifiedbyRepeatMasker,
chimpanzee.Ofthe∼50locationsdetectedwithRP11–395L14
andtheGC-contentandisochoreclassificationweredeterminedby
in any of the four tested hominid species, only seven are GESTALT.TheverticalbarsareexonsofthePGM5gene(seeFanetal.,
commontoallfourspecies,∼13arespecies-specificlocations, 2002formoredetails).
1656 Genome Research
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
2q Fusion: Structure, Paralogy, and Evolution
mans(Carrolletal.2001).Incontrast,theYclasswasactive fromthecanonicaltelomericrepeatappearstoberandomly
earlierduringhominidevolution;insertionsofYelementsare distributedacrossthefusionsiteinbothalleles(notshown).
rarelypolymorphicinhumans,becausetheYclassis∼1000 Two short arrays of degenerate telomere repeats, in ad-
timeslessactivenowthantheYa5andYb8subfamilies(Roy- ditiontothearraysmarkingthefusionsite,arefoundwithin
Engeletal.2001). 2qFus.Theyare181bpand248bplong,and17kband21kb
FourAluinsertionsintheregionareinformativeforin- distalofthefusionsite,respectively.Interstitialarraysofde-
ferring phylogeny (Fig. 3). (1) We find an AluY element in generatetelomerearraysarecommoninthehumangenome,
2q11.2-Athatisnotpresentatthecorrespondingsiteinthe particularly in subtelomeric regions (Riethman et al. 2001).
2qFusparalogousblock.OtherAluYelementsarecommonto Like the array at the fusion site, these arrays are highly di-
both blocks. Thus, the duplication that spawned these two vergedfromtheprototypictelomericrepeats(70%and86%
blocks must have occurred during AluY activity. (2 and 3) identicalto[TTAGGG] ,respectively).ASATR1(satellite)re-
n
Withintheregionsharedby2qFus,9q13,and9pter,wefind peatclusterwithintheblockcommonto2qFus,9pter,9q13,
an AluYb8 inserted only in some alleles of 9pter and an and9p11.2-B(asterisksinFig.3)alsoshowshighvariabilityin
AluYa5 element inserted uniquely in 2qFus (sequence from length,especiallywhencomparedwiththeoverallhighiden-
9p11.2 for this portion of the paralogous region is unavail- tityoftheseblocks.
able).The2qFusand9ptercopiesareotherwiseverysimilar
(Table1),buttheduplicationthatgeneratedthesetwocopies
must have occurred before these Alu elements inserted. The DISCUSSION
AluYb8elementispresentintwoofthreesequencedclones Thegrosscharacteristicsofthechromosomalfusionthatgave
thatoverlapthisregionof9pter(inRP11–59O6[AL158832] rise to human chromosome 2 were apparent 20 years ago,
andRP13–39F9[AL591968],butlackingfromRP11–174M15 whenYunisandPrakashalignedthehigh-resolutionbanding
[AL356244]). These clones represent allelic variants of 9pter patternsofhuman,chimpanzee,gorilla,andorangutanchro-
becausetheiroverlapsarecontiguousand(cid:1)99.7%identical. mosomes(YunisandPrakash1982).Theidentitiesofthefu-
(4) Another AluYb8 element is common to both 2qFus and sion partners were confirmed 10 years later when human
22qter in their region of homology. Its presence in both chromosome-2 specific DNA was observed to “paint” chim-
blocksindicatesthatsequencewastransferredbetween22qter panzeechromosomes12and13(Jauchetal.1992;Wienberg
and 2qFus (or its unfused predecessor) after the AluYb8 ele- etal.1992).Becausethefusedchromosomeisuniquetohu-
mentwasinserted.Theimplicationsoftheseobservationsare mans and is fixed, the fusion must have occurred after the
discussedbelow. human–chimpanzeesplit,butbeforemodernhumansspread
Threerepetitiveelementscrossbreakpointsofhomology around the world, that is, between ∼6 and ∼1 million years
andthereforeprovidecluestotheancestralandderivedstates ago(Mya;ChenandLi2001;Yuetal.2001)(Fig.5).Thisgross
oftheduplicativetransfers.(1)AnL1PBaelementcrossesthe karyotypicchangemayhavehelpedtoreinforcereproductive
red-to-lightbluebreakpointin9q13,butistruncatedinthe barriersbetweenearlyHomosapiensandotherspecies,asthe
9pterand2qFussequences;consistentwiththecreationofan F1offspringwouldhavehadreducedfertilitybecauseofthe
isochoretransitionin9pter/2qFus(Figs.1and4).(2)AnAluJb riskofunbalancedsegregationofchromosomesduringmeiosis.
elementistruncatedin19pteratthedarkblue-to-redbreak-
pointofhomologywith2qFus,butcrossesthebreakpointin
Molecular Characteristics of theFusion
2qFus.ThebreakpointalsocreatesanL-to-H-2isochoretran-
sitionin19pter,butleavestheH1–2isochoreintactin9pter/ Whenobservedatthesequencelevel,theancestralchromo-
2qFus(notshown).(3)AnL1MEelementistruncatedbythe somes appear to have undergone a straightforward fusion.
duplication from 2qFus to 2q11.2 (red-to-light green break- The sequence of RP11–395L14, like the cosmid partially se-
point);itcrossesthebreakpointin2qFus.Thiscaseistheonly quencedbyIjdoetal.(1991),showstwohead-to-headarrays
oneencounteredinwhichthedirectionoftransferindicated ofdegeneratetelomererepeatsatthe2qfusionsite,withno
bytherepeat-elementisoppositethatinferredfromtheiso- other sequence between the arrays. This observation indi-
chore-transitionpattern. catedthatthetwoancestralchromosomeshadjoinedend-to-
end within the terminal telomeric repeats, with subsequent
inactivationofoneofthetwocentromeres.Kasaietal.(2000)
Sequence Variation Across the 2q13–2q14.1
showed using FISH that the chromosomes underwent no
FusionSite
grossalterationinstructure:Therelativeorderof38cosmids
Thehead-to-headarraysofrepeatsatthefusionsiteinRP11– derived from 2q12–2q14 was the same on human chromo-
395L14 have degenerated significantly (14%) from the near some2andtheshortarmsofchimpanzeechromosomes12
perfectarraysof(TTAGGG) foundattelomeres.Comparison and13.Althoughthesequenceisnotyetavailablefromthe
n
ofthefusionsiteinRP11–395L14withan1873-bpsequence terminal regions of chimpanzee chromosomes 12p and 13p
fromadifferentindividual(M73018)(Ijdoetal.1991)reveals withwhichtocomparetohuman2q13–2q14.1,thehuman
ahighdegreeofvariationinthelengthandsequenceofthe sequence is very similar to two extant human subtelomeres
head-to-head arrays of degenerate telomere repeats (not (9pter and 22qter) (Fig. 3, Table 1). Very little, if any, distal
shown).Overall,thetwosequencesshowonly90%sequence material is unaccounted for in the two comparisons. Al-
identity.Moredifferencesareobservedwithinthedegenerate thoughneither9pternor22qterhasbeensequencedintothe
telomerearrays(88%identity)thaninthesequencesimme- telomeric arrays, the available sequences for these chromo-
diately flanking them (97.6% identity; 94.9% when each of somesmatch2qFustowithin21kband1.4kbofthearrayat
thebasesininsertionsanddeletions,whichrangeinsizefrom thefusionsite,respectively,andPCRassaysindicatethatho-
1to8bp,arecountedasmismatches).Only48%ofthe127 mologywith9pterextendstoatleast8.4kbfromthearray.
repeats in RP11–395L14 and 46% of the 158 repeats in Ifthefusionoccurredwithinthetelomericrepeatarrays
M73018 are perfect TTAGGG or TTGGGG units. Deviation less than ∼6 Mya, why are the arrays at the fusion site so
Genome Research 1657
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
Fan et al.
Millionyearsago(Mya) have joined either within terminal
A orsubterminalarraystoformchro-
14 12 10 8 6 4 2 0 mosome2.
MRCA
Human Segmental Duplications
By using PCR analyses of a hybrid
Chimpanzee 1.24%(cid:1)–0.07% panel, genomic sequence align-
ment, and FISH, we demonstrate
Gorilla 1.63%–0.08% that (cid:1)360 kb of the region sur-
rounding the fusion site is dupli-
Orangutan 3.08%–0.11% catedatleastonceelsewhereinthe
Fusion genome. These paralogous seg-
B
ments are distributed primarily in
subtelomeric and pericentromeric
2q11.2-A<-->2qFus-Prox
locations, consistent with the dis-
tributionofsegmentalduplications
9q13-->9pter,2qFus-Prox exchanges9pter,2qFus-Prox found in a recent whole genome
survey(Baileyetal.2001)andear-
Inv(9q13,9pter) 9q13-->9p11.2-Aand-B lierFISHanalyses(Ijdoetal.1991;
Trask et al. 1993; Hoglund et al.
2q11.2-A-->2q11.2-B 1995; Martin-Gallardo et al. 1995;
Ning et al. 1996; Lese et al. 1999;
Ciccodicola et al. 2000; Park et al.
22qter-->2qFus-Dist homogenizingexchanges
2000; Bailey et al. 2002; Martin et
al. 2002). Subtelomeric homology
Figure5 Estimatedtimingofduplications,inversions,andrelocationsof2qFus-paralogousblocks spans ∼258 kb. The long blocks
basedonsequenceidentitymeasuresandfluorescenceinsituhybridizationanalysesofhominids.(A)
Phylogenyofhominids.Thebranchlengthsandestimatedspeciationdatesarebasedonsequence shared by 9pter or 22qter on the
analysesof53autosomalintergenicnonrepetitiveDNAsegmentsanalyzedbyChenandLi(2001).The proximal and distal side of the fu-
speciationdatesaredrawnatthemidpointofestimatedranges:human–chimpanzee,4.6–6.2Mya; sion site, respectively, account for
human–gorilla,6.2–8.4Mya;human–orangutan,12–16Mya.MRCAmarkstheestimateofthetimeof thebulkofthishomology.Inaddi-
themostrecentcommonancestorofallmodernhumans(Yuetal.2001).Theaverage(cid:2)SDpercent
tion, highly dispersed, multicopy
sequencesubstitution(Jukes-Cantormodel,excludingindels)betweenhumanandeachofthethree
blocks comprise the 68 kb directly
other hominids is given at the right (Chen and Li 2001). (B) Estimated timing of events involving
2qFus-paralogousblocks.Theblocksareidentifiedbytheircurrentpositionsinhumans(i.e.,2qFus-Dist surrounding the fusion site. These
and2qFus-Proxaretheregionsformingthedistalandproximalsidesofthe2qfusionsite,respectively; blocksarerelativelyshortandshow
bothwereatchromosometipswhentheduplicativetransfersoccurred).Ancestralandderivedstates, 93%–99% identity to various sub-
whenindicated,areinferredfrombreaksthatdisruptgenes,specificrepetitiveelements,orisochore telomeres.Thiscomplexpatternof
patterns;thecopycarryingthefull-lengthgene/elementand/orlackinganisochoretransitionatthe
homologyamongpresent-daysub-
breakpointisassumedtorepresenttheancestralversion.
telomeres and the fusion site indi-
cates that various DNA segments
degenerate?Thearraysare14%divergedfromcanonicaltelo- hadduplicatedamongsubtelomericregions,includingthose
mererepeats(notshown),whereasnoncodingsequencehas of the fusion partners, before the fusion took place (see fol-
diverged<1.5%inthe∼6Myasincechimpanzeeandhumans lowing).
diverged(ChenandLi2001)(Fig.5).Therearethreepossible Verylargesegmentsof2qFusalsohavehomologywith
explanations: (1) Given the many instances of degenerate nontelomericsites.Theseinterstitialparalogsarelesssimilar
telomeric arrays within the subtelomeric regions of human to the 2qFus sequence than are the subtelomeric paralogs
chromosomes (Riethman et al. 2001), the chromosomes (Table 1) and presumably result from earlier duplication
joined at interstitial arrays near, but not actually at, their events(seefollowing).Thefusionregionand2q11.2shareat
ends.Inthiscase,materialfromtheveryendsofthefusion least100kbastheresultoflargeintrachromosomalduplica-
partnerswouldhavebeendiscarded.(2)Thearrayswereorigi- tions.Intrachromosomalduplicationshavealsogeneratedat
nally true terminal arrays that degenerated rapidly after the least three large interstitial blocks of homology on chromo-
fusion.Thishighrateofchangeisplausible,giventheremark- some9,inadditionto9pter.
ably high allelic variation observed at the fusion site. The Manyothercross-hybridizingsiteswereobservedinthe
arrays in the BAC and the sequence obtained by Ijdo et al. genomes of nonhuman primates (Fig. 2), reflecting the evo-
(1991)differby12%,whichishighevenifsomedifferences lutionary mobility of sequences homologous to the region
areascribedtoexperimentalerror.(3)Somearraydegeneracy surroundingthefusionsite.
could be a consequence of sequencing errors. We have not The size and high similarity of these duplications have
been able to PCR successfully across the fusion site, which beenproblematicfortheautomatedassemblyofhumange-
would be required to assess the contribution of sequencing nome sequence across these regions. For example, RP11–
errorstothismeasureoffusion-sitesequencepolymorphism. 15J10hasmigratedfrom2q11.2,9q13,9p23,9q21,and9q12
However,explanation2issupportedbythehighvariability in various versions of the genome assembly. It contains Se-
amongalleliccopiesofotherinterstitialtelomericrepeatsand quence-tagged Sites (STSs) that have been assigned to chro-
associated regions sequenced by Mondello et al. (2000) mosomes 2, 9, 7, and X, but none is single copy in the ge-
(AF236886andAF236885).Consideringthehighmutability nome.BasedonourFISHandhybrid-panelresults,thisclone
ofinterstitialtelomererepeatarrays,thefusionpartnerscould most likely derives from 9p11.2. RP11–143M1 has migrated
1658 Genome Research
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
2q Fusion: Structure, Paralogy, and Evolution
from9q13,to9p22,to9pter,itstruelocation.Thenumbersin some3containingolfactoryreceptorgenes(Brand-Arponet
Table1providejustificationforsomeofthisconfusion:9pter al.1999).
and9q13are∼98%identicaloveraspanof>150kb.Wehave Twoduplicationsofmaterialfrom9q13to9p11.2were
no explanation for why several 2qFus-related clones have thenextevolutionaryeventstooccurinvolving2qFusblocks
been assigned at one time or another to 9p22–9p23 (RP11– on chromosome 9. FISH signals appear at 9p11.2 only after
15J10,(cid:1)403A15,(cid:1)143M1and(cid:1)174M15);noneproducesa human and chimpanzee diverged (Fig. 2), and the 9p11.2-A
FISH signal there. Unfortunately, some of these localization and -B blocks are more similar to sequence in 9q13 than in
errorshavebeenpropagatedinpublications(e.g.,Mahetal. 9pteror2qFus(Table1).Oneduplicationinvolveda(cid:1)42-kb
2001), which augments the confusion. These examples and segment,andtheothera(cid:1)110-kbsegment.(Wesurmisethat
the deceptive Y-hybrid results we encountered illustrate the theseblocksderivefrom9p11.2,buttheymayrepresentad-
needformultiplemappingmethodstoaddressthechallenges ditionalcopiesfrom9q13,asFISHsignalsareequallybrightin
encounteredinthestudyofsegmentalduplications. 9p11.2and9q13.)Theseblocksadjoininthe9q13sequence,
but are distinct in the regions represented by the 9p11.2-A
and-Bsequences(Fig.3).Theblockscouldhavetransposed
The History of the ParalogousSequences independently, or together and then been separated by the
insertionofothermaterial.Assumingthattherehasbeenno
LargeDuplicationsandPericentromericInversions furtherexchangebetweentheblocksinthesetwobands,the
Basedonoursequencecomparisons,theoldesteventinvolv- degree of their divergence (1.0% and 1.2%) also dates the
ing 2qFus paralogous blocks was the duplicative exchange duplication(s) soon after the human–chimpanzee split (Fig.
between 2q11.2-A and the progenitor of the centromere- 5).Afterhumanandchimpanzeediverged,thehuman9q12
proximalsideofthefusionsite(Fig.5).Thesesequenceshave heterochromatic region expanded, placing the 9q13 paralo-
sincedivergedbyatleast4%.TheFISHresultsindicatethat,at goussegmentmuchfurtherfromthecentromereonthehu-
thetimeofthisduplication,bothregionswerelocatedonthe man chromosome than the chimpanzee ortholog. Chromo-
p arm of the ancestral chromosome that was later to be a some 9 also underwent a second inversion along the chim-
fusionpartner.Iftherehasbeennoectopicrecombinationor panzeebranchafterthechimpanzee–humansplit.Although
geneconversionbetweenthesetworegionssincetheoriginal oneinversionbreakpointmapsatcytogeneticresolutionclose
duplication,andthetwocopieshavedivergedataratetypical tothe9p11.2paralog,thissequenceisunlikelytobeinvolved
for the hominid noncoding DNA (Chen and Li 2001), then intherearrangement,becauseitappearsatthislocationonly
this intrachromosomal duplication predates hominid diver- onhumanchromosome9.Theserearrangementsmayexplain
gence(16–20Mya)(Fig.5).OurFISHdataareconsistentwith whyMartinetal.(2002)assignedthisparalogousblocktothe
thistiming:Theblockispresentinbothlocationsinallfour sitecorrespondingto9p11.2insteadof9q13inchimpanzee
hominidsanalyzed.AluYelementsintheblocksarealsocon- (seeearlier).
sistent with this timing: Together, the presence of several Thetwochromosomesthatjoinedtoformhumanchro-
AluY insertions common to both blocks and one AluY ele- mosome 2 each underwent pericentromeric inversions in
ment in 2q11.2, but not 2qFus, dates this duplication some hominidevolution(Fig.2).Twoofthefourbreakpointsmap
timeduringtheperiodofthetranspositionalactivityofthis nearthetelomericregionsthatwereinvolvedinthefusion,
Aluclass,thatis,earlyinhominidevolution(Roy-Engeletal. butthesequenceswithhomologywiththefusionsiteappear
2001). tolieoutsideoftheinvertedsegments.
Thenexteventwastheduplicativetransferofa(cid:1)150-kb
SubtelomericExchanges
blockfromwhatarenow9q13andtheancestorof9pteror
the2q-formingchromosome.Severallinesofevidenceindi- Ourstudyalsoaddstothecomplexpictureofinterchromo-
catethattheancestralcopyofthisblockisnowin9q13.The somal subtelomeric duplications. Duplications among sub-
transfer of material from 9q13 to 2qFus/9pter disrupted an telomeresgeneratedablockcommontothechromosomedes-
L1PBa element, the PGM5 gene (Fan et al. 2002), and an L tined to become human 2p and the ancestor of 9pter, and
isochore.Thesefeaturescrossthebreakpointin9q13,butare another block common to the chromosome destined to be-
truncated in 2qFus/9pter (Fig. 4). The divergence between comehuman2qandtheancestorof22qter.Althoughweare
9q13 and these other blocks is now ∼2%. This divergence not able to infer the direction of the 9pter–2qFus transfer
indicates that this duplication predated the gorilla– fromtheavailableinformation,22qterrepresentstheances-
chimpanzee–humansplit(Fig.5).FISHdatawouldindicatea tral state and 2qFus the derived state: The breakpoint is
morerecentdate,becauseonlyonelocationislabeledingo- marked by an isochore transition in 2qFus, not 22qter, and
rilla and orangutan, compared with sites corresponding to theACRgeneisintactin22qter,buttruncatedin2qFus(Fan
both 9q13 and 9pter in human and chimpanzee (not 9p11 etal.2002;Martinetal.2002).Thedivergencebetweenthese
and9pter,asreportedforchimpanzeebyMartinetal.[2002]). pairs of blocks (1.1% and 1.4%, respectively) dates both
The location in gorilla coincides, at cytogenetic resolution, events at or around the time of chimpanzee–human specia-
withabreakpointofthe9pter–9q13inversionthatoccurred tion. This similarity is surprising, however, given the age of
after human and chimpanzee branched off from gorilla. the duplications indicated by FISH analyses of other
Giventheirsequencedivergence,itispossiblethatthe9q13 hominids. The 2q clone containing the 2qFus/22qter-
and9pterblocksbeganasatandemduplicationinthecom- homologyblockhybridizestobothofthesesitesinhuman,
mon ancestor of human, chimpanzee, and gorilla, but were chimpanzee, gorilla, and orangutan, dating the duplication
visibly separated by the pericentromeric inversion that oc- beforehominiddivergence.However,Martinetal.(2002)ob-
curredlaterintheancestorofhumanandchimpanzee(Fig. servedsignalsonlyonthechromosome-22equivalentsitein
2).TwocloselyjuxtaposedcopieswouldnotbevisiblebyFISH orangutan and an Old World monkey when using a clone
inmetaphasechromosomes.Wehaveobservedasimilarstag- derivedfromchromosome22containingthe2qFus/22qteras
ingofthestepsleadingtotwocopiesofaportionofchromo- theirFISHprobe.Itisthereforepossiblethatthesignalswesee
Genome Research 1659
www.genome.org
Downloaded from genome.cshlp.org on January 22, 2023 - Published by Cold Spring Harbor Laboratory Press
Fan et al.
on the 2q ancestor in orangutan represent an independent tionofthe9p11–q13regionreportedbyLukusaetal.(2000).
duplicationinorangutanofadifferentsegmentofthe2qFus Furtheranalyseswillbeneededtodetermineiftheseblocksof
sequence.Eventakingthemoreconservativeview—thatthe homologyboundtheduplicatedsegments.
duplication from 22qter to the 2qFus ancestor occurred just The2qFus-paralogousblocksarealsogoodcandidatesfor
before the human–chimpanzee–gorilla split—the two blocks involvement in recombination events that cause other de
must have undergone homogenizing ectopic exchanges at novorearrangementsofhumanchromosomes.Forexample,
leastupuntilthefusioneventtoreconcilethefactthatthese adeletionofthematerialbetween2q11and2qFushasbeen
sequences are now only 1.4% different. The fact that the notedinapatientwithacutemyeloidleukemiaintheMitel-
blocksin2qFusand22qternowcarrythesameAluYb8inser- man catalog of chromosome abnormalities (http://cgap.nci.
tionisstrongevidencethattheseblocksexchangedsequence nih.gov/chromosomes/CytSearchForm).Thecatalogalsocon-
since humans and chimpanzee diverged. Members of the tainsatleast60casesfromawidevarietyoftumorsinwhich
AluYb8familyhavebeenactivelyretrotransposingonlysince oneofthebandscontaining2qFusparalogyisjoinedtouni-
human–chimpanzeedivergenceandoccuralmostexclusively dentifiedmaterialtoformanunbalancedrearrangement.
inthehumangenome(Carrolletal.2001). It has also been suggested that interstitial telomeric se-
Theevolutionarymobilityofsubtelomericregionsisfur- quences are sites of preferential chromosome breakage, am-
therunderscoredbythegrossdifferencesinthelocationand plification,andrecombination(Bertonietal.1994;Boutouil
number of subtelomeric blocks observed among hominids. etal.1996;Slijepcevicetal.1996;Simietal.1998;Desmazeet
Considering the extensive polymorphism and recurrent ex- al.1999).Someinternaltelomericrepeatsmapatcytogenetic
changes among subtelomeres (Mefford et al. 2001; Mefford resolution together with mapped fragile sites (Musio and
and Trask 2002), it may be unreasonable to expect that a Mariani1999).Theinvertedtelomericrepeatarraywasacan-
linearlybranchingpedigreeofsubtelomericduplicationscan didatefortheFRA2B,whichislocatedin2q13(Williamsand
everbededuced. Howell 1977; Sutherland and Mattei 1987), but published
data(Ijdoetal.1992)andourownexperiments(CF,YF,and
Breakpoints
BT;unpublishedresults)showthattheFRA2Bsitemapsproxi-
Are there sequences at the breakpoints of homology blocks malofthe614-kbregiondescribedhere.
thatmightshedlightontheduplicationandexchangepro- Intheaccompanyingpaper(Fanetal.2002),wecharac-
cesses that have acted on these regions? Half of the break- terize11geneswithinthe2qFussequence.Asaconsequence
pointsdefiningthepairsofmajorparalogousblocksinTable of the various intra- and interchromosomal duplications
1canbepinpointedatthesequencelevelbecausesequenceof documentedhere,9ofthesegenesarepresentinthehuman
bothpartnersisavailablewherethehomologybreaksdown. genome in more than one copy. Thus, in addition to their
We observe no element that is common to the available historicalcontributionstothegrossstructuralchangesamong
breakpoints of paralogous segments. Several occur in LINE hominidchromosomesandpossibleinvolvementinchromo-
elements,andothersareinnonrepeatsequencesthathaveno somal rearrangements in humans, duplications and rear-
homologywitheachother.Fourbreakpointsappeartooccur rangementsof2qFus-paralogousblocksalsohavefunctional
within a common L1PBa element, but all concern the same relevance.
events—thedisplacementof9q13homologyintheancestor
of 2qFus and 9pter by sequences common to multiple telo-
METHODS
meres(seealsoFanetal.2002).Theduplicationthatgener-
atedcopieson9pterand2qFusoccurredafterthisevent,so
Database Mining and SequenceAnalyses
thatbothoftheselocationssharethesamebreakpointwith
9q13anditscopyin9p11.2.Overall,thediversityamongthe The sequence of RP11–395L14 served as the entry point for
this study. Homologous sequences were obtained iteratively
breakpointsindicatesthattheduplicationsandexchangesoc-
from GenBank (Benson et al. 2002) by BlastN (http://www.
curred by mechanisms involving random double-strand
ncbi.nlm.nih.gov/BLAST/).Finishedsequencesfromdifferent
breaksinDNAratherthanspecialcommonsequences.
cloneswereassembledintothesamecontigonlyiftheirover-
lapwascontiguousand(cid:1)99.7%identical,areasonableallow-
Paralogy and (Deleterious)Rearrangements
anceforpolymorphismandsequencingerrors.Wescreened
Wehaveprovidedtwoexamplesinwhichblocksparalogous for interspersed repeats with RepeatMasker (http://ftp.
to the fusion site are potentially involved in gross chromo- genome.washington.edu/cgi-bin/RepeatMasker). We used
somal rearrangements that have occurred during hominid GESTALT (http://bioinformatics.weizmann.ac.il/GESTALT/)
(Glusman and Lancet 2000) to characterize GC and repeat
evolution.Theselargeblocksofhomologymayalsosporadi-
content. Pairwise alignments were performed with BLAST2,
callymediategrossrearrangementsinhumans.Bands9p11.2
without repeat masking and with gap-initiation and gap-
and 9q13 contain the breakpoints of common pericentro- extension parameters adjusted to minimize breaking long
meric inversion polymorphisms in humans (Samonte et al. matches into pieces at sites of insertion/deletions. Percent
1996).Thehighlysimilarblocksidentifiedhereinthesebands identities of paralogous blocks were calculated from the
((cid:1)40-kbblocksof99%)couldmediatehomologousrecombi- BLAST2 output as follows (Linardopoulou et al., in prep.).
nation and cause some of these inversions. This hypothesis First,thenumberofnucleotidesubstitutionsbetweenthetwo
could be tested by comparing the sequence of the common sequenceswascountedanddividedbythenumberofaligned
andinvertedformsofchromosome9;thebreakpointsshould bases(bothnumbersexcludegaps).Thisobservedproportion
(p) was entered in the Jukes-Cantor equation to estimate K,
mapwithintheparalogousblocks.Inaddition,wewouldex-
the number of nucleotide substitutions per site. The Jukes-
pectthetwointeractingblockstolieinoppositeorientation
Cantor equation takes into account that multiple substitu-
onthechromosome.Thetentativeorientationsoftheblocks tionsmighthaveoccurredatthesamesiteandisasfollows:
in9q13and9p11.2-A(Fig.3)areconsistentwiththisexpec- K=(cid:1)(3/4)ln[1–(4p/3)](JukesandCantor1969).Thepercent
tation.Theseblocksmayalsobeinvolvedintheformationof identity of the aligned sequences is therefore 100%*(1(cid:1)K).
adicentricchromosome9withtandemhead-to-tailduplica- Thenumberandsizeofgapsinalignmentscausedbyinser-
1660 Genome Research
www.genome.org