genomic instability within centromeres of interspeciﬁc … · 2007-12-13 · with no pouch or...

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.082313

Genomic Instability Within Centromeres of Interspecific Marsupial Hybrids

Cushla J. Metcalfe,*,1 Kira V. Bulazel,* Gianni C. Ferreri,* Elizabeth Schroeder-Reiter,†

Gerhard Wanner,† Willem Rens,‡ Craig Obergfell,* Mark D. B. Eldridge§ andRachel J. O’Neill*,2

*Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, ‡Cambridge Resource Centre forComparative Genomics, Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB2 1TN,

United Kingdom, †Department Biologie I, Ludwig-Maximilians-Universitat Munchen, 80638 Munich, Germany and§Australian Museum, Sydney, New South Wales 2010, Australia

Manuscript received September 23, 2007Accepted for publication October 16, 2007

ABSTRACT

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to insta-bility that can lead to specific types of chromosome rearrangements important in species incompatibility.Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent andextensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centro-meres are the primary target for destabilization in cases of genomic instability, such as interspecific hy-bridization, and participate in the formation of novel chromosome rearrangements. Here we use standardcytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electronmicroscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus 3 Macropus agilis). Theparental complements share the same centric fusions relative to the presumed macropodid ancestralkaryotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having largercentromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomalinstability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus)complement. This instability included amplification of a satellite repeat and a transposable element,changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons andmechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.

IT has been noted since the 1970s that chromosomerearrangements within some plant and animal line-

ages are nonrandom. For example, within the primatelineage, fissions predominate within the Old Worldmonkeys, pericentric inversions within the great apesand Robertsonian translocations within the lemurs(Dutrillaux 1979). In the human lineage, there is astriking correspondence between the position of evolu-tionary breakpoints conserved in mammals, humanfragile site locations, and the distribution of tandemrepeats (Ruiz-Herrera et al. 2006). Numerous studies(e.g., McClintock 1987 for Zea spp.) have shown thatmultiple chromosomal rearrangements of the sametype can occur in different individuals simultaneously(reviewed in King 1993). These data suggest that ‘‘hotspots’’ in the genome are predisposed to instability andmay be subject to genomic rearrangements.

The family Macropodidae exhibits recent and exten-sive chromosome evolution, in contrast with most other

marsupials, which are generally karyotypically con-servative (reviewed in Hayman 1990; Eldridge andMetcalfe 2006). Chromosome evolution within macro-podids has been extensively studied; the majority ofmacropodids have been karyotyped and the evolution-ary trajectory of chromosome rearrangements has beendetermined (Hayman 1990; Eldridge and Close 1993;Bulazel et al. 2007). Within the genus Macropus(kangaroos and wallabies), rearrangements involvingthe centromere predominate, in particular centricfusions (Rofe 1978; Hayman 1990) and whole-armreciprocal translocations (WARTs) (reviewed in O’Neill

et al. 2004). However, the mechanism responsible forthe rapid karyotypic diversification within this group ofmammals has not been fully explored.

Instances of rapid genomic change can result froman increased mutation rate caused by genome destabi-lizing events, such as inter- and/or intraspecific hybrid-ization or exposure to environmental mutagens andstress (Fontdevila 1992). This increase in mutationrate has been observed to coincide with an increase inthe local activity of transposable elements, retroele-ments, and other repeated DNAs (see Lim and Simmons

1994; O’Neill et al. 1998; Labrador et al. 1999;Fontdevila 2005; Ungerer et al. 2006). In addition

1Present address: Laboratoire Evolution, Genomes et Speciation(UPR9034), Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France.

2Corresponding author: Department of Molecular and Cell Biology,U-2131, 354 Mansfield Rd., Room 323, University of Connecticut, Storrs,CT 06269. E-mail: [email protected]

Genetics 177: 2507–2517 (December 2007)

to a bias in sequence classes involved in rearrangement,extensive genome sequence comparisons and compar-ative cytogenetics now point to specific chromosomefeatures, such as centromeres, that can also influencethe number, position, and type of rearrangement.

Our previous work in macropodid hybrids showedthat a retroviral sequence located at the centromere hadundergone demethylation and amplification, concom-itant with chromosome remodeling (O’Neill et al.1998). Subsequent analyses using cross-species chro-mosome painting of four other macropodid hybridindividuals (hybrids) showed that the rearrangementsobserved in these genomes were also restricted to cen-tromeres (O’Neill et al. 2001).

In our previous work, it was not determined whetherthe observed rearrangements were shared in otherhybrids of the same type or whether the rearrangementswere the result of interchromosomal segmental dupli-cations of centromeric sequences, non-allelic recombi-nation between sequences at centromeres on differentchromosomes, or whether they resulted from the trans-position and/or amplification of mobile DNA or otherrepeated DNAs. In this study, the genomes of four inter-specific hybrids from a cross between two macropodidspecies not previously studied, Macropus rufogriseus(maternal component) and Macropus agilis (paternalcomponent), were assayed for chromosome rearrange-ments and genomic instability using standard cyto-logical staining techniques, cross-species chromosomepainting, DNA probe analyses, as well as ultrastruc-tural analyses of centromeres via scanning electronmicroscopy.

These data show that the centromere is a significantcontributing factor in chromosome aberration in all ofthe hybrid genomes examined. The current analysesshow that, in these hybrids, some centromeres differstructurally from parental chromosomes and are thesite of extensive genome rearrangements accompaniedby DNA amplification of retroelement sequences andsatellites. These rearrangements include a broad arrayof abnormalities typical of genomic instability, includ-ing fissions, isochromosomes, whole-arm reciprocaltranslocations, and minichromosomes. Remarkably, re-arrangements were found only within the maternalcomplement and all were associated with the maternallyderived centromeres. This study extends previous workand, for the first time, clearly defines the centromere asthe site of genomic instability, anomalous chromosomestructures, and structural variants.

MATERIALS AND METHODS

Animals and karyotypes: Five M. rufogriseus 3 M. agilis hy-brids were available. RA1190 is a normal XY male, and RA1122and ‘‘new’’ RA are normal XX females. RA1118 is a XX animalwith no pouch or penis and a small, empty but well-developedscrotum. RAX0 was a female with no pouch, a small, empty

scrotum, rudimentary female reproductive tract, and largeamounts of fat in the body cavity.

Three normal parental animals (A1843, R1188, and R3242)were examined. The male M. agilis (A1843) is the father of allhybrids. The M. rufogriseus animal, R1188, is the mother ofRA1188. The mother of the remaining hybrids is from thesame population as R1188 but could not be identified bymicrosatellite analysis (data not shown). An unrelated M.rufogriseus male (R3242) was also examined.

Cell culture: Ear biopsies were obtained from animals heldat Macquarie University, Fauna Park. Primary fibroblast cul-tures were established from ear biopsies and maintained usingstandard techniques (Cooper et al. 1977). Cultures were pas-saged no more than 10 times, with the exception of R3242(.15 passages). Metaphases were prepared using standardtechniques from all cultures. Cytogenetic techniques wereperformed on metaphases from the three parental animals(A1843, R1188, and R3242) and four hybrids (RA1190,RA1122, RA1188, and ‘‘new’’ RA).

C-banding: Metaphases from each species were C bandedusing the technique of Sumner (1972) as modified byEldridge (1991).

Scanning electron microscopy: Chromosomes were isolatedaccording to the ‘‘drop/cryo’’ technique (for details seeMartin et al. 1994). Briefly, cell suspensions as per standardchromosome preparations were dropped onto cold laser-marked slides (Laser Marking, Fischen, Germany). Just priorto fixative evaporation, 20 ml of 45% acetic acid was applied,and the specimens were covered with a cover slip and frozenupside down on dry ice for 15 min. The cover slip was priedoff, and specimens were immediately transferred into fixative(2.5% glutaraldehyde, 75 mm cacodylate buffer, 2 mm MgCl2,pH 7.0).

For DNA staining, chromosomes were incubated for 30 minat room temperature with platinum blue (½CH3CN�2Pt oligo-mer, 10 mm, pH 7.2) and washed with distilled water and thenwith 100% acetone prior to critical point drying (for details seeWanner and Formanek 1995).

Specimens were washed in distilled water, dehydrated in100% acetone, and critical point dried from CO2. Slides weremounted onto stubs and carbon coated by evaporation.Specimens were examined at accelerating voltages from 15to 30 kV with an Hitachi S-4100 field emission scanning electronmicroscope equipped with a yttrium aluminum garnet (YAG)-type backscattered electron (BSE) detector (Autrata). Second-ary electron and BSE images were recorded simultaneouslywith Digiscan hardware (Gatan).

Preparation of chromosome paints and fluorescence in situhybridization: Flow sorting and chromosome paint produc-tion of Petrogale xanthopus (2n ¼ 22 karyotype) chromosomepaints were performed according to the protocol previouslydescribed (Rens et al. 1999). Highly pure chromosomesamples were collected, with the exception of the Y chromo-some, which was too small to be collected. Pools of individuallyisolated chromosome DNA were subjected to degenerateoligo primed (DOP)–PCR amplification for incorporationof either biotin– or digoxigenin (DIG)–dUTP (Roche) asper the manufacturer’s instructions. Cross-species chromo-some painting was performed as described by O’Neill et al.(1999) with the following modifications. P. xanthopus geno-mic DNA was sonicated to between 200 and 500 bp and wasused for suppression during a preannealing step prior tohybridization. Biotin-labeled probes were detected using avidin–fluorescein-5-isothiocyanate (FITC) (Vector Labs) and digox-igenin probes were detected with antidigoxigenin rhodamine(Roche). Metaphases were captured on an Olympus AX70microscope equipped with a Photometrics Sensys CCD camera

2508 C. J. Metcalfe et al.

and analyzed using the Genus Cytovision software (AppliedImaging).

Five chromosome paint ‘‘sets’’ were used corresponding toP. xanthopus chromosomes 1 and 10; 6, 9, and 7; 5 and 8; 3 and4; and 2 and the X chromosome. The five paint sets cor-respond to M. rufogriseus and M. agilis chromosomes 1p and1q; 6p, 6q and 7; 3p and 3q; 4 and 5; and chromosome 2 andthe X chromosome, respectively. A total of 3094 metaphaseswere scored, 376–479 for each parent and hybrid individual.Cross-species chromosome painting was also performed andsubject to analysis on an unrelated M. rufogriseus male todetermine if observed hybrid-specific rearrangements werefound within the parental species.

Dot-blot analyses of sat23 and KERV: Dot-blot analyseswere performed on each parent and hybrid animal, and serialdilutions of DNA concentrations ranging from 500 to 0.005ng/ml were denatured for 5 min at 99�, snap cooled, andspotted onto Hybond N1 (Amersham, Piscataway, NJ) mem-brane and fixed according to the manufacturer’s protocol.Blots were prehybridized and hybridized according to Ferreri

et al. (2005) at 65� overnight, washed in 13 SSC, 0.1% SDS at65�, and exposed to film overnight at �80�. Cytb probe wasproduced by PCR as per Bulazel et al. (2007) and hybridizedas for sat23 and KERV.

Fluorescence in situ hybridization (sat23 and KERV): Thesat23 (pGEM-T vector containing 1.5 copies of the 178-bpsatellite) and KERV-1 (pGEM-T vector containing 1.5 kbspanning the gag-pro-pol region of the retrovirus) probes werelabeled with biotin–dUTP and digoxigenin–dUTP, respect-ively, through PCR amplification of insert DNA using 100 ngeach of the plasmid primers SP6 and T7. PCR products werecleaned with a microcon 100, and 2 ml (of 10 ml) wasresuspended in 8 ml of hybridization buffer (50% formamide,10% dextran sulfate, 50 mm Na2HP04, 23 SSC) and denaturedat 80� for 10 min. Following denaturation in 70% formamide/23 SSC at 70� for 2 min and processing through a serialethanol series at �20�, slides were hybridized in the presenceof probe overnight at 37�. For metaphases from hybrids, post-hybridization washes were performed as per Bulazel et al.(2006). For metaphases from M. agilis, low-stringency post-hybridization washes (three washes of 50% formamide/23SSC for 5 min each; three washes of 23 SSC for 5 min each)

were performed at room temperature. The biotinylated probewas detected with antibiotin rhodamine (Molecular Probes,Eugene, OR) and DIG-labeled probe was detected with FITC-anti-DIG antibody (Molecular Probes). Metaphases werecounter stained with 49,6-diamidino-2-phenylindole (DAPI).Images were captured as described above.

RESULTS

Karyotypes: A 2n ¼ 22 karyotype is considered an-cestral for macropodids (kangaroos and wallabies) sincea homologous complement is found in representa-tives of four genera (Petrogale, Thylogale, Setonix,Dorcopsis) and the karyotypes of all other macropodidspecies can be readily derived from this 2n ¼ 22 kar-yotype, principally via centric fusions (Eldridge andMetcalfe 2006). The karyotype of M. rufogriseus sharesa suite of three centric fusions with two other Macropusspecies (Rofe 1978). M. agilis is presumed to also havethe same complement, but only on the basis of conven-tional staining (Sharman 1961). Chromosome paintingconfirms that the M. agilis complement does have thesame suite of chromosome rearrangements (data notshown).

Four hybrids produced from a cross between M. agilisand M. rufogriseus were confirmed as interspecific off-spring on the basis of cytological examination of chro-mosome complements. In each case, the maternal andpaternal complements are readily identifiable by Xchromosome morphology differences and the markedcentromere length differences for all chromosomes(Lowry et al. 1995; Bulazel et al. 2006).

Previous studies of marsupial hybrids indicated apredominance of centromere-associated rearrangementswithin macropodid hybrids (O’Neill et al. 1998, 2001).To examine gross constitutive heterochromatin amountsat the centromeres of hybrids in this study, C-bandingwas performed. C-banding is a technique that specifi-cally stains blocks of constitutive heterochromatin,typically at centromere locations. C-banding patterns(dark-staining regions) are markedly different betweeneach parental animal (Figure 1, A–C), with extensive cen-tromeric heterochromatin in M. rufogriseus and littledetectable centromeric heterochromatin in M. agilis.The origin of each homologous chromosome in thehybrids was clearly identifiable by its C-banding pattern;the M. rufogriseus-derived autosomes within the hybridsare clearly identified as the chromosomes with ex-tended, dark-staining centromere regions.

Ultrastructural analyses: In preliminary fluorescentmicroscopic (DAPI) investigation of the chromosomemorphology within the hybrid genomes, some centro-meres appear elongated and segmented (Figure 2). Incontrast to maternal centromeres, some hybrid centro-meres appear to have two constrictions, lending thecentral chromatin a ‘‘knob-like’’ appearance (Figure 2).For structural analysis with higher resolution, hybrid

Figure 1.—C-band karyotype of metaphase chromosomesfrom (A) M. rufogriseus, (B) M. agilis, and (C) M. rufogriseus 3M. agilis (RA1120). Chromosome numbers are indicatedbelow C.

Hybrid Centromere Instability 2509

chromosomes were investigated with scanning electronmicroscopy (SEM). Specific DNA staining for SEM withplatinum blue (Pt blue) revealed that the DNA distri-bution is uneven along these hybrid chromosomes(Figure 3, A and B). DNA distribution in the centromereis similar, if narrower, to that of the chromosome arms.In the secondary electron (SE) image, chromatids arehardly distinguishable, but in the BSE images they areclearly distinguishable by a longitudinal ‘‘signal-free’’space (Figure 3B). However, at the centromere the chro-matids are joined in an area of compact chromatin, asindicated by an unseparated signal in the BSE image(Figure 3B). Chromosomes exhibited a generally loos-ened chromatin structure; parallel fibers and chromo-meres are visible throughout the whole chromosome(Figure 3B).

Three-dimensional ultrastructural analysis of elon-gated centromeres with high-resolution SEM showedthat parallel fibers are interspersed with chromomeresof varying sizes (Figure 4). This diverges slightly fromthe centromere ultrastructure observed in other centricchromosomes in plants and animals, for which thecentromere is characterized by exposed parallel matrixfibers (Wanner and Formanek 1995, 2000; Sumner

1998). Platinum blue staining revealed that DNA israther heterogeneously distributed along the hybridchromosomes in a network of varying local concentra-tions (Figure 4). Strong BSE signal regions correspondwith chromomeres and compact structural regions(Figure 4). Centric chromosomes typically show dis-tinctly less DNA content at the centromere, indicatedby a significantly weaker BSE signal at the primaryconstriction with respect to the chromosome arms(Wanner and Formanek 2000). The DNA distributionfor the hybrid chromosomes with elongated centro-meres, however, is uninterrupted on each chromatid;the strong Pt blue signal reflects a high chromatindensity in these chromosomal regions.

Centromere sequences in hybrid genomes: We havepreviously isolated two sequences found specifically atactive centromere locations within Macropus. The firstsequence, sat23, is a 178-bp satellite that is capable ofbinding the centromere protein CENP-B both in vitroand in vivo and most likely represents the primary sat-ellite dictating centromere function within this groupof mammals (Bulazel et al. 2006). The second, KERV-1,is a retrovirus that is found in the genomes of a widerange of marsupials (Ferreri et al. 2005). It is found at

higher copy number at the active centromere (O’Neill

et al. 1998; Ferreri et al. 2004) as well as in low copynumber at breaks of synteny between the conservedchromosome segments within marsupials (Ferreri et al.2004).

To test for a correlation between the copy numbers ofthese two centromere-predominant sequences and theabnormally extended centromeres observed within thehybrid genomes, dot-blot analyses were performed toassay for copy-number variance in relation to the parentfrom which the maternal complement was derived(M. rufogriseus). The titration dilution used in this assaywas necessarily low due to the extreme high copynumber of these sequences at the centromeres withinM. rufogriseus (Bulazel et al. 2006) compared withM. agilis. Thus, as M. agilis carries both KERV-1 andsat23 at its centromeres in significantly lower copynumber than M. rufogriseus (Bulazel et al. 2006, 2007),all copies in the former species were essentially diluted tonondetectable levels in this assay. Dot-blot hybridizationlevels were normalized with cytB and intensities wereexamined on the Bio-Rad (Hercules, CA) Gel Doc EQ,Quantity One software package. Table 1 shows that eachhybrid exhibited an average approximately twofold in-crease over the M. rufogriseus parent in copy number ofboth sat23 and KERV-1 sequences.

The most likely location of the hybrid-specific ampli-fied copies is within the centromeres of the M. rufogriseuscomplement as no homogenously staining regions out-side of the active centromere were observed within thehybrid karyotypes. This was confirmed by fluorescencein situ hybridization of both DNA probes (sat23 andKERV-1) to metaphase chromosomes of each hybrid(Figure 5A). On hybrid metaphases, sat23 was observedat all of the centromeres of M. rufogriseus-derivedchromosomes, while KERV-1 was observed at all of thecentromeres of M. rufogriseus-derived chromosomes,albeit in virtually undetectable copy numbers on the Xchromosome. Interestingly, a subset of cells in eachhybrid displayed minichromosomes containing sat23DNA (Figure 5B). It is not known whether these arecapable of forming kinetochores or are capable of stablemitotic division.

Due to the extremely low copy number for bothsat23 and KERV-1 at the centromeres of M. agilis(Bulazel et al. 2007), low stringency conditions wererequired to detect these sequences in the hybrid ge-nomes on the paternal complement (data not shown).

Figure 2.—Abnormal centromere structuresin M. rufogriseus 3 M. agilis hybrids. DAPI-stainedmetaphase chromosomes from two cells ofM. rufogriseus 3 M. agilis (RA1120) highlightthe abnormal centromere structures of theM. rufogriseus-derived chromosomes (arrows indi-cate the ‘‘knob-like’’ structures) detected by grosskaryotype analyses.


These hybridization conditions resulted in spurioussignal throughout both complements and were thusuninformative.

Cross-species chromosome painting: To test whetherthere were other structural abnormalities present withinthe karyotypes of these hybrids, cross-species chromo-some painting was performed using two-color fluores-cence in situ hybridization and dual-peak localization ofchromosome paints derived from P. xanthopus. This

species was used for cross-species analyses as it carriesthe ancestral macropodid karyotype (Eldridge et al.1991, 1992). Each paint probe was used in a two-probehybridization to delineate de novo rearrangements ineach parent, each of the four hybrids, and an unrelatedmale M. rufogriseus cell line that had undergone a highnumber of passages.

No rearrangements for any chromosome paint pairwere observed in either of the two parental animals or inthe unrelated M. rufogriseus male (Table 2A). In con-trast, each hybrid carried a mosaic suite of rearrange-ments at varying frequencies (Table 2B), with mostrearrangements classified as unique (Table 2C). Table 2lists the frequency of each observed rearrangement bychromosome paint pair. A x2 test was used to determinewhether the number of rearrangements observed inthe hybrids was statistically significant. The number ofrearrangements was found to be significantly differentin hybrids compared with the parents (x2 ¼ 21, d.f. ¼ 1,P ¼ 3.214 3 10�6). The M. rufogriseus-derived chromo-some in the hybrids was easily identified both by inverseDAPI, where the M. rufogriseus chromosome showedextensive staining at the centromere, and by a lackof hybridization of the chromosome paints at theM. rufogriseus-derived centromere. Each of the observedrearrangements in the hybrids involved the M. rufogriseus-derived centromere and included isochromosomes,WARTs, and fissions (Figure 6). However, each hybridcell line was mosaic for cells with abnormal chromo-somes derived from independent rearrangement events(i.e., de novo for each cell) and karyotypically normalcells. While only one tissue (fibroblast) was available forthis study, this level of mosaicism within one tissue typeimplies that the instability may manifest late in devel-opment and is likely continuing at a low frequency.

DISCUSSION

Modified centromeres are correlated with chromo-some aberrations: The most remarkable feature ofthe chromosome instability manifested in all fourM. rufogriseus 3 M. agilis marsupial hybrids is that theinstability was confined to the centromeres of the ma-ternally derived, i.e., M. rufogriseus, chromosomes. Whilethe hybrids that we examined were of the same type ofcross and generated from the same M. agilis maleparent, they did not share the same M. rufogriseus femaleparent, excluding parental instability as a contributingfactor to the chromosome aberrations observed in thehybrid genomes. All hybrids displayed a low frequencyof de novo rearrangements involving a M. rufogriseus-derived centromere, specifically with chromosome armsof the M. rufogriseus complement. While we cannotexclude the possibility that the observed instability maybe due partially to a maternal effect, we propose that thisinstability may also be an inherent feature of the struc-

Figure 3.—Scanning electron micrographs of M. rufogriseus 3M. agilis hybrid chromosomes stained specifically for DNAwith platinum blue. Some centromeres are elongated andexhibit an uneven DNA distribution similar, if narrower, tothat on the chromosome arms (A). Parallel fibers and chro-momeres are distributed throughout the chromosome,including the centromere (B, the SE image). DNA distribu-tion shows distinguishable chromatids joined at a small regionin the centromere (B, the BSE image); DNA distribution isuninterrupted longitudinally along the chromatid arms (B,the BSE image).


ture of the M. rufogriseus centromere in a hybrid orotherwise destabilizing environment.

The increased frequency of chromosome rearrange-ments in these hybrids is specific only to the centromerethat within the parental species (M. rufgogriseus) con-tains higher copy numbers of both centromeric sequen-ces, sat23 and KERV, as determined by fluorescencein situ hybridization (FISH) (Bulazel et al. 2006,2007) (Figure 5) and dot-blot analyses (Table 1). Theserearrangements consist of a broad spectrum of kar-

yotypic instabilities, including WARTs, fissions, isochro-mosomes, and minichromosomes. FISH localizationof both the KERV-1 element and the sat23 satelliterepeat suggests that, in the hybrids, amplification hasoccurred at the M. rufogriseus-derived centromere, sup-porting the SEM data. The amplification of KERV-1 inall four of these hybrids is analogous to that previouslyobserved in another macropodid hybrid (O’Neill et al.1998) and implies a general mechanism for geno-mic instability involving retroelement amplification in

TABLE 1

Copy number quantitation of centromere sequences: dot-blot analyses of sat23 and KERV-1 in M. rufogriseus,M. agilis, and M. rufogriseus 3 M. agilis hybrids

sat23 KERV-1

Normalized value Fold change Normalized value Fold change

ParentalsM. rufogriseus (R1188) 1892.8 2323.3M. agilis (A1843) 0 0

HybridsRA 1190 2398.5 2.534 2850.7 2.454RA 1122 1887.9 1.995 1217.2 1.048RA 1118 2327.3 2.459 2067.2 1.779New RA 2346.5 2.479 2533 2.181

Average hybrids 2.37 1.87

Normalized values and fold change compared to M. rufogriseus are indicated.

Figure 4.—High-resolution stereo micrographpairs of a M. rufogriseus 3 M. agilis hybrid chro-mosome centromere stained with platinum blue.(Top) SE images showing centromere topogra-phy. (Bottom) BSE images showing DNA distribu-tion. Stereo viewing allows recognition of thespatial distribution of structural elements: chro-momeres of varying sizes (top, circles) are inter-spersed with parallel fibers (top, arrows).Chromomeres (top, circles) correspond with lo-cal increases in DNA concentration (bottom,circles).


marsupial hybrid karyotypic instability specific to cen-tromere locations.

Transposable elements (TEs) have been shown to bedirectly responsible for chromosome rearrangementsin both plants and insects (Zhang and Peterson 1999;Evgen’ev et al. 2000; Caceres et al. 2001). In Drosoph-ila, for example, there is a high correlation between thepresence of a particular TE and chromosome rearrange-ments (Lim 1988; Evgen’ev et al. 2000). A more detailedstudy of genome rearrangements in maize showed that aduplication/deletion event can be produced by a singletransposition event involving a full-length TE and a TEfragment (Zhang and Peterson 1999). However, thereare fewer examples of direct involvement of TEs in mam-malian genome remodeling. Inversions in primates areknown to be induced by recombination of some transpos-able elements (Schwartz et al. 1998; Kehrer-Sawatzki

et al. 2002). Gerbils of the genus Taterillus have under-gone rapid and extensive chromosome repatterning withconcomitant amplification of LINE-1 elements. A clearlack of correlation between LINE-1 accumulation andchromosomal breakpoints, however, suggests that theseelements were not directly involved in the chromosomalchanges (Dobigny et al. 2004). The authors suggest thatduring intense genome repatterning some epigeneticfeatures, such as DNA methylation, are relaxed, allowingTE amplification (Dobigny et al. 2004). The amplifiedKERV-1 element at the hybrid centromere may thereforebe directly involved in the various chromosome rear-rangements observed or, alternatively, the chromosomerearrangements observed may be concomitant withgenomic stress during which epigenetic controls, suchas DNA methylation or histone modification, are relaxed.In the Macropus hybrids, it is unclear whether epigeneticrestructuring of the centromere allowed the KERV-1 andrepeat amplification observed or whether the amplifica-tions resulted in chromatin remodeling.

Hybrid centromeres are structurally modified:Knobs were first described in maize by McClintock

1929). In maize, they appear in pachytene cells as darklystaining heterochromatic regions at intercalary posi-tions and are composed of repeat elements and retro-transposable elements (Ananiev et al. 1998). Knobsrepresent a structural manifestation in meiosis of thesechromosomal elements. In rice, a correlation has beenobserved between the size of the knob and the amountof repeat DNA, suggesting that a minimum amount oftandemly repeated DNA may be required to induceknob-like heterochromatic structure that can be vi-sualized by microscopic methods (Cheng et al. 2001).In plants, centromeres are characterized by extremelylarge blocks of tandem repeats and retrotransposons(Houben and Schubert 2003). It is interesting that, inthis respect, the centromeres of these marsupial speciesand their hybrids resemble plant chromosomes. To ourknowledge, however, there are no reports of elongatedor ‘‘segmented’’ plant centromeres.

The observed segmented or ‘‘knob-like’’ structures inmitotic metaphase chromosomes at the M. rufogriseus-derived centromeres in the hybrids were visible withinelongated centromeres and not consistently found inthe same position on a particular chromosome. Thismay indicate that the instability at these centromericloci is not fixed but is in a dynamic state of chromatinremodeling. Three-dimensional ultrastructural analysisshows that, although these centromeres do not contain‘‘knobs’’ in the dimension suggested by light micro-scopic data, they not only are elongated with respect toother chromosomes in their complement, but also arecharacterized by more chromomeres and a denserchromatin configuration with respect to other plantand animal mitotic centromeres (Wannerand Formanek

1995, 2000; Sumner 1998). Similar centromere elonga-tions in human chromosomes have been described for

Figure 5.—sat23 and KERV-1FISH to metaphase chromosomesof M. rufogriseus 3 M. agilishybrids. (A) Hybridization (fromleft) of sat23 (red), KERV-1(green) to metaphase chromo-somes (inverted DAPI) ofRA1118 (merge). (B) Minichro-mosome from ‘‘new RA’’ contain-ing both sat23 (red) and KERV-1(green) DNA (merge).


cases of cytosine hypomethylation such as observed with5-azacytidine (methylase antagonist) treatment and ICF(immunodeficiency, centromeric instability, and facialabnormalities) syndrome (Sumner 2003; Miniou et al.1994) and have been attributed to decondensation ofcentromeric heterochromatin. Our results showing thatthe elongated hybrid centromeres are DNA rich sup-port an amplification event rather than a decondensa-tion event. ISH and dot-blot analysis confirm that theelongated centromeres are indeed manifestations of asignificant increase in DNA repeats. Further immuno-detection experiments are necessary to determine thedegree of methylation in the hybrid centromeres.

Centromere evolution and hybrid dysgenesis: Recentobservations with regards to the rapid evolution of acentromeric protein in plants and animals (Malik andHenikoff 2001; Talbert et al. 2004) suggest thatgenomic conflict in the form of centromere drive(Henikoff et al. 2001; Henikoff and Malik 2002;

Malik and Henikoff 2002) may be responsible forthe centromere instability at the M. rufogriseus-derivedcentromeres. Centromere protein C (CENP-C) is a largeprotein that binds to vertebrate centromeric DNA(Sugimoto et al. 1994) with low sequence conservationexcept for a CENP-C motif (Talbert et al. 2004). Theobservation that both centromeres and CENP-C arerapidly evolving (Talbert et al. 2004), even betweenclosely related species, suggests that a disconnect be-tween CENP-C and centromeric sequence between thetwo parental centromeres may be responsible for thecentromere instability observed in the marsupial hy-brids (O’Neill et al. 1998, 2001; this study). However, itis not clear why this instability should be confined to theM. rufogriseus-derived centromere.

The most obvious difference between the M. rufogriseusand M. agilis karyotypes is the difference in the amountof constitutive heterochromatin at the centromeres,as evidenced by C-banding. The large amounts of

TABLE 2

Summary of chromosome rearrangements in M. rufogriseus 3 M. agilis hybrids

Chromosome1p and 1q

Chromosome6p, 6q 7

Chromosome3p and 3q

Chromosome4 and 5

Chromosome2 and X Totals

Scored Abnormal Scored Abnormal Scored Abnormal Scored Abnormal Scored Abnormal Scored Abnormal

A. ParentalsR1188 93 0 91 0 100 0 100 0 79 0 463 0A1843 100 0 93 0 107 0 50 0 101 0 451 0R3242 86 0 74 0 100 0 55 0 100 0 415 0Totals 279 0 258 0 307 0 205 0 280 0 1329 0Grand total

Scored 1329Rearranged 0

B. Hybrids—all rearrangementsRA1118 104 0 85 0 98 0 142 3 50 0 479 3RA1122 A 102 9 91 4 100 0 88 0 82 1 463 14RA1190 102 2 87 0 79 0 79 0 100 0 447 2‘‘New’’ RA 89 0 80 6 70 0 100 2 37 2 376 10Totals 397 11 343 10 347 0 409 5 269 3 1765 29Grand total


C. Hybrids—unique rearrangements onlyRA1118 104 0 85 0 98 0 142 3 50 0 479 3RA1122 A 102 4 91 3 100 0 88 0 82 1 463 8RA1190 102 2 87 0 79 0 79 0 100 0 447 2‘‘New’’ RA 89 0 80 1 70 0 100 2 37 0 376 3Totals 397 6 343 4 347 0 409 5 269 1 1765 16Grand total


Scored cells containing abnormal (i.e., rearranged) chromosomes in both parents (R1188, A1843) and a high passage cell linefor M. rufogriseus (R3242) (A) and four M. rufogriseus 3 M. agilis hybrids (as indicated) (B and C). Hybrid data are divided into thetotal number of rearrangements observed (B) and those that are unique (i.e., different types of de novo rearrangements within aparticular hybrid). Each chromosome or chromosome pair targeted by chromosome paints from P. xanthopus are indicated. Mini-chromosomes were not scored. All rearrangements observed in the hybrids involved the centromere of the M. rufogriesus-derivedchromosome (see results).


heterochromatin at the M. rufogriseus centromeres maybe the result of past centromeric drive activity. Talbert

et al. (2004) propose a centromeric drive model, basedon karyotypic drive in female meiosis (Pardo-Manuel

De Villena and Sapienza 2001), to explain the appar-ent anomaly of rapidly evolving sequence but conservedfunction in centromeres and centromeric proteins.They suggest that, because only one product of femalemeiosis ends up in the final meiotic product, centro-meres ‘‘compete’’ during female meiosis. A centromerethat is ‘‘stronger’’ will have a greater chance of endingup in the single female meiotic product. However, dif-ferences in centromeric drive in male meiosis, where allproducts end up in the final meiotic product, may resultin reduced male fertility. Mutations in CENP-C thatrestore centromere parity in male meiosis will thereforebe selected for in males. Recurrent cycles of selectionfor ‘‘stronger’’ centromeres in females and ‘‘restoring’’CENP-C in males would result in the observed rapidevolution of CENP-C and centromeres (Talbert et al.2004). They further suggest that addition of satelliterepeats at the centromere would attract more centro-mere histones (CenH3 or CENP-A), thus binding moremicrotubules during cell division and creating a ‘‘stron-ger’’ centromere (Talbert et al. 2004).

Genome repatterning in the four marsupial hybrids islimited to the M. rufogriseus centromere and correlateswith the presence of the larger amounts of heterochro-matin at these locations. Previous studies indicated thatlarge heterochromatic blocks at centromeres of thisspecies contain CENP-B-binding sequences. However,these centromeres do not form a large kinetochoreregion spread across these heterochromatic sequences,but, contrary to the centromere drive model, form asmall, confined kinetochore akin to that of species withsmall centromere regions (Bulazel et al. 2006). Theapparent restructuring of chromatin fibers at hybridM. rufogriseus centromeres may be indicative of moreextensive remodeling at these loci, although kineto-chore size and CENP-A affinity have not been assayed.However, one backcross hybrid has been previouslyexamined (Lowry et al. 1995), the karyotype of whichdid not indicate preferential inheritance of theM. rufogriseus-derived centromere.

An alternative hypothesis for the chromatin remodel-ing and destabilization of these centromeres is thatthere is an incompatibility within the RNA interference(RNAi) machinery. RNA silencing or RNA interfer-ence occurs in a wide variety of eukaryotic organisms(Meister and Tuschl 2004) and is involved in generegulation, transposable element mobilization suppres-sion, and epigenetic regulation of heterochromatin.RNA interference appears to be involved in the reg-ulation of centromere function and architecture, inparticular with the epigenetic maintenance of hetero-chromatic regions (Grewal and Jia 2007). While it hasbeen better studied in yeast (Volpe et al. 2002, 2003)and Drosophila (Pal-Bhadra et al. 2004), there is evi-dence that RNAi is also involved in maintenance ofheterochromatic and centromeric regions in mammals(Fukagawa et al. 2004; Kanellopoulou et al. 2005;

Figure 6.—De novo chromosome aberrations in M. rufogri-seus 3 M. agilis hybrids. Examples of chromosome rearrange-ments identified through cross-species chromosome paintingin RA1122: (A) fission, (B) translocation, and (C) isochromo-some. The parental origin is labeled as M.r. (M. rufogriseus)and M.a. (M. agilis).


Bouzinba-Segard et al. 2006). It has been proposedthat incompatibility between short interfering RNAs(Lippman and Martienssen 2004) or piwi-interactingRNAs (Brennecke et al. 2007) between maternal andpaternal species within a hybrid genome may lead totransposon activation and/or disruption of heterochro-matic structure and ultimately to hybrid dysgenesis.Such activation could lead to copy-number increasesand chromosome instability, as well as chromatin re-modeling, all features of our hybrid system. The differ-ence seen between M. rufogriseus- and M. agilis-derivedcentromeres may simply be an effect of a larger regionin M. rufogriseus in which disruption of epigenetic main-tenance could occur.

Conclusions: Here we have identified centromericinstability and remodeling in hybrids within a marsupialgenus known for rapid chromosome evolution pre-dominately involving centromere-involved rearrange-ments. We observed the same instability in all fourhybrids, which was confined to the maternally derivedcentromeres containing large blocks of heterochroma-tin that are expanded relative to the paternally derivedcentromere. The inherent instability that we have iden-tified in this region of the genome may have a role in therapid chromosome evolution and speciation withinmacropodids.

We thank Macquarie University, Fauna Park, for care of animals; theUniversity of Connecticut Mentor Connection for participation ofsummer high school students in aspects of this research; Patricia C. M.O’Brien for flow sorting chromosomes; Sabine Steiner for excellenttechnical assistance; and M. J. O’Neill for critical evaluation of thismanuscript. We also thank two anonymous reviewers for their helpfulcomments. C.J.M., K.B., G.C.F., and R.J.O. were supported by aNational Science Foundation CAREER award (MCB-0093250).M.D.B.E. was supported by the Australian Research Council andMacquarie University, Sydney, Australia. W.R. was supported withfunding from the Wellcome Trust.

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