Centromere Repositioning
G.Montefalcone, S.Tempesta, M.Rocchi, and N.Archidiacono
Istituto di Genetica, Via Amendola 165/A, 70126 Bari
ABSTRACT
Primate pericentromeric regions have been recently shown to exhibit
an extraordinary evolutionary plasticity. In the present paper
we report an additional peculiar feature of these regions which
we discovered while analysing, by FISH, the evolutionary conservation
of primate phylogenetic chromosome IX. If the position of the
centromere is not taken into account a relatively small number
of rearrangements must be invoked to account for interspecific
differences. If the centromere, conversely, is included in the
analysis a paradox emerges: the position of the centromere seems
to have undergone, in some species, an evolutionary history independent
from the surrounding markers. Additional rearrangements are then
necessary to reconcile the order of the markers with centromere
position. Alternatively, the evolutionary emergence of neocentromeres
can be postulated.
INTRODUCTION
The molecular structure and evolution of the eukaryotic centromere
was remained very elusive. Despite its importance in cell division,
the nature of the centromere remains poorly understood. Typically,
the centromeres of primate chromosomes are composed of long arrays
of alphoid sequences, organized in tandemly repeated monomers
of approximately 171bp (Maio 1991; Willard and Waye 1987; Choo
et al. 1997). The evolution of alphoid DNA has been very rapid.
Comparative Fluorescence In Situ Hybridization (FISH) studies
in great apes using human alphoid probes have revealed substantial
divergence in both the nature of the sequence as well as its location
among chromosomes belonging to the same phylogenetic group (Archidiacono
et al. 1995; Warburton et al. 1996). Pericentromeric regions exhibit
even more complex evolution. We have investigated the organization
and recent evolution of the pericentromeric region of chromosome
10, chosen as a model since it was the only chromosome for which
a detailed physical map was available (Jackson et al. 1999). The
results have indicated that this region has undergone an unprecedented
level of rearrangements including duplications, transpositions,
inversions and deletions. Although the data are limited, this
plasticity seems to be a general feature of many different pericentromeric
regions (Murphy and Karpen 1998; Eichler et al. 1999). Here we
report a study on the evolutionary organization of the phylogenetic
chromosome IX in primates suggesting an additional peculiar property
of these regions: in some species the centromere position exhibits
an evolutionary history which appear to be independent from the
flanking chromosomal markers.
RESULTS
Nine primate species were studied:
- HSA (Homo sapiens);
- 3 great apes: common chimpanzee (Pan troglodytes,
PTR), gorilla (Gorilla gorilla, GGO), and orangutan (Pongo
pygmaeus, PPY);
- 1 Cercopithecidae (Old World Monkey, OWM): silvered leaf-monkey
(Presbytis) cristata, PCR);
- 4 Platyrrhinae (New World Monkeys, NWM): dusky titi (Callicebus
molloch, CMO, Callicebinae), spider monkey (Ateles geoffroyi,
AGE, Atelinae); common marmoset (Callithrix jacchus, CJA,
Callitrichinae), and squirrel monkey (Saimiri sciureus,
SSC, Saimirinae).
The Presbytis cristata (PCR) was chosen as the sole representative
of the Cercopithecidae since previous unpublished data from our
laboratory, based on Partial Chromosome Paints (PCP) and appropriate
YAC probes, have shown that chromosome IX of PCR (Colobinae),
CAE (Cercopithecus aethiops, Cercopithecinae), and MMU
(Macaca mulatta, Cercopithecinae) appear perfectly alike
(data not shown).
Fig. 1a shows a sample of DAPI banded chromosome IX from each
species. In AGE, SSC, and CJA the chromosome IX lies uninterrupted
within a larger chromosome (Sherlock et al. 1996; Morescalchi
et al. 1997). In both AGE and SSC the additional cytogenetic material
is positioned at one side, with the centromere defining the boundary.
In CJA this chromosome is encompassed on both sides by additional
cytogenetic material of different chromosomal origin, with the
centromere lying within chromosome IX.
Evolution of chromosome IX in great apes have been investigated
by Yunis and Prakash (1982) using banding techniques. Data on
evolutionary conservation of chromosome IX in Old and New World
monkeys have been obtained using whole chromosome paints, which,
however, are not able to detect intrachromosomal rearrangements
(Sherlock et al. 1996; Morescalchi et al. 1997).
Twelve human probes distributed along chromosome 9 were utilized
in the study (Table 1 and Figure 1b). Each probe was used in FISH
experiments on each species. Partial Chromosome Paints (PCP) specific
for 9p (PCP #502) and 9q (PCP #29) (Antonacci et al. 1995) have
been also used to grossly define the constitution of chromosome
IX in the different species (Figure 1c). In several instances
cohybridization experiments were performed in order to assess
with certainty the relative order of probes. An example is shown
in Figure 1d where cohybridization experiments using probes
M and N against metaphases from PCR and CMO were performed to
unambiguously determine order. The results obtained have been
summarized in Figure 2, bottom part. The position of each probe
has been reported on the left of the chromosome IX ideograms,
using the corresponding letter (see Table 1).
The order of the twelve markers was found to be identical in PCR
(OWM), CMO and AGE (both NWM) and therefore was assumed to descend
unchanged from an hypothesized Primate Common Ancestor (PCA, Figure
2). A paracentric inversion spanning markers A->H defines a
Pongidae Ancestor (PA) whose cromosomal constitution was retained
in GGO and PPY. A further pericentric inversion (see Figure 2)
give rise to HPA (HSA/PTR common ancestor) whose constitution
is unchanged in HSA. PTR derives from HPA through a pericentric
inversion. One breakpoint of this inversion is detected by marker
B (YAC 945F5) (Figure 1e). The splitting of this probe in PTR
has been previously reported by Nickerson and Nelson (1998). The
reconstruction of the evolutive pathways linking present day great
apes to PA are in perfect agreement with data from Yunis and Prakash
(1982). The markers' arrangement found in SSC and CJA can be derived
from the PCA by hypothesizing a specific inversion in each lineage.
The breakpoints of the inversion leading to SSC occurred between
probes C/D and M/N respectively. One breakpoint of the inversion
leading to CJA falls between probes D/E; the second lies inside
marker B (YAC 945F5)(Figure 1e), which is the marker also involved
in the inversion leading to PTR (see above).
The hypothesized phylogenetic pathways illustrated in Figure 2
do not take into account, intentionally, the position of the centromere.
If the centromere is included in the analysis, indeed, a paradox
emerges: in several instances its evolutive history seems to behave
independently from the surrounding markers. The position of the
centromere sorts the species under study into five groups: HSA-PTR-GGO-PPY,
PCR, CMO-SSC, AGE, and CJA, as indicated in Figure 2 by a black
line underlining each group. The differences in centromere position
among the groups can not be easily reconciled with each other:
an additional series of rearrangements must be postulated to fully
account for the differences we have documented, as discussed below.
DISCUSSION
We have studied the evolutionary conservation of chromosome
IX in nine primate species, using 12 molecular markers whose mapping
in humans is well documented. Figure 2 summarizes the most parsimonious
set of chromosomal inversions we propose to explain the constitution
of chromosome IX in each species. Primate centromeric and pericentromeric
regions have been shown to exhibit extraordinary evolutionary
plasticity. Our findings add further complexity to the already
complex evolutionary history of these chromosomal regions. The
position of the centromere in some species, indeed, appears to
have followed an independent evolutive path in respect to the
flanking markers. Two different hypotheses can be proposed to
reconcile these discrepancies:
(i) Additional inversions have occurred in the evolutionary history
of chromosome IX of these species. The ultimate results of these
rearrangements would be the repositioning of the centromere leaving
unchanged the markers' order.
(ii) Alternatively, the evolutionary emergence of neocentromeres
can be hypothesized.
A detailed series of hypothetical inversions needed to relocate
the centromere to its present-day location through chromosomal
rearrangements is schematized in Figure 3. In several instances
the inversion breakpoints involve pericentromeric and telomeric
regions. In two instances (PCR and CJA) the mechanism acts in
a flip-flop mode (double inversion), the breakpoints in the pericentromeric
region being once distal and the second time proximal to the centromere
(or vice versa), so that the only detectable result would be the
repositioning of the centromere.
In light of the data recently reported by du Sart et al. (1997)
and Barry et al. (1999), the hypothesis of neocentromere emergence
can not a priori be readily eliminated. The fact that all primate
centromeres are defined by the presence of considerable amounts
of alpha-satellite does not negate this hypothesis. It has been
suggested that the accumulation of alpha satellite DNA at centromeres
may simply be a consequence of its function and not a prerequisite
to its origin (review by Eichler 1999) . One obvious consequence
of the birth of a neocentromere is the inactivation of the previously
active centromere. Such centromere inactivation is a common event
among human dicentric chromosomes resulting from chromosomal rearrangement
(Sullivan and Willard 1998). What about the relics of these events?
The incredible plasticity of these regions and our poor knowledge
of primate genomes have made the identification of these remnants
difficult. The only available example in this respect is the human
ancestral centromere present at 2q21. This region was the domain
of a normal centromere which was inactivated following the telomere-telomere
fusion of the two ancestral chromosomes (phylogenetic IIp and
IIq) which gave rise to the present-day human chromosome 2 (Ijdo
et al. 1992). The fusion occurred at most 3-5 million years ago,
which is the estimated date of the human-chimpanzee divergence
(Andrews 1992; Li 1997). Despite its recent origin, relics of
alphoid sequences are hardly detectable at this site (Avarello
et al. 1992; Baldini et al. 1993), nor is there any evidence of
C-banded material commonly associated with centromeric regions.
These considerations suggest that the degradation of the ancestral
centromere toward simple DNA has been extremely rapid. Relic sequences
after such centromere inactivation events, therefore, can be very
difficult to identify. The actual involvement of the two mechanisms
(birth of a neocentromere and flip-flop processes) to centromere
repositioning can not be easily distinguished at present. The
flip-flop model might explain why pericentromeric and telomeric
sequences share sometimes common sequences (Jackson et al. 1999;
Puechberty et al. 1999).
A additional interesting observation we have documented concerns
the two breakpoints identified in PTR and CJA, both lying inside
the YAC 945F5 (Figure 1e). Both breakpoints appear to be asymmetrically
located within the YAC, as revealed by the substantial difference
in the intensity ratio between the two FISH signals, and are similarly
oriented in respect to the flanking markers. We have documented,
in a recent study, that the YAC 695H10 detects a breakpoint in
the phylogenetic chromosome IV of PTR and MMU (Macaca mulatta)
(Marzella et al. 1999). It could be suggested that the breakpoint
sites detected by YACs 945F5 and 695H10 has been utilized more
that one time during evolution as a consequence of sequence intrinsic
features. This conclusion, however, requires validation at the
molecular level. Recurrence of chromosomal rearrangements due
to intrinsic sequence features is now well documented in humans
(Christian et al. 1999, and references therein).
Concluding remarks. It is becoming increasingly apparent that
there are peculiar regions of the primate genome which exhibit
an extraordinary degree of evolutionary plasticity. Such regions
are in stark contrast to the bulk of euchromatic DNA which appears
evolutionary stable. High evolutionary plasticity has been documented
on centromeric and pericentromeric domains (Archidiacono et al.
1995; Jackson et al. 1999), and on the chromosome Y-specific chromosomal
segment (Archidiacono et al. 1998). It is noteworthy that these
regions share a very low or absence of meiotic recombination (Puechberty
et al. 1999). At present we are investigating the evolutionary
history of additional primate chromosomes in order to establish
if the paradox documented for the centromere of chromosome IX
is shared by other centromeres. Murphy and Karpen (1998) have
proposed that the centromere function could be the result of an
epigenetic mark. This hypothesis is very appealing in explaining
the emergence of neocentromeres. In this respect studies at the
molecular level on the phenomena we have documented, now in progress
in our laboratory, could be crucial in substantiating this hypothesis.
METHODS
Probes. YACs are from the CEPH megalibrary; PAC 835J22 is
from the PAC library described by Ioannou and de Jong (1996).
YAC and PAC clones have been kindly provided by the YAC Screening
Centre, Milan (http://www.spr.it/iger/home.html). The PAC 835J22
was identified by primers specific for the ABL locus at 9q34 (see
our Web site http://bioserver.uniba.it/fish/Cytogenetics/webbari/YAC-TUMORS/project/abl-bcr.html).
All probes used are listed in Table 1.
Cell lines. Human metaphase spreads were obtained from
PHA-stimulated peripheral blood lymphocytes of a normal human
donor. Cell lines from nine primates species have been previously
described (Archidiacono et al. 1998).
FISH. Probes were labeled with biotin by nick-translation
and hybridized in situ essentially as described by Lichter et
al. (1990) with minor modifications. Detection was performed using
avidin-conjugated Cy3 (Amersham). Chromosome identification was
obtained by simultaneous DAPI staining. Co-hybridization experiments
were accomplished by labeling the second probe with FluorX-dCTP
(Amersham). Digital images were obtained using a Leica DMRXA epifluorescence
microscope equipped with a cooled CCD camera (Princeton Instruments,
NJ). Cy3, FluorX, and DAPI fluorescence signals, detected using
specific filters, were recorded separately as gray scale images.
Pseudocoloring and merging of images were performed using the
Adobe PhotoshopTM commercial software.
Figure 1
Examples of DAPI-banded phylogenetic chromosome IX from each
species under study (a). Chromosome IX in AGE, SSC, and
CJA are part of a larger chromosome. In all cases, however, chromosome
IX is uninterrupted. The square parentheses indicate the portion
of chromosome IX. Some chromosomes are presented in an inverted
orientation , in respect to the position of the centromere, to
match the orientation reported in Fig. 2. The actual chromosome
number in each species is also reported, on the right of the species
acronym. (b) FISH signal of the 12 probes on human chromosome
9. The examples have been arranged from left to right in increasing
mapping distance from 9pter. (c) Example of FISH signals
(green) of PCP #29, specific for human 9q, on PCR (left) and SSC
(right). (d) Example of a cohybridization experiment performed
to establish the relative order of probes M (red) and N (green)
in PCR and CMO. The DAPI-banded chromosome IX without signals
is on the left, to better show the morphology and centromere position.
(e) The figure shows the splitting of probe B in PTR and
CJA (see text). In all Figures the arrows point to the centromere.
Figure 2
The diagram schematically summarizes the results obtained
by hybridizing the 12 markers on each species under study. GGO
and PPY turned out to be identical and have been grouped. Regions
homologous to the human 9p (red) and 9q (green) are shown on the
left of each ideogram which shows, on the right, the G-banding
pattern. PCA stands for the hypothesized Primate Common Ancestor,
PA for Pongidae Ancestor, HPA for HSA-PTR Ancestor. The not detailed
cytogenetic material from different chromosome(s) present in AGE,
SSC, and CJA is in brown. Close horizontal lines indicate heterochromatin
blocks. The hypothesized pericentric or paracentric inversions
are indicated by square parentheses spanning the inverted cytogenetic
segment. The split signals of marker B (YAC 945F5) are indicated
as B' and B". In both cases signal of B" is much stronger
than B' (see text and Fig. 1e).
Figure 3
The Figure schematically describes the most parsimonious series
of hypothetical rearrangements that would be needed to reconcile
the observed marker order and the position of the centromere s.
They are based on the assumption that in PCA the centromere was
positioned telomeric to marker A. This conclusion is drawn exclusively
from the constraint imposed by the maximum parsimony. The inversions
are indicated by square parenthesis. The inversions not present
in Figure 2 have been specifically introduced to account for the
paradoxal position of the centromere and are indicated by an asterisc.
In AGE and SSC the centromere is positioned at the boundary between
chromosome IX and the chromosome segment brought there by an interchromosomal
rearrangement. It can not be excluded, therefore, that the centromere
of these two species has originated from a different chromosome.
The orientation of the chromosomes has been reported to match
the orientation reported in Fig. 2.
Table 1 Probes used in the study
| Probe | cM | cR | |
| E | YAC 816E6 | 0 | 3 |
| F | YAC 922A5 | 36 | |
| G | YAC 823G12 | 57 | 134-139 |
| H | YAC 763A12 | 60 | 172 |
| D | YAC 748D2 | 65 | |
| C | YAC 906G6 | 84 | |
| B | YAC 945F5 | 87 | 318 |
| A | YAC 747B3 | 93-94 | 338 |
| I | YAC 750C6 | 117 | 414 |
| L | YAC 756E10 | 128 | 426 |
| M | YAC 758F1 | 136-143 | 458 |
| N | PAC 835J22 | ABL locus | |
Table 1
The FISH probes are reported according to their order along
human chromosome 9. The order has been confirmed by data derived
from STSs lying inside each YAC (MIT database) and reported in
the 3rd column (genetic data, in cM) and in the 4th column (radiation
hybrids data, in cR). An upper case letter identifies each probe
(1st column), and was arranged so that the ascending sequence
from A to N corresponds to the hypothesized physical order in
the ancestral chromosome IX (Fig. 2). The YACs 763A12 and 748D2
has been chosen because they are very close to the centromere
on p and q side respectively (see MIT database).
ACKNOWLEDGEMENTS
The financial support of AIRC, Telethon (Grant E.672 and E.962),
and cofin98-MURST is gratefully acknowledged.
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