1 Department of Biology, University of Bologna, via Selmi 3, 40126 Bologna,
Italy
2 Department of Cell Biology, Institute of Experimental Medicine, Academy of
Sciences of the Czech Republic, CZ-12800 Prague 2, Czech Republic
3 Laboratory of Gene Expression, 1st Faculty of Medicine, Charles University,
Albertov 4, CZ-12800 Prague 2, Czech Republic
4 Institute of Cell and Molecular Biology, University of Edinburgh, Swann
Building, The King's Boulevard, Mayfield Road, Edinburgh EH9 3JR,
Scotland
* These authors contributed equally to this work
Author for correspondence (e-mail:
gdelval{at}alma.unibo.it
)
Accepted 10 March 2002
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Summary |
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Key words: Alpha-satellite DNA, CENP-C, CENP-B, Centromere, Chromatin-immunoprecipitation
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Introduction |
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Recent observations indicate that the alpha-satellite DNA may play a
pivotal role in the organization of the human centromeres. In fact, at least
three important centromeric proteins bind the alpha-satellite DNA. CENP-B, a
nonessential protein situated in the central domain, recognizes a specific 17
bp sequence (CENP-B box) within the alpha-satellite repeats
(Cooke et al., 1990;
Muro et al., 1992
;
Pluta et al., 1992
;
Yoda et al., 1992
) and may
facilitate the packaging of the alpha-satellite arrays.
Poly(ADP-ribose)polymerase (PARP) binds to a 9 bp sequence (pJ
box) in
alpha-satellite DNA (Earle et al.,
2000
). PARP binds preferentially to active centromeres and to a
sequence in cloned human neocentromere DNA, but its role at centromeres is not
yet known. CENP-A is an essential histone H3-like protein, which localizes to
active centromeres at the inner plate of the kinetochore
(Earnshaw and Rothfield, 1985
;
Warburton et al., 1997
).
CENP-A is specifically associated with alpha-satellite DNA in vivo although it
is not yet clear whether there are sequences that it preferentially recognizes
(Vafa and Sullivan, 1997
).
Other proteins have been found in the inner plate of the kinetochore, which
participate in centromere organization. For instance, CENP-C is an essential
factor involved in the assembly of the kinetochore and in the correct
segregation of sister chromatids (Saitoh
et al., 1992; Tomkiel et al.,
1994
; Fukagawa and Brown,
1997
; Kalitsis et al.,
1998
; Fukagawa et al.,
1999
). Because of its localization, it has been long hypothesized
that it might recognize the centromeric DNA
(Sugimoto et al., 1999
).
Several in vitro studies have established that it is possible to define a
minimal domain of CENP-C that binds DNA
(Sugimoto et al., 1994
;
Yang et al., 1996
;
Sugimoto et al., 1997
). This
binding domain seems to overlap with the domain required for the targeting of
CENP-C to the centromere in vivo (Yang et
al., 1996
). However, several attempts to identify in vitro
putative CENP-C-binding sequences have failed. This scenario is complicated by
the observation that CENP-C, CENP-A and PARP also associate with active
neocentromeres that apparently do not contain repetitive sequences typical of
conventional centromeres, suggesting that their localization to centromere may
occur through epigenetic mechanisms (Choo,
1997
; Depinet et al.,
1997
; Choo, 2000
;
Sullivan, 2001
).
To study the role of CENP-C in centromere formation, we took advantage of a
chromatin-immunoprecipitation assay (ChIP) that allows the analysis of
specific DNA-protein complexes in vivo
(Orlando et al., 1997). This
technique has been successfully used to study the DNA-binding activity of more
than 40 proteins, including general transcription factors, trans-activators,
repressors and structural components of chromatin
(Solomon et al., 1988
;
Dedon et al., 1991
;
Strutt and Paro, 1998
;
Chen et al., 1999
;
Hsu et al., 1999
;
Parekh and Maniatis, 1999
;
Tanaka et al., 1999
;
Orlando, 2000
;
Partridge et al., 2000
;
He et al., 2001
). Moreover, a
thorough study by Toth and Biggin indicates that formaldehyde crosslinking
occurs mainly at the protein-DNA interface and reflects the in vivo binding of
a specific factor to its cognate DNA sites
(Toth and Biggin, 2000
). On
the basis of previous results showing that CENP-C can both bind chromosomal
DNA in vitro and co-localize with centromeric DNA in interphase nuclei, we
decided to use the ChIP assay to examine the dynamics of how CENP-C associates
to the centromeric DNA sequences in vivo.
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Materials and Methods |
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UV crosslinking
2x108 HeLa cells were grown for 24 hours in DMEM
supplemented with 2 mM L-glutamine, 10% FCS and 20 µM
5-bromo-2-deoxyuridine. Cells were trypsinized, collected in a 50 ml falcon
tube, washed three times with cold 1xPBS and resuspended in 8 ml of
1xPBS. They were transferred into a 100 mm petri dish, kept on ice and
directly exposed for 15 minutes to a source of 366 nm UV light positioned at a
distance of about 3 cm from the dish. Cells were finally collected, washed
once with 1xPBS, resuspended in 2 ml of FA lysis buffer and from here on
treated by following the same steps of the formaldehyde crosslinking
procedure.
Dot blot and Southern blot analysis of immunoprecipitated DNA
Total input DNA and immunoprecipitated DNA samples were digested with
EcoRI, resolved on 1.5% agarose gel and transferred to a
Hybond-N+ filter (Amersham Pharmacia Biotech). The filter was
pre-hybridized and hybridized with a solution containing 7% SDS and 0.5 M
Na3PO4, pH 7.0. Probes were
[-32P]-labeled with the Megaprime kit (Amersham) and added
to the hybridization solution at a specific activity of 2x109
cpm/µg. The alpha-satellite DNA probes
(Archidiacono et al., 1995
)
used for the hybridization were: (1) pZ7.6B (680 bp) detecting chromosome 7;
(2) pZ21.A (850 bp) detecting chromosomes 13 and 21; (3) pZ17-1.6A (1.02 kb)
detecting chromosome 17; (4) pZ5.1 (680 bp) detecting chromosomes 1, 5 and 19;
(5) pZ8.4 (1.2 kb) detecting chromosome 8; (6) pDMX1 (2 kb) detecting
chromosome X. Other probes recognizing non-centromeric sequences were: BLUR-8
(300 bp) detecting Alu repeats (Jelinek et
al., 1980
); and p(291)LSau (291 bp) detecting beta-satellite
(Agresti et al., 1987
). Dot
blot analysis was performed by transferring total input DNA and the
immunoprecipitated DNA samples onto a Hybond-N+ filter (Amersham)
with a Bio-Dot Apparatus (Bio-Rad). The filter was pre-hybridized and
hybridized using the same conditions as for the Southern blots.
Semiquantitative PCR
The region containing part of the human renin gene promoter was amplified
by using primer REF: 5'-CAGCTGTTGCTTTTCCTGCC-3' and RER:
5'-AAACAGCACTGTCAGGGCTA-3'. Amplification conditions were as
follows: 94°C for 30 seconds, 58°C for 15 seconds and 72°C for 30
seconds. 10 µl aliquots of the PCR products were taken after 30, 35 and 40
cycles respectively, run on 2% agarose gel and visualized by ethidium
bromide.
Construction and expression of CENP-C mutants
Expression constructs encoding the HA-tagged truncations of CENP-C were
generated by PCR as follows. HA::23/943: primer 23F,
5'-AAAGGGGGATCCGCACGTGACATTAA-CACAGAG-3' and primer
943R,
5'-AAAGGGGAATT-CTCATCTTTTTATCTGAGTAAAAAG-3';
HA::192/537: primer 192F,
5'-AAAGGGGGATCCATGCTGCCTTCAA-GTACAGAGG-3' and primer
537R,
5'-AAAGGGGA-ATTCTCACTCCTCTGATTTTACCACCC-3';
HA::23/410: primer 23F and primer 410R,
5'-AAAGGGGAATTCTCA-TGGTTTTCTGCATTCTTGG-3'.
PCR products were digested with BamHI and EcoRI and directionally cloned into the pCDNA3.1 vector, in frame with a HA-tag located at the N-terminal of the proteins. For each mutant, 20 µg of expression construct were used to transfect HEK293T cells by means of the lipofectamin method (Roche). HEK293T cells were grown in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine. Expression levels of the truncated proteins were tested by western blot. Transfected cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Nadeoxycholate, 0.1% SDS) and sonicated. 20 µg of total cell extract were separated on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The filter was blocked with TBS (150 mM NaCl, 25 mM Tris-HCl pH 8.0) and 8% nonfat dry milk. After incubation with the specific antibodies, the filter was washed three times with TBST (TBS plus 0.1% Tween-20) and incubated with anti-rabbit peroxidase-conjugated secondary antibodies (Amersham). Proteins were revealed by ECL (Amersham).
Ultrastructural immunocytochemistry
Subconfluent HeLa cells, grown as previously described, were fixed
overnight in 4% paraformaldehyde in 0.2 M PIPES, pH 7.0 at 4°C. After
rinsing in cold PBS, the cells were scraped and pelleted at low speed.
Pelleted cells were infused with a mixture of sucrose and polyvinylpyrrolidone
before freezing, as described (Tokuyasu,
1989). Thin cryosections were cut with a Drukker diamond knife
(Drukker Co., Almere, The Netherlands) on a Reichert Ultracut S ultramicrotome
(Leica) equipped with cryoattachment.
Grids with sections were first incubated in 0.2% BSA-c (Aurion, Wageningen,
The Netherlands) to reduce unspecific labeling. For the CENP-B and CENP-C
mapping, the respective sera were diluted 1:100 in PBS containing 0.1% BSA-c.
The bound antibodies were revealed by 6 or 10 nm protein A-gold complexes
(Aurion). In the co-localization experiments, consecutive immunolabeling of
the two CENPs was performed as described
(Griffiths, 1993). The results
of parallel labelings (i.e. single labeling, as well as double labeling with
interchanged sizes of the gold complexes for the two CENPs), gave consistent
results. In the negative control experiments, the primary antibodies were
omitted; the positive control involved an unrelated rabbit p80-coilin antibody
kindly provided by E. K. Chan, The Scripps Research Institute, La Jolla, CA
(Andrade et al., 1993
).
Antibodies and protein A-gold complexes were incubated for 60 minutes. After
the labeling was completed, sections were fixed with 1% glutaraldehyde in PBS
for 20 minutes and treated for 15 minutes in 2% OsO4 in PBS.
Sections were extensively washed in water and postembedded in 0.3% uranyl
acetate in polyvinyl alcohol (Tokuyasu,
1989
). Grids were observed by EM Zeiss 109.
To estimate the mutual distribution of CENP-C and CENP-B, the most external CENP-B (or CENP-C) particles were joined by straight lines and the CENP-C (or CENP-B) particles inside and outside the formed polygon were counted. Data from 65 centromeres were evaluated using the Wilcoxon rank test.
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Results |
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Although the formaldehyde concentration used in this work has been widely
employed to study protein-DNA interactions in vivo we cannot exclude the
possibility that it may induce covalent links between proteins and thus may
have the potential to connect proteins to DNAs that they do not directly
contact (Strahl-Bolsinger et al.,
1997). To demonstrate that the results obtained with the
formaldehyde crosslinking reflect a direct interaction of CENP-C to the
alpha-satellite DNA, we used an alternative procedure based on an in vivo UV
crosslinking assay (Rzepecki et al.,
1998
). It is well established that UV light can generate covalent
links either between dimers of pyrimidines or between DNA and proteins
directly associated to it (Rzepecki et
al., 1998
; Toth and Biggin,
2000
). HeLa cells were grown for 24 hours in the presence of
5-bromo-2-deoxyuridine before being exposed for 15 minutes to a 366 nm UV
light. 5-bromo-2-deoxyuridine can replace thymidine during DNA replication and
is more efficient than thymidine at forming covalent links when exposed to UV
light. Since the alpha-satellite DNA possesses a high A+T content, the use of
5-bromo-2-deoxyuridine should increase the chances of crosslinking CENP-C to
the alpha-satellite DNA. Cells were lysed and chromatin was then
immunoprecipitated and processed by following the same steps described for the
formaldehyde crosslinking. Immunoprecipitated DNAs, transferred onto a nylon
filter by dot blot, were hybridized with a cocktail of the
[
-32P]-labeled pZ7.6B and pZ21.A probes.
Fig. 1D shows that both
anti-CENP-B and anti-CENP-C antibodies can specifically immunoprecipitate the
alpha-satellite DNA. The results of the UV crosslinking indicate that CENP-C,
as well as CENP-B directly contacts the alpha-satellite DNA in vivo.
The organization of alpha-satellite DNA arrays is highly structured and can differ from one chromosome to another. Moreover, each chromosome carries particular arrays of alpha-satellite DNA sequences that can be revealed by using specific DNA probes. To demonstrate the general nature of the CENP-C binding to the alpha-satellite DNA of human chromosomes, a set of filters carrying the immunoprecipitated DNA samples, along with internal standards, were hybridized with chromosome-specific centromeric DNA probes. The results of Fig. 2A show that CENP-C binds the alpha-satellite DNA of all tested chromosomes.
|
The different localization of CENP-B and CENP-C in the centromere of
metaphase chromosomes (Cooke et al.,
1990; Saitoh et al.,
1992
; Sugimoto et al.,
1999
) suggests that the two proteins may recognize different
subtypes of alpha-satellite DNAs. Conversely, our results indicate that both
CENP-B and CENP-C seem to bind the same alpha-satellite DNA higher order
repeats. However, the dot blot analysis does not allow a precise
discrimination between different subfamilies of alpha-satellite DNA of a given
chromosome. To address this issue we have compared the profile of the
alpha-satellite DNA immunoprecipitated by both anti-CENP-B and anti-CENP-C
antibodies after digestion with EcoRI, known to display the
long-range periodicity of the alpha-satellite DNA of chromosomes 7, 13 and 21.
The DNA, run on a 1.5% agarose gel and blotted onto a nylon membrane, was
hybridized with a cocktail of the pZ7.6B and pZ21A DNA probes.
Fig. 2B shows that both CENP-B
and CENP-C recognize the same dimeric, tetrameric and pentameric subtypes of
alpha-satellite DNA.
The DNA region associated with CENP-C are situated within the
alpha-satellite arrays
Although anti-CENP-C antibodies specifically immunoprecipitate chromatin
fragments containing the alpha-satellite DNA, we cannot exclude the
possibility that such an interaction may occur in different DNA regions within
these fragments that flank or are interspersed in the alpha-satellite arrays.
To address this question we have modified the ChIP assays as follows. Before
reversal of the crosslinking, DNA was digested with EcoRI when the
IgCENP-CDNA complexes were still immobilized on the
trisacryl-proteinA beads (see Materials and Methods). Furthermore, the
complexes were thoroughly washed with FA buffer to remove the DNA fragments
released by EcoRI. The digestion of the immunoprecipitated DNA would
be expected to generate two alternative and mutually exclusive patterns
depending on where CENP-C binds the DNA. If CENP-C binds the DNA within the
alpha-satellite, then the alpha-satellite arrays would remain part of the
complexes retained on the beads and would be further recovered and purified.
On the contrary, if CENP-C binds sequences adjacent to or interspersed in the
alpha-satellite arrays, then the alpha-satellite DNA would be released from
the beads upon EcoRI digestion, lost during the extensive washings
and thus undetected by Southern blot hybridization. Although the digestion was
not complete, probably because chromatin proteins crosslinked to DNA partially
prevent the access of the endonuclease to its cognate restriction sites,
Fig. 3 shows that the
alpha-satelliteCENP-C complexes were always retained on the beads in
spite of the fact that the EcoRI digestion was performed either
before or after the reversal of crosslinking
(Fig. 3, compare lanes 6 and 7,
and, at a longer exposure, lanes 8 and 9). As a control, we analyzed CENP-B
that binds DNA inside the alpha-satellite. As expected, the profile of the
alpha-satellite DNA recovered by anti-CENP-B antibodies is unchanged in both
conditions (lanes 4,5). Moreover, the DNA profiles of the alpha-satellite DNA
bound by CENP-B and CENP-C were practically identical (lanes 4,8). Taken
together these results suggest that CENP-C binds DNA within the
alpha-satellite DNA arrays.
|
Definition of the CENP-C domain needed to bind the alpha-satellite
DNA in vivo
To define the CENP-C domain required to bind the alpha-satellite DNA, we
analyzed the binding activity of specific CENP-C mutants in vivo. We have used
these mutants essentially as controls to confirm that the DNA-binding domain
of CENP-C defined on the basis of in vitro results was also involved in the in
vivo binding of CENP-C to DNA The CENP-C mutants were chosen and designed
based on in vitro results showing that the CENP-C DNA-binding activity resides
in the central region of the protein
(Sugimoto et al., 1994;
Yang et al., 1996
;
Sugimoto et al., 1997
),
approximately between amino acid 433 and 520
(Yang et al., 1996
).
Specifically, we have generated three HA-tagged CENP-C mutants either carrying
(HA::23-943; HA::192-537) or missing (HA::23-410) the putative DNA-binding
region. All truncated CENP-C mutant proteins
(Fig. 4A, left) were expressed
at a comparable level in transfected HEK293T cells and could be recognized by
both anti-CENP-C and anti-HA antibodies in western blot
(Fig. 4A, right). According to
previous studies (Yang et al.,
1996
), all three mutants accumulate in the nucleus and specific
staining of the centromeres could be clearly detected only in the cells
expressing the mutants carrying the putative DNA-binding region (HA::23-943;
HA::192-537) (data not shown). To investigate the binding activity of the
truncated proteins to the alpha-satellite DNA we have applied the ChIP assay
to HEK293T cells transfected with each of the described constructs. DNA,
immunoprecipitated with anti-HA antibodies, was analyzed with a mixture of the
pZ7.6.B and pZ21.A alpha-satellite DNA probes. Decreased amounts of total
input DNA were also included in the filter. As shown in
Fig. 4B (left), anti-HA
antibodies recover the alpha-satellite DNA from cells transfected with the
HA::23-943 and HA::192-537 constructs but not from cells transfected with the
HA::23-410 one. To exclude that the overexpression of the different mutant
proteins may affect the interaction of CENP-C with the alpha-satellite DNA,
transfected cells were also subjected to immunoprecipitation with anti-CENP-C
antibodies. The anti-CENP-C antibodies cannot discriminate between endogenous
and truncated CENP-C proteins and thus, if transfection did not alter
chromatin organization, the amount of recovered alpha-satellite DNA should be
roughly identical in all cell samples. The results of
Fig. 4B (left) show that this
was the case. Furthermore, the background level, determined by the Alu
hybridization, is similar in all DNA samples either immunoprecipitated with
anti-HA or anti-CENP-C antibodies (Fig.
4B, right). The relative enrichment of both alpha-satellite DNA
and Alu sequences is illustrated in the two graphs in
Fig. 4B (bottom). Finally, we
have verified whether the overexpression of CENP-C mutants might in someway
alter the typical EcoRI profile observed with the pZ7.6.B and pZ21A
probes. The results of Fig. 4C
indicate that the wild-type and CENP-C mutants, HA::23-943 and HA::192-537,
bind the same subfamilies of alpha-satellite DNA, whereas the HA::23-410
mutant cannot. In addition, this excludes the possibility that the simple
overexpression of the CENP-C proteins can interfere with their correct loading
onto the centromeric DNA.
|
Our results show that the differences in the amount of alpha-satellite DNA recovered by the CENP-C mutants seem to reflect their intrinsic ability to bind the alpha-satellite DNA and confirm that the central region of the CENP-C is indeed required to perform this function in vivo.
CENP-B and CENP-C localize to alpha-satellite arrays of different
centromere domains
The fact that both CENP-B and CENP-C can be crosslinked to the same
alpha-satellite subfamilies suggests that CENP-C and CENP-B may co-localize
within the alpha-satellite arrays. This, however, appeared to be in contrast
with what has been previously observed by immunoelectron microscopy
(Cooke et al., 1990) and
immunofluorescence (Sugimoto et al.,
1999
), where CENP-B and CENP-C seem to occupy distinct structural
domains in the kinetochore/centromere complex of mitotic chromosomes. To
investigate this apparent discrepancy we have used a modified version of the
ChIP assay. First, crosslinked HeLa cell extracts were immunodepleted, by
using anti-CENP-B antibodies, for the DNACENP-B complexes. One round of
immunoprecipitation with anti-CENP-B antibodies was sufficient to abate the
crosslinked DNACENP-B complexes to background levels
(Fig. 5, compare lane 6 with
lane 2). Following the depletion of the DNACENP-B complexes, the
extract was subjected to immunoprecipitation with anti-CENP-C antibodies. The
amounts of DNACENP-C complexes recovered before and after the
immunodepletion of the CENP-BDNA complexes were compared.
Fig. 5 shows that the amount
and the profile of alpha-satellite DNA immunoprecipitated by anti-CENP-C
antibodies are not affected by immunodepletion of the DNACENP-B
complexes (Fig. 5, compare
lanes 4 and 5). A similar outcome was obtained for the DNACENP-B
complexes in the reverse experiment (data not shown). These results strongly
suggest that CENP-B and CENP-C localize to distinct non-overlapping centromere
domains composed of the same subfamilies of higher order alpha-satellite
repeats.
|
Previous immunoelectron analysis performed on mitotic chromosomes has shown
that CENP-B and CENP-C seem to occupy distinct structural domains in the
kinetochore (Cooke et al.,
1990). However, the relative distribution of the two proteins at
the mitotic kinetochore-centromere complex might be masked by the folding of
the chromatin fiber in the complex. To overcome this problem, we analyzed the
relative distribution of the two proteins by double-label immunoelectron
microscopy on ultrathin cryosections of interphase HeLa cells.
In this analysis only one section per given cell was considered and this section was more than two orders of magnitude thinner than the cell nucleus. Each micrograph of Fig. 6 shows the centromere of an indiscriminate chromosome from a single cell nucleus. Double labeling of CENP-B (10 nm) and CENP-C (6 nm) proteins with colloidal gold particles resulted in their co-localization in clusters in which the two labels were not randomly interspersed (Fig. 6A-F). A morphometric analysis (see Materials and Methods) showed that CENP-B was concentrated in the interior of the clusters, while CENP-C localized predominantly to the periphery. Specifically, an average of 7.17±0.62 (mean±s.e.) of CENP-B-associated gold particles were located in the interior, while 2.54±0.35 were found in the exterior of these domains. Vice versa, 1.48±0.23 of CENP-C associated particles were located in the interior, while 11.47±0.97 CENP-C were in the exterior. According to the Wilcoxon rank test, the difference between CENP-B and CENP-C localization in the centromeric domain analyzed is significant, with P<0.05. Similar results were obtained if the colloidal gold probes were switched (data not shown). These data reinforce the notion that CENP-B and CENP-C occupy distinct domains of the interphase centromere.
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Discussion |
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These results are quite striking since CENP-C does not bind specific DNA
sequences in vitro. A simple explanation for this discrepancy may reside in
the fact that CENP-C-binding activity might be fully manifested as a result of
interactions with other centromere proteins in situ. This hypothesis is
corroborated by recent findings from gene targeting experiments in mice and
conditional loss-of-function analysis in the chicken DT40 cell line, which
have shown that CENP-A and CENP-H are necessary to correctly position CENP-C
at active centromeres (Howman et al.,
2000; Fukagawa et al.,
2001
). These data support the model that CENP-C localizes to the
centromere through contacts with auxiliary proteins rather than recognizing
the kinetochore DNA directly. However, a direct interaction of CENP-C, with
either CENP-A or CENP-H, has not been demonstrated and, so far, experiments to
prove this point have been unsuccessful. Obviously, it is possible that other
centromere proteins concur to target CENP-C to the centromere through this
mechanism. However, the situation might be more complex than predicted. In
fact, we cannot exclude the possibility that these proteins may contribute to
the generation of a particular DNA structure or impose modifications upon
specific DNA sequences that would allow CENP-C to be recruited and assembled
into the centromere chromatin. Observations by several groups suggest that
specific centromeric DNA sequences may play a crucial role in the constitution
of a functional human centromere. For instance, Masumoto and colleagues have
demonstrated that alphoid DNA from the alpha21-I locus could give rise to
artificial chromosomes in HT1080 cells, whereas alphoid DNA from the 21-II
locus could not (Masumoto et al.,
1998
). This strongly suggests that specific blocks of alpha
satellite DNA are endowed with peculiar features that allow them to seed
centromere formation in vivo. The fact that specific blocks of DNA may satisfy
the structural and/or sequence requirements for the assembly of an active
centromere-kinetochore complex is further supported by recent finding showing
that a 330 kb CENP-A-binding domain of the neocentromere found on the human
marker chromosome mardel (10) possesses an A+T content (>60%) similar to
that of alpha-satellite DNA (Lo et al.,
2001
).
Although we cannot at present clarify the exact mechanism of recruitment of CENP-C to the centromere, our approach has allowed the analysis of the CENP-C DNA-binding activity as an intrinsic component of the kinetochore and shows that CENP-C contacts the alpha-satellite DNA in vivo. This result is important for two reasons: first, it provides a methodological approach to investigate the dynamic of how this protein takes part in the assembly of a functional centromere in vivo; and second, it further supports the idea that the alpha-satellite is the kinetochore DNA in normal chromosome.
Another important observation of this work arises from comparison of the
DNA sequences recognized by CENP-B and CENP-C. Although CENP-C and CENP-B seem
to recognize the same sets of alpha-satellite (although it is possible that
CENP-C might recognize other types of alpha-satellite sequences in addition to
those also recognized by CENP-B), they appear to be organized topologically in
distinct centromere subdomains containing alpha-satellite DNA. This is
supported by two results: first, immunodepletion with an anti-CENP-B antibody
of the DNACENP-B complexes from the crosslinked cell extract does not
affect the recovery of the DNACENP-C complexes, indicating that the two
proteins are not associated to the same alpha-satellite DNA fragments; and
second, immunolocalization of CENP-B and CENP-C on interphase chromosomes
shows, at the high resolution offered by double-label immunoelectron
microscopy, that the tendency of the two antigens to occupy distinct domains
is statistically significant. We believe that the best interpretation of these
data is that the ChIP assay shows that CENP-B and CENP-C occupy separate
alpha-satellite domains, and this is supported by the immunoelectron
microscopy analysis. Our results suggest a subtle and composite organization
of the centromere proteins relative to alpha-satellite DNA at the centromere.
In fact, both CENP-B and CENP-C bind to the same family of repeated DNA, but
only CENP-C binds to the DNA that ends up at the surface of the chromosome
associated with the active kinetochore. The mechanism of segregation of CENP-B
and CENP-C to different regions of alpha-satellite is not known, but it
appears that, in addition to DNA sequence, other factors are likely to be
involved. In this contest, as previously mentioned, the localization of CENP-C
to the centromeres requires at least CENP-A and CENP-H. The knockout of the
CENP-A gene in mice (Howman et al.,
2000) or the inhibition of its homologue in C. elegans by
RNA interference (Moore et al., 2001;
Oegema et al., 2001
),
abolishes the ability of CENP-C to target the kinetochore. By comparison,
disrupting CENP-C in C. elegans has no effect on CENP-A
(Oegema et al., 2001
).
Therefore, CENP-A appears to act upstream of the assembly pathway that drives
CENP-C to the kinetochore. Given that the presence of CENP-A with the
centromeric DNA is a pre-requisite for the CENP-C localization to the
centromere, then the absence of CENP-C from the inactive centromere of stable
dicentric chromosomes (Earnshaw et al.,
1989
; Page et al.,
1995
; Sullivan and Schwartz,
1995
; Fisher et al.,
1997
; Page and Shaffer,
1998
; Sullivan and Willard,
1998
) may be causally related to the lack of this preliminary step
of the kinetochore assembly. The mechanisms that precisely specify where
functional mammalian centromeres form within arrays of centromeric
heterochromatin, and how the centromere identity might be propagated at a
specific chromosomal site, remain largely unknown. Recent observations
indicate that the centromere identity might be established by epigenetic
mechanisms (Karpen and Allshire,
1997
; Wiens and Sorger,
1998
). A centromeric epigenetic mark could be specified by
exclusive protein binding, histone modifications, spatial or temporal
organization of chromosomal processes, or through the activity of specific
centromere-identity loading factors
(Sullivan et al., 2001
;
Sullivan, 2001
). In this
contest, it has been suggested that CENP-A might modify chromatin organization
to favor the deposition of other centromere-kinetochore proteins, including
CENP-C (Van Hooser et al.,
2001
). However, mistargeting of CENP-A to DNA outside the
conventional centromere regions is not sufficient per se to induce the
formation of functional neocentromeres, although it seems to re-direct CENP-C
to these sites. This suggests that other factors that include particular DNA
sequences or structures may be required for this process.
It is possible that there are different subclasses of alpha-satellite sequences, some of which can contain CENP-C DNA recognition sites. The epigenetic determination of centromere identity and propagation in fact does not rule out the possibility that sequence composition, such as enrichment for repeated sequences or an A+T sequence bias, also has a role in these processes. Although it is clear that the organization of a functional centromere requires the contribution of several factors assembled in a hierarchical fashion, our results strongly indicate that the binding of CENP-C to the alpha-satellite DNA in vivo may represent an important step in this process and provide a more comprehensive view of how the architecture of centromeres at human chromosomes is achieved.
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