Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
* Author for correspondence (e-mail: g44478a{at}nucc.cc.nagoya-u.ac.jp)
Accepted 4 June 2003
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Summary |
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Key words: Centromere, Alpha-satellite, Mammalian artificial chromosome, Heterochromatin, Histone acetylation
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Introduction |
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Our understanding of the molecular mechanisms that specify the sites of
functional centromeres in higher eukaryotes is still incomplete
(Shelby et al., 2000). Many
observations and analyses of human chromosomes have indicated that the
mechanisms specifying the sites for functional centromeres are highly complex.
Extensive stretches of alpha satellite (alphoid) DNA, consisting of tandemly
repeated, 171 bp units, are present in the centromeric regions of all normal
human chromosomes (Willard and Waye,
1987
; Choo et al.,
1991
; Marcais et al.,
1993
). These sequences colocalize with protein components of the
centromere/kinetochore domains (Masumoto
et al., 1989a
), including CENP-A, a centromere-specific histone H3
variant (Palmer et al., 1991
;
Sullivan et al., 1994
);
CENP-B, a CENP-B box (a 17 bp sequence found in alphoid DNA)-binding protein
(Earnshaw et al., 1987
;
Masumoto et al., 1989b
;
Cooke et al., 1990
), and
CENP-C, an inner kinetochore protein
(Saitoh et al., 1992
).
However, the presence of alphoid DNA is not sufficient to create a functional
centromere structure. On stable dicentric chromosomes, CENP-A, CENP-C and
CENP-E [a kinesin-like motor protein found at the kinetochore
(Yen et al., 1991
;
Wood et al., 1997
)] do not
assemble on the inactive centromere despite the presence of alphoid DNA and
CENP-B (Sullivan and Schwartz,
1995
; Warburton et al.,
1997
; Fisher et al.,
1997
). A marker chromosome containing a neocentromere, which lacks
detectable alphoid DNA and CENP-B (du Sart
et al., 1997
), was found to be mitotically stable, and most of the
known centromere proteins assembled at the site of the neocentromere
(Saffery et al., 2000
). One
possible explanation for these phenomena is that centromere activity is
maintained by a chromatin assembly mechanism rather than by the primary DNA
sequence (Steiner and Clarke,
1994
; Williams et al.,
1998
; Murphy and Karpen,
1998
; Wiens and Sorger,
1998
).
On all normal human chromosomes, however, functional centromere structures
are formed and maintained on alphoid DNA arrays. The mechanisms that specify
the location of centromeres are not straightforward, because simple mechanisms
do not explain these paradoxical events. Despite these discrepancies, several
groups have succeeded in generating mammalian artificial chromosomes (MACs)
with functional centromeres that depend on type I alphoid sequences for de
novo events (Harrington et al.,
1997; Ikeno et al.,
1998
; Masumoto et al.,
1998
; Henning et al.,
1999
; Ebersole et al.,
2000
; Mejia et al.,
2001
; Grimes et al.,
2002
; Ohzeki et al.,
2002
). However, MAC formation as the predominant effect was
observed in approximately 30-40% of cell lines, and in a subpopulation of
cells the multimerized alphoid YAC DNAs integrated into host chromosomes. The
different fates of the introduced YAC DNA in different cells suggest that
epigenetic controls, such as those that regulate chromatin assembly, are
involved in this process.
In this study, to investigate how epigenetic mechanisms direct the assembly of chromatin on human centromeres, we isolated a cell sub-line in which the transfected alphoid YAC DNA was integrated into a host chromosome and was stably maintained without changes in the overall DNA structure of the introduced alphoid YAC array during subcloning. We demonstrate that the suppressed state of centromeres on the alphoid construct can be changed, and that this is accompanied by CENP-A, -B and -C assembly, as shown under two different conditions: long-term culture with selection for the YAC-borne marker, and treatment with an inhibitor of histone deacetylase, Trichostatin A (TSA). Under selective pressure or treatment with TSA the transcription of a marker gene located in the YAC arm was increased. These processes, which change the chromatin structure, might be involved in the mechanisms of epigenetic change of the suppressed state of centromeres at ectopic alphoid DNA loci.
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Materials and Methods |
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Fluorescence in situ hybridization (FISH)
Standard techniques were employed for FISH
(Masumoto et al., 1989a).
Probes used were chromosome 16 Painting Probe DIG-labeled (Roche Diagnostics
K. K.), p11-4 alphoid DNA (Ikeno et al.,
1994
) for the
21-I loci and pYAC4
(Burke et al., 1987
) for YAC
arm DNA. Images were captured using a cooled-CCD camera (PXL, Photometrics
Ltd) and analyzed by IPLab software (Signal Analytics).
Indirect immunofluorescence and simultaneous staining by FISH
Indirect immunofluorescence and simultaneous staining by FISH were carried
out as previously described (Masumoto et
al., 1989a). Antibodies used were anti-CENP-A (mAN1)
(Masumoto et al., 1998
),
anti-CENP-B (2D8D8) (Ohzeki et al.,
2002
), and anti-CENP-C (CGp2)
(Ikeno et al., 1998
). In the
7C5HT1-19 and del.22HT1-3 cell lines, more than 50 and 30 metaphase cells from
each preparation were analyzed, respectively. Intensities of CENPs and YAC
signals on integrated alphoid YACs were analyzed by NIH image (National
Institute of Health, USA) and IPLab software (Signal Analytics). Signal
intensities with the chromosome 16 painting probe in captured images were
measured using IPLab. Each value was normalized to that of the host chromosome
16.
Mitotic stability of re-formed minichromosomes
The cell line 7C5HT1-19 was cultured in selective medium for 60 days and
subclones were established during the next 30 days. To test minichromosome
stability, the subclones were cultured for a further 60 days in nonselective
medium and analyzed by FISH.
Chromatin immunoprecipitation (ChIP)
Cells were crosslinked for 5 minutes in 0.25% formaldehyde. The reaction
was stopped by addition of glycine to 417 mM and cells were washed three times
with TBS and frozen in liquid nitrogen. Chromatin was fragmented by sonication
to a size of 100-500 bp in sonication buffer (5 mM Hepes pH 8.0, 1.5 µM
aprotinin, 10 µM leupeptin, 1 mM DTT, 40 µM MG132) using Bioruptor
Closed Type Sonicator (Cosmo Bio Co., Ltd).
The soluble chromatin was pre-cleaned with normal mouse IgG (Santa Cruz) and immunoprecipitated with anti-CENP-A (mAN1) or anti-CENP-B (mouse monoclonal antibodies raised against human CENP-B, 2D8D8 and 5E6C1) antibodies in IP buffer (30 mM Hepes pH 8.0, 150 mM NaCl, 1 mM EGTA, 2 mM MgCl2, 2 mM ATP, 1.5 µM aprotinin, 10 µM leupeptin, 1 mM DTT, 0.1% NP-40, 40 µM MG132). Protein G Sepharose (Amersham) blocked with 5% BSA was added, and the antibody-chromatin complex was recovered by centrifugation. The ChIP assay using anti-acetylated histone H3 (Upstate Biotech) was carried out basically according to the manufacturer's protocol. The recovered DNA and the soluble chromatin (as input) were quantitated by real-time PCR. The percentage recovery of DNA fragments and the ratio of enrichment were calculated as described below. The statistical significance of the results obtained was confirmed using the t-test (level of significance=0.05).
Real-time PCR
The amount of immunoprecipitated DNA and the dilution of the input DNA were
measured by real-time PCR using the iCycler IQTM Multi-Color Real
Time PCR Detection System (Bio-Rad). The following primers were used to
amplify LYS2, the left alphoid junction, the right alphoid junction,
bsr, the alphoid 11mer and 5S ribosomal DNA:LYS2-1,
5'-ATGACTAACGAAAAGGTCTGGATA-3'; LYS2-3,
5'-ATCATGAGGCACATCGAGCTGAGG-3'; ARM2,
5'-GCGGCCGCCCAATGCATTGGTACC-3'; mCbox-3,
5'-AGATTTTAGATGATGATATTCCCG-3'; ARM4,
5'-ACCATACCCACGCCGAAACAAGCG-3'; mCbox-2,
5'-GCTT(T/G)GAGGATTTCGTTGGAAGCG-3'; bsr-F,
5'-TCCATTCGAAACTGCACTACCA-3'; bsr-R,
5'-CAGGAGAAATCATTTCGGCAGTAC-3'; 11-10R,
5'-AGGGAATGTCTTCCCATAAAAACT-3'; mCbox-4,
5'-GTCTACCTTTTATTTGAATTCCCG-3'; 5SDNA-F1,
5'-CCGGACCCCAAAGGCGCACGCTGG-3' 5SDNA-R1,
5'-TGGCTGGCGTCTGTGGCACCCGCT-3'. Except for the bsr gene,
amplifications were carried out using the QuantiTect SYBR Green PCR Kit
(QIAGEN). Quantitation of bsr was carried out using the Taq-Man probe
method with a bsr-FAM probe, the primer
5'-Fam-AATGGCTTCTGCACAAACAGTTACTCGTCCTamra-3' and AmpliTaq Gold
DNA polymerase (AP Biosystems).
Trichostatin A treatment of the 7C5HT1-19 cell line
Aliquots of 2.44x106 cells were seeded in 10 ml of medium
with 250, 500 or 1000 ng/ml of Trichostatin A (TSA) (Wako). After 2 days, the
medium was changed. Cell samples for cytological and ChIP analyses were
prepared 7 days after removal of the TSA.
Western blot analysis of the TSA-treated 7C5HT1-19 cell line
Extraction of cellular histones was performed basically according to the
method of Yoshida et al. (Yoshida et al.,
1990). The extracted cellular histones from 5x104
cells were applied to a 12% SDS-polyacrylamide gel or Triton-acid urea gel,
electrophoresed, and blotted onto a PVDF membrane (Immobilon, Millipore). The
membrane was probed with anti-acetylated histone H3 and H4 (Ac Lys 12)
antibodies (Upstate Biotechnology) or anti-CENP-A antibody (mAN1), followed by
secondary antibody, anti-rabbit IgG HRP conjugate or anti-mouse IgG HRP
conjugate (Bio-Rad). Blots were developed using ECL (Amersham Pharmacia).
RT-PCR and real-time RT-PCR
RT-PCR was carried out using a One-step RNA PCR kit (AMV) (TaKaRa)
according to the manufacturer's protocol, using total RNA prepared with the SV
Total RNA Isolation system (Promega). Primers for bsr amplification
were BSR-1 (5'-ATGAAAACATTTAACATTTCTCAA-3') and BSR-4
(5'-TTAATTTCGGGTATATTTGAGTGG-3'). Primers for human
ß-actin amplification were obtained from Clontech. For each
reaction 125 ng of total RNA was used. Products were electrophoresed on
agarose gels, and stained with ethidium bromide. Quantitation of
transcriptional products was also performed by real-time RT-PCR using the
iCycler IQTM Multi-Color Real Time PCR Detection System (Bio-Rad).
Real-time RT-PCR was carried out using a QuantiTect SYBR Green RT-PCR kit
(QIAGEN) according to the manufacturer's protocol. Primers for bsr
amplification were bsr-F and bsr-R. Primers for human ß-actin
amplification were hActin-a
(5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3') and hActinc
(5'-GTCATCTTCTCGCGGTTGGCCTTGGGGTTCAG-3'). For each reaction, 20 or
4 ng of total RNA was used. Each quantity of bsr transcript was
normalized to that of the endogenous ß-actin transcript, and
these ratios compared to that of the 7C5HT1-19 cell line at day 0
(transcription level). Relative copy numbers of the bsr gene were
quantitated by real-time PCR using genomic DNA. Each transcription level was
normalized using the relative copy number of the bsr gene (relative
transcription). Total RNA from the TSA-treated 7C5HT1-19 cell line was
prepared after 2 days of TSA treatment.
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Results |
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Disassembly of centromere/kinetochore components at the integration
site of alphoid YAC
A chromosome with two functional centromeres (a dicentric chromosome) is
mitotically unstable, and can be stabilized by `inactivation' of one of the
two centromeres. At the inactive centromeres of stable dicentric chromosomes,
most known centromere proteins are disassembled, although alphoid DNA and
CENP-B are still present (Earnshaw and
Migeon, 1985). We next examined the assembly of the
centromere/kinetochore components CENP-A, CENP-B and CENP-C at the site of
integration of the alphoid YAC construct by indirect immunofluorescent
staining and simultaneous FISH analysis
(Fig. 2A-F). The intensity of
each signal relative to that of the native chromosome 16 centromere was
measured and plotted (Fig.
2G-L, and for CENP-B also in
Fig. 2P). In 17-20% of
7C5HT1-19 cells, CENP-A, -B and -C signals were detected at YAC integration
sites (Fig. 2A,C,E,Q,
Table 1) but signal intensities
were very weak compared with those of the native chromosome 16 centromere
(Fig. 2G,I,K). In about 80% of
the cells, CENP-A, -B and -C signals were almost undetectable at the YAC
integration site, despite the presence of 26-29 copies of the YAC arm
fragments and insert alphoid arrays (70 kb) keeping the initial YAC DNA
structures (a total length of alphoid arrays corresponds to 2 megabases
containing CENPB binding motif, CENP-B box)
(Fig. 1B,
Fig. 2B,D,F,H,J,L,Q). These
variegated assemblies of CENPs are also observed at synthetic
21-I
alphoid BAC integration sites (Ohzeki et
al., 2002
). In contrast, in the B13HT1 cell line, which was
obtained by transfection with the
21-II alphoid YAC and which could not
generate active centromeres or MACs, CENP-A, -B, and -C signals specific to
the integrated multimerized
21-II DNA were not detected
(Fig. 2M-O). These results
suggest that the suppressed state of chromatin at the alphoid YAC integration
site is stable, similar to what has been observed in inactive centromeres of
dicentric chromosomes.
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Minichromosome formation associated with an activated centromere on
the alphoid YAC site
We used a system containing blasticidin S (BS) and the blasticidin S
resistance gene (bsr) to obtain alphoid YAC DNA transformants. BS
inhibits protein synthesis globally, and the bsr gene product
inactivates BS by deamination (Dinos and
Kalpaxis, 1997). Although introduced alphoid YACs were stably
maintained at the same integration sites in all 7C5HT1-19 cells even after 80
days in culture in nonselective medium, when the subclone 7C5HT1-19 was
cultured for long periods in medium containing BS the number of YAC
integration sites that showed CENP-A, -B and -C signals increased
(Fig. 2Q), and a chromosome
fragment containing the YAC integration site was frequently observed
(Fig. 3A,B). The results of
FISH analysis, shown in Fig.
3A, indicate that the proportion of 7C5HT1-19 cells containing a
re-formed minichromosome increased with the length of time in culture (from
30% at day 60 to 58% at day 80), while YAC-specific signals decreased at the
host chromosome ends (from 64% at day 60 to 32% at day 80). After 80 days of
growth in selective medium, 58% of cells contained such a minichromosome
signal including the YAC site and 17.1% of cells showed integration of the
alphoid YAC at a telomere region of a host chromosome other than chromosome
16. A total of 14.6% of cells still showed the same integration of the alphoid
YAC into the chromosome 16 ends by FISH analysis
(Fig. 3A,B). Some structural
instability and rearrangement involving the exposed double strand breaks might
have occurred even in this fibrosarcoma-derived cell line (HT1080) in which a
high telomere seeding activity was indicated
(Holt et al., 1996
;
Tsutsui et al., 2003
).
However, no growth difference was detected between 7C5HT1-2 and 7C5HT1-19. On
all the re-formed minichromosomes (100%), CENP-A, -B and -C signals were
detected with specific antibodies, along with
21-I YAC signals
(Fig. 2Q, Fig. 3D). In addition, the
re-formed minichromosomes were very stably maintained as a single
extra-chromosomal copy after further culturing for 60 days without the
selective drug (Fig. 3E). These
results indicate that re-formed minichromosomes contain functional
centromere/kinetochore structures. However, in a cell line containing
21-II alphoid YAC DNA (B13HT1), CENP-A, -B, and -C signals were not
detected with the multimerized
21-II YAC integration site and
chromosome fragments were never released from the integration site under the
same conditions (Table 1).
In contrast to the de novo MAC on which no signals derived from host
chromosomal fragments were detected (Ikeno
et al., 1998; Masumoto et al.,
1998
; Ohzeki et al.,
2002
), most reformed minichromosomes (93%) in 7C5HT1-19 cells
contained a small portion of the chromosome 16 arm DNA at various levels
(Fig. 3B, middle panel), but
with less signal intensity than normal chromosome 16 short arms detected with
a chromosome 16 painting probe (Fig.
3C). Thus, in these cases, chromosome breakage might have occurred
randomly between the YAC integration site and the native centromere of
chromosome 16 (Fig. 3C).
Real-time PCR analysis showed that, although the copy number of alphoid YAC in each cell line changed only 0.8- to 1.2-fold after 80 days in culture, the level of transcription of the bsr gene as analyzed by real-time RT-PCR decreased to one tenth the original level after 80 days in culture without selection and increased about threefold under selective conditions (Fig. 3F). Thus, the relative transcriptional level of the bsr gene increased during selection, although this transcriptional activity is still very weak compared with that of the endogenous ß-actin gene. All these results strongly suggest that a functional centromere/kinetochore chromatin structure was re-assembled in parallel with the small increase in the transcriptional activity of the BS resistance gene on the YAC arm through culture under selective conditions.
Direct assembly of CENP-A and -B on introduced 21-I alphoid
DNA
To monitor the assembly of CENP-A and CENP-B on introduced alphoid YAC DNA
at the molecular level, we carried out chromatin immunoprecipitation (ChIP)
assays on 7C5HT1-2 cells, 100% of which contain a single stable MAC
(Fig. 1). Cells were fixed with
formaldehyde and sonicated to reduce the size of DNA to 100-500 bp, and then
immunoprecipitated with mouse monoclonal antibodies against CENP-A or -B. The
precipitated DNA samples were quantitated by real-time PCR with the primer
sets shown in Fig. 4A. All of
the primer pairs could amplify the target sequences in a
concentration-dependent manner in real-time PCR using sequential dilutions
from 10-1 to 10-4 of input DNA
(Fig. 4B). Immunoprecipitation
with all the antibodies tested allowed very low and similar levels of recovery
of control 5S ribosomal DNA on which no centromere protein assembly was
observed (Ikeno et al., 1998)
(Fig. 4C). Therefore, we used
the recovery ratios of 5S ribosomal DNA to the input DNA as an internal
control for normalization of the recovery ratio of the DNA fragment from each
cell line. The relative enrichment of each DNA fragment compared to the
recovery of 5S ribosomal DNA by the immunoprecipitation analysis is indicated
in Fig. 4D,E,F. By
immunoprecipitation with mouse normal IgG, no specific chromatin site was
precipitated (Fig. 4D, enrichment ratios are 0.5- to 1.4-fold) in any of the cells tested.
21-I alphoid arrays were specifically and highly enriched 6.7- to
13-fold and 13- to 25-fold by immunoprecipitation with anti-CENP-A and
anti-CENP-B antibodies, respectively, in all cell lines analyzed and under all
conditions (Fig. 4E,F). This
result is consistent with many cytological observations showing centromere
protein assembly on
21-I alphoid sequences
(Ikeno et al., 1994
;
Ikeno et al., 1998
;
Masumoto et al., 1998
).
However, in this ChIP analysis, it is difficult to distinguish 21-I
alphoid sequences on introduced YAC DNA from
21-I alphoid DNA at the
native centromere of chromosome 21. We have demonstrated de novo assembly of
CENP-A on introduced alphoid DNA derived from synthetic (thus distinguishable)
21-I type alphoid using another ChIP and competitive PCR assay
(Ohzeki et al., 2002
). Thus,
in the present study, we designed primers that bridge the boundaries between
the insert alphoid DNA and the YAC left or right arm (the left alphoid
junction or the right alphoid junction, respectively). Both the left and the
right alphoid junctions containing alphoid DNA with the CENP-B box as its
principal component were also enriched 4.9- to 6.0-fold and 4.2- to 4.5-fold
with anti-CENP-A and anti-CENP-B antibodies, respectively, in 7C5HT1-2 cells,
which contain a single stable MAC (Fig.
4E,F). In contrast, the LYS locus on the YAC left arm and
the bsr locus on the right arm were only slightly enriched with these
antibodies (0.8- to 1.8-fold). The results indicate that, despite the
multimeric structure of the alphoid YAC DNA, CENP-A and CENP-B actually
associated only with the 70 kb alphoid arrays on the de novo MAC, and CENP-A
and CENP-B did not assemble on the YAC arms.
In contrast, in the 7C5HT1-19 cell line, the left and right alphoid
junctions were enriched only 1.8- to 2.3-fold with anti-CENP-A and anti-CENP-B
antibodies, indicating that CENPA and CENP-B assembly on the ectopic alphoid
array of the integrated YAC was also reduced at molecular level
(Fig. 4E,F). However, when
7C5HT1-19 cells were cultured for 80 days with BS selection, both of the
alphoid junctions were enriched 3.6- to 9.2-fold by immunoprecipitation with
anti-CENP-A and anti-CENP-B antibodies
(Fig. 4E,F). The reassembly of
CENP-A and CENP-B, as shown by the ChIP analysis, coincided well with
cytological observations of the same cell lines cultured for 80 days with BS
selection. All these results indicate that in 7C5HT1-19 cells, the extent of
CENPA and -B assembly on integrated alphoid YAC DNA was reduced, reflecting
centromere inactivity, and increased when minichromosome formation was induced
by long-term culture with BS selection. However, even under conditions in
which a 3.6- to 9.2-fold enrichment of the alphoid junctions was achieved, DNA
sequences on both YAC arms (bsr and LYS sites) were not
significantly immunoprecipitated with anti-CENP-A and anti-CENP-B antibodies
(1.0- to 2.0-fold), suggesting that epigenetic CENP-A and CENP-B re-assembly
remained restricted to the 70 kb 21-I alphoid DNA array
(Fig. 4E,F).
Minichromosome formation and CENP re-assembly induced by inhibition
of histone deacetylase
What is the mechanism responsible for the re-assembly of centromere
components? To study this question, cells showing relatively higher
transcription activity of the bsr gene were selected by long-term
culture with blasticidin S (BS). In treated cells, the chromatin structure
might differ from its original state. Therefore, one possible explanation is
that during long-term culture in medium containing BS, structural changes in
chromatin might have occurred at the ectopic alphoid YAC site that would allow
the assembly of functional centromere/kinetochore components. To test this
hypothesis, we used a histone deacetylase inhibitor, Trichostatin A (TSA)
(Yoshida et al., 1990), to
alter the state of chromatin at YAC integration sites. TSA treatment causes
histone hyperacetylation and converts a transcriptionally inactive chromatin
structure into an active one at many loci
(Bartsch et al., 1996
). The
results of western blotting indicated that the levels of acetylation of
histone H3 and H4 in the cell line 7C5HT1-19 were increased by TSA treatment
for 2 days (Fig. 5A). In
contrast to what was observed for histone H4, using Triton-acid urea (TAU)
gel, neither the mobility nor the intensity of CENP-A was affected directly,
even by 1000 ng/ml of TSA treatment, in our experiment
(Fig. 5B). The effect of TSA
treatment on minichromosome re-formation and CENP re-assembly was analyzed
after 7 days in culture after removal of TSA, because high concentrations of
TSA cause cell cycle arrest in the G1 and G2 phases (data not shown). During
this period, the levels of histone H3 and H4 acetylation in 7C5HT1-19 cells
were reduced to the levels seen in untreated cells
(Fig. 5A). FISH analysis
combined with indirect immunofluorescence staining showed that the assembly of
centromere proteins at the alphoid YAC sites occurred in 54-75% of 7C5HT1-19
cells cultured after TSA treatment, and minichromosomes with active centromere
components also formed in 17-30% of the cells
(Fig. 5C,
Table 1). Similar results were
obtained following treatment with sodium butyrate (data not shown), another
histone deacetylase inhibitor (Candido et
al., 1978
). The ChIP analysis indicated that TSA treatment of
7C5HT1-19 cells caused a 5.7- to 11-fold enrichment of both alphoid junctions
on the YAC site by immunoprecipitation with anti-CENP-A and anti-CENP-B
antibodies (Fig. 4E,F).
However, DNA sequences on both YAC arms (bsr and LYS sites)
were not significantly enriched (1.3- to 2.1-fold). Thus the same phenomenon,
indicating epigenetic CENP-A and CENP-B re-assembly and activation of a
centromere restricted to the ectopic 70 kb
21-I alphoid DNA array, was
produced by two different treatments of the cells: long-term culture with BS
selection or short-term culture with TSA.
Centromere activation associated with the activation of transcription
of a YAC marker gene
What is the common mechanism by which both treatments affect the ectopic
alphoid YAC sites? During long-term culture with BS selection, the relative
transcriptional level of the bsr gene on the YAC arm increased. We
therefore analyzed the effect of TSA on the chromatin structure at the
introduced alphoid YAC locus by using a ChIP assay with an antibody against
acetylated histone H3 (Fig.
5D). The extent of recovery of alphoid DNA, 5S ribosomal DNA and
the sequences of the YAC were assessed in recovered DNA by real-time PCR (e.g.
Fig. 4A,B). Acetylated histone
H3 assembles anywhere on DNA, so that in this analysis we directly compared
each ratio of the recovered DNA to the input DNA. With control normal IgG, all
the tested DNA fragments were recovered at low levels (less than
0.014±0.008%, Fig. 5D).
The ratio of recovery of bsr sequences derived from the YAC vector on
the artificial chromosome with an active centromere in 7C5HT1-2 cells was very
high (4.7%) with the anti-acetylated histone H3 antibody compared with the
recovery of 5S ribosomal DNA (0.39% in Fig.
5D), genomic alphoid DNA (0.054%), the left and the right alphoid
junctions (0.044, 0.070%, respectively) and LYS locus (0.048%). On
the other hand, the ratio of recovery of the bsr gene from the YAC
integration site in 7C5HT1-19 cells with anti-acetylated histone H3 was low
(1.9%, 1/2.5 of that of the bsr gene in 7C5HT1-2 cells). However,
after TSA treatment recovery increased 4.6-fold (8.7%). In contrast, those of
5S ribosomal DNAs (0.27-0.35%), genomic alphoid, the left and the right
alphoid junctions and LYS locus (less than 0.075%) were changed by
1.3- to 2.4-fold, with very low levels even after the TSA treatment. After 80
days of culture with BS selection, the ratio of recovery of the bsr
gene increased to 4.9%, reaching the same level as with artificial
chromosomes. The patterns of the recovery of each DNA sequences on the YAC in
7C5HT1-2 cells were similar but increased by 1.2- to 1.6-fold after TSA
treatment (data not shown). All these results indicate that histone H3
associated only with the bsr locus was hyperacetylated on the
artificial chromosome containing the active centromere, on the YAC integration
site in cells treated with TSA and on the YAC site in cells after 80 days of
the culture with the selective condition. The level of transcription of the
bsr gene as analyzed by real-time RT-PCR increased with a higher TSA
concentration (Fig. 5E). Thus,
these results indicate that TSA treatment changes the chromatin structure of
the bsr gene, which is close to the insert alphoid DNA on the YAC
site, into a weak transcription-capable state.
De novo assembly of active centromere/kinetochore components without
chromosome rearrangements
We examined several different cell lines (7C5HT2-1, del.20HT3-26) in which
21-I YAC and its derivative YACs and BACs were integrated at other
sites. TSA treatment also caused minichromosomes to form at varying frequency,
accompanied by CENP-A, -B and -C signals from the
21-I YAC integration
sites in these cell lines (Table
1). When the 10 kb
21-I alphoid DNA YAC was integrated at
an interstitial site (del.22HT1-3 cell line; about 98 copies of multimerized
YAC DNA were integrated into a single site of a host chromosome, thus totaling
980 kb of
21-I alphoid DNA but interrupted by YAC arm sequences about
every 10 kb of
21-I alphoid array, data not shown), no assembly of
CENP-A and -C was detected on the YAC sites with the specific antibodies
(Fig. 6A,E,H,L,N,
Table 1). However, TSA
treatment induced the de novo assembly of CENP-A and CENP-C at this 10 kb
alphoid YAC integration site without chromosome breakage
(Fig. 6B,F,I,M,N,
Table 1). Even before TSA
treatment, CENPB signals were detected at YAC integration sites in 83% of
cells (Fig. 6C,J,N,
Table 1); however, of these,
62% showed very weak CENP-B assembly which could barely be distinguished from
background levels (Fig. 6N).
After TSA treatment, the intensity of the CENP-B signals at the YAC
integration sites increased significantly
(Fig. 6D,K,N). Therefore, prior
chromosome breakage is not required for centromere chromatin reassembly at the
alphoid YAC sites, and only the structural change of chromatin by
hyperacetylation induced by TSA treatment is needed. No abnormalities were
observed at the centromeres on host chromosomes after TSA treatment and no
extra assembly of CENP-A, -B and -C was detected on the chromosome arm regions
other than at the YAC integration site
(Fig. 6G).
|
YAC integration sites derived from 21-II alphoid YAC (B13HT1) never
formed minichromosomes after TSA treatment, nor did centromere components
assemble there (Fig. 5C,
Table 1), while transcription
from the selectable marker gene increased
(Fig. 5E). Thus, centromere
component reassembly occurs universally and specifically at the
21-I
alphoid YAC/BAC integration sites of HT1080 cell lines. The centromere
competence of the
21-II alphoid YAC integration site might be different
from that of
21-I alphoid YAC integration sites, which show strong
CENP-B binding.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Centromere components assemble on 70 kb 21-I alphoid arrays
but not on YAC arms in a stably propagated artificial chromosome
Our stable MACs consist of multimers of introduced 21-I alphoid YAC
DNA. Cytological and ChIP analyses indicated that on a stable MAC with an
active centromere, CENP-A and CENP-B assembled only on 70 kb
21-I
alphoid arrays of the insert but not on sequences located 3 kb or 8 kb from
the alphoid junctions on the YAC arms (Fig.
7A). CENP-A is also associated with neocentromeres. These findings
suggest that assembly might not depend on specific DNA sequences
(Lo et al., 2001
). However, a
DNA sequence underlying a neocentromere cannot undergo de novo centromere
formation (Saffery et al.,
2001
). We have demonstrated that a synthetic alphoid construct
with canonical CENP-B boxes is active for de novo centromere assembly
including CENP-A, and can promote MAC formation, whereas the same construct
with point mutations affecting every CENPB box does not exhibit these
activities (Ohzeki et al.,
2002
). Thus, functional centromeres cannot form de novo on alphoid
DNA without CENP-B boxes. Alphoid DNA with CENP-B boxes may have a specific
capacity for assembling centromere chromatin.
|
In our present study, the bsr gene on the YAC arm was transcribed
and acetylated histone H3 was assembled at this locus. Based on such an
alphoid YAC multimer, a functional centromere structure was assembled and
propagated as a stable MAC. Therefore, non-CENP-A chromatin intervals on the
YAC arms between 70 kb alphoid repeats with CENP-A chromatin might not
interrupt the functional kinetochore structures. These results might support
the idea of a repeat subunit model of mammalian kinetochore at the molecular
level (Zinkowski et al., 1991;
Henikoff et al., 2000
).
Interestingly, interruption of CENP-A chromatin clusters with histone H3
chromatin was observed on the extended chromatin fiber of mitotic chromosomes
in both human and Drosophila centromeres
(Blower et al., 2002
).
Suppressed states of centromeres and reactivation at ectopic alphoid
loci
At the site of integration of the alphoid YAC in 7C5HT1-19 cells the extent
of CENP-A and -B assembly on the alphoid array was decreased compared with
that on a stable MAC with an active centromere
(Fig. 7B). These results, and
the observation of the stability of the integrated alphoid YAC sequences at
the same locus, indicated that centromere functions are lost or suppressed at
the ectopic alphoid loci in this cell line, as in the inactive centromeres of
dicentric chromosomes. Thus it is likely that a chromatin change makes the
binding sites, including the CENP-B box, inaccessible. On the ectopic alphoid
YAC loci, the transcription of bsr and the level of acetylated
histone H3 on this gene also decreased
(Fig. 7B). Therefore, not only
was centromeric protein assembly inhibited, but many chromatin functions were
also suppressed at the ectopic alphoid locus. At such suppressed (inactive)
sites on the alphoid YAC loci, centromere chromatin was reassembled by
long-term culture in selective medium. The results of TSA treatment also
suggest that these reassemblies of components of the functional
centromere/kinetochore structure were correlated with structural chromatin
changes caused by histone acetylation, allowing a detectable increase in
transcription of the nearby bsr gene to take place. We speculate that
transcriptionally competent chromatin at the intervals between the 70 kb
alphoid DNA arrays at the ectopic locations, or a hyperacetylated state of the
histones due to TSA, would both be expected to break such a suppressed
(inactivated) chromatin state at the loci by opening and remodeling the
chromatin structure. Thus, one possible explanation for reassembly of
centromere components is that the chromatin structure in the integrated
suppressed alphoid DNA state might be changed by constant weak gene expression
on the YAC arms or TSA-promoted hyperacetylation of histones as a common
effect. CENPs and kinetochore proteins could be reassembled dependent on the
capacity of open chromatin structure on 21-I alphoid YAC DNA even with
the 10 kb alphoid insert, so that a functional centromere/kinetochore
structure is re-formed (Fig.
7C). Alternatively, TSA may affect the transcription of genes that
are presumed to contribute to centromere activation or centromeric protein
assembly. However, as described below, several observations indicate that the
latter possibility is incapable of explaining the reassembly phenomenon. BS
selection, which has no relation to the transcription level of the endogenous
presumptive centromere-related genes, also induces ectopic alphoid DNA
activation to form a centromere. Centromere assembly on introduced
21-I
alphoid DNA also occurred by selection of a nearby neomycin-resistance gene
instead of the bsr gene (Ohzeki
et al., 2002
). Therefore, the former possibility is more likely:
our analyses indicate that the centromere activity of
21-I alphoid DNA
at an ectopic locus can be altered as a result of changes in the chromatin
structure of nearby genes.
Centromere activity and transcription
In fission yeast, components of the kinetochore, including yeast CENP-A,
assemble on the central core of the centromere and the resulting complex is
surrounded by heterochromatin (Saitoh et
al., 1997; Takahashi et al.,
2000
; Partridge et al.,
2000
; Bernard et al.,
2001
; Nakagawa et al.,
2002
). TSA-induced high levels of transcription from genes
inserted in centromeric flanking regions cause chromosome instability and
conformational changes in centromeric heterochromatin
(Ekwall et al., 1997
).
Moreover, the TSA-induced expression of the inserted genes was metastable and
frequently reverted to a repressed state in the absence of TSA. This result
suggests that functional centromeres, including heterochromatin regions, might
be affected by changes in chromatin structure caused by strong transcriptional
activity. However, in our experiment, TSA treatment has the opposite effect.
In our system, marker gene transcription is regulated by the SV40 early
promoter, which is not strong in this cell line (1/10-1/20 the level of that
of the endogenous ß-actin gene per 30 copies of YAC arms, as
indicated by RT-PCR, Fig. 3F). This level of activity presumably suffices to allow the reassembly and
maintenance of a functional centromere in our system. Strong transcriptional
activity driven by the CMV and PGK promoter, however, drastically decreased
the efficiency of artificial chromosome formation in our system (H. Nakashima
and H.M., unpublished). Strong transcriptional activity or differences in
chromatin structure as a result of the different promoter and enhancer between
the 70 kb alphoid intervals may inhibit the active structure of centromeres or
heterochromatin on artificial chromosomes. In both fission yeast and human
cells, chromatin structures at the periphery of CENP-A-assembled core
centromere domains affect centromere function
(Nakagawa et al., 2002
). In
budding yeast, there is no evidence for epigenetical inactivation of the
functional centromere, however, the phenotype of the chl4 deletion
mutant suggests that even in budding yeast, established centromeres are
propagated in an epigenetic manner
(Mythreye and Bloom, 2003
). In
addition, one centromere component, Cbf1, is a transcriptional regulator, and
centromeric activity is also inhibited by strong transcription of genes
inserted nearby (Bram and Kornberg,
1987
; Hill and Bloom,
1987
). Interestingly, Ams2, a GATA-type transcriptional regulator,
is present in the central regions of fission yeast centromeres
(Chen et al., 2003
).
Centromere chromatin associated with pericentromeric
heterochromatin
If endogenous centromere activity is affected positively or negatively by
the chromatin structure that induces weak or strong transcription, chromosome
loss and breakage leading to severe aneuploidy might be caused. Conversely,
centromeric chromatin structure might inhibit transcriptional regulation of
flanking genes. Therefore, to stabilize centromere activity it would be
reasonable to place centromeres in specific chromosomal regions away from
transcriptional activities.
Indeed, normal human centromeres are located on megabase-sized alphoid
arrays in normal human cells. Furthermore, the long alphoid arrays in
endogenous chromosomes are surrounded by large regions of inactive
heterochromatin. In mouse and Drosophila centromeres, large tracts of
pericentromeric heterochromatin are also observed adjacent to centromeric
satellite DNAs on which CENPs assemble
(Mitchell et al., 1993;
Blower and Karpen, 2001
). It is
possible that the structure of endogenous centromeres is protected by
pericentromeric heterochromatin (Choo,
2001
). Even when the cells were treated with TSA, which induced
the reactivation of suppressed centromeres in the integrated alphoid YAC,
endogenous centromeres did not exhibit abnormalities in centromere component
assembly. Our results also indicated that CENP-A might not be acetylated by
treatment with TSA. Thus, once assembled, CENP-A chromatin may be more
TSA-resistant or -insensitive than histone H3 chromatin. The findings of
Taddei et al. supports this: TSA treatment was found to cause the disassembly
of HP1 from regions of pericentromeric heterochromatin but did not impair
centromeric protein assembly (Taddei et
al., 2001
).
Assembly of the centromeric proteins CENP-A and CENPC into chromatin was
observed in a limited area on the MACs, whereas CENP-B was detected almost
throughout the MACs, so that not all the alphoid DNA arrays in the approx. 30
copies of introduced alphoid YAC DNA might be involved in kinetochore
assembly. The remainder of the alphoid array on the multimerized YAC DNA might
be important for the formation of structures other than the kinetochore, such
as pericentromeric heterochromatin or other states of chromatin such as a
stable artificial chromosome. Actually, anti-CENPA antibodies have been
reported to stain only one-half to two-thirds of the alphoid regions of normal
human centromeres analyzed by fiber FISH
(Blower et al., 2002). In
fission yeast, pericentromeric heterochromatin is involved in sister chromatid
cohesion (Bernard et al., 2001
;
Nonaka et al., 2002
).
A more detailed understanding of centromere assembly will require further investigation of centromere structure and function with respect to protein assembly, epigenetic modifications of histones, and DNA modifications (such as methylation), as well as the organization of heterochromatin. Our alphoid YAC construct integrated into ectopic chromosomal locations will provide a powerful tool not only for the analysis of artificial chromosomes, but also for the generation of novel views of the functional organization of centromeres, heterochromatin and chromosomes in mammals, which are very difficult to analyze.
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Acknowledgments |
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