1 CREST Research Project, Kansai Advanced Research Center, Communications
Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492,
Japan
2 Department of Biology, Graduate School of Science, Osaka University,1-1
Machikaneyama, Osaka 560-0043, Japan
3 Division of Biological Science, Graduate School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-8602, Japan
* Author for correspondence (e-mail: yasushi{at}crl.go.jp)
Accepted 28 April 2003
![]() |
Summary |
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Key words: Heterochromatin, HP1, PML, Centromere, CENP-B
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Introduction |
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Although the molecular details that define euchromatin and heterochromatin
are not fully understood, it is now clear that heterochromatin protein 1 (HP1)
is a major constituent of heterochromatin and plays a key role in its
formation and maintenance. HP1 is highly conserved from yeasts to humans
(Eissenberg and Elgin, 2000).
HP1 was first identified as a gene product of an allele of Su(var)2-5
in Drosophila melanogaster (James
and Elgin, 1986
; Eissenberg et
al., 1990
). In Drosophila, HP1 is localized to
pericentric and telomeric heterochromatin. When mutated HP1 was expressed in
Drosophila, abnormal condensation and segregation of chromosomes was
observed (Kellum and Alberts,
1995
). Involvement of HP1 in heterochromatin formation is now
known to occur through its interaction with SUV39H1, the histone
methyltransferase that methylates histone H3 at lysine 9. HP1 binds to histone
H3, which has been methylated at lysine 9 by SUV39HI, and in turn it recruits
SUV39H1 to the DNA, which further propagates methylation along the chromatin
(Jenuwein, 2001
). This
relationship between HP1 and SUV39H1 is conserved in their
Schizosaccharomyces pombe homologues Swi6 and Clr4
(Bannister et al., 2001
),
suggesting evolutionary conservation of this mechanism of heterochromatin
formation.
In humans, three subtypes of HP1 have been identified, HP1,
HP1ß, and HP1
(Singh et al.,
1991
; Saunders et al.,
1993
; Ye and Worman,
1996
). They share significant similarities in their amino acid
sequences (see Fig. 7). HP1
consists of two highly conserved regions, the N-terminal chromo domain (CD)
(Paro and Hogness, 1991
) and
the structurally related C-terminal chromo shadow domain (CSD)
(Aasland and Stewart, 1995
;
Cowieson et al., 2000
). These
two domains are connected by a less well conserved hinge region (IVR) (see
Fig. 7). The CSD is a unique
motif found only in the HP1 family; the CD motif, however, is also found in
Su(var)3-9 family proteins and Polycomb (Pc) family proteins
(Jacobs et al., 2001
).
|
HP1 seems to have pleiotropic functions in the genetic activities of
chromosomes. It has been shown that mammalian HP1 homologues have a number of
interacting partners such as the CAF-1 subunit p150
(Murzina et al., 1999), SP100
(Lehming et al., 1998
;
Seeler et al., 1998
),
TIF1-ß (KAP-1) (Le Douarin et al.,
1996
; Ryan et al.,
1999
), Ku70 (Song et al.,
2001
), lamin B receptor (Ye
and Worman, 1996
; Ye et al.,
1997
), SUV39H1 (Aagaard et al.,
1999
), Ki-67 (Kametaka et al.,
2002
; Scholzen et al.,
2002
), histone H1-like protein
(Nielsen et al., 2001
),
methylated histone H3 (Bannister et al.,
2001
; Lachner et al.,
2001
) and INCENP (Ainsztein et
al., 1998
). These proteins have a wide variety of functions
involved in chromatin assembly, transcriptional regulation, telomere
maintenance, and nuclear membrane formation. Many of the HP1 interacting
partners listed above bind to the CSD of HP1, but interaction of lysine
9-methylated histone H3 with the CD region
(Lachner et al., 2001
) and
INCENP with the IVR region (Ainsztein et
al., 1998
) have also been documented.
HP1 subtypes have shown heterogeneous patterns of intracellular
localization in accordance with the presence of a wide variety of interacting
molecules. Localization of HP1 subtypes during the cell cycle have been
extensively studied in human and mouse cells
(Wreggett et al., 1994;
Horsley et al., 1996
;
Furuta et al., 1997
;
Minc et al., 1999
;
Yamada et al., 1999
;
Minc et al., 2000
;
Sugimoto et al., 2001
). In
interphase nuclei, HP1 subtypes are localized in heterochromatin
(Wreggett et al., 1994
;
Minc et al., 1999
;
Minc et al., 2000
) in
accordance with their interaction with SUV39H1
(Aagaard et al., 1999
), and
localized at the PML nuclear bodies in interphase nuclei
(Everett et al., 1999
) in
accordance with their interaction with SP100, a component of the PML nuclear
body (Lehming et al., 1998
;
Seeler et al., 1998
); HP1
subtypes are also localized at the periphery of nucleoli
(Minc et al., 1999
). In
metaphase, HP1
is localized at the centromeric region
(Minc et al., 1999
;
Yamada et al., 1999
;
Minc et al., 2001
), as is its
interacter INCENP (Ainsztein et al.,
1998
). Furthermore, live cell imaging of GFP-fused HP1
throughout the cell cycle in human cells demonstrated that HP1
is
colocalized with DsRed-fused CENP-B at the centromere during metaphase
(Sugimoto et al., 2001
).
Compared with the reproducible results reported for HP1
, localization
of HP1ß and
at the metaphase centromere has been debatable,
depending on cell types and antibodies used
(Wreggett et al., 1994
;
Minc et al., 1999
;
Saffery et al., 1999
;
Minc et al., 2000
;
Minc et al., 2001
).
To determine molecular domains of HP1 subtypes required for their
intracellular localization, we examined human HP1 subtypes and various
truncation constructs in living HeLa cells, and determined the molecular
domains responsible for their localization to centromeres and PML nuclear
bodies. Here we report that HP1ß associates with interphase centromeres
but is replaced at the centromere by HP1 as the cell enters metaphase.
The N-terminal region of HP1ß including the CD, is responsible for
localization to the interphase centromere and the C-terminal region of
HP1
including the CSD and a portion of the IVR, is responsible for
localization to the metaphase centromere. Simultaneous observations of the
N-terminal fragment of HP1ß and the C-terminal fragment of HP1
in
living cells revealed that during late prophase the HP1ß fragment
dissociated from the centromere and as the cells entered metaphase the
HP1
fragment accumulated at the centromere. Thus, localization of human
HP1ß and HP1
to centromeric heterochromatin during different
phases of the cell cycle is mediated by distinct regions of these proteins.
These results suggest specific roles for HP1ß and HP1
at the
interphase and metaphase centromere, respectively.
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Materials and Methods |
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YFP and CFP fusion constructs
The open reading frames (ORF) of human HP1 (GenBank accession
number: NM_012117.1), HP1ß (GenBank accession no.: NM_006807.2) and
HP1
(GenBank accession no.: AB030905.1) were cloned by RT-PCR using
mRNA isolated from HeLa cells as the PCR template. Total RNA was isolated from
HeLa cells using TORIZOL (Invitrogen) according to the manufacturer's
instructions. Complementary DNAs were amplified with Pyrobest DNA
polymerase (TaKaRa, Ohtsu, Japan) using specific primers for the ORF of each
gene; the EcoRI linker was added to the 5' primer, and a stop
codon plus the BamHI linker was added to the 3' primers.
Synthesized cDNAs were digested with EcoRI and BamHI, then
cloned into the pEYFP-C1, pECFP-C1 or pDsRed2-C1 vector (Clontech, Palo Alto,
CA, USA). Truncated constructs of human HP1 were produced by PCR-based
amplification using its ORF as the template. All clones were checked for their
nucleotide sequence using ABI310 or ABI377 (Applied Biosystems Inc., Foster
City, CA, USA).
The plasmids pGFP-CB1-160c and pCFP-CB1-160c, containing the N-terminal
fragment of human CENP-B fused with GFP and CFP, respectively, were generated
as follows: The N-terminal fragment of CENP-B (2 to 481 bp) was excised
from pETCBN-160 (Kitagawa et al.,
1995) with NcoI (the NcoI digestion product was
blunt-ended using Klenow) and BamHI. A BamHI-
HindIII adaptor was prepared by annealing oligonucleotides
5'-GATCCATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGTAGTCGACA-3' and
5'-AGCTTGTCGACTACCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATG-3'. The
CENP-B fragment with the adaptor was cloned into SalI (the
SalI digestion product was blunt-ended using
Klenow)-HindIII-cut pMTID13S
(Stuart et al., 1984
) to
create pMTCB1-160c. Then, to create the plasmid pGFP-CB1-160c, the N-terminal
fragment of CENP-B was excised from pMTCB1-160c with XbaI and
HindIII (the HindIII digestion product was blunt-ended using
Klenow) and introduced into XbaI-BclI-digested and
blunt-ended pGFP-C1 (Clontech). To create the plasmid pCFP-CB1-160c, the
N-terminal fragment of CENP-B was excised from the plasmid pGFP-CB1-160c with
SalI and BamHI, and introduced into
SalI-BamH-digested pECFP-C1 (Clonetech).
Transfection
Transfection reactions were carried out with LipofectAmine PLUS
(Invitrogen). HeLa cells cultured in a 35 mm glass-bottom culture dish
(MatTek, Ashland, MA, USA) were transfected with 250 ng of plasmid DNA, and
incubated at 37°C for 1.5 hours. For co-transfection with two or three
kinds of plasmid DNA, 125 ng or 85 ng DNA of each plasmid were applied,
respectively. Transfected cells were incubated in fresh DMEM medium containing
10% calf serum for 1 or 2 days before observation.
Indirect immunofluorescent staining
Indirect immunofluorescent staining was carried out essentially as
described previously (Haraguchi et al.,
2000). Cells cultured in a 35 mm glass-bottom dish were fixed with
a mixture of 3.7% formaldehyde and 0.2% glutaraldehyde in DMEM culture medium
for 20-30 minutes at room temperature. Fixed cells were then treated twice
with 0.1% sodium borohydrate in phosphate-buffered saline (PBS) for 15 minutes
at room temperature. The cells were then permeabilized with 0.1% Triton X-100
in PBS for 5 minutes, followed by incubation in 1% bovine serum albumin in PBS
at room temperature for 1 hour. Anti-centromere human antisera and anti-PML
mouse monoclonal antibody were then added at a dilution of 1:100 and 1:200,
respectively, and incubated for 3 hours at room temperature. After the first
antibody reaction, cells were washed five times with PBS for 10 minutes, and
stained for 3 hours at room temperature with the relevant fluorescent
secondary antibodies: Cy3-conjugated goat anti-mouse IgG or Cy5-conjugated
goat anti-human IgG, both at a dilution of 1:200. The stained cells were
washed five times with PBS for 10 minutes and then taken sequentially with
20%, 40%, 60% and 80% glycerol in PBS containing 0.5 µg/ml DAPI and 2.5%
DABCO. Finally, cells were mounted in 90% glycerol for microscopic
observation.
Live cell observation
observation of live cells was carried out as previously described
(Haraguchi et al., 1997;
Haraguchi et al., 1999
).
Briefly, cells cultured in a 35 mm glass-bottom dish were stained with 100
ng/ml Hoechst 33342 for 30 minutes and then washed twice with DMEM containing
10% calf serum. The Hoechst 33342-stained cells were then cultured in phenol
red-free DMEM supplemented with 10% fetal bovine serum (Invitrogen),
antibiotics (50 unit/ml penicillin and 50 unit/ml streptomycin; purchased from
Invitrogen), and 20 mM Hepes pH 7.4. These cells were incubated in a
CO2 incubator for at least 30 minutes and then placed on a
microscopic stage in a temperature-controlled room. Mineral oil was layered
onto the culture medium to avoid evaporation during microscopic observation.
After 30 minutes of culture on the microscope stage, observation was carried
out using a DeltaVision microscope system (Applied Precision, Issaquah, WA,
USA) kept at 37°C in a temperature-controlled room
(Haraguchi et al., 1999
). The
oil-immersion objective lens UApo40x/NA1.35 (Olympus Opitcal, Tokyo, Japan)
was used for the observation.
Non-fixed chromosome spread
HeLa cells cultured in a 60 mm culture dish were transfected with 750 ng
DNA for 1.5 hours using LipofectAmine PLUS. The transfected cells were then
cultured in DMEM supplemented with 10% calf serum at 37°C for 24 hours.
Thymidine was added to the culture at a final concentration of 2.5 mM to
synchronize the cell cycle. After incubation in the thymidine-containing
medium for 18 hours, the cells were washed twice with DMEM and cultured in
DMEM supplemented with 10% calf serum at 37°C for 8 hours to allow cells
to proceed into mitosis. Mitotic cells were enriched by collecting cells that
detached from the dish by gentle pipetting. Cells with no mitotic enrichment
were collected using a rubber scraper. For hypotonic treatment, the collected
cells were washed once with 75 mM KCl that had been pre-warmed to 37°C,
and incubated for 20 minutes at 37°C. Mitotic cells were collected by
pipetting, resuspended in 5 ml of 75 mM KCl and kept on ice. Then an equal
volume of ice-cold buffer A (15 mM Hepes pH 7.4, 80 mM KCl, 20 mM NaCl, 0.5 mM
EGTA, 2 mM EDTA, 0.5 mM spermidine, 0.2 mM spermine and 0.1%
2-mercaptoethanol) (Hiraoka et al.,
1990) containing 0.1% Triton X-100 (buffer A-T) was added to the
cells while on ice. Cells were centrifuged, washed and then resuspended with
buffer A-T at 4°C. The cells were placed on a glass slide, DAPI added for
DNA counterstaining, and the cells were covered with a coverslip. Chromosome
spreads were prepared by gently tapping the coverslip.
Fluorescence microscopy
Fluorescence microscope images were obtained on a Peltier-cooled CCD using
a DeltaVision microscope system. Details of the microscope system have been
described previously (Haraguchi et al.,
1997; Haraguchi et al.,
1999
). For high-resolution analysis of fixed specimens, the
oil-immersion objective lens PlanApo60x/NA1.4 (Olympus Opitical) was used.
Three-dimensional optical section images were taken at 0.5 µm focus
intervals and computationally processed by an iterative deconvolution method
(Agard et al., 1989
).
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Results |
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|
Localization of YFP-fused HP1 subtypes was also examined on spread
preparations of metaphase chromosomes. When metaphase chromosome spreads were
prepared from cell populations enriched with mitotic cells HP1 was
clearly present at the centromere (Fig.
2a) whereas HP1ß and
showed faint or no localization
at the centromere (Fig. 2c and
e, respectively). In contrast, when chromosome spreads were
prepared from whole cell populations with no enrichment, HP1ß showed a
somewhat increased tendency to be associated with the centromere
(Fig. 2d) although its
localization was much fainter than that of HP1
. Quantitative data is
summarized in Table 1. The
apparent centromere localization of HP1ß was the result of interphase
cells being present in the preparations whereas localization of HP1
and
was not significantly affected by the presence of interface cells.
This would suggest that relocalization of HP1ß occurs during the cell
cycle and may explain the discrepancies in the reported localization of
HP1ß at the metaphase centromere (see Discussion).
|
|
Taken together, these results show that YFP-fused HP1 subtypes change their
intracellular localization from interphase to metaphase. All HP1 subtypes
associate with interphase centromeres, but only HP1 shows strong
association with metaphase centromeres. These intracellular localization
patterns of YFP-fused HP1 are consistent with the previous observations
obtained using antibodies or GFP-fusion constructs
(Minc et al., 1999
;
Sugimoto et al., 2001
) (see
Introduction). Thus, we determined molecular domains required for the
intracellular localization using truncation constructs of the YFP-fusion HP1
subtypes in HeLa cells.
Molecular domains in HP1 necessary for their intracellular
localization
As HP1 was uniquely localized to the metaphase centromere, we
determined the necessary molecular domains of HP1
required for this
localization. To this end, we constructed a series of truncated HP1
peptides tagged with YFP (Fig.
3A). First, we divided HP1
into three domains, namely the
N-terminal region, consisting of residues 1-75, which contains the CD; the
C-terminal region consisting of residues 121-191, which contains the CSD; and
the IVR region, consisting of residues 76-120. However, none of these domains
was found to be localized to the metaphase centromere (data not shown).
|
We then examined residues 1-170 containing the CD and the IVR and residues
76-191 containing the IVR and the CSD. We found that the latter peptide was
localized to the metaphase centromere (Fig.
3A; image data not shown). Further experiments using truncations
of HP1 determined that the peptide consisting of residues 101-180 was
localized to the metaphase centromere, whereas the peptide consisting of
residues 111-180 was not (Fig.
3B), indicating that residues 101-180 of HP1
is the minimum
region necessary for localization of HP1
to the metaphase centromere.
Interestingly, residues 98-176 of HP1ß and residues 83-170 of HP1
,
which correspond to residues 101-180 of HP1
, were also localized to the
metaphase centromere (Fig. 4B,
Fig. 5B) in spite of the fact
that neither full-length HP1ß nor HP1
was localized to the
metaphase centromere (Fig. 1B),
suggesting that this region of HP1 has a fundamental role in the localization
to the metaphase centromere.
|
|
HP1 peptides that were localized to the centromere at metaphase were not
localized to the centromere in interphase (HP1 101-180, compare
Fig. 3C with
Fig. 3B; HP1ß 98-176,
compare Fig. 4C with B;
HP1
83-170, compare Fig. 5C with
B). Instead they localized exclusively to PML nuclear bodies (data
not shown). The shorter fragments containing only the CSD rgion were also
present (HP1
121-191 in Fig.
3D; HP1ß 117185 in
Fig. 4D; HP1
111-173 in
Fig. 5D). Thus, the CSD portion
of HP1 is sufficient for localization to PML nuclear bodies in interphase,
whereas the additional IVR residues are required for localization to the
metaphase centromere.
As the residues of HP1 that were localized to the metaphase centromere were
not localized to the interphase centromere, we examined the molecular domains
of HP1 necessary for localization to the centromere within the interphase
nucleus. Our results showed that in HP1 and HP1ß the N-terminal
fragments containing the CD were localized to the interphase centromere, but
this fragment in HP1
was poorly localized (HP1
1-75 in
Fig. 3C; HP1ß 1-76 in
Fig. 4C; HP1
1-75 in
Fig. 5C). These results
indicate that the domains of HP1 required for centromeric localization during
interphase are different from those required for centromeric localization
during metaphase.
Simultaneous observations of HP1 and HP1ß in living
cells
Of the three HP1 subtypes HP1ß was localized to the interphase
centromere most strongly, whereas HP1 was the only subtype localized to
the metaphase centromere. In order to examine the switching of HP1
and
HP1ß during the cell cycle, we attempted to observe HP1
and
HP1ß simultaneously in living cells. To avoid possible interaction
between HP1
and HP1ß through their CSD
(Le Douarin et al., 1996
;
Ye et al., 1997
), we used the
N-terminal fragment of HP1ß (residues 1-76) and the near C-terminal
fragment of HP1
(residues 101-180); these fragments do not interact
with each other. Fig. 6 shows
an example of HeLa cells expressing the residues 1-76 of HP1ß fused with
YFP, the residues 101-180 of HP1
fused with DsRed2, and the N-terminal
fragment of CENP-B fused with CFP. Between interphase and prophase, the
HP1
fragment was not localized to the centromere, but instead localized
to PML nuclear bodies (Fig. 6,
frames at 0:00-1:44). However, the HP1ß fragment did localize to the
centromere, stained with CENP-B-CFP, during this period
(Fig. 6, frames at 0:00-1:02).
In late prophase, the HP1ß fragment dissociated from the centromere
(Fig. 6, frames at 1:44-2:01),
and, in the same cell, the HP1
fragment accumulated at the centromere
just prior to metaphase (Fig.
6, frame at 2:29). During anaphase, the HP1
fragment
dissociated from the centromere (Fig.
6, frame at 2:34) and the HP1ß fragment began to accumulate
first on the chromosome (Fig.
6, frame at 2:45), and then on the centromere
(Fig. 6, frame at 2:51).
Accumulation of the HP1
fragment at the midbody was also observed
during cytokinesis (Fig. 6,
frames at 2:45 and 2:51). Full-length HP1
also showed accumulation at
the midbody (data not shown). By the next interphase, the HP1
fragment
reformed foci with PML nuclear bodies and the HP1ß fragment relocalized
to centromeric regions (an example for later interphase is shown in
Fig. 6, frame at 16:09). These
results demonstrate that as a result of the interaction of different domains
of human HP1 proteins with interphase and metaphase centromeric regions,
HP1ß is localized to the centromere during interphase and is replaced by
HP1
during metaphase (summarized in
Fig. 7; see Discussion).
|
![]() |
Discussion |
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HP1 proteins in the interphase nucleus
In interphase, all three human HP1 subtypes localize to centromeric regions
and other heterochromatin regions to varying extents. Of the subtypes,
HP1ß most preferentially localized to heterochromatin. We determined that
the N-terminal fragment containing the CD was the molecular domain of HP1
required for localization to interphase centromeric heterochromatin.
Localization of the CD region of HP1 to heterochromatin probably reflects the
fact that the CD of HP1 binds to lysine 9 methylated histone H3
(Bannister et al., 2001;
Lachner et al., 2001
) while a
CSD interacting partner, SUV39H1, methylates lysine 9 of histone H3
(Rea et al., 2000
). It is now
hypothesized that the CD of HP1 recognizes modified histones, the `histone
code', and recruits a variety of proteins through interaction with the CSD to
establish chromatin structure (Jenuwein
and Allis, 2001
).
Our results also showed that all three human HP1 subtypes were localized to
the PML nuclear bodies, as previously reported
(Everett et al., 1999).
HP1
and
were preferentially localized to PML nuclear bodies.
The PML nuclear body is an intranuclear structure that contains a number of
proteins, such as PML, SP100, ISG20, PIC1/SUMO1, LYSP100, PLZF, INT6, CBP,
RB1, RFP and ribosomal protein P (Lamond
and Earnshaw, 1998
; Seeler and
Dejean, 1999
). It has been shown that HP1 interacts with SP100,
one of the major components of the PML nuclear body
(Lehming et al., 1998
;
Seeler et al., 1998
;
Seeler and Dejean, 1999
). In
this study, we determined that the CSD is the molecular domain of HP1 required
for localization to PML nuclear bodies. This is consistent with the fact that
the CSD portion of HP1 interacts with SP100
(Lehming et al., 1998
;
Seeler et al., 1998
).
HP1 proteins in the metaphase centromere
In metaphase, HP1 localized to the centromere whereas HP1ß and
HP1
did not. This result is consistent with previous observations
obtained from antibodies or GFP fusion constructs
(Sugimoto et al., 2001
).
However, it has also been reported that HP1ß localizes to the centromere
of some, but not all, metaphase chromosomes
(Saffery et al., 1999
) and
that HP1
is localized to the metaphase centromere
(Minc et al., 2000
). Furuta et
al. (Furuta et al., 1997
)
demonstrated that centromere localization of HP1ß is observed at
metaphase, but is most prominent at anaphase, suggesting cell cycle
regulations. We also occasionally observed some faint centromere staining for
HP1ß or HP1
in metaphase chromosome spreads that also contained
some interphase cells (Fig. 2;
Table 1), suggesting that their
localization may be affected by unknown factors from interphase cells. Since
our experiments of HP1 truncations showed that all HP1 subtypes are
potentially capable of binding to the metaphase centromere, centromere
localization of HP1 is probably regulated during mitosis by protein
modification or interaction with other proteins. Also their localization may
depend on heterochromatin structures, which vary in different cell types.
We defined the domain of HP1 that is required for its localization
to the metaphase centromere. This domain consists of a portion of the IVR and
the CSD region, as opposed to the CD which is required for localization to the
interphase centromere (Fig. 7). Replacement of an HP1ß peptide containing the CD region with an
HP1
peptide containing the IVR/CSD region at the centromere during
mitosis was clearly demonstrated by observation of live cells (see
Fig. 6). Interestingly, the
corresponding IVR/CSD domain of HP1ß and HP1
was also localized to
the metaphase centromere whereas full-length HP1ß or HP1
was not.
These results show that HP1ß and HP1
potentially have the
capability of localizing to the metaphase centromere, but the neighboring
domains somehow interfere with metaphase centromere localization. These
results also suggest that their localization at metaphase centromere is
regulated by small variations of amino acid sequences or modifications such as
phosphorylation. Putative phosphorylation sites regulated during the cell
cycle have been reported (Minc et al.,
1999
) although there is no direct evidence that phosphorylation is
involved in HP1 localization.
Centromeres are likely to be reorganized during progression through
mitosis. The human centromere/kinetochore consists of many structural units
(Clarke et al., 1999): the
sister chromatids cohesion element (INCENP and Rad21), centromeric chromatin
element (CENP-B), inner kinetochore element (CENP-A, CENP-C), outer
kinetochore element (CENP-F), and fibrous corona (CENP-E, dynein, dynactin).
Additionally, the spindle checkpoint apparatus, including Mad, Bub and other
proteins, is localized on the outer kinetochore element. It should be noted
that a portion of the IVR, in addition to the CSD, of HP1 is required for its
localization to the metaphase centromere. INCENP is known to interact with HP1
through the IVR (residues 64-111)
(Ainsztein et al., 1998
). Our
results show that neither the IVR nor the CSD alone is sufficient for
localization to the metaphase centromere. Thus, INCENP is not sufficient for
localizing HP1 to the metaphase centromere, and other protein molecules
interacting with the CSD appear to be required, in combination with INCENP or
other unknown IVR interactive molecules, for this effect.
In accordance with the specific localization of HP1 to the metaphase
centromere, some cellular events are known to be specifically associated with
HP1
. In highly invasive, metastatic breast carcinoma cell lines,
HP1
expression is decreased at both mRNA and protein levels, whereas
expression of HP1ß or HP1
is unaffected
(Kirschmann et al., 2000
).
Conversely, the protein level of HP1
increases under anti-IgM induced
apoptotic conditions in a human Burkitt lymphoma cell line
(Brockstedt et al., 1998
).
HP1
interacts with INCENP (Ainsztein
et al., 1998
), and INCENP interacts with Aurora B kinase
(Adams et al., 2001
). Deletion
of Scc1/Rad21 in DT40 cells eliminates the localization of INCENP to the inner
centromeric domain (Sonoda et al.,
2001
). Furthermore, deletion of Swi6 (S. pombe HP1)
causes loss of sister centromeric cohesion
(Bernard et al., 2001
). These
results would suggest that the HP1
localized to the mitotic centromeric
heterochromatin may contribute to the stability of sister chromatid cohesion
or activation of the kinetochore checkpoint. Reduced HP1
at the
metaphase centromere may also be a cause of chromosome instability in cancer
cells.
HP1 subtypes interact with a variety of molecules. This generates a heterogeneous pattern of intracellular localization and dynamic relocalization during the cell cycle. Molecular dissection of their domains provides a simpler view of their localization through interaction with specific binding partners.
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