Comprehensive analysis of the ICEN (Interphase Centromere
Complex) components enriched in the CENP-A chromatin of
human cells
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11
Comprehensive
2006
riginal
Izutatoet
Article
al.Publishing
analysis
of the ICEN
Blackwell
Malden,
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1365-2443
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Hiroshi Izuta1,†, Masashi Ikeno2,†, Nobutaka Suzuki2,†, Takeshi Tomonaga3, Naohito Nozaki4,
Chikashi Obuse5, Yasutomo Kisu6, Naoki Goshima6, Fumio Nomura3, Nobuo Nomura6 and
Kinya Yoda1,*
1
Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1101, Japan
3
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
4
Kanagawa Dental College, Inaoka-cho,Yokosuka-ku, Kanagawa 238-8580, Japan
5
Department of Gene Mechanism, Graduate School of Biostudies, Kyoto University,Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
6
Biological Information Research Center, Advanced Industrial Science and Technology, Koto-ku,Tokyo135-0064, Japan
2
The centromere is a chromatin structure essential for correct segregation of sister chromatids, and
defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the
presence of 40 proteins (ICEN1–40), along with CENP-A, -B, -C, -H and hMis6, by proteomic
analysis. Here we report analysis of seven ICEN components with unknown function. Centromere
localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells.
Depletion of each of these proteins by short RNA interference produced abnormal metaphase
cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the
ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from
all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results
suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of
CENP-H and hMis6 onto the centromere.
Introduction
The centromere has a pivotal role in accurate chromosome segregation. Kinetochores are formed on the centromeres in mitosis, where spindle microtubules attach to
them to generate the physical forces necessary for chromosome movements, and they are also required for spindle checkpoint regulation (Cleveland et al. 2003). Defects
in kinetochore function lead to chromosome instability
(CIN) and aneuploidy, which often results in birth defects
and/or human cancers (Lengauer et al. 1998; Kops et al.
2004). CENP-A is a histone H3 variant widely conserved
among species from S. cerevisiae, Schz. pombe, C. elegans and
Drosophila to mammals (Sullivan et al. 1994; Stoler et al.
Communicated by: Fuyuki Ishikawa
*Correspondence: E-mail: i45156a@cc.nagoya-u.ac.jp
†
These authors contributed equally to this work
1995; Buchwitz et al. 1999; Henikoff et al. 2000; Takahashi et al. 2000).The putative histone-fold domain located
in the C-terminal region is essential for targeting to the
centromeric region (Shelby et al. 1997; Black et al. 2004).
CENP-A can replace histone H3 to form stable nucleosomes by in vitro reconstitution (Yoda et al. 2000). It has
been widely accepted that formation of nucleosomes
containing CENP-A defines functional centromeres
(Cleveland et al. 2003). In fission yeast, the centromeric
regions are composed of the central cores, defined by the
presence of CENP-A chromatin, and the centromeric
heterochromatin which is responsible for cohesion of
sister centromeres (Pidoux & Allshire 2004). These two
centromeric structures may also be important for mammalian centromeres. In interphase nuclei, many proteins
are assembled around the CENP-A nucleosomes to form
a large chromatin complex. To date, six centromeric
proteins have been reported in human cells: CENP-A,
DOI: 10.1111/j.1365-2443.2006.00969.x
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2006) 11, 673–684
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H Izuta et al.
CENP-B, CENP-C, CENP-H, hMis6/CENP-I and
hMis12 (Dobie et al. 1999; Sugata et al. 2000; Nishihashi
et al. 2002; Goshima et al. 2003; Liu et al. 2003). These
proteins seem to be basic components for centromere
architecture, since they can be detected at the centromeric
region throughout the cell cycle. In human chromosome 21, the centromeric DNA is composed of a highly
regular type-I α-satellite array and a flanking type-II αsatellite array (Ikeno et al. 1994).The type-I array is mainly
composed of a conserved dimer repeat (171 bp × 2)
containing one CENP-B box in one of the dimer units.
The CENP-B box in the type-I array is necessary for de
novo formation of artificial chromosomes (Ohzeki et al.
2002). The type-II array is composed of a simple monomer repeat with a relatively variable sequence lacking a
CENP-B box. Both arrays are of the order of megabases
in length. CENP-A chromatin is predominantly formed
on 30–50% of the length of the type-I array (Ando et al.
2002), and it is speculated that centromeric heterochromatin is formed outside the CENP-A chromatin region,
although the precise structure remains to be elucidated.
We have developed a method to isolate from HeLa
interphase nuclei the centromere complexes that contain
CENP-A, CENP-B and CENP-C, using monoclonal
antibodies against CENP-A (Ando et al. 2002).To isolate
the DNA–protein complex and analyze the purified
proteins, we avoided sonication or cross-linking reagents
such as formaldehyde that are usually used in conventional chromatin immunoprecipitation (ChIP) analysis in
yeast (Aparicio 1999). To extract the centromeric chromatin into the soluble fraction in a native state, using the
mildest conditions, we only cleaved chromatin DNA using
micrococcal nuclease (MNase) and salt (0.3 M NaCl)
treatment. To discriminate this method from other conventional ChIP methods we call this method “Native
Chromatin Immunoprecipitation” (NChIP). As the CENPA/B/C chromatin complex purified with NChIP using
anti-CENP-A antibodies contained CENP-H, hMis6
and hMis12 as well as CENP-A, CENP-B and CENPC, which constitute all the reported structural components of the centromere, we call this complex the ICEN
( Interphase CENtromere complex). Approximately 70%
of the DNA segments in the ICEN are composed of the
type-I α-satellite sequence, indicating that the minimum
purity of the ICEN can be regarded as 70%. By proteomic analysis we have studied these ICEN components
extensively and revealed 40 proteins. We have further
shown that two of these, DDB1 and BMI1, are actually
located in the centromeric regions (Obuse et al. 2004b).
In this paper we have named these proteins ICEN1–
ICEN40, as shown in Table 1. To reveal the function
of each ICEN component in relation to centromere/
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Genes to Cells (2006) 11, 673–684
kinetochore function, we have examined these proteins
in detail using newly prepared antibodies, various
EGFP-tagged genes, and siRNA transfection. We have
revealed that seven ICEN proteins, ICEN22, 24, 32, 33,
36, 37 and 39, are novel components of the centromeric
chromatin complex, and have a role in kinetochore
function. ICEN22, 32, 33, 37 and 39 are necessary for
loading of both CENP-H and hMis6 proteins onto the
centromeric region. On the basis of these results we propose that, although fragmented to < 30 mer chromatin,
the ICEN may represent a large centromere chromatin
complex, and therefore may contain many other protein
components with important roles for structure and function of the centromeric regions.
Results
EGFP tagged proteins of ICEN22, 24, 32, 33, 36, 37
and 39 localize to centromeres in interphase and in
metaphase
To observe localization of the ICEN components in a
cell, we tagged the EGFP gene or its derivative to the
NH2- or COOH-terminus of the genes of ICEN22, 24,
32, 33, 36, 37 and 39, and transfected each of the fused
genes into HT1080 cells as described in Experimental procedures. Each of the stable transformant cells were immunolabeled with anti-GFP antibodies and anti-CENP-A
antibodies. Figure 1 shows that EGFP-ICEN22, 24, 32,
33, 36, 37 and 39 all co-localize with CENP-A in interphase and metaphase cells. These results suggest that
ICEN22, 24, 32, 33, 36, 37 and 39 localize to centromeres in interphase and in metaphase.
Endogenous ICEN39/PANE1 distributes both in
cytoplasm and in nucleus and also localizes to
centromeres in interphase
In order to study the ICEN 39/PANE1 protein we prepared monoclonal antibodies (3G8) against the synthetic
peptides as shown in Experimental procedures. The
molecular mass of this protein is expected to be 19.7 kDa
from the amino acid sequence (Obuse et al. 2004b) and
the antibody recognized an 18 kDa protein in the purified ICEN fraction with Western blotting after SDSPAGE (Fig. 2A). To examine the distribution of ICEN
39 protein, HeLa cells were fractionated to a cytoplasmic
fraction (#1 in Fig. 2B) and two nucleus fractions, 0.3 m
NaCl extract (#2) and bulk chromatin (#3), as illustrated
in Fig. 2B. As shown in Fig. 2C, ICEN39 distributed
both in the cytoplasmic fraction (Fig. 2C, lane 1) and in the
two nucleus fractions (Fig. 2C, lanes 2 and 3). ICEN39
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Comprehensive analysis of the ICEN
Table 1 List of ICEN components
ICEN#
Annotation
GENE ID
1
2
3
4
5
6
7
8
9
KIAA0853
Rsf1, HBXAP-8
KIAA1429
WD 11
KIAA0224, PRP16
FACT p140
CENP-C
hSNF2H
uvDDB-1
23091
51773
25962
55023
9785
11198
1060
8467
1642
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ASR2A
HMKLP1, KNL-5
FACT p80, SSRP-1
ZFP91
KIAA0095, NP93
cullin-4A
FLJ22009
MgcRacGAP
HSPA5
CENP-I, hMis6
CENP-B
HSPA8
FLJ13111
FLJ23109
MLF1IP, CENP-50
51593
9493
6749
80829
9688
8451
79801
29127
3309
2491
1059
3312
80152
79872
79682
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
WTAP
RNF1
HPC3
HDAC-1
PHP2
BMI1
RNF2
BM039
BC007071
fibrillarin
CENP-H
MGC11266
FKSG14
HPI gamma
PANEI
CENP-A
9589
6015
57332
3065
1912
648
6045
55839
91687
2091
64946
79172
64105
11335
79019
1058
Localization
CEN
CEN
Function
References
ChRMD
(Loyola et al. 2003)
RNH
ChRMD
KTF
ChRMD
HtCh
(Belotserkovskaya et al. 2003)
(Loyola et al. 2003)
(Obuse et al. 2004b;
Jia et al. 2005)
ChRMD
(Belotserkovskaya et al. 2003)
HtCh
(Jia et al. 2005)
CK
CEN
CEN
KTF
HtCh
CEN
KTF
this work
CEN
KTF
this work,
(Minoshima et al. 2005)
CENt
CEN
CEN
CEN
CEN
CEN
CEN
CEN
PcG
PcG
HtCh
PcG
PcG
PcG
KTF
KTF
KTF
KTF
KTF
HtCh
KTF
KTF
(Obuse et al. 2004b)
this work
this work
this work
this work
this work
KTF, kinetochore function; ChRMD, chromatin remodeling; RNH, RNA helicase; CK, cytokinesis; HtCh, heterochromatin; CEN,
centromere localization; CENt, transient centromere localization; PcG, polycomb group protein.
in the bulk chromatin fraction (Fig. 2C, lane 3) all localized to the centromere region, since NChIP of the
bulk chromatin with anti-CENP-A antibodies depleted
ICEN39 from the sup fraction (Fig. 2C, lane 4) and recovered to the ICEN fraction (Fig. 2C, lane 5). Immuno-stain
of MRC-5 (human normal fibroblast) cells in Fig. 2D
showed that dot-like signals of ICEN39 are detected
both in interphase cells (Fig. 2D,b,f ) and these signals colocalize with CENP-C (Fig. 2D,d,h), although the intensity of the dots in interphase 1 (Fig. 2D,b) was very weak.
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2006) 11, 673–684
675
H Izuta et al.
Figure 1 EGFP-tagged ICEN22, 24, 32,
33, 36, 37 and 39 proteins localize to centromeric regions both in interphase and metaphase. EGFP gene or VenusA206K gene
was tagged to the NH2-terminus or COOHterminus of each of the ICEN22, 24, 32,
33, 36, 37 and 39 genes, as described in Experimental procedures (the ICEN number
for each expression gene is written at the
left of each panel). Stable transformants were
established. The cells were fixed with cold
acetone (−20 °C), and stained with antiGFP antibodies (green) and anti-CENP-A
antibodies (red). DNA was stained with
DAPI (blue).
The intensity of ICEN39 in cytoplasm and nucleus is
variable in interphase 1 and 2 (Fig. 2D,b,f). These results
suggest that the endogenous ICEN39/PANE1 distributes both in cytoplasm and in nucleus and also localizes
to centromereric regions in interphase.
Depletion of ICEN22, 24, 32, 33, 36, 37 or 39
proteins by short RNA interference inhibits the
kinetochore function of congression to the
equatorial regions in metaphase
To examine comprehensively the physiological role of
the ICEN components for chromosome segregation, we
prepared siRNA for the ICEN genes and transfected
into HeLa cells. In Fig. 3A we examined sequencespecific siRNA activity to deplete target protein, 2 days
after transfection of GFP-DNA in the absence (upper
panel) or presence (lower panel) of siRNA. The results
indicate that each of the transfected siRNAs effectively
suppresses production of the target protein, but not the
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Genes to Cells (2006) 11, 673–684
off-target protein (si24 RNA against EGFP-22 DNA;
Fig. 3A bottom left panel). As shown in Fig. 3B, abnormal metaphase cells carrying misaligned chromosomes
were observed in cells treated with siRNA for each ICEN
component. As control experiments we also examined
siRNA depletion of hMis6 (ICEN19) and CENP-H
(ICEN35). As shown in Fig. 3C, both CENP-H and
hMis6 signals disappeared in hMis6 (ICEN19)- and/or
CENP-H (ICEN35)-depleted cells, while CENP-A
and CENP-C localization were unaffected at 4 days after
siRNA transfection. Kinetochore function was severely
inhibited and abnormal cells with misaligned chromosomes appeared at an extremely high frequency
(Fig. 3C,D).To establish the frequency of abnormal cells,
we examined 100–150 metaphase cells for misaligned
chromosomes, and the results are summarized in Fig. 3D.
In the cells transfected with siRNA for ICEN32, 33 and
37, the frequency of abnormal metaphase cells (79 ± 2%,
59 ± 6% and 67 ± 6%, respectively) were high as was that
observed in the cells treated with siRNA for CENP-H
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Comprehensive analysis of the ICEN
Figure 2 Endogenous ICEN39/PANE1
protein distributes in both the cytoplasm
and the nucleus and also co-localizes with
the centromere protein in interphase.
(A) Western-blot analysis of the ICEN
fraction (lane 1), that is the pellet fraction after immunoprecipitation with
anti-CENP-A antibodies, and the nonimmune IgG fraction (lane 2). ICEN39
protein in each fraction was detected
with newly prepared anti-ICEN39 monoclonal antibodies (3G8). Lane M shows
a molecular weight marker. ( B) Procedures
for fractionation of the cells to cytoplasm
fraction and nucleus fractions and for purification of the ICEN were shown. Each
fraction number (#1–#5) corresponds to
the lane number in C. (C) Distribution of
ICEN39 protein in fractionated samples
#1–#5 in (B).The proteins in each fraction
were separated by a 12.5% SDS-PAGE and
the ICEN39 protein in each fraction was
detected with Western blotting using 3G8
antibodies. Ratios of the amount of the
applied samples were as follows; cytoplasm
(lane 1): 0.3 M NaCl nuclear extract (lane 2):
bulk chromatin (lane 3): bulk chromatin after
IP (lane 4): ICEN (lane 5) = 0.2 : 1: 1 : 1: 9.
(D) Indirect immunofluoresence microscopy observation of MRC-5 cells (human
fibroblast). The cells were fixed with cold
acetone (−20 °C) and stained with 3G8
antibodies (green) and anti-CENP-C
antibodies (red). DNA was stained with
DAPI.
and hMis6. While the effect of siRNA for ICEN39 is
medium (34 ± 5%) and for ICEN 22, 24 and 36 were
low (19 ± 2%, 14 ± 3% and 15 ± 5%) but significantly
higher than mock cells (4 ± 2%) (Fig. 3D). These results
suggest that ICEN22, 24, 32, 33, 36, 37 and 39 are
required for proper kinetochore function.
Depletion of each of the seven ICEN proteins by
siRNA transfection causes aneuploidy
To check if depletion of ICEN proteins causes aneuploidy as the result of a mitotic defect, FISH analysis
using several centromere probes was performed after
transfection with siRNA for each of the seven ICEN
genes to a diploid colorectal cancer cell line, RKO.
Figure 4A shows examples of aneuploidy observed
by FISH with the DNA sequence from pericentromeric
region of chromosome 7 (CEP7) probe on RKO cells,
3 days after transfection with siRNA. Compared to
control cells, after transfection with siRNA for ICEN
33, 37 or 39, the cells frequently contain more than
two centromere signals, suggesting that the cells have
failed to segregate chromosome 7 in the previous mitosis
(Fig. 4A). To calculate the frequency of aneuploidy,
the cells carrying more than two centromeres of CEP7,
CEP12 and CEP15 were counted. As shown in Fig. 4B,
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2006) 11, 673–684
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H Izuta et al.
Figure 3 siRNA depletion of ICEN22, 24, 32, 33, 36, 37 and 39 proteins. (A) siRNA suppressed expression of EGFP fused genes. The
siRNA sequence for each ICEN gene was selected and its knockdown ability was assessed as suppression activity against over-expression
of the EGFP fused gene, 2 days after transfection. 0.25–0.5 µg of each EGFP-ICEN DNA (ICEN number written at the top of each
panel) was transfected without (upper panel) or with (lower panel) 20 pmoles of each homologous siRNA. For EGFP-ICEN22 DNA
transfection, we additionally co-transfected with heterologous siRNA (for ICEN24) as a control experiment to show that suppression of
the over-expression was sequence-specific. (B, C) Depletion of ICEN proteins with short RNA interference produced abnormal cells
carrying misaligned chromosomes. Each siRNA for ICEN gene (number written at the left side of each panel) and H2O (mock) was
transfected into HeLa cells and incubated at 37 °C for 4 days. The cells were fixed with 2% paraformaldehyde at room temperature and
stained with anti-β-tubulin (green) and anti-CENP-C antibodies (red). DNA was stained with DAPI (blue). (C) siRNA for ICEN19
(hMis6)-transfected cells were stained with anti-hMis6 (green) and anti-CENP-C antibodies (red) (upper panel), or anti-CENP-H
(green) and anti-hMis6 antibodies (red) (lower panel). siRNA for ICEN35 (CENP-H)-transfected cells were stained with CENP-H
(green), CENP-A (red) or hMis6 (red) antibodies. (D) Frequencies of abnormal M-phase cells with misaligned chromosomes as shown
in B and C were counted. The experiments were repeated 2 to 3 times for each siRNA. Mean values and standard deviations are shown
by vertical bars and horizontal lines, respectively.
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Genes to Cells (2006) 11, 673–684
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Comprehensive analysis of the ICEN
Depletion of ICEN22, 32, 33, 37 or 39 proteins by
siRNA transfection inhibits CENP-H and hMis6
loading onto centromeres, but depletion of ICEN24
or 36 does not
Figure 4 Depletion of ICEN22, 24, 32, 33, 36, 37 or 39 protein
by siRNA transfection induces aneuploidy in the diploid cell line
RKO. (A) FISH using the centromere probe CEP7 was
performed 3 days after transfection of siRNA for ICEN into a
diploid colorectal cancer cell line, RKO. Cells transfected with
ICEN33, 37, 39 siRNA showed more than two centromere
signals (33, 37, 39), whereas untransfected control cells were
diploids (ctl). (B) Frequency of polyploidy in RKO cells
transfected with ICEN22, 24, 32, 33, 36, 37, and 39 siRNA.
CEP7, 12 and 15 signals were examined in at least 200 cells each.
Cells with a single stained centromere were not included in the
analysis because it is difficult to judge whether the single staining
is due to monoploidy or incomplete staining.
the frequency of aneuploidy for each chromosome after
transfection with siRNA for ICEN22, 24, 32, 33, 36, 37
or 39 was increased by 1.2–2.5 times compared with that
of control cells. These results suggest that the damage
to kinetochore function due to the depletion of each of
the seven ICEN proteins caused defects in chromosome
segregation and resulted in aneuploidy. It is noteworthy
that the frequency of aneuploidy in si36 cells, in spite of
the low frequency of abnormal cells with misaligned
chromosomes as shown in Fig. 3D, is as high as that
in si32 and si37 cells that show high frequency of misaligned chromosomes.
As shown in Fig. 5, we next examined the effect of siRNA
depletion of each of the seven ICEN proteins on loading
of the known centromeric proteins onto centromeres.
While loading of CENP-A and CENP-C onto centromeres was unaffected by depletion of any of these proteins (Fig. 5A and Fig. 3B, respectively), we observed that
in interphase and/or metaphase cells depleted of ICEN22,
32, 33, 37 or 39, both CENP-H and hMis6 signals were
often lost from the centromeric regions.The frequencies
of loss of the CENP-H and hMis6 signals in interphase
and/or metaphase cells were 40–76% for CENP-H and
70–78% for hMis6 (data not shown). Interestingly, the
abnormal cells carrying lagging chromosomes produced
by siRNA for ICEN 22, 32, 33, 37 or 39-transfection
had often lost all the CENP-H and hMis6 signals, as shown
in Fig. 5. CENP-H and hMis6 signals in cells depleted
of each ICEN were measured for at least 50 normal and
abnormal metaphase cells in each case, and the results
are summarized in Fig. 6, demonstrating that the appearance of misaligned chromosomes for si22, 32, 33, 37 and
39 clearly correlates with the loss of CENP-H and hMis6
signals in metaphase cells. These results suggest that
ICEN22, 32, 33, 37 and 39 proteins are necessary for
CENP-H and hMis6 loading onto centromeres, and that
abnormal cells with lagging chromosomes may be produced as a consequence of the loss of CENP-H and
hMis6 at the centromeric regions. On the other hand,
depletion of ICEN24 or 36 proteins does not affect the
loading of CENP-H or hMis6.
Discussion
In this paper, we have reported a comprehensive analysis of the ICEN components, and the results are summarized in Table 2. Centromeric localization of seven
ICEN proteins (ICEN22, 24, 32, 33, 36, 37 and 39) was
shown using EGFP-tagged genes, and that of endogenous ICEN39 with antibodies (Figs 1 and 2). We surveyed kinetochore function for 16 ICEN components
(data not shown), as well as hMis6 (ICEN19) and CENPH (ICEN35), by depletion using short RNA interference and found that the seven proteins with unknown
function (ICEN22, 24, 32, 33, 36, 37 and 39), in addition to ICEN19 and 35 (hMis6 and CENP-H), had
a role in kinetochore function. We deduced this from
the fact that abnormal metaphase cells carrying misaligned chromosomes appeared at a high frequency
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2006) 11, 673–684
679
H Izuta et al.
Figure 5 CENP-H and hMis6 loading to
centromeres were inhibited by siRNA
depletion of ICEN 22, 32, 33, 37 and 39,
but not by that of ICEN24 and 36. HeLa
cells were transfected with siRNAs for each
ICEN gene (the ICEN number for each
siRNA is written at the left of each panel).
The cells were fixed with 2% paraformaldehyde, 4 days after transfection with
each siRNA, and immunolabeled with (A)
anti-CENP-H (green) and anti-CENP-A
(red) antibodies and (B) anti-hMis6 (green)
antibodies. DNA was stained with DAPI
(blue). Normal metaphase cells (top row)
and abnormal metaphase cells produced by
siRNA transfection (second and lower row)
are shown. Note that CENP-A signals are
unaffected by the siRNA treatment, but
CENP-H and hMis6 signals disappear predominantly from the abnormal metaphase cells
with misaligned chromosomes in samples
22, 32, 33, 37 and 39.
after siRNA depletion (Fig. 3D). In addition, depletion
of each of the seven ICEN proteins frequently induced
aneuploidy in RKO cells (Fig. 4). Misaligned chromosomes in ICEN22, 32, 33, 37 or 39-depleted cells were
closely related to loss of CENP-H and hMis6 signals
from all centromere regions (Figs 5 and 6), suggesting
that ICEN22, 32, 33, 37 and 39 are necessary for
loading of CENP-H and hMis6 onto centromeres.
ICEN39/PANE1
ICEN39 is the same as PANE1 (Proliferation Associated
Nuclear Element 1) that is induced upon transformation
of mouse mammary epithelium by an activated βcatenin (Renou et al. 2003). This protein was reported
to localize in both the cytoplasm and the nucleus, and its
nuclear localization is predominant in actively proliferating cells (Renou et al. 2003). We also observed that the
intensity of the ICEN39 signals in nuclei and centromeres are variable (Fig. 2D), which may be dependent
on the state of the cell.These results suggest that this protein might have a role at the centromeric regions in the
regulation of cell proliferation.
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Genes to Cells (2006) 11, 673–684
ICEN22, 32, 33, 37 and 39
CENP-H and hMis6 associate with each other to form
a complex and are dependent on each other for centromere
localization (Fig. 3C) (Nishihashi et al. 2002). Centromere
localization of the CENP-H/hMis6 complex also requires
ICEN22, 32, 33, 37 and 39 (Figs 5 and 6). These results
suggest that the CENP-H/hMis6 complex functionally
associates with each of the ICEN22, 32, 33, 37 or 39 proteins, implying that some or all of these proteins might
physically associate with each other to form a complex,
and that the stoichiometry of these proteins might be
important for centromere targeting and kinetochore function. Tomonaga et al. (2005) reported that CENP-H overexpression in mouse 3T3 cells inhibited the loading of
endogenous CENP-H onto the centromeric regions,
which could be explained if over-expression of CENP-H
disturbed the stoichiometry of the multiprotein complex.
ICEN24/KLIP1/MLF1IP/CENP-50
The frequency of abnormal cells carrying misaligned
chromosomes in ICEN24 siRNA-transfected cells is rather
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Comprehensive analysis of the ICEN
Figure 6 CENP-H and hMis6 signals were selectively lost in
abnormal cells with misaligned chromosomes in the cells
transfected with siRNAs for ICEN22, 32, 33, 37 and 39, but not
with siRNA for ICEN24 and 36. Frequencies of CENP-H signals
(A) and hMis6 signals (B) for normal metaphase cells () and
abnormal metaphase cells ( ) are shown.
low (14 ± 3%) compared to that found in the case of
ICEN32, 33 or 37 (60–80%) (Fig. 3D), implying that
the production of misaligned chromosomes in this case
might be attributable to a different cause. Centromere
localization of ICEN24 was confirmed using GFP-tagged
genes (Fig. 1). It is noteworthy that ICEN24 protein
does not participate in CENP-H and hMis6 loading onto
centromeres (Figs 5 and 6). It could therefore be considered that inhibition of kinetochore function by depletion of ICEN 24 may involve a pathway different from
the CENP-H/hMis6 complex pathway. A series of protein interaction pathways originating from hMis12 were
reported to take part in kinetochore functions (Obuse
et al. 2004a). The hMis12 interacting proteins, HEC1
and Zwint1, are components of the outer kinetochore
and HP1α and HP1γ are centromeric chromatin components. Another five proteins are unknown, and none
corresponds to ICEN components except HP1γ (ICEN38).
We detected hMis12 signals on the centromeres of the
lagging chromosomes produced by ICEN24 siRNA
(data not shown). ICEN24 was also identified as KLIP1/
MLF1IP/CENP-50 (Pan et al. 2003; Hanissian et al. 2004;
Minoshima et al. 2005). Studies of CENP-50-deficient
DT40 cells indicate that the CENP-50 gene is nonessential, except that recovery from mitotic arrest induced
by nocodazole treatment is delayed substantially (Minoshima
et al. 2005). In human cells, ICEN24 depletion produces
misaligned chromosomes (Fig. 3) and causes aneuploidy
(Fig. 4B), which is different from the situation with
CENP-50 in the chicken. Like human ICEN24, chicken
CENP-50 does not participate in CENP-H/hMis6
loading onto centromeres (Fig. 5). ICEN24/CENP-50
was isolated by two-hybrid selection using MgcRacGAP
as a fishing bait, suggesting that ICEN24/CENP-50
directly interacts with MgcRacGAP (Minoshima et al.
2005). The ICEN includes MgcRacGAP (ICEN17) as
one of its components (Table 1) (Obuse et al. 2004b).
These results suggest that ICEN24 may physically,
and therefore functionally, interact with MgcRacGAP.
We showed that the MgcRacGAP/hMKLP1 complex
localized to centromeres from interphase to early metaphase, and had a role in kinetochore function (our
unpublished observation). Recently, it was also reported
that the Ect2/Cdc42/mDia3 and MgcRacGAP signaling pathway positively regulated the dynamics of spindle
microtubule/kinetochore interaction (Yasuda et al. 2004;
Oceguera-Yanez et al. 2005).
Aneuploidy and cancer
The frequency of misaligned chromosomes (Fig. 3D)
and aneuploidy (Fig. 4B), both of which may be produced by the defect of kinetochore function owing to
siRNA transfection, were not necessarily correlated with
each other. In fact, the frequency of misaligned chromosomes in si22, 24 or 36 cells were only a little higher than
that of control cells (Fig. 3D), but that of aneuploidy is
high (Fig. 4B). This tendency is especially conspicuous
in si36 cells. On the other hand in si33 cells, the frequency of aneuploidy is low, in spite of that of misaligned
chromosomes being relatively high. This discrepancy
may be because the cells become lethal if they suffer substantial damage to their chromosome. Conversely, if the
damage is minimal, the cells may be able to stay alive
with the accumulation of aneuploid chromosomes,
which leads to the development of cancer. It is interesting to investigate whether the expression and/or the
function of ICEN components are deregulated in human
cancers.
Future scope for the ICEN
We have previously reported that DDB1 (ICEN9) is a
component of the centromere complex and suggested that
DDB1 might take part in regulation of heterochromatin
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2006) 11, 673–684
681
H Izuta et al.
Centromere signals for†
siRNA ICEN
Abnormal
Metaphase*
CENP-A
CENP-C
CENP-H
hMis6
Mock
22
24
32
33
36
37
39
35 (cenp-h)
19 (hMis6)
–
+
+
++++
+++
+
++++
++
+++++
+++++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
+
–
–
+
–
–
–
–
+
–
+
–
–
+
–
–
–
–
Table 2 Summary of the results after
transfection of siRNA for the seven ICEN
genes to HeLa cells
*Level of abnormal cells with misaligned chromosomes from Fig. 3C; – < 10%, + 10–
20%, ++ 20–40%, +++ 40–60%, ++++ 60–80%, +++++ > 80%. †Samples with
CENP-H and hMis6 signals < 40% were marked as “–” from Fig. 6.
dynamics around the centromeric region (Obuse et al.
2004b). Jia et al. (2005) have recently reported that Rik1,
Cul4, Swi-6, HDAC and Clr-4 are necessary for heterochromatin formation at the mating type locus and the
pericentromeric region of Schizosaccaromyces pombe, and
that Rik1 is functionally and structurally related to DDB1
and interacts with Cul4. As shown in Table 1, the ICEN
contains DDB1 (ICEN 9), cullin-4 A (ICEN15), HP1γ
(ICEN38), which is one of the Swi6 homologs, and
HDAC1 (ICEN28). These results strongly suggest that
the ICEN contains a set of proteins necessary for centromeric heterochromatin formation. We have shown in
this study that seven ICEN components localize to the
centromeres and have a role in kinetochore function.
These results strongly support the idea that, although
fragmented, the ICEN represents a large CENP-A chromatin complex and therefore may contain many other
protein components with important roles for structure
and function of the centromeric regions. We still have
many ICEN components with unknown centromere
functions, and we believe that studies of these proteins
will open up a new horizon in the structure and function
of centromeres.
Experimental procedures
Bulk chromatin and NChIP
Methods for preparation of bulk chromatin from HeLa cells and
NChIP were fundamentally the same as previously reported
(Obuse et al. 2004b; Yoda & Ando 2004).
Antibodies
Monoclonal antibodies against ICEN39 (clone 3G8) were prepared as described previously (Kimura et al. 1994). A mixture of
amino- and carboxyl-terminal peptides (MSVLRPLDKLPGLNTAC, and CSLLRSSEGPSLEDL, respectively) was
used as antigens. Antibodies against CENP-A, CENP-C, CENPH, hMis6 and hMis12 were described previously (Obuse et al.
2004b). Anti-CENP-A monoclonal antibodies clone 3–19, antiCENP-C from guinea pig, anti-CENP-H and anti-hMis12 from
rabbit, and anti-hMis6 from rat were used. Anti-GFP antibodies
(rabbit) were obtained from MBL and anti-β-tubulin (mouse
monoclonal) from Sigma.
Western blotting
The methods for Western blotting were as described previously
(Ando et al. 2002). Five to ten microliters of the sample was
separated using a 12.5% SDS-PAGE. The separated proteins
were transferred to a PVDF membrane (Millipore), and immunoreaction was for 10–12 h at 4 °C.
Strains and media
HeLa S3 cells and HT1080 cells were grown at 37 °C in DMEM
(Sigma) for monolayer culture or RPMI 1640 (Nissui, Japan) for
suspension culture, supplemented with 5% or 10% calf serum and
antibiotics. MRC-5 and RKO cells were grown in Iscove’s modified Dulbecco’s medium (IMDM < Invitrogen) supplemented
with 10% fetal calf serum and antibiotics.
682
Genes to Cells (2006) 11, 673–684
Construction of the EGFP-ICEN fusion gene
Coding regions of ICEN24, 33, 37 and 39 were amplified by RTPCR from HT1080 total RNA, using the following primers:
24: forward: 5′-AAGCTTCGATGGCCCCGCGGGGGCGGCGGCGGCCG-3′
reverse: 5′-GTCGACTCATCCCTGGTCAAGGAGCTTCTC-3′
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Comprehensive analysis of the ICEN
33: forward: 5′-GAATTCTATGGATTCTTACAGTGCACCAGA-3′
reverse: 5′-GTCGACTCAATTTGAAAAATTGCCAGTTCT3′
37: forward: 5′-GAATTCTATGAATCAGGAGGATCTAGATCC-3′
reverse: 5′-GTCGACTTACTGATGGAAAGCTTCTAATCT3′
39: forward: 5′-CACCATGTCGGTGTTGAGGCCCCTG3′
reverse: 5′-TCACAGGTCCTCCAAGGGAGGG-3′
PCR products of ICEN24, 33 and 37 were cloned into pDrive
vector (QIAGEN) and re-cloned into pEGFP-C1 vector (Clontech) after digestion with EcoRI and SalI for ICEN33 and 37, and
HindIII and SalI for ICEN24. The PCR product of ICEN39 was
cloned into pCR2.1-TOPO vector (Invitrogen) and re-cloned
into pEGFP-C1 after digestion with EcoRI. Coding sequences
for ICEN22, 32 and 36 were amplified by PCR from human
FLJ cDNA clones (Ota et al. 2004) and cloned into pDONR201
vector (Invitrogen).These clones were recloned into pDESTMNVenusA206K vector or pDESTMC-VenusA206K vector, as
described in the company’s instruction manual. pDESTMNVenusA206K-ICEN22 and ICEN32 and pDESTMC-VenusA206KICEN36 were used in this experiment. Venus protein is a
derivative of EGFP protein (Nagai et al. 2002).
Transfection of EGFP-ICEN genes and/or siRNA
and isolation of stable transformants
Transfection of DNA and/or RNA was performed using
Lipofectamine 2000 (Invitrogen) according to the company’s
instruction manual.To isolate stable transformant cells, 1 µg DNA
was transfected into HT1080 cells (1 × 106) and cultured for a
few weeks with DMEM medium containing 400 µg/mL of
G418.
Depletion of ICEN proteins by short RNA
interference
The following short double-stranded RNAs were obtained from
Invitrogen as stealth siRNA:
ICEN22: 5′-AGUUGAGUGGCCAAACAAGGACGAU-3′
ICEN24: 5′-GCAAGCCUAUUGACGUGUUCGACUU-3′
ICEN32: 5′-GCUGCCCTGUUAGACAUCAUUUAUA -3′
ICEN33: 5′-GCCACAAGAUUAGUUCGUGUUUCAA-3′
ICEN36: 5′-CCUUGCAGAGAAACCCACUGUGUAA-3′
ICEN37: 5′-GACUGAAGACGUUCUCAUAACAUUA-3′
ICEN39:5′-UUGACCUGAUCGUGUUUGUGGUUAA-3′
ICEN19 (HMIS6): 5′-AUCAUCAGCAUGUUCAUAAUCUCCC-3′
ICEN35 (CENP-H): 5′-AACAAUUUCCUUAAGGGCAGGAUCC-3′
We usually used four-well slide glasses (Nalge Nunc) coated
with 0.01% poly L-lysine (mol. wt. 150 000–300 000, Sigma).
Twenty picomoles of each siRNA and 1 µL of Lipofectamine
2000 were used in 0.5 mL DMEM medium.
Optical-microscopy observations
The cells were fixed with 2% paraformaldehyde for 30 min at
room temperature or with cold acetone (98%, −20 °C) for 30 min,
and stained with antibodies as indicated in each figure. Second
antibodies were conjugated with FITC (green) or TRITC (red),
and DNA was stained with DAPI (blue). The cells were observed
with a BX51 microscope (Olympus) and images were taken with
a CoolSNAP monochrome camera (Roper Scientific Ltd) under
Openlab version 2 (Improvision Ltd).
FISH analysis
After culture, slides were washed twice with PBS, incubated in
75 mm KCl for 10 min, fixed in 3 : 1 methanol: acetic acid for
10 min at room temperature, and treated with 0.1 mg/mL of RNase
A in 2 × SSC for 30 min at 37 °C.After washing in PBS, slides were
dehydrated by passage through an ethanol series (70%, 85% and
100%), then incubated in 2 × SSC/0.1% Nonidet P-40 solution
for 30 min at 37 °C, and dehydrated again.Target DNA was denatured for 5 min at 73 °C in 70% formamide/2 × SSC (pH 7.3).
Probes (10 µL) to the pericentromeric regions of chromosome 7
(CEP7 Spectrum GreenTM), chromosome 12 (CEP12 Spectrum
OrangeTM), and 15 (CEP15 Spectrum GreenTM) (Vysis, Downers
Grove, IL, USA) were also denatured for 5 min at 73 °C, then
hybridized to the target DNA by incubation overnight at 37 °C. Posthybridization washes were performed 3 times in 50% formamide/
2 × SSC (pH 7.0) for 10 min at 45 °C, once in 2 × SSC and in
2 × SSC/0.1% Nonidet P-40 solution for 5 min at 45 °C.
Hybridization signals were observed and analyzed with Leica
QFISH (Leica QFISH; Leica Microsystems,Tokyo, Japan). At least
200 nuclei of each sample were evaluated for chromosome counts.
Acknowledgements
We thank Hua Yang, Shouhei Goto, Perpelescu Marinela and
Masumi Ishibashi for technical assistance. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas
for K.Y. and by Grants-in-aid for Scientific Research (C) for M. I.
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Received: 7 March 2006
Accepted: 15 March 2006
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.