T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 183 No. 5 805–818
S. Erhardt, B.G. Mellone, and C.M. Betts contributed equally to this paper.
Correspondence to Aaron F. Straight: firstname.lastname@example.org
B.G. Mellone ’ s present address is Dept. of Molecular and Cell Biology, University
of Connecticut, Storrs, CT 06269.
Abbreviations used in this paper: APC, anaphase-promoting complex; ? -ME,
? -mercaptoethanol; BTP, bromothenylpteridine; CENP, centromere protein; CID,
centromere identifi er; CLD, CID localization defi cient; CYCA, cyclin A; DOTAP,
1,2-dioleoyl-3-trimethylammonium-propane; dsRNA, double-stranded RNA; FZR,
FZY related; FZY, fi zzy; IF, immunofl uorescence; LAP, localization and puri-
fi cation; LPC, leupeptin, pepstatin, and chymostatin; Rod, rough deal; TMR*,
tet ramethyl rhodamine*.
The online version of this article contains supplemental material.
Faithful chromosome segregation during mitosis and meiosis is
essential for the proper inheritance of the genome. During cell
division, chromosomes attach to the spindle through a unique
chromosomal structure termed the kinetochore. Kinetochores
monitor proper chromosome attachment to the spindle through
the mitotic checkpoint and couple spindle and motor protein
forces to move chromosomes in prometaphase and anaphase
( Cleveland et al., 2003 ). Centromeres are specialized regions of
the chromosome that serve as the structural and functional foun-
dation for kinetochore formation. Centromeric DNA sequences
are not evolutionarily conserved, and in most eukaryotes, spe-
cifi c DNA sequences are neither necessary nor suffi cient for
centromere formation. Thus, centromere formation is thought
to be epigenetically controlled through chromatin ( Carroll and
Straight, 2006 ).
An excellent candidate for an epigenetic mark that speci-
fi es centromere identity is the centromere protein A (CENP-A)
family of centromere-specifi c histone H3 variants ( Cleveland
et al., 2003 ). CENP-A is present at centromeres throughout the
cell cycle and is essential for the recruitment of kinetochore
proteins, establishment of spindle attachments, and normal chro-
mosome segregation in all eukaryotes, which is consistent with
it functioning as the foundation for the kinetochore ( Carroll and
Straight, 2006 ). Furthermore, CENP-A mislocalization to non-
centromeric regions produces functional ectopic kinetochores,
suggesting that CENP-A chromatin is suffi cient for centromere
formation ( Heun et al., 2006 ).
Despite the central role played by CENP-A in kinetochore
assembly and function, little is known about the mechanisms
that regulate its deposition specifi cally into centromeric chromatin.
study, we isolated factors required for centromere prop-
agation using genome-wide RNA interference screening
for defects in centromere protein A (CENP-A; centromere
identifi er [CID]) localization in Drosophila melanogaster .
We identifi ed the proteins CAL1 and CENP-C as essen-
tial factors for CID assembly at the centromere. CID,
CAL1, and CENP-C coimmunoprecipitate and are mutu-
ally dependent for centromere localization and function.
entromeres are the structural and functional
foundation for kinetochore formation, spindle
attachment, and chromosome segregation. In this
We also identifi ed the mitotic cyclin A (CYCA) and the
anaphase-promoting complex (APC) inhibitor RCA1/
Emi1 as regulators of centromere propagation. We show
that CYCA is centromere localized and that CYCA and
RCA1/Emi1 couple centromere assembly to the cell cycle
through regulation of the fi zzy-related/CDH1 subunit of
the APC. Our fi ndings identify essential components of
the epigenetic machinery that ensures proper specifi ca-
tion and propagation of the centromere and suggest a
mechanism for coordinating centromere inheritance with
Genome-wide analysis reveals a cell cycle – dependent
mechanism controlling centromere propagation
Sylvia Erhardt , 1,2,4 Barbara G. Mellone , 1,2 Craig M. Betts , 3 Weiguo Zhang , 1,2 Gary H. Karpen , 1,2 and Aaron F. Straight 3
1 Department of Genome Dynamics, Lawrence Berkeley National Laboratory and 2 Department of Molecular and Cell Biology, University of California, Berkeley,
Berkeley, CA 94720
3 Department of Biochemistry, Stanford Medical School, Stanford, CA 94305
4 Zentrum f ü r Molekulare Biologie, Universit ä t Heidelberg, D-69120 Heidelberg, Germany
© 2008 Erhardt et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
JCB • VOLUME 183 • NUMBER 5 • 2008 806
We have focused detailed experiments on the four CLD
genes that when depleted displayed the strongest levels of CID
loss from centromeres ( Fig. 1 A and Fig. S1 B). The CLD1 gene
encodes the Drosophila homologue of the essential CENP-C
( Heeger et al., 2005 ). Centromere localization of CENP-C
depends on CENP-A in many eukaryotes ( Carroll and Straight,
2006 ), but in this study, we observe that CENP-A and CENP-C
are reciprocally dependent for centromere localization. CLD2
is encoded by the CG5148 gene and was recently identifi ed
as CAL1 in a screen for mitotic defects in Drosophila tissue
culture cells ( Goshima et al., 2007 ). CAL1 contains a putative
ubiquitin interaction domain, and homology searching iden-
tifi ed clear homologues in drosophilids (Fig. S2, available at
et al., 2007 ) but not in other eukaryotes. Interestingly, CLD3
encodes the Drosophila cyclin A (CYCA) protein, and CLD4
encodes the regulator of CYCA, RCA1 (Emi1 in vertebrates;
Lehner and O ’ Farrell, 1989 ; Dong et al., 1997 ; Machida and
Dutta, 2007 ). Although mitotic cyclins have recently been local-
ized to centromeres ( Bentley et al., 2007 ; Nickerson et al., 2007 ),
neither CYCA nor RCA1 has been implicated in centromere
assembly or maintenance. RCA1 protects CYCA from degradation
by inhibiting the fi zzy (FZY)-related (FZR)/CDH1 anaphase-
promoting complex (APC [APC FZR/CDH1 ]; Grosskortenhaus and
Sprenger, 2002 ), suggesting that loss of CID after RCA1 deple-
tion is the result of premature CYCA degradation.
Localization and dynamics of CLD proteins
To determine the localization of CLD proteins, we analyzed the
distributions of GFP-CLD fusions in live and fi xed Drosophila
S2 cells ( Fig. 1 B and Fig. S3 A, available at http://www.jcb.org/
cgi/content/full/jcb.200806038/DC1) and confi rmed localization
of CYCA and CENP-C by antibody staining in untransfected cells
(Fig. S3 A). We performed time-lapse analysis of living cells
stably expressing GFP-tagged proteins to determine the dynamics
of CLD proteins during the cell cycle ( Fig. 1 B and Videos 1 – 5).
As previously reported, CENP-C colocalized with CID through-
out the cell cycle (Fig. S3 A and Video 2; Heeger et al., 2005 ).
CYCA was recently shown to localize to centromeres in mouse
spermatocytes during late diplotene and prometaphase of meio-
sis I ( Nickerson et al., 2007 ) but has not been previously re-
ported to be centromeric in Drosophila embryos or somatic cells
( Lehner and O ’ Farrell, 1989 ). We analyzed CYCA localization
in Drosophila cells and found that, in addition to its broader
cellular distribution, CYCA is enriched at centromeres in both
interphase and mitosis ( Fig. 1 B and Fig. S3, A and B). Costaining
with antibodies to CYCA and different cell cycle markers (not
depicted) and time-lapse analysis of CYCA-GFP (Video 4) dem-
onstrated that centromere enrichment increased signifi cantly
after entry into mitosis and decreased after entry into anaphase,
which is coincident with the general degradation of CYCA
( Lehner and O ’ Farrell, 1989 ). GFP-RCA1 displayed no obvi-
ous concentration at centromeres or chromatin and instead was
distributed throughout the nucleus in early M phase, disappeared as
M phase continued, and reappeared early in G1 ( Fig. 1 B , Fig. S3 A,
and Video 5) in a manner similar to that reported in cycle 16
Drosophila embryos ( Grosskortenhaus and Sprenger, 2002 ).
In Schizosaccharomyces pombe , mutations in Mis6 cause loss of
Cnp1/CENP-A ( Takahashi et al., 2000 ) from centromeres. The
vertebrate Mis6 homologue CENP-I promotes proper localiza-
tion of newly synthesized CENP-A but is not essential for main-
taining previously assembled CENP-A ( Okada et al., 2006 ). Mis16,
the S. pombe homologue of the Drosophila melanogaster p55
subunit of CAF1 (chromatin assembly factor 1) and RbAp46/48
(human retinoblastoma-associated protein 46 and 48), is neces-
sary for CENP-A localization in fi ssion yeast and human cells
( Hayashi et al., 2004 ). The Drosophila p55 protein facilitates
CENP-A nucleosome assembly in vitro ( Furuyama et al., 2006 ),
although the specifi city of this reaction for CENP-A is unknown.
In fi ssion yeast, another histone chaperone, Sim3, is required
for Cnp1/CENP-A deposition at centromeres; however, Sim3 also
acts as a chaperone for histone H3, so it is unlikely to provide
centromere specifi city ( Dunleavy et al., 2007 ).
Currently, the best candidate proteins for regulating the
specifi city of CENP-A assembly in eukaryotes are S . pombe
Mis18 and its homologues and binding partners in metazoans
(Mis18- ? , Mis18- ? , and M18BP1/KNL2). Depletion of these
proteins in S. pombe , Caenorhabditis elegans , or human cells re-
sults in a failure to incorporate CENP-A at centromeres ( Hayashi
et al., 2004 ; Fujita et al., 2007 ; Maddox et al., 2007 ). In human
cells, these proteins only localize to centromeres during late ana-
phase to telophase/G1 ( Fujita et al., 2007 ; Maddox et al., 2007 ).
Recent data demonstrating that human CENP-A assembly occurs
between telophase and the following G1 and that the Drosophila
centromere identifi er (CID) assembles into the centromere during
anaphase ( Jansen et al., 2007 ; Schuh et al., 2007 ) suggest that
activity or removal of the Mis18 complex and mitotic exit may
be necessary for centromere formation. However, it is unclear
whether known centromere-localized factors regulate CENP-A
transcription, translation, nuclear import, chromatin assembly, or
maintenance. The identifi cation of factors required for CENP-A
localization, without bias for a particular model or biological pro-
cess, is a strategy that is likely to provide new insights. In this
study, we report a genome-wide RNAi screen that identifi ed new
factors required for Drosophila CID localization to centromeres.
This screen revealed signifi cant interdependence between novel
and known CENPs for centromere assembly and a novel link be-
tween centromere propagation and the cell cycle machinery.
Genome-wide screen for CID localization –
defi cient (CLD) genes
The generation of double-stranded RNA (dsRNA) collections
homologous to ? 24,000 genes and predicted genes in the Dro-
sophila genome ( Kiger et al., 2003 ) allowed us to perform a
genome-wide RNAi screen to identify factors required for nor-
mal centromere localization of CID (Fig. S1, available at http://
www.jcb.org/cgi/content/full/jcb.200806038/DC1; see Materials
and methods). We directly screened for loss of CID by immuno-
fl uorescence (IF) after dsRNA depletion, allowing rapid, unbi-
ased identifi cation of genes that specifi cally affected centromere
propagation rather than relying on an indirect phenotype such
as chromosome missegregation or cell lethality.
807CENTROMERE PROPAGATION MECHANISMS • Erhardt et al.
Figure 1. Identifi cation and localization of
CLDs. (A) IF of cells depleted of the top four posi-
tive hits from the screen. Cells were stained for
DNA, CID, and HP1. CID localization to the
centromere (bottom) is highly reduced or absent
after RNAi depletion of all four candidates or
CID in comparison with the control (left), and HP1
staining is normal in all cases. Notice that in ad-
dition to CID depletion, RCA1 and CYCA RNAi
result in endoreduplication and an increase in
nuclear size. (B) CLD dynamics through mitosis.
Still images from time-lapse analysis of stable S2
cell lines expressing mCherry-tubulin (red) and
GFP-CID, – CENP-C, -CAL1, -CYCA, and -RCA1;
relative times in minutes from the start of the
video are indicated in each frame. See Videos
1 – 5 (available at http://www.jcb.org/cgi/
content/full/jcb.200806038/DC1). (C) Local-
ization of GFP-CAL1, CID, and Rod. Metaphase
chromosome spreads of S2 cells stably express-
ing GFP-CAL1 were stained with anti-CID (red),
anti-GFP (green), and anti-Rod (blue) antibodies.
The GFP-CAL1 signal overlapped signifi cantly
with the outer kinetochore protein Rod, whereas
only a little overlap was observed with CID, in-
dicating that CAL1 is located close to the outer
kinetochore in mitotic chromosomes, which is in
contrast to localization to CID chromatin in inter-
phase. Inset shows magnifi cation of image. Bars:
(A) 15 μ m; (B and C) 5 μ m.
JCB • VOLUME 183 • NUMBER 5 • 2008 808
Figure 2. Mitotic defects caused by CLD depletion. (A) FACS analysis of control and CLD-depleted Kc167 cells. The graph shows the distribution of the
DNA content of control and dsRNA-treated cells after a 4-d incubation with dsRNA. Ploidy is shown on the x axis. A high number of polyploid cells ac-
cumulated after CYCA and RCA1 depletion. A milder effect on ploidy was observed in CENP-C – depleted cells. (B) The number of defective mitoses was
quantifi ed after a 4-d incubation with control (no RNA; n = 38), CID ( n = 53), CENP-C ( n = 50), CAL1 ( n = 58), CYCA ( n = 31), or RCA1 ( n = 34) dsRNA.
n represents the number of cells examined in each condition. The percentage of cells with abnormal prometaphase or metaphase fi gures is shown on the
left, and the percentage of cells with abnormal anaphase or telophase fi gures is shown on the right. The data are taken from a single experiment, but
a duplicate experiment yielded similar results. (C) Still frames from time-lapse experiments of mitotic defects associated with RNAi depletion of CLDs in
cells expressing mCherry-tubulin and H2B-GFP. Control cells (left) displayed accurate and timely chromosome segregation. CID-, CENP-C – , and CAL1-
depleted cells showed a dramatic mitotic delay, little to no chromosome movement, abnormally elongated and defective spindles, and chromosome
809 CENTROMERE PROPAGATION MECHANISMS • Erhardt et al.
missegregation; nevertheless, cytokinesis occurred. CYCA depletion caused defective spindles, missegregation of chromosomes, and cytokinesis defects.
RCA1 depletion predominantly caused a cell cycle arrest; most cells did not divide after RNAi treatment. Times are minutes from the start of the video
(see Videos 6 [control], 7 [CID-RNAi], 8 [CENP-C – RNAi], 9 [CAL1-RNAi], and 10 [CYCA-RNAi], available at http://www.jcb.org/cgi/content/full/
jcb.200806038/DC1). Bars, 5 μ m.
GFP-CAL1 colocalized with CID at centromeres in inter-
phase cells, which is consistent with previous localization data
( Goshima et al., 2007 ), and also displayed diffuse staining asso-
ciated with the nucleolus, around which Drosophila centro-
meres typically cluster ( Fig. 1 B and Fig. S3, A and C). In
metaphase cells, GFP-CAL1 associated with centromeres and
also displayed weak staining throughout the nucleus. Colocal-
ization with the outer kinetochore protein rough deal (Rod;
Scaerou et al., 1999 ) suggested that GFP-CAL1 is associated
with the outer kinetochore in mitotic chromosomes ( Fig. 1 C ).
However, GFP-CAL1 colocalizes with CID in interphase when
there is no outer kinetochore. In time-lapse experiments (Video 3),
GFP-CAL1 was highly concentrated at centromeres during in-
terphase and prophase, became less prominent at centromeres at
metaphase, and then increased again starting in late anaphase.
These results indicate that CAL1 is a constitutive CENP with
unique centromere and kinetochore localization dynamics.
CLD protein depletion results in
chromosome segregation defects
Defects in centromere and kinetochore assembly cause chromo-
some missegregation, mitotic delays, and defects in mitotic spindle
assembly. FACS analysis showed little change in DNA content
after CID and CAL1 RNAi as compared with control cells
( Fig. 2 A ), although CAL1 depletion caused a fourfold increase in
the mitotic index (control = 0.9 ± 1.7% of mitotic cells and CAL1
RNAi = 3.7 ± 1.6% of mitotic cells; error = SD; P < 0.005).
In contrast, depletion of CENP-C, CYCA, or RCA1 caused a
signifi cant increase in cell ploidy. CYCA- and RCA1-depleted
cells showed progressive accumulation of polyploid cells by
FACS analysis resulting in a > 4N DNA content ( Fig. 2 A ) and a
large increase in nuclear size in 85% of CYCA and 88% of
RCA1 cells compared with 1% of control cells. These changes
in DNA content and nuclear size are consistent with endo-
reduplication of DNA without intervening cell division previously
reported for CYCA- and RCA1/Emi1-depleted Drosophila and hu-
man cells ( Mihaylov et al., 2002 ; Machida and Dutta, 2007 ).
Analysis of fi xed cells after depletion of CID, CAL1, or
CENP-C demonstrated that > 80% of mitoses had defects in pro-
metaphase and metaphase chromosome alignment, and > 60% of
cells showed defective anaphases and telophases with missegre-
gated or lagging anaphase chromosomes (P < 0.01 compared with
controls; Fig. 2 B ). 2.7% of CYCA- and 1.8% of RCA1-depleted
cells entered mitosis, of which 80% and 66%, respectively, had
abnormal anaphases and telophases with missegregated and
lagging chromosomes (P < 0.01 compared with controls). The
defects in prometaphase and metaphase stages after CYCA and
RCA1 depletion were not signifi cantly different from controls.
Time-lapse analysis of untreated S2 cells stably expressing
mCherry-tubulin and histone H2B-GFP showed normal pro-
gression through mitosis ( Fig. 2 C , control; and Video 6, available
Consistent with previous observations, CID-depleted cells were
delayed in mitosis with defective spindles, abnormal chromo-
some condensation, and severe chromosome segregation defects
( Fig. 2 C and Video 7; Blower and Karpen, 2001 ; Blower et al.,
2006 ; Heun et al., 2006 ). CENP-C and CAL1 were identifi ed
recently in a mitotic defects screen in Drosophila S2 cells
( Goshima et al., 2007 ), and our time-lapse analysis showed that
CENP-C or CAL1 depletion resulted in defective spindles and
chromosome condensation, little or no chromosome movement,
and unequal chromosome segregation ( Fig. 2 C and Videos 8 and 9).
The CYCA- ( Fig. 2 C and Video 10) and RCA1-depleted
( Fig. 2 C ) cells that entered mitosis displayed defective spindles,
defective chromosome segregation, a mitotic delay with con-
densed chromosomes, and failed cytokinesis resulting in multi-
nucleate cells (control = 0.9%, CYCA = 4.5%, and RCA1 = 5%
multinucleate cells; Fig. 2 C ). The mitotic defects we observe
after CYCA and RCA1 depletion are likely to be the result of
both the loss of centromere function and deregulation of the cell
cycle caused by the loss of CYCA-dependent kinase activity.
Genetic and physical interactions
We examined the interdependence between CLDs for centro-
mere targeting by depleting each gene and localizing the other
CLD proteins. Consistent with the results from our screen, epis-
tasis analysis showed that CID is dependent on all the CLDs for
its localization ( Figs. 1 A and 3 A ). The depletion of CID,
CAL1, CYCA, or RCA1 all resulted in the loss of CENP-C
from centromeres ( Fig. 3 A ). In addition, CAL1 depletion caused
diffuse localization of CENP-C throughout the nucleus, sug-
gesting a delocalization of CENP-C from the centromere rather
than loss of CENP-C protein. CID, CENP-C, CYCA, or RCA1
depletion resulted in signifi cant loss of CAL1 from centromeres,
although CENP-C depletion produced residual, diffuse CAL1
staining, suggesting delocalization and not the loss of CAL1
( Fig. 3 B ). The impact of CYCA and RCA1 depletion was weaker
than observed for CID and CENP-C depletion ( Fig. 3 B ). CYCA
enrichment at interphase centromeres was lost after CID,
CENP-C, or CAL1 RNAi, and depletion of RCA1 resulted in a
general decrease in CYCA staining ( Fig. 3 C ). Similar relation-
ships were observed when we examined centromere localiza-
tion dependencies in mutant embryos (Fig. S4 A, available at
onstrating the importance of these factors for centromere local-
ization in animals. We conclude that the centromere localizations
of CID, CAL1, CENP-C, and CYCA exhibit signifi cant inter-
dependence ( Fig. 3 E ).
Previous studies in human cells demonstrated that CENP-A
and CENP-C coimmunoprecipitate, although a direct interaction
has not been demonstrated ( Ando et al., 2002 ; Foltz et al., 2006 ).
JCB • VOLUME 183 • NUMBER 5 • 2008 810
sion proteins, and Western blotting with CID, CENP-C, CAL1,
and CYCA antibodies ( Fig. 3 D ). We observed reciprocal co-
immunoprecipitation of CID, CENP-C, and CAL1, suggesting
that they are present in one or more complexes; however, we did
We examined the physical interactions between CID and CLDs
by digesting chromatin from cell lines expressing GFP-CID or
GFP-CLDs to primarily mono- and dinucleosomes with micro-
coccal nuclease (Fig. S4 B), immunoprecipitating the GFP fu-
Figure 3. CLDs are interdependent for centromeric localization and are physically associated. (A) CLD depletions cause CID and CENP-C mislocaliza-
tion. Control cells showed strong centromeric localization of CID (red) and CENP-C (green; merged panel across top; DAPI shown in gray). Depletion of
CID, CENP-C, CAL1, CYCA, and RCA1 all resulted in absent or reduced centromeric staining of CID and CENP-C. Note that CAL1 depletion resulted
in diffuse mislocalization of CENP-C in the nucleus, which is in contrast to the elimination of CENP-C observed after CID depletion. In addition, residual
centromeric CID staining was observed after CENP-C depletion. (B) CID or CLD depletion causes loss of centromeric CAL1. Control cells showed strong
colocalization between CID (red) and GFP-CAL1 (green), whereas cells depleted for CID, CENP-C, CYCA, or RCA1 displayed severely reduced centro-
meric CAL1. (C) Centromeric enrichment of CYCA (green) was visible in control cells and was lost in cells depleted for CID (red) after CID, CENP-C, or
CAL1 RNAi (DNA is shown in gray). RCA1 depletion resulted in a general decrease in CYCA staining. (D) Coimmunoprecipitation of CLDs. GFP-CLD
fusions were immunoprecipitated from stable cell lines expressing GFP-CID, GFP – CENP-C, or GFP-CAL1 or from the parent Kc167 cell line (control).
Immunoprecipitates were Western blotted with antibodies against CID (top), CENP-C (middle), and CAL1 (bottom). The position of the GFP fusion and
the endogenous protein is labeled on the right. (E) Summary of epistatic and physical relationships. CID, CAL1, and CENP-C physically interact and
are interdependent for centromere localization. CID, CAL1, and CENP-C require RCA1 and CYCA for centromere localization, and CYCA requires all
three for centromere enrichment. Bars, 5 μ m.
811 CENTROMERE PROPAGATION MECHANISMS • Erhardt et al.
because mitotic Cdk activity and geminin fail to accumulate,
resulting in aberrant relicensing and reinitiation of replication
origins ( Arias and Walter, 2007 ; Machida and Dutta, 2007 ).
The recent observation that human CENP-A assembly oc-
curs in late mitosis/G1 ( Jansen et al., 2007 ) raised the possibility
that the loss of CID, CAL1, and CENP-C after CYCA or RCA1
depletion could result indirectly from their effects on cell cycle
progression and replication. RNAi depletion of geminin causes
endoreduplication without affecting CYCA levels ( Mihaylov
et al., 2002 ). After depletion of geminin from cultured Drosophila
cells, CID and CENP-C localization to centromeres was unper-
turbed ( Fig. 5 A ). Furthermore, in normally occurring endoredu-
plicated embryonic nuclei and highly polytenized salivary gland
cells (not depicted), we observed CID localization to centromeres
( Fig. 5 B ). We conclude that mislocalization of CID, CENP-C,
and CAL1 after CYCA or RCA1 depletion is not caused by the
dilution of CID at centromeres after endoreplication.
RCA1 and CYCA – Cdk1 both function to inhibit the activities
of the APC. We investigated the roles of RCA1, CYCA, and the
APC in centromere assembly in more detail by determining
whether the APC activators FZR/CDH1 and FZY/CDC20 af-
fected CID, CAL1, and CENP-C centromere localization or nu-
clear protein levels. RCA1 or CYCA depletion resulted in CID
and CENP-C mislocalization ( Fig. 6, A and B ) as well as a sig-
nifi cant reduction in CID protein levels in nuclear extracts,
although RCA1 depletion had a stronger effect than CYCA
depletion on CID protein levels ( Fig. 6 C ). RNAi depletion of
FZY or FZR alone did not reduce CID or CENP-C localization
or levels (Fig. S5 A, available at http://www.jcb.org/cgi/content/
full/jcb.200806038/DC1). However, simultaneous depletion of
CYCA and FZR or RCA1 and FZR rescued the CID and CENP-C
centromere mislocalization phenotypes ( Fig. 6, A and B ) and
restored CID protein levels ( Fig. 6 C ). In contrast, simultaneous
depletion of FZY/CDC20 and RCA1 or CYCA had no signifi -
cant effect on CID or CENP-C mislocalization ( Fig. 6 B ), which is
consistent with previous observations that RCA1 primarily inhibits
FZR/CDH1 and not FZY/CDC20 in Drosophila ( Grosskortenhaus
and Sprenger, 2002 ). We conclude that the APC activator
FZR plays a role in the CYCA- and RCA1-dependent regula-
tion of centromere assembly.
CYCA could control centromere propagation either through
CYCA – Cdk1 phosphorylation of centromere-specifi c substrates
or through inhibitory phosphorylation of the APC. Cells depleted
for both CYCA and FZR have low CYCA levels that are equiva-
lent to the amounts observed after RCA1 depletion ( Fig. 6 C ).
However, these cells still display normal centromere localization
of CID and CENP-C ( Fig. 5 A ), demonstrating that normal levels
of CYCA and its associated kinase activity are not required for
centromere propagation. In Drosophila , the primary roles for
CYCA – Cdk1 kinase and RCA1 are to inhibit premature APC ac-
tivation so that A- and B-type cyclin can accumulate ( Dienemann
and Sprenger, 2004 ). In the absence of either inhibitor, mitotic cy-
clins are prematurely destroyed ( Grosskortenhaus and Sprenger,
2002 ; Mihaylov et al., 2002 ). Deletion of the fi rst 55 amino acids
FZR/CDH1 activity regulates centromere
not detect association of CYCA with any of the immunoprecipi-
tated proteins (unpublished data). The stable cell lines express
the GFP-CLD fusions at or below the level of the endogenous
protein, indicating that detecting the interactions between these
proteins is not the result of overexpression (Fig. S4 C). A previ-
ous study suggested that CID exists as half octamers or “ hemi-
somes ” consisting of one copy each of CID, H4, H2A, and H2B
( Dalal et al., 2007 ). However, our copurifi cation of equal amounts
of tagged and untagged CID from cells expressing lower amounts
of tagged CID than endogenous CID is consistent with a Dro-
sophila centromeric nucleosome containing more than one mole-
cule of CID. We conclude that the interdependency of CID,
CENP-C, and CAL1 centromere localization refl ects the physi-
cal association of these proteins. At this time, it is unclear
whether the interactions are direct and whether these proteins
are present in one or more complexes.
CAL1 and CENP-C regulation of CID
We analyzed the effects of CAL1 and CENP-C depletion on lo-
calization of newly synthesized CID to delineate roles for CAL1
and CENP-C in CID assembly versus maintenance. Newly syn-
thesized CID was distinguished from preexisting CID by fusing
CID to a modifi ed O 6 -alkylguanine-DNA alkyltransferase
also known as the SNAP tag, which selectively reacts with
O 6 -benzylguanine derivatives. This tagging method has previously
been used to monitor new CENP-A assembly in human cells
( Jansen et al., 2007 ). We depleted CAL1 and CENP-C from Kc167
cells expressing SNAP-tagged CID and blocked preexisting
SNAP-tagged CID in these cells with bromothenylpteridine (BTP).
We then allowed time for the new synthesis of CID in the ab-
sence of CAL1 or CENP-C followed by labeling of SNAP-CID
with a red fl uorescent O 6 -benzylguanine derivative (tetramethyl
rhodamine* [TMR*]). We observed that CENP-C or CAL1 de-
pletion prevented the centromere localization of newly synthe-
sized CID ( Fig. 4 ). We conclude that CENP-C and CAL1 are
required for centromeric deposition of newly synthesized CID.
Currently, we cannot rule out an additional role for CENP-C and
CAL1 in the maintenance of CID in centromeric chromatin.
Centromere defects after CYCA or RCA1
depletion do not result from overreplication
A surprising result of our analysis was the requirement for
CYCA and RCA1 in centromeric localization of CID, CENP-C,
and CAL1. In metazoan cells, mitotic Cdk activity prevents re-
replication by inhibiting the assembly of prereplication complexes
( Arias and Walter, 2007 ). APC-dependent proteolysis of mitotic
cyclins and geminin allow prereplication complex assembly in
anaphase followed by a single round of replication in S phase.
A central role for both CYCA and RCA1 in cell cycle control is
to inhibit the activity of the APC. CYCA in complex with either
Cdk1 or Cdk2 (CYCA – Cdk) directly phosphorylates and inac-
tivates the APC activator FZR/CDH1, and RCA1 binds to FZR
and prevents the interaction of APC FZR/CDH1 with APC substrates
( Zachariae et al., 1998 ; Jaspersen et al., 1999 ; Lukas et al.,
1999 ; Grosskortenhaus and Sprenger, 2002 ). In the absence of
CYCA or RCA1/Emi1, cells overreplicate their genomes
JCB • VOLUME 183 • NUMBER 5 • 2008 812
Figure 4. CENP-C and CAL1 are required for centromeric localization of newly synthesized CID. (A) Centromeric localization of newly synthesized SNAP-
CID is defective after CENP-C or CAL1 depletion. Localization of DNA (DAPI), total SNAP-CID (SNAP Ab), and newly synthesized SNAP-CID (TMR*) are
shown from representative images of cells treated with DOTAP transfection reagent only (control, top), CENP-C dsRNA (CENP-C RNAi, middle), and CAL1
dsRNA (CAL1 RNAi, bottom), respectively. Total SNAP-CID (SNAP Ab) and newly synthesized SNAP-CID (TMR*) overlap in a typical punctate centromere
pattern in control cells that is not visible in CENP-C – or CAL1-depleted cells. Each image in the right panels shows an individual cell from the merge panels
(dashed circle) at increased magnifi cation. (B) Quantifi cation of frequencies of cells with different intensities of TMR* labeling at centromeres in control
cells or after CENP-C (CENP-C RNAi) or CAL1 depletion (CAL1 RNAi). Centromeric TMR* signal intensities were categorized into strong (green), medium
(yellow), weak (red), or no signal (blue). The total numbers of cells used in the analysis are indicated above each graph. Most control cells display strong
to medium TMR* signals at centromeres, representing normal centromere localization of newly synthesized CID. In contrast, the majority of cells contained
weak or no TMR* signal after CENP-C and CAL1 RNAi, suggesting defective localization of newly synthesized CID and a role for these proteins in CID
assembly. Bars, 15 μ m.
813 CENTROMERE PROPAGATION MECHANISMS • Erhardt et al.
CENP-A in yeast and fl ies and that CENP-A at centromeres
is protected from proteolysis ( Collins et al., 2004 ; Moreno-
Moreno et al., 2006 ). Our results suggest that APC FZR/CDH1 activ-
ity also controls centromere propagation by coupling centromeric
chromatin assembly to the cell division cycle. In the absence of
CYCA and RCA1, the premature activation of APC FZR/CDH1
may result in the premature degradation of an APC substrate
required for centromere propagation. CID levels are signifi -
cantly reduced when CAL1 or RCA1 is depleted from cells
( Fig. 6 C ), and FZR/CDH1 RNAi rescued the reduction in
from the amino terminus of CYCA ( Δ 55-CYCA) removes the de-
struction box and stabilizes CYCA by preventing its APC-mediated
ubiquitination and destruction by the proteasome ( Kaspar et al.,
2001 ). We observed that expression of nondestructible CYCA
in Drosophila cells depleted of RCA1 suppressed the loss of CID
from centromeres (Fig. S5, C and D). Together, these observations
demonstrate that proper regulation of APC FZR/CDH1 activity is essen-
tial for controlling centromere propagation.
Previous studies showed that proteolysis facilitates for-
mation of a single centromere by degrading noncentromeric
Figure 5. Defects in centromere assembly
after CYCA and RCA1 depletion do not result
from overreplication. (A) Geminin RNAi causes
overreplication without loss of CID or CENP-C
from centromeres. Fluorescence images of
DNA, CID, and CENP-C in control cells and
cells depleted of geminin. Large nuclei are in-
dicative of overreplication in geminin-depleted
cells without disruption of CID or CENP-C local-
ization. The graph shows quantifi cation of CID
and CENP-C levels at centromeres with and
without geminin depletion ( n = 4; error bars
represent SEM). (B) Endoreduplication naturally
occurs in Drosophila embryos and larvae. CID
(red) is present at the centromere in embry-
onic cells with endoreduplicated chromosomes
stained for histone H3Ser10 phosphorylation
(green). Bars: (A) 15 μ m; (B) 5 μ m.
JCB • VOLUME 183 • NUMBER 5 • 2008 814
We have screened for genes that regulate the centromeric local-
ization of CENP-A/CID. This is the fi rst example of a genome-
wide RNAi screen for mislocalization of an endogenous
chromosomal protein and provides the distinct advantage that
the primary screen output is a direct readout of the phenotype of
interest. This approach identifi ed novel and known factors that
control the assembly of centromeric chromatin and link centro-
mere assembly and propagation to the cell cycle.
Although centromere assembly has been described as a
hierarchical process directed by CENP-A, our data show that
CID, CENP-C, and CAL1 are interdependent for centromere
propagation, which is consistent with experiments in vertebrate
CID protein levels that occurred after RCA1 depletion ( Fig. 6 C ).
CID destabilization after CAL1 depletion could not be rescued
by depletion of either FZR/CDH1 or the APC subunit CDC27
(Fig. S5 A), and CID localization to centromeres in CAL1-
depleted cells was not suppressed by the expression of Δ 55-CYCA
(Fig. S5 B). These results suggest that CID loss in CAL1-depleted
cells does not result from APC FZR/CDH1 degradation. The level of
CAL1 at centromeres declines between metaphase and late
anaphase, and CAL1 levels increase in the absence of FZR
( Fig. 6 C ). In contrast, CENP-C protein levels were not
strongly affected by inhibition of CID, CAL1, CYCA, RCA1,
and/or APC FZR/CDH1 ( Fig. 6 C ). Our data suggest that CYCA,
CID, and CAL1 are possible APC FZR/CDH1 substrates that con-
trol the propagation of the centromere during cell division.
Figure 6. Rescue of centromere assembly by APC inhibition. (A) CYCA and RCA1 disruption of CID localization is rescued by FZR depletion. Localization
of DNA (top), CID (middle), and CENP-C (bottom) in Kc167 cells treated with CYCA, RCA1, CYCA + FZR, or RCA1 + FZR dsRNAs. Control cells were not
treated with dsRNA. (B) Quantifi cation of CID and CENP-C fl uorescence intensity levels at centromeres after RNAi treatment. Quantifi cation of FZY-depleted
cells is also included, showing specifi city of the rescue by FZR depletion ( n = 2; error bars represent SEM). (C) Protein levels of CID (31 kD), CENP-C
(160 kD), CAL1 (120 kD), and CYCA (60 kD) after RNAi depletion in Kc167 cells. Nuclear extracts from Kc167 cells treated with no dsRNA (control) or
dsRNA specifi c for CID, CENP-C, CAL1, CYCA, RCA1, FZR, CYCA + FZR, or RCA1 + FZR were Western blotted with antibodies against CLD genes or tubulin
(50 kD) as a control. (D) Model for the role of CLD genes in cell cycle regulation of centromere assembly. The green background indicates proteins that
localize to the centromere, orange indicates that the proteins are not competent for centromere assembly, and double arrowheads indicate that the protein
or complex is required for the centromere localization of CYCA and CID-, CAL1-, and CENP-C – interdependent assembly. The APC substrate could be CYCA
(red arrow) or CID, CAL1, or an unidentifi ed substrate, X (blue arrow). Bar, 15 μ m.
815 CENTROMERE PROPAGATION MECHANISMS • Erhardt et al.
APC in G2 to allow mitotic cyclin accumulation. An APC FZR/CDH1
substrate could repress centromere assembly until anaphase/G1,
when proteolysis would remove the repression in a manner
analogous to replication licensing. If an APC FZR/CDH1 substrate
acted solely as a negative regulator of centromere assembly,
FZR/CDH1 depletion should prevent CID assembly at centro-
meres, and premature APC FZR/CDH1 activation by CYCA or RCA1
depletion might cause an increase of CID at centromeres as a
result of premature assembly. We observed that neither CDH1
nor CDC20 depletion alone impacted CID, CAL1, or CENP-C
assembly at centromeres or the overall levels of these proteins
but that premature APC activation resulted in failed centromere
assembly ( Fig. 6, A and B ; and Fig. S5 A).
A simple interpretation of our results is that CYCA – Cdk1
or another APC FZR/CDH1 substrate acts during G2/metaphase be-
fore APC FZR/CDH1 activation to make centromeres competent for
assembly during anaphase and/or G1 ( Fig. 6 D ). Premature re-
moval of the APC FZR/CDH1 substrate would cause failure to re-
license the centromeres for assembly in the next G1 phase. When
compared with the process of replication licensing, in which the
positive regulator CDC6 and the negative regulators geminin
and CYCA are all substrates of APC FZR/CDH1 , the model of a sin-
gle APC FZR/CDH1 substrate that controls centromere licensing or
propagation may be oversimplifi ed. We observed that defective
centromere localization of CID and CENP-C after CYCA or
RCA1 depletion was not rescued by CDC20 depletion, but we
cannot rule out a role for APC FZY/CDC20 in centromere propagation
because premature APC FZR/CDH1 activation could mask a subse-
quent role for FZY/CDC20, which is activated at the metaphase/
We do not yet know whether the localization of CYCA
at centromeres is important for the regulation of centromere
assembly. In Drosophila , it has been demonstrated that the sub-
cellular localization of CYCA is not important for proper pro-
gression through the cell cycle; however, these experiments did
not directly address whether mislocalization of CYCA prevented
the association of CYCA with centromeres ( Dienemann and
Sprenger, 2004 ). It will be interesting to determine whether CID,
CENP-C, and CAL1 localization require centromere-localized
CYCA – Cdk1 activity or whether any of these proteins are a di-
rect target of CYCA – Cdk1.
Our results suggest that CID or CAL1 levels are indirectly
controlled by APC activity. Interestingly, the human M18BP1
has recently been proposed to act as a “ licensing factor ” for
centromere assembly. Although no clear homologues of M18BP1/
KNL2 have been identifi ed in Drosophila , both CAL1 in fl ies
and M18BP1/KNL2 in other species are interdependent with
CENP-A for centromere localization ( Fujita et al., 2007 ; Maddox
et al., 2007 ). Strikingly, levels of CAL1 and M18BP1/KNL2 are
reduced on metaphase centromeres and increase coincident with
CENP-A loading in late anaphase/telophase ( Jansen et al., 2007 ;
Schuh et al., 2007 ). Further analysis is required to determine
whether CAL1 and M18BP1/KNL2 function analogously in
centromere assembly. It will be important to determine whether
fl y homologues of other Mis18 complex components are associ-
ated with CAL1 and important for centromere assembly. Identi-
fying the APC substrates involved in centromere assembly will
cells showing interdependence between the CENP-H – CENP-I
complex and CENP-A ( Okada et al., 2006 ). However, studies in
C . elegans and vertebrates have not detected a role for CENP-C
in CENP-A chromatin assembly, suggesting that CENP-C plays
a more prominent role in regulating centromere propagation in
fl ies ( Oegema et al., 2001 ; Okada et al., 2006 ; Kwon et al., 2007 ).
Collectively, these results suggest that CENPs that depend
on CENP-A for their localization may “ feed back ” to control
CENP-A assembly. Histone variants are assembled into chroma-
tin both by histone chaperones (e.g., the histone H3.3 – specifi c
chaperone HIRA [histone regulatory A] that provides specifi c-
ity to the CHD1 chromatin-remodeling ATPase) and by histone
variant – specifi c ATPases (e.g., Swr1 that can use the general
chaperone Nap1 or the specifi c chaperone Chz1 to assemble
H2A.Z; Krogan et al., 2003 ; Kobor et al., 2004 ; Mizuguchi et al.,
2004 ; Jin et al., 2005 ; Konev et al., 2007 ; Luk et al., 2007 ).
CENP-C or CAL1 might facilitate centromere-specifi c CID lo-
calization by providing centromere specifi city to a chromatin-
remodeling ATPase in a manner analogous to HIRA or might
direct the localization of chromatin assembly factors to the cen-
tromere. It will be interesting to determine what factors associate
with CAL1 and CENP-C as a route to elucidating the mecha-
nisms of centromere assembly and propagation.
The loading of CENP-A in human somatic cells and in
Drosophila embryos occurs after anaphase initiation ( Jansen et al.,
2007 ; Schuh et al., 2007 ) when APC FZR/CDH1 activity is high
( Morgan, 2007 ). Ubiquitin-mediated proteolysis facilitates for-
mation of a single centromere by degrading noncentromeric
CENP-A ( Collins et al., 2004 ; Moreno-Moreno et al., 2006 ), and
subunits of the APC are localized to kinetochores ( Jorgensen et al.,
1998 ; Kurasawa and Todokoro, 1999 ; Topper et al., 2002 ;
Acquaviva et al., 2004 ). Our results demonstrate that normal
regulation of APC FZR/CDH1 activity is required for centromere
propagation, providing a link between centromere assembly and
cell cycle regulation.
We propose two alternative models for the role of
APC FZR/CDH1 in centromere function. The fi rst model is that CYCA
is the relevant substrate of APC FZR/CDH1 and that the kinase ac-
tivity of the CYCA – Cdk1 complex is required for the localiza-
tion of CID, CENP-C, and CAL1 to the centromere ( Fig. 6 D ,
red arrow). CYCA is normally degraded as cells proceed through
mitosis, suggesting that CYCA – Cdk1 would likely act during G2
or early M to phosphorylate a substrate involved in centromere
assembly. The CID and CENP-C localization defect caused by
CYCA depletion was rescued by the simultaneous depletion of
FZR/CDH1 even though the levels of CYCA protein remained
low in the double depletion. The rescue of the CID and CENP-C
localization defect in cells with low CYCA protein suggests that
maintaining high levels of CYCA – Cdk1 activity is not required
for centromere propagation, but we cannot rule out that the re-
sidual CYCA protein in these cells is suffi cient to rescue the
centromeric phenotype when APC activity is compromised by
The second model that is consistent with our observations
is that one or more APC FZR/CDH1 substrates ( “ X ” ) regulate the in-
terdependent localization of CID, CENP-C, and CAL1 to the
centromere ( Fig. 6 D , blue arrow). RCA1 and CYCA inhibit the
JCB • VOLUME 183 • NUMBER 5 • 2008 816
CENP-C antibodies were generated by cloning the fi rst 2,196 bp of
the DmCENP-C gene into the pET100/D-TOPO vector (Invitrogen). Protein
was expressed in BL21-star cells and purifi ed using the pETQIA expressionist
kit (QIAGEN). Polyclonal antibodies were produced in guinea pigs by Co-
vance, and crude serum was used for immunostaining. The fi rst 176 amino
acids of CAL1 and the fi rst 124 amino acids of CID were fused to GST using
a modifi ed pGEX-6P vector (EMD). Fusion proteins were expressed in BL21
(DE3) pLysS cells and purifi ed with glutathione agarose (Sigma-Aldrich).
Polyclonal antibodies were produced in rabbits, and antisera were affi nity
purifi ed against the antigen after removal of the GST tag with PreScission
Immunoprecipitation from Kc167 cells
Approximately 8 × 10 10 cells stably expressing GFP-tagged CLD proteins
at levels equal to or lower than the endogenous levels were harvested and
washed in PBS. Nuclei were isolated by lysing the cells in 25 ml of nuclear
extraction buffer (20 mM Hepes, pH 7.7, 50 mM KCl, 2 mM MgCl 2 , 5 mM
? -mercaptoethanol [ ? -ME], 1% Triton X-100, 1 mM PMSF, 2 mM benzamidine-
HCl, and 10 μ g/ml LPC [leupeptin, pepstatin, and chymostatin]) followed
by a wash in 25 ml of nuclear wash buffer (20 mM Hepes, pH 7.7, 50 mM
KCl, 2 mM MgCl 2 , 5 mM ? -ME, 1 mM PMSF, 2 mM benzamidine-HCl, and
10 μ g/ml LPC). Nuclei were resuspended in 3 ml of micrococcal nuclease
buffer (20 mM Hepes, pH 7.7, 50 mM KCl, 2 mM MgCl 2 , 5 mM ? -ME,
3 mM CaCl 2 , 1 mM PMSF, 2 mM benzamidine-HCl, and 10 μ g/ml LPC), and
chromatin was digested with 0.1 U/ μ l micrococcal nuclease (Worthington
Biochemical) for 30 min at 25 ° C. 3 ml of 2 × extraction buffer (20 mM
Hepes, pH 7.7, 575 mM KCl, 5 mM EGTA, 5 mM EDTA, 5 mM ? -ME,
20% glycerol, 0.1% Igepal-CA630, 1 mM PMSF, 2 mM benzamidine-HCl,
and 10 μ g/ml LPC) was added to stop the reactions. The lysate was soni-
cated twice for 30 s and cleared by centrifugation at 10,000 g for 15 min.
Cleared lysate was added to 30 μ l of anti-GFP resin (0.5 mg/ml polyclonal
rabbit anti-GFP antibody coupled to protein A – Sepharose beads) and incu-
bated for 2 h at 4 ° C. The anti-GFP resin was washed three times in 20 mM
Hepes, pH 7.7, 300 mM KCl, 2.5 mM EGTA, 2.5 mM EDTA, 5 mM ? -ME,
10% glycerol, 0.05% Igepal, 1 mM PMSF, 2 mM benzamidine-HCl, and
10 μ g/ml LPC, boiled in 70 μ l of SDS sample buffer, and analyzed by
Approximately 4 × 10 7 cells were harvested and washed once in PBS. The
cells were lysed in 20 mM Hepes, pH 7.7, 500 mM NaCl, 5 mM EDTA,
5 mM EGTA, 0.5% Igepal, 1 mM PMSF, 2 mM benzamidine-HCL, and
10 μ g/ml LPC. Protein concentrations were equalized by Bradford assays,
and 30 μ g of total protein was loaded per sample. Proteins were transferred
to a nitrocellulose membrane in 10 mM 3-(cyclohexylamino)-1-propane sul-
fonic acid, 0.1% SDS, and 20% methanol for 45 min for CID and CYCA or
for 90 min for CENP-C and CAL1. Affi nity-purifi ed antibodies were used at
a concentration of 1 μ g/ml.
Kc167 cells were grown in 6-well dishes and harvested at 24, 48, 72, and
96 h after RNAi treatment. Cells were washed once in PBS and fi xed by
dropwise addition into 70% ethanol while mixing. Cells were washed twice
in PBS. The cells were stained by the addition of 100 μ l of 100 μ g/ml RNaseA
and 400 μ l of 50 μ g/ml propidium iodide. DNA content was measured using
a FACS analyzer (FACScan; BD). FACS data were analyzed with FlowJo
software (Tree Star, Inc.).
dsRNA was prepared using a kit (MegaSCRIPT T7; Applied Biosystems)
according to the manufacturer ’ s procedures. Templates were generated
by PCR from genomic DNA using the following primers: CYCA reverse,
5 ? -GCCAAGAAATCGAATGTGGT-3 ’ ; CYCA forward, 5 ? -ATTTCACGT-
CATGGTTCTCTT-3 ’ ; RCA1 reverse, 5 ? -TTTCAATCGCCACACAGTAG-3 ’ ;
RCA1 forward, 5 ? -GCCTCGCTTATGAAAACCC-3 ’ ; CAL1 forward,
5 ? -TGGATGCCAGGAAAGTTAGT-3 ’ ; CAL1 reverse, 5 ? -CTATAGGGATTGT-
TGATATCAGC-3 ’ ; CENP-C forward, 5 ? -TGGTAAACTATTTGGGTCTCTC-3 ’ ;
CENP-C reverse, 5 ? -GGTACCAGTTCGTTCTCCA-3 ’ ; CID forward,
5 ? -ACCGTGCAGCAGGAAAG-3 ’ ; and CID reverse, 5 ? -CCCCGGTCG-
CAGATGTA-3 ’ . 10 6 logarithmically growing S2 cells were plated in 1 ml
of serum-free medium, and 15 μ g dsRNA was added to the culture.
Control wells received water instead of dsRNA. After 1 h of incubation,
1.5 ml of serum-containing medium was added, and incubation pro-
ceeded for 4 d. Samples of 100 μ l were taken every day and were sub-
jected to indirect IF analysis.
be necessary to distinguish between these models and to deter-
mine how these proteins epigenetically regulate centromere as-
sembly and couple this essential process to the cell cycle.
Materials and methods
Genome-wide RNAi screen
Logarithmically growing Kc167 cells were trypsinized, washed, and resus-
pended in serum-free medium at 1 × 10 6 cells/ml, and 10 μ l of cells was
plated to each well of the 384-well plate containing dsRNA. Cells were in-
cubated for 1 h at room temperature before 35 μ l of Schneider ’ s medium
(Invitrogen) with 1 × antibiotics (Invitrogen), and 10% FCS (Omega) was
added to each well and incubated for an additional 4 d at 25 ° C. Cells
were fi xed for 5 min at room temperature in 100% methanol and washed
twice in 1 × TBS with 0.1% Triton X-100 (TBST). Cells were treated for 30
min with blocking solution (TBST containing 2% BSA), which was replaced
by 10 μ l of blocking solution containing chicken anti-CID antibody (1:100
dilution) and mouse anti-HP1 (1:400 dilution) and incubated overnight at
4 ° C. Cells were washed twice for 5 min with blocking solution, and 10 μ l
of secondary antibodies (Alexa 568 anti – chicken and Alexa 488 anti –
mouse antibody at 1:400 dilutions) was added and incubated for 1 h at
room temperature. Secondary antibodies were washed away three times
with TBST. DNA was stained with 2 μ g/ml Hoechst 33342 in TBS for 10 min
at room temperature and washed with TBS. Plates were imaged using a
40 × objective using an automated plate-imaging microscope (ImageXpress;
MDS Analytical Technologies).
A MetaMorph (available at http://straightlab.stanford.edu/analysis)
journal was written to segment nuclei based on DNA staining. Cell area
was estimated by a 10-pixel dilation from the nuclei. Centromere staining
was identifi ed within the cells through a morphological top hat fi lter with a
5-pixel diameter. The integrated intensity of the centromere and cell body
staining was collected. Each well was assigned a score equal to the sum of
the cell body intensity divided by the sum of the centromere intensity. Posi-
tives were verifi ed by manual image analysis and retesting (Fig. S1).
Indirect IF on fi xed cells
S2 cells were settled on glass slides and fi xed with either 4% PFA, 4% for-
malin, or 100% methanol for 10 min. After three washes with PBS for 10 min
each, cells were blocked in either 5% milk or 2% BSA in PBST (PBS with
0.2% Triton X-100) for 10 min before primary antibody incubation over-
night at 4 ° C followed by three washes of 10 min in PBST. To detect the
kinetochore localization of GFP-CAL1, a stable line expressing GFP-CAL1
was treated with 0.5% sodium citrate for 7 min followed by centrifugation
on a slide using cytospin (Shandon) at 2,900 rpm for 10 min; cells were
then fi xed, washed, blocked in PBST with 5% milk, and incubated over-
night at 4 ° C with anti-CID and anti-Rod antibodies. After washes and incu-
bation with anti – chicken and anti – rabbit secondaries, cells were fi xed
again for 5 min with 4% formalin, washed, and incubated overnight with
Alexa 488 – conjugated anti-GFP antibody (Invitrogen). All of the secondary
antibodies used were Alexa 488, Alexa 546, or Alexa 647 (Invitrogen)
conjugates, and they were incubated at 1:500 dilutions for 45 min at room
temperature. After three 10-min washes in PBS, cells were mounted in
2.5% 1,4-diazabicyclo[2.2.2]octane and 1 μ g/ml DAPI in 50% glycerol.
The dilutions and antibodies used were 1:300 chicken anti-CID ( Blower
and Karpen, 2001 ), 1:300 guinea pig anti – CENP-C, 1:500 rabbit anti-
GFP (Invitrogen), 1:10 mouse anti-CYCA (Developmental Studies Hybridoma
Bank), 1:500 mouse antitubulin (Sigma-Aldrich), 1:500 rabbit anti-PH3
(H3S10ph; Millipore), 1:500 mouse antifi brillarin (Cytoskeleton, Inc.), and
1:500 rabbit anti-Rod ( Scaerou et al., 1999 ). All images were taken on a
microscope (Deltavision Spectris; Applied Precision, LLC) and deconvolved
using softWoRx (Applied Precision, LLC). Images were taken as z stacks of
0.2- or 0.3- μ m increments using a 100 × oil-immersion objective.
Embryos were collected either overnight or for 1 h, aged, and
dechorionized with 50% bleach for 2 min. Embryos extensively washed in
100 mM NaCl + 0.5% Triton X-100 were fi xed with formaldehyde satu-
rated with heptane for 20 min and hand devitillinized on double sticky
tape with a 35-gauge needle in PBTA (1% BSA + 0.2% Triton X-100 and
0.05% NaN 3 in PBS). Antibodies were diluted in PBTA and incubated
overnight. After three 10-min washes in PBTA, secondary antibodies were
incubated for 2 h. After three washes in PBS, embryos were mounted in
Vectashield containing DAPI and imaged on a Deltavision microscope as
stated in the previous paragraph. The mutant embryos used for this study
were y , ry (control), cycA C8LR1 , cenpC prl-41 (C. Lehner, University of Zurich,
Zurich, Switzerland), rca1 IX , and pBacCG5148 .
817 CENTROMERE PROPAGATION MECHANISMS • Erhardt et al.
For the experiments to test nondegradable CYCA rescue of CAL1
depletion, cells were transfected with 5 μ g pCopia-GFP – ? 55-CYCA alone
or in combination with 5 μ g dsRNAi against CAL1 using the DOTAP trans-
fection reagent. 4 d after incubation, cells were fi xed and stained with
anti-GFP and anti-CID antibodies.
Online supplemental material
Fig. S1 depicts the workfl ow of the genome-wide siRNA screen for cen-
tromere propagation and the quantifi cation of CID levels after RNAi
depletion of the CLD genes. Fig. S2 shows the alignment of CAL1 ho-
mologues in several drosophilid species. Fig. S3 shows the localization
of CLDs in interphase and mitosis. Fig. S4 shows the effect of cenpC , cal1 ,
cycA , and rca1 mutants on centromeres in Drosophila embryos, the frag-
mentation of chromatin used in the LAP-CLD purifi cations, and the expres-
sion levels of LAP-CLD fusions in Kc167 cells. Fig. S5 shows the rescue of
centromeric CID localization in RCA1-depleted cells by nondegradable
CYCA. Videos 1 – 5 show time-lapse videos of Drosophila S2 cells express-
ing mCherry-tubulin and GFP-CID, GFP – CENP-C, GFP-CAL1, GFP-CYCA,
and GFP-RCA1, respectively. Videos 6 – 10 show time-lapse videos of
Drosophila S2 cells expressing GFP-H2B and mCherry-tubulin after dsRNA
depletion with control, CID, CENP-C, CAL1, and CYCA RNA, res pec-
tively. Online supplemental material is available at http://www.jcb.org/
We thank Alison Farrell and Abby Dernburg for critical reading of the manu-
script and Michael Rape for helpful discussions. We are grateful to Pat Brown,
Yoav Soen, and Bob Marinelli for assistance with high throughput imaging.
We are also grateful to Norbert Perrimon, Nadire Ramadan, and Bernard
Mathey-Prevot ( Drosophila RNA Interference Screening Center, Harvard, Cam-
bridge, MA) for assistance with dsRNA screening, Gohta Goshima for re-
agents, Christian Lehner for cenpC mutant fl ies, and the Developmental Studies
Hybridoma Bank for CYCA antibodies developed by Christian Lehner. We
also thank Aki Minoda and David Seo for general technical help and the
Straight and Karpen laboratories for discussion and support.
This work was supported by National Institutes of Health grants to
G.H. Karpen and A.F. Straight (R01 GM066272 and R01 GM074728,
respectively), grants from the Wellcome Trust to S. Erhardt, grants from Philip
Morris USA, Inc. and Philip Morris International to B.G. Mellone, and grants
from the Susan G. Komen Breast Cancer Foundation to W. Zhang. A.F.
Straight is a Gordon Family Scholar supported by the Damon Runyon Cancer
Submitted: 5 June 2008
Accepted: 30 October 2008
Acquaviva , C. , F. Herzog , C. Kraft , and J. Pines . 2004 . The anaphase promoting
complex/cyclosome is recruited to centromeres by the spindle assembly
checkpoint. Nat. Cell Biol. 6 : 892 – 898 .
Ando , S. , H. Yang , N. Nozaki , T. Okazaki , and K. Yoda . 2002 . CENP-A, -B, and -C
chromatin complex that contains the I-type alpha-satellite array consti-
tutes the prekinetochore in HeLa cells. Mol. Cell. Biol. 22 : 2229 – 2241 .
Arias , E.E. , and J.C. Walter . 2007 . Strength in numbers: preventing rereplication
via multiple mechanisms in eukaryotic cells. Genes Dev. 21 : 497 – 518 .
Bentley , A.M. , G. Normand , J. Hoyt , and R.W. King . 2007 . Distinct sequence
elements of cyclin B1 promote localization to chromatin, centrosomes,
and kinetochores during mitosis. Mol. Biol. Cell . 18 : 4847 – 4858 .
Blower , M.D. , and G.H. Karpen . 2001 . The role of Drosophila CID in kineto-
chore formation, cell-cycle progression and heterochromatin interactions.
Nat. Cell Biol. 3 : 730 – 739 .
Blower , M.D. , T. Daigle , T. Kaufman , and G.H. Karpen . 2006 . Drosophila
CENP-A mutations cause a BubR1-dependent early mitotic delay without
normal localization of kinetochore components. PLoS Genet . 2 : e110 .
Carroll , C.W. , and A.F. Straight . 2006 . Centromere formation: from epigenetics
to self-assembly. Trends Cell Biol. 16 : 70 – 78 .
Cheeseman , I.M. , and A. Desai . 2005 . A combined approach for the localization
and tandem affi nity purifi cation of protein complexes from metazoans.
Sci. STKE . doi:10.1126/stke.2662005p11.
Clark , A.G. , M.B. Eisen , D.R. Smith , C.M. Bergman , B. Oliver , T.A. Markow ,
T.C. Kaufman , M. Kellis , W. Gelbart , V.N. Iyer , et al . 2007 . Evolu-
tion of genes and genomes on the Drosophila phylogeny. Nature .
450 : 203 – 218 .
Cleveland , D.W. , Y. Mao , and K.F. Sullivan . 2003 . Centromeres and kinetochores:
from epigenetics to mitotic checkpoint signaling. Cell . 112 : 407 – 421 .
Quantitation of defective mitoses and mitotic index
S2 cells were depleted of CID, CLD-2, CENP-C, RCA1, or CYCA by RNAi.
After 4 d, cells were fi xed for 10 min with 10% formalin in PBS and stained
for DNA (DAPI), CID, CENP-C, tubulin, or H3S10Ph by indirect IF. Mitotic
cells were scored as defective or normal based on chromosome and spin-
dle morphology compared with control cells, and the signifi cance of the
differences was determined using the ? 2 test.
SNAP tag labeling of newly synthesized CID
Drosophila Kc cells stably expressing SNAP-tagged CID (SNAP-CID) were
transfected with 5 μ g dsRNA against CENP-C or CAL1 using the 1,2-dio-
leoyl-3-trimethylammonium-propane (DOTAP) transfection reagent (Roche)
or transfection reagent alone (control). SNAP-CID was quenched with BTP
after 72 h of RNAi depletion. BTP was removed, and cells were allowed to
grow for another 24 h to allow synthesis of unlabeled SNAP-CID protein
followed by TMR* labeling. Cells were fi xed with 4% formaldehyde in
PBST for 5 min, and total SNAP-CID was detected by IF with a rabbit
? -SNAP polyclonal antibody (Covalys) used at 1:500.
Time-lapse analysis and GFP constructs
Time-lapse videos were performed using a Deltavision Spectris microscope.
Cells were mounted using the hanging drop method ( Heun et al., 2006 ).
For the mitotic time-lapse videos, cells expressing mCherry-tubulin and
H2B-GFP in prophase/prometaphase (gift of G. Goshima and R. Vale,
University of California, San Francisco, San Francisco, CA) were imaged
every 1 or 2 min until cytokinesis for a total of 30 – 45 min with the excep-
tion of CID RNAi, in which, in some severe cases, cells were imaged for
90 – 120 min and did not undergo cytokinesis. Cells were imaged 3 – 4 d
after RNAi treatment except for CYCA and RCA1 RNAi, in which cells
were imaged after 1 – 2 d of treatment. 5 – 10 videos of randomly selected
prophase cells were imaged for each RNAi experiment. 80 – 100% of vid-
eos per RNAi experiment showed mitotic defects with the exception of
CYCA and RCA1 RNAi, in which the percentage of defective mitoses was
? 60%. Videos of GFP-CID, GFP-CDL2, GFP – CENP-C, and GFP-CYCA in
mCherry-tubulin – expressing cells were imaged every 2 min until cytokine-
sis was completed. Videos were edited in Photoshop (Adobe) to reduce
video size, and the time in minutes in the still images refl ects the actual
elapsed time during image acquisition.
GFP constructs were generated for all fi ve CLD genes after PCR am-
plifi cation with a template PCR system (Expand Long; Roche) from either
Drosophila Genomic Resource Center clones or from Drosophila Kc167 cell
poly-A RNA using the following primers fl anked by an AscI site at the 5 ? end
and a PacI site at the 3 ? end: CID forward, 5 ? -GCATCATATGCAGCACGCT-
GTTTCCGCTG-3 ? ; CID reverse, 5 ? -GCATGCTAGCGCTTTTTTGGAACAGT-
GTGACCG-3 ? ; CG5148 forward, 5 ? -ATGGCGAATGCGGTGGTG-3 ? ;
CG5148 reverse, 5 ? -TTACTTGTCACCGGAATTATTCTCG-3 ? ; CENP-C for-
ward, 5 ? -ATGTCGAAGCCCCAGAAC-3 ? ; CENP-C reverse, 5 ? -CTAACTGC-
GTATACACATCAG-3 ? ; RCA1 forward, 5 ? -ATGAGCGCCTATTATCGGCG-3 ? ;
RCA1 reverse, 5 ? -CTAAAAGCAGAGCCGCTTGAGCGAGTT-3 ? ; CYCA
forward, 5 ? -ATGGCCAGTTTCCAGATCCAC-3 ? ; CYCA reverse, 5 ? -ATGTCC-
GTGACGGATGTTCAGTC-3 ? ; and Δ 55-CYCA forward, 5 ? -AACAATGT-
GCCGCGTCCG-3 ? . PCR products were cloned into pCopia – localization
and purifi cation (LAP) digested with AscI and PacI. pCopia-LAP was gener-
ated by replacing EGFP with the LAP tag ( Cheeseman and Desai, 2005 )
and modifying the polylinker such that AscI/PacI cloning would result in the
N-terminal LAP (GFP) tagging of genes. The Copia promoter was PCR am-
plifi ed from the pCoPuro plasmid ( Iwaki et al., 2003 ) with the primers for-
ward (5 ? -GCATCATATGGGCAAATGGGTTTAGGATTGGG-3 ? ) and reverse
(5 ? -GCATGCTAGCGGAAGGTCGTCTCCTTGTGAGG-3 ? ) and was cloned
into the EGFP vector using the NdeI and NheI sites. The plasmid for nonde-
gradable cyclin expression was generated by ligating Δ 55-CYCA into the
AscI and PacI sites of pCopia-LAP to generate pCopia-GFP – ? 55-CYCA.
S2 cells were transfected with Cellfectin (Invitrogen) according to
the manufacturer ’ s instructions. Stable lines were obtained by cotransfect-
ing the LAP tag constructs with the plasmid pHygro (Invitrogen) and by se-
lection in the presence of 100 μ g/ml hygromycin B (Invitrogen).
Nondegradable CYCA rescue experiments
Kc167 cells were depleted of CYCA and RCA1 by treatment with dsRNA.
Immediately after the incubation of cells with dsRNA, the cells were trans-
fected with plasmids expressing GFP, pCopia – GFP-CYCA, or pCopia-GFP –
? 55-CYCA with FuGene 6 (Roche) according to the manufacturer ’ s
instructions. After 4 d, the cells were fi xed for IF and stained for DNA, GFP,
and CID. CID centromere intensities were measured in the GFP-positive and
JCB • VOLUME 183 • NUMBER 5 • 2008 818 Download full-text
Lehner , C.F. , and P.H. O ’ Farrell . 1989 . Expression and function of Drosophila
cyclin A during embryonic cell cycle progression. Cell . 56 : 957 – 968 .
Luk , E. , N.D. Vu , K. Patteson , G. Mizuguchi , W.H. Wu , A. Ranjan , J. Backus , S.
Sen , M. Lewis , Y. Bai , and C. Wu . 2007 . Chz1, a nuclear chaperone for
histone H2AZ. Mol. Cell . 25 : 357 – 368 .
Lukas , C. , C.S. Sorensen , E. Kramer , E. Santoni-Rugiu , C. Lindeneg , J.M.
Peters , J. Bartek , and J. Lukas . 1999 . Accumulation of cyclin B1 requires
E2F and cyclin-A-dependent rearrangement of the anaphase-promoting
complex. Nature . 401 : 815 – 818 .
Machida , Y.J. , and A. Dutta . 2007 . The APC/C inhibitor, Emi1, is essential for
prevention of rereplication. Genes Dev. 21 : 184 – 194 .
Maddox , P.S. , F. Hyndman , J. Monen , K. Oegema , and A. Desai . 2007 . Functional
genomics identifi es a Myb domain – containing protein family required for
assembly of CENP-A chromatin. J. Cell Biol. 176 : 757 – 763 .
Mihaylov , I.S. , T. Kondo , L. Jones , S. Ryzhikov , J. Tanaka , J. Zheng , L.A. Higa ,
N. Minamino , L. Cooley , and H. Zhang . 2002 . Control of DNA replica-
tion and chromosome ploidy by geminin and cyclin A. Mol. Cell. Biol.
22 : 1868 – 1880 .
Mizuguchi , G. , X. Shen , J. Landry , W.H. Wu , S. Sen , and C. Wu . 2004 . ATP-
driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin
remodeling complex. Science . 303 : 343 – 348 .
Moreno-Moreno , O. , M. Torras-Llort , and F. Azorin . 2006 . Proteolysis re-
stricts localization of CID, the centromere-specifi c histone H3 variant of
Drosophila , to centromeres. Nucleic Acids Res. 34 : 6247 – 6255 .
Morgan , D.O. 2007 . The Cell Cycle: Principles of Control. New Science Press,
London. 297 pp.
Nickerson , H.D. , A. Joshi , and D.J. Wolgemuth . 2007 . Cyclin A1-defi cient mice
lack histone H3 serine 10 phosphorylation and exhibit altered aurora B
dynamics in late prophase of male meiosis. Dev. Biol. 306 : 725 – 735 .
Oegema , K. , A. Desai , S. Rybina , M. Kirkham , and A.A. Hyman . 2001 . Functional
analysis of kinetochore assembly in Caenorhabditis elegans . J. Cell Biol.
153 : 1209 – 1226 .
Okada , M. , I.M. Cheeseman , T. Hori , K. Okawa , I.X. McLeod , J.R. Yates III ,
A. Desai , and T. Fukagawa . 2006 . The CENP-H-I complex is required
for the effi cient incorporation of newly synthesized CENP-A into centro-
meres. Nat. Cell Biol. 8 : 446 – 457 .
Scaerou , F. , I. Aguilera , R. Saunders , N. Kane , L. Blottiere , and R. Karess . 1999 .
The rough deal protein is a new kinetochore component required for accu-
rate chromosome segregation in Drosophila . J. Cell Sci. 112 : 3757 – 3768 .
Schuh , M. , C.F. Lehner , and S. Heidmann . 2007 . Incorporation of Drosophila
CID/CENP-A and CENP-C into centromeres during early embryonic
anaphase. Curr. Biol. 17 : 237 – 243 .
Takahashi , K. , E.S. Chen , and M. Yanagida . 2000 . Requirement of Mis6 cen-
tromere connector for localizing a CENP-A-like protein in fi ssion yeast.
Science . 288 : 2215 – 2219 .
Topper , L.M. , M.S. Campbell , S. Tugendreich , J.R. Daum , D.J. Burke , P. Hieter ,
and G.J. Gorbsky . 2002 . The dephosphorylated form of the anaphase-pro-
moting complex protein Cdc27/Apc3 concentrates on kinetochores and
chromosome arms in mitosis. Cell Cycle . 1 : 282 – 292 .
Zachariae , W. , M. Schwab , K. Nasmyth , and W. Seufert . 1998 . Control of cyclin
ubiquitination by CDK-regulated binding of Hct1 to the anaphase pro-
moting complex. Science . 282 : 1721 – 1724 .
Collins , K.A. , S. Furuyama , and S. Biggins . 2004 . Proteolysis contributes to the
exclusive centromere localization of the yeast Cse4/CENP-A histone H3
variant. Curr. Biol. 14 : 1968 – 1972 .
Dalal , Y. , H. Wang , S. Lindsay , and S. Henikoff . 2007 . Tetrameric structure of cen-
tromeric nucleosomes in interphase Drosophila cells. PLoS Biol. 5 : e218 .
Dienemann , A. , and F. Sprenger . 2004 . Requirements of cyclin A for mitosis are
independent of its subcellular localization. Curr. Biol. 14 : 1117 – 1123 .
Dong , X. , K.H. Zavitz , B.J. Thomas , M. Lin , S. Campbell , and S.L. Zipursky .
1997 . Control of G1 in the developing Drosophila eye: rca1 regulates
cyclin A. Genes Dev. 11 : 94 – 105 .
Dunleavy , E.M. , A.L. Pidoux , M. Monet , C. Bonilla , W. Richardson , G.L.
Hamilton , K. Ekwall , P.J. McLaughlin , and R.C. Allshire . 2007 . A NASP
(N1/N2)-related protein, Sim3, binds CENP-A and is required for its de-
position at fi ssion yeast centromeres. Mol. Cell . 28 : 1029 – 1044 .
Foltz , D.R. , L.E. Jansen , B.E. Black , A.O. Bailey , J.R. Yates III , and D.W.
Cleveland . 2006 . The human CENP-A centromeric nucleosome-associ-
ated complex. Nat. Cell Biol. 8 : 458 – 469 .
Fujita , Y. , T. Hayashi , T. Kiyomitsu , Y. Toyoda , A. Kokubu , C. Obuse , and M.
Yanagida . 2007 . Priming of centromere for CENP-A recruitment by hu-
man hMis18alpha, hMis18beta, and M18BP1. Dev. Cell . 12 : 17 – 30 .
Furuyama , T. , Y. Dalal , and S. Henikoff . 2006 . Chaperone-mediated assem-
bly of centromeric chromatin in vitro. Proc. Natl. Acad. Sci. USA .
103 : 6172 – 6177 .
Goshima , G. , R. Wollman , S.S. Goodwin , N. Zhang , J.M. Scholey , R.D. Vale ,
and N. Stuurman . 2007 . Genes required for mitotic spindle assembly in
Drosophila S2 cells. Science . 316 : 417 – 421 .
Grosskortenhaus , R. , and F. Sprenger . 2002 . Rca1 inhibits APC-Cdh1(Fzr) and is
required to prevent cyclin degradation in G2. Dev. Cell . 2 : 29 – 40 .
Hayashi , T. , Y. Fujita , O. Iwasaki , Y. Adachi , K. Takahashi , and M. Yanagida .
2004 . Mis16 and Mis18 are required for CENP-A loading and histone
deacetylation at centromeres. Cell . 118 : 715 – 729 .
Heeger , S. , O. Leismann , R. Schittenhelm , O. Schraidt , S. Heidmann , and C.F.
Lehner . 2005 . Genetic interactions of separase regulatory subunits reveal
the diverged Drosophila Cenp-C homolog. Genes Dev. 19 : 2041 – 2053 .
Heun , P. , S. Erhardt , M.D. Blower , S. Weiss , A.D. Skora , and G.H. Karpen .
2006 . Mislocalization of the Drosophila centromere-specifi c histone
CID promotes formation of functional ectopic kinetochores. Dev. Cell .
10 : 303 – 315 .
Iwaki , T. , M. Figuera , V.A. Ploplis , and F.J. Castellino . 2003 . Rapid selection of
Drosophila S2 cells with the puromycin resistance gene. Biotechniques .
35 : 482 – 484 , 486.
Jansen , L.E.T. , B.E. Black , D.R. Foltz , and D.W. Cleveland . 2007 . Propagation
of centromeric chromatin requires exit from mitosis. J. Cell Biol.
176 : 795 – 805 .
Jaspersen , S.L. , J.F. Charles , and D.O. Morgan . 1999 . Inhibitory phosphoryla-
tion of the APC regulator Hct1 is controlled by the kinase Cdc28 and the
phosphatase Cdc14. Curr. Biol. 9 : 227 – 236 .
Jin , J. , Y. Cai , B. Li , R.C. Conaway , J.L. Workman , J.W. Conaway , and T. Kusch .
2005 . In and out: histone variant exchange in chromatin. Trends Biochem.
Sci. 30 : 680 – 687 .
Jorgensen , P.M. , E. Brundell , M. Starborg , and C. Hoog . 1998 . A subunit of the
anaphase-promoting complex is a centromere-associated protein in mam-
malian cells. Mol. Cell. Biol. 18 : 468 – 476 .
Kaspar , M. , A. Dienemann , C. Schulze , and F. Sprenger . 2001 . Mitotic degrada-
tion of cyclin A is mediated by multiple and novel destruction signals.
Curr. Biol. 11 : 685 – 690 .
Kiger , A.A. , B. Baum , S. Jones , M.R. Jones , A. Coulson , C. Echeverri , and N.
Perrimon . 2003 . A functional genomic analysis of cell morphology using
RNA interference. J. Biol. 2 : 27 .
Kobor , M.S. , S. Venkatasubrahmanyam , M.D. Meneghini , J.W. Gin , J.L.
Jennings , A.J. Link , H.D. Madhani , and J. Rine . 2004 . A protein complex
containing the conserved Swi2/Snf2-related ATPase Swr1p deposits his-
tone variant H2A.Z into euchromatin. PLoS Biol. 2 : E131 .
Konev , A.Y. , M. Tribus , S.Y. Park , V. Podhraski , C.Y. Lim , A.V. Emelyanov , E.
Vershilova , V. Pirrotta , J.T. Kadonaga , A. Lusser , and D.V. Fyodorov .
2007 . CHD1 motor protein is required for deposition of histone variant
H3.3 into chromatin in vivo. Science . 317 : 1087 – 1090 .
Krogan , N.J. , M.C. Keogh , N. Datta , C. Sawa , O.W. Ryan , H. Ding , R.A. Haw ,
J. Pootoolal , A. Tong , V. Canadien , et al . 2003 . A Snf2 family ATPase
complex required for recruitment of the histone H2A variant Htz1. Mol.
Cell . 12 : 1565 – 1576 .
Kurasawa , Y. , and K. Todokoro . 1999 . Identifi cation of human APC10/Doc1 as a
subunit of anaphase promoting complex. Oncogene . 18 : 5131 – 5137 .
Kwon , M.S. , T. Hori , M. Okada , and T. Fukagawa . 2007 . CENP-C is involved in
chromosome segregation, mitotic checkpoint function, and kinetochore
assembly. Mol. Biol. Cell . 18 : 2155 – 2168 .