Research Article
4341
Mitotic chromosome interactions of Epstein-Barr
nuclear antigen 1 (EBNA1) and human EBNA1-binding
protein 2 (EBP2)
Vipra Kapur Nayyar*, Kathy Shire* and Lori Frappier‡
Department of Molecular Genetics, University of Toronto, Toronto, Canada M5S 1A8
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 14 October 2009
Journal of Cell Science 122, 4341-4350 Published by The Company of Biologists 2009
doi:10.1242/jcs.060913
Summary
The Epstein-Barr nuclear antigen 1 (EBNA1) protein enables
the stable persistence of Epstein-Barr virus episomal genomes
during latent infection, in part by tethering the episomes to the
cellular chromosomes in mitosis. A host nucleolar protein,
EBNA1-binding protein 2 (EBP2), has been shown to be
important for interactions between EBNA1 and chromosomes
in metaphase and to associate with metaphase chromosomes.
Here, we examine the timing of the chromosome associations
of EBNA1 and EBP2 through mitosis and the regions of EBNA1
that mediate the chromosome interactions at each stage of
mitosis. We show that EBP2 is localized to the nucleolus until
late prophase, after which it relocalizes to the chromosome
periphery, where it remains throughout telophase. EBNA1 is
associated with chromosomes early in prophase through to
Keywords: Epstein-Barr virus, Segregation, Chromosome periphery,
Nucleolus, EBNA1, EBP2
Introduction
Epstein-Barr virus (EBV) genomes stably persist in the nuclei of
proliferating cells as double-stranded circular DNA plasmids. This
persistence, which occurs during latent infection, involves the
replication of the EBV genomes once per cell cycle coupled with
a mitotic segregation mechanism in which the EBV genomes are
tethered to the cellular chromosomes (reviewed by Frappier, 2004;
Frappier, 2010). The replication and segregation of EBV DNA
requires a single viral protein, Epstein-Barr nuclear antigen 1
(EBNA1), in addition to cellular factors. EBNA1 contributes to the
initiation of EBV DNA replication by binding to four recognition
sites present in the dyad-symmetry (DS) element of the latent origin
of replication, oriP (Koons et al., 2001; Rawlins et al., 1985). This
interaction destabilizes nucleosomes at the origin and aids the
recruitment of cellular replication factors to the origin (AvolioHunter et al., 2001; Dhar et al., 2001; Julien et al., 2004; Norseen
et al., 2008; Schepers et al., 2001). The segregation function of
EBNA1 involves EBNA1 binding to 20 recognition sites in the
family of repeats (FR) element of oriP, as well as the interaction
of EBNA1 with the cellular mitotic chromosomes (Krysan et al.,
1989; Lupton and Levine, 1985). EBNA1 binds specific sequences
in the FR and DS elements through its C-terminal DNA-binding
and dimerization domain (Fig. 1), although this domain is not
sufficient for the functional activities of EBNA1 (Ambinder et al.,
1991; Bochkarev et al., 1996; Summers et al., 1996).
Given their importance in EBV persistence, the mechanisms by
which EBNA1 interacts with mitotic chromosomes and mediates
the segregation of EBV episomes has been the subject of several
studies. EBNA1, EBV episomes and oriP-based plasmids have all
been found to be tightly associated with metaphase chromosomes
(Grogan et al., 1983; Harris et al., 1985; Petti et al., 1990; Simpson
et al., 1996). EBNA1 and the FR element have been shown to be
required for the attachment of oriP-containing plasmids to mitotic
chromosomes, although EBNA1 interacts with mitotic
chromosomes whether or not it is bound to oriP (Kanda et al., 2001;
Kapoor et al., 2005; Wu et al., 2000). Functional characterization
of EBNA1 mutants has shown that the DNA replication and
segregation functions of EBNA1 are distinct in terms of their amino
acids requirements. In particular, deletion of the Gly-Arg-rich
sequence between residues 325-376 (Fig. 1) abrogates the ability
of EBNA1 to stably maintain oriP plasmids in human cells without
affecting the replication of these plasmids, a pattern that is consistent
with a specific role in plasmid segregation (Wu et al., 2002). This
mutation also decreases the association of EBNA1 with metaphase
chromosomes, which is consistent with the chromosome-tethering
model of segregation (Wu et al., 2002). A similar but more subtle
effect on plasmid segregation was observed when residues 8-67
were deleted (Wu et al., 2002). This N-terminal region includes a
second Gly-Arg-rich sequence between residues 33 and 53 (Fig.
1), although deletion of this sequence alone had no detectable effect
on any EBNA1 function (Wu et al., 2002). Both Gly-Arg-rich
sequences of EBNA1 have been shown to have a propensity to bind
to mitotic chromosomes when excised from EBNA1 and fused to
other proteins (Hung et al., 2001; Marechal et al., 1999). However,
the degree to which each is involved in chromosome interactions
in the context of the native EBNA1 protein is unclear. The similarity
of the sequences in these Gly-Arg regions to AT hook sequences
has led to the hypothesis that these sequence might directly contact
telophase and partially colocalizes with chromosomal EBP2 in
metaphase through to telophase. Using EBNA1 deletion
mutants, the chromosome association of EBNA1 at each stage
of mitosis was found to be mediated mainly by a central glycinearginine region, and to a lesser degree by N-terminal sequences.
These sequence requirements for chromosome interaction
mirrored those for EBP2 binding. Our results suggest that
interactions between EBNA1 and chromosomes involve at least
two stages, and that the contribution of EBP2 to these
interactions occurs in the second half of mitosis.
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Fig. 1. Schematic representation of EBNA1 and EBNA1 mutants. The EBNA1
used in these studies has a small version of the Gly-Ala repeat region, which is
known to vary in length in different EBV isolates and does not contribute to
the known EBNA1 functions (Wu et al., 2002; Yates and Camiolo, 1988). The
indicated deletions were made within this context. Positions of the Gly-Argrich regions, nuclear localization sequence (NLS) and DNA-binding and
dimerization domains are indicated.
chromosomal DNA (Sears et al., 2004), although the same sequences
are known to interact with at least a few different cellular proteins
(Holowaty et al., 2003; Shire et al., 1999; Snudden et al., 1994;
Van Scoy et al., 2000; Wang et al., 1997).
To date, one cellular protein, EBNA1-binding protein 2 (EBP2),
has been found to have a role in EBNA1-mediated plasmid
segregation. This protein was discovered as an EBNA1 binding
partner in a two-hybrid screen, and residues 325-376 of EBNA1 are
important for this interaction (Shire et al., 1999). It was subsequently
shown that EBP2 enables EBNA1 to segregate FR-containing
plasmids in yeast by facilitating the interaction of EBNA1 with yeast
mitotic chromatin (Kapoor and Frappier, 2003; Kapoor et al., 2001).
In keeping with this finding, EBP2 associates with human metaphase
chromosomes, and silencing of EBP2 expression results in a large
decrease in the amount of EBNA1 associated with metaphase
chromosomes (Kapoor et al., 2005; Wu et al., 2000). In addition, the
expression of human EBP2 in murine Sp2/0 cells enables EBNA1
to stably maintain oriP-based plasmids in these cells (Habel et al.,
2004). However, it is not clear whether EBP2 enables the initial
association of EBNA1 with the chromosomes, or whether it is required
to maintain or stabilize the EBNA1-chromosome interaction.
EBP2 is highly conserved in eukaryotes (Henning and Valdez,
2001; Shire et al., 1999), where it forms part of the nucleolus
(Andersen et al., 2002; Chatterjee et al., 1987). Studies in
Saccharomyces cerevisiae show that yeast EBP2 has an essential
role in processing 27S pre-rRNA to 25S rRNA (Huber et al., 2000;
Tsujii et al., 2000) and, in keeping with this role, EBP2 is also
required for cell proliferation in human cells (Kapoor et al., 2005).
In mammals, the nucleolus disassembles at the beginning of mitosis
and the components disperse, some of which then associate with
the cellular chromosomes to form a peripheral layer (Gautier et al.,
1992; Hernandez-Verdun and Gautier, 1994). The timing of
redistribution of specific nucleolar proteins appears to be highly
ordered, although only a small number of nucleolar proteins have
been followed through mitosis (Leung et al., 2004; Zatsepina et al.,
1997). Our initial observations suggest that EBP2 belongs to the
class of nucleolar protein that redistributes to the chromosomes in
mitosis (Kapoor et al., 2005; Wu et al., 2000), but nothing is known
about the timing of this movement. In addition, controversy on the
localization of EBP2 was raised when one group failed to detect
EBP2 on mitotic chromosomes (Sears et al., 2004).
Although it is well established that EBNA1 associates with
metaphase chromosomes, few studies have examined the timing
with which EBNA1 associates with host chromosomes through
mitosis. In addition, it remains controversial as to whether or not
EBNA1 binds chromosomes in interphase (Daikoku et al., 2004;
Ito et al., 2002; Kanda et al., 2007; Kanda et al., 2001; Ritzi et al.,
2003). In this study, we address the timing of the association of
EBP2 and EBNA1 with host chromosomes through mitosis to try
to understand how EBP2 contributes to EBNA1-mediated
segregation and how relocalization of EBP2 from the nucleolus
compares with that of other nucleolar proteins. We also investigate
the contribution of the two Gly-Arg-rich regions of EBNA1 to
interactions with EBP2, and to chromosome association at different
stages of mitosis.
Results
EBP2 relocalizes late in prophase to the chromosomal
peripheral layer
To date, the subcellular localization of EBP2 has only been
examined in interphase- and in metaphase-blocked cells. We wanted
to assess the localization of EBP2 throughout mitosis more carefully,
in the absence of agents that block progression through the cell
cycle. Imaging of mitotic cells is often hampered by their round
morphology. To circumvent this problem, we chose to conduct our
studies in Rat2 cells, which have a relatively flat morphology
throughout mitosis and hence are suitable for mitotic imaging (Bakal
et al., 2005). Endogenous EBP2 in log-phase cells was visualized
using polyclonal antibody raised against highly purified human
EBP2, and cells at various stages of mitosis were identified by the
DNA morphology after DAPI staining.
The localization of EBP2 was first examined in prophase cells,
which were identified according to their spaghetti-like DNA strands
and also, in some cases, by the presence of phosphorylated histone
H3 (Fig. 2A). In all prophase cells examined, EBP2 was localized
to one or two discreet regions of the nucleus, which is consistent
with its known nucleolar localization in interphase (Fig. 2A,B top
panel). B23 (also called nucleophosmin) was examined as a marker
for a nucleolar protein that is known to redistribute to the cellular
chromatin during prophase (Dundr et al., 1997). In prophase cells
where B23 was localized to the nucleolus, EBP2 always showed
similar localization as B23, confirming that EBP2 is largely
nucleolar in prophase (Fig. 2A, middle panel). However, prophase
cells were also evident in which B23 was dispersed over the host
DNA (Fig. 2A, bottom panel), consistent with previous reports that
B23 leaves the nucleolar region during prophase (Dundr et al., 1997;
Zatsepina et al., 1997). In these cells, EBP2 remained largely
localized to the nucleolar region, although a small amount of staining
outside of this region was also detected. These results indicate that
EBP2 remains in the nucleolus after the redistribution of B23.
Rat2 cells at various stages of mitosis were also imaged by
deconvolution of optical stacks (Fig. 2B, left panels). The majority
of EBP2 was observed to be associated with the condensed
chromosomes in metaphase, anaphase and telophase, where it
localized predominantly to the edges of the chromatin. To verify
that this localization is not particular to rodent EBP2, the same
experiments were performed with CFPAC cells, a human
pancreatic adenocarcinoma cell line also known for its flat
morphology in mitosis (Levesque et al., 2003). EBP2 localization
in these cells throughout mitosis was found to be identical to that
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EBNA1 and EBP2 chromosome interactions
Fig. 2. Localization of endogenous EBP2 at
various stages of mitosis. (A)Rat2 cells in
prophase were stained with antibodies against
EBP2 and either phosphorylated histone H3
(top panel) or B23 (bottom 2 panels) and
counterstained with DAPI. (B)Rat2 or CFPAC
cells were stained with antibodies against
EBP2 (green) and counterstained with DAPI
(blue). Single planes from deconvolved optical
stacks are shown for cells at the indicated
stages of mitosis.
in Rat2 cells (Fig. 2B, right panels). The results indicate that EBP2
relocalizes to the chromosomes very late in prophase and forms
part of the chromosomal peripheral layer in metaphase through
to telophase.
Comparison of EBNA1 and EBP2 localization throughout
mitosis
We next wanted to compare the localization of EBNA1 and EBP2
throughout mitosis, and determine whether EBNA1 expression
affected the localization of EBP2. For these experiments, we
generated Rat2 cells expressing EBNA1 fused to a dimeric form
of the HcRed fluorescent tag. Log-phase fixed cells were visualized
for Red-tagged EBNA1 and endogenous EBP2 (using anti-EBP2
antibody) and categorized into mitotic phases based on morphology
of the DAPI-stained DNA. Deconvolved images are shown in Fig.
3. In prophase (Fig. 3A), EBNA1 fluorescence largely corresponded
to the condensed DNA. By contrast, most of the EBP2 was
localized to the nucleolar region, with a small amount exhibiting
more dispersed staining, as observed in the absence of EBNA1.
Tracings showing a degree of correspondence of EBNA1, EBP2
and DAPI staining are shown for each image (right panels), as
determined from a line drawn diagonally through the image
(indicated in merged images) using ImageJ software. The results
indicate that EBNA1 does not alter the localization of EBP2 and
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Fig. 3. Localization of HcRed-EBNA1 and endogenous EBP2 at various stages of mitosis. Rat2 cells expressing HcRed-tagged EBNA1 (shown in red or pink)
were stained with antibodies against EBP2 (green) and counterstained with DAPI (blue). Images are single planes from deconvolved optical sections of prophase
(A), metaphase (B), anaphase (C) and telophase (D) cells. Line graphs on the right in A were obtained using ImageJ software from a line drawn diagonally across
the cells (indicated on overlay images). Line tracings of DAPI, EBNA1 and EBP2 staining are shown in blue, red and green, respectively. (E)ImageJ line graphs
from multiple images at each stage of mitosis were used to determine Pearson coefficients of correlation for EBNA1 and EBP2. Average values + s.d. are shown.
Correlation coefficients are also shown for endogenous EBNA1 and EBP2 on metaphase chromosome spreads from Akata cells (Fig. 4).
that most of the EBNA1 is associated with host chromatin in
prophase.
In metaphase through to telophase, EBNA1 was tightly associated
with the host chromosomes, resulting in diffuse staining over the
entire chromatin mass (Fig. 3B-D). EBP2 behaved as it did in the
absence of EBNA1, with a significant portion of the protein
associating with the edges of the chromatin, beginning in metaphase
and extending through to telophase. For each stage of mitosis,
several images were analysed by ImageJ software and Pearson
coefficients of colocalization were determined for six deconvolved
images at each stage of mitosis (where a value of ‘1’ indicates perfect
colocalization). The average values (shown in Fig. 3E) confirm that,
although there is little correspondence of EBNA1 and EBP2 in
prophase, the overlap in the localization of these proteins
significantly increases, starting in metaphase and continuing into
anaphase and telophase.
EBNA1 and EBP2 localization on metaphase chromosomes
from Burkitt’s lymphoma cells
We also examined the localization of EBNA1 and EBP2 in
metaphase in the context of latent EBV infection. EBV establishes
latent infection in B cells, in some cases contributing to the
development of Burkitt’s lymphoma. The round morphology of B
cells severely impairs the imaging of these cells; however,
localization of protein to mitotic chromosomes can be assessed by
imaging spreads of the mitotic chromosomes. Therefore, Akata
EBV-positive Burkitt’s lymphoma cells were blocked in metaphase,
and the chromosomes were spread on slides and stained with
antibodies against endogenous EBNA1 and EBP2. Images from
several cells were analysed by deconvolution and three layers from
the deconvolved Z-stacks of a representative chromosome spread
are shown in Fig. 4, starting from the middle of the chromosomes
(top row) and moving towards the outer layer (bottom row). Line
scans taken diagonally across the images are also present, showing
the degree of correspondence of DAPI, EBNA1 and EBP2 staining.
EBNA1 exhibited diffuse staining that corresponded well to the
DAPI staining, and was most intense in the middle layer of the
chromosomes. EBP2 staining also largely corresponded to the DAPI
staining, and in many cases with EBNA1 peaks. However, EBP2
was more prevalent in the outer layer of the chromosomes than in
the middle layer, which is consistent with the peripheral
chromosome staining observed in Rat2 and CFPAC cells. It is
important to note, however, that at each layer through the
chromosomes, there are yellow dot, which are indicative of an
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EBNA1 and EBP2 chromosome interactions
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Fig. 4. Localization of endogenous EBNA1 and EBP2 on Akata metaphase chromosomes. Akata cells were blocked in mitosis with colcemid, dropped onto slides,
fixed, stained with antibodies against EBNA1 (red) and EBP2 (green) and counterstained with DAPI. Microscopy was performed in which multiple Z-stacks were
collected and used to generate a deconvolved image. Three layers of the deconvolved stacks are shown from the middle of the image (top) moving outwards
(bottom). Line graphs (right) were generated in ImageJ from a line drawn diagonally through each image between the upper right-hand and lower-left hand corners.
Line tracings of DAPI, EBNA1 and EBP2 staining are shown in blue, red and green, respectively.
interaction between a proportion of EBNA1 and EBP2. Line scans
from three different chromosome spreads were used to calculate
Pearson coefficients of colocalization for EBNA1 and EBP2 (Fig.
3E). The average coefficient of 0.38 was similar to that observed
in the later stages of mitosis in Rat2 cells, and is consistent with
colocalization of a proportion of the two proteins.
Contributions of EBNA1 regions to host chromosome
interactions
Both the N-terminal (residues 34-52) and central (residues 325376) Gly-Arg-rich regions of EBNA1 have been shown to have
some capacity to bind metaphase chromosomes when excised from
EBNA1, and the central Gly-Arg region has been shown to be
important for association of EBNA1 with chromosomes from
metaphase-blocked cells. However, the degree to which the two
Gly-Arg regions contribute to chromosome interactions of native
EBNA1 at various stages of mitosis has not been examined. We
addressed this by expressing EBNA1 deletion mutants (without
tags) in human nasopharyngeal carcinoma (CNE2Z) cells, staining
log-phase cells for EBNA1 using EBNA1-specific monoclonal
antibody and examining cells at various stages of mitosis according
to the DNA morphology after DAPI staining (Fig. 5). CNE2Z cells
were used because they are derived from an EBV-positive
nasopharyngeal carcinoma (a tumour that is highly associated with
latent EBV infection), but lose the EBV genomes during growth
in culture. The localization of EBNA1 in CNE2Z was
indistinguishable from that observed above for Rat2, showing very
tight correlation to the condensed DNA at prophase through to
telophase (Fig. 5).
Comparison of the localization of EBNA1 mutants (shown in
Fig. 1) in prophase cells showed that EBNA1 proteins lacking the
N-terminal Gly-Arg region (8-67 or 34-52) remained closely
associated with the condensed DNA throughout mitosis, although
a small amount of staining outside the chromosomes was observed
for 8-67 at each stage of mitosis (Fig. 5). A more obvious effect
on EBNA1 localization was observed when the central Gly-Arg
region was deleted in EBNA1 mutant 325-376. This mutant
exhibited considerable staining both on and outside the
chromosomes at all stages of mitosis. Deletion of both of the GlyArg regions in 8-67325-376 or 34-52325-376 resulted in a
diffuse staining pattern that did not correspond to the chromosomes
at any stage of mitosis (Fig. 5). Note that the intensity of EBNA1
staining was similar for all mutants, indicating that there were no
major differences in expression levels. The results indicate that
sequences 325-376 are predominantly responsible for chromosome
interactions throughout mitosis and that sequences 34-52 make a
minor contribution to chromosome attachment that can partially
substitute for the 325-376 sequence in its absence.
The contribution of EBNA1 residues 8-67 and 325-376 to
metaphase chromosome attachment was also assessed by
biochemical fractionation of colcemid-blocked cells (Fig. 6A, right
panel). In these experiments, cells expressing the EBNA1 proteins
were lysed in isotonic buffer and subjected to centrifugation to
separate soluble proteins from insoluble chromatin-bound proteins.
Consistent with the microscopy data, this method showed a tight
association of EBNA1 with the chromosomal pellet and that
deletion of residues 325-376 results in the release of a large
proportion of EBNA1 from the chromosomes. EBNA1 8-67,
however, remained largely localized to the chromosomal pellet, with
a release of a smaller proportion of EBNA1 into the soluble fraction.
Note that, because the fractionation method requires that EBNA1
remains associated with the chromosomes during centrifugation,
this should result in the release of proteins weakly associated with
the chromosomes, and hence show a higher proportion of proteins
in the soluble fraction than is evident by microscopy. The
fractionation results confirmed that residues 325-376 have a major
role in chromosome attachment and that the N-terminal sequences
have a more modest role.
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Fig. 5. Localization of EBNA1 mutants throughout mitosis. (A)Log-phase CNE2Z cells expressing wild-type EBNA1or the indicated EBNA1 mutant were stained
with anti-EBNA1 antibody (green) and counterstained with DAPI (red). Examples of prophase, metaphase and telophase cells are shown for each EBNA1 protein.
Is EBNA1 associated with chromatin in interphase?
There have been mixed reports in the literature as to whether
EBNA1 is associated with cellular chromatin in interphase. For
example, biochemical fractionation performed by Kanda and coworkers (Kanda et al., 2001) support the conclusion that EBNA1
is bound to chromatin, whereas those performed by Daikoku and
colleagues (Daikoku et al., 2004) indicate that EBNA1 is largely
soluble. However, these experiments were performed using different
buffer conditions (salt and detergent concentrations), which might
account for this discrepancy. To further investigate the possible
association of EBNA1 with interphase chromatin, we performed
biochemical fractionation of log-phase cells using the same
conditions that we used to detect EBNA1 binding to mitotic
chromosomes (isotonic conditions) and compared this with the same
fractionation performed in buffer systems used by Kanda and
colleagues [hypotonic sucrose buffer (HSB)] and Daikoku and coworkers [CSK with 0.5% Triton X-100 (mCSK)]. EBNA1 pelleted
with the chromosomes in interphase in isotonic and HSB buffers,
but not in mCSK buffer. When biochemical fractionation was
performed on metaphase-blocked cells, similar results were
obtained: the mCSK buffer resulted in the solubilization of EBNA1.
Given that the interaction of EBNA1 with metaphase chromosomes
is well established, the results indicate that mCSK buffer disrupts
the interaction of EBNA1 with chromosomes, and hence does not
provide reliable information on whether or not EBNA1 is associated
with interphase chromatin. However, the results with both isotonic
and mCSK buffers are consistent with EBNA1 binding to interphase
chromatin, although association with other insoluble components
is also possible.
We also performed biochemical fractionations (using isotonic
buffer) with log-phase cells expressing EBNA1 mutants, to assess
whether sequences involved in chromosome attachment in mitosis
also mediate the putative chromatin interactions in interphase. We
found that, as in mitosis, residues 325-376 have a major role in
interphase chromosome interactions, whereas deletion of residues
8-67 had a much smaller effect. The results suggest that residues
325-376 have an important role in chromosome interactions
throughout the cell cycle.
Relationship between EBP2 binding and mitotic chromosome
attachment of EBNA1
To better understand the relationship between the mitotic
chromosome interactions of EBNA1 and EBP2 binding, we
examined the ability of the EBNA1 mutants used in the localization
EBNA1 and EBP2 chromosome interactions
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Fig. 6. Biochemical fractionation experiments for chromosome interactions.
(A)Whole-cell lysates (W) generated from log-phase (left) or mitotic-blocked
(right) HeLa cells expressing EBNA1, or 8-67 or 325-376 mutants were
separated into soluble (S) and insoluble pellet (P) fractions by centrifugation.
Equal cell equivalents of each fraction were subjected to western blotting
using anti-EBNA1 antibody. (B)Comparison of biochemical fractionation of
log-phase and mitotically blocked (M) cells performed in isotonic, hypotonic
sucrose (HSB) and modified CSK (mCSK) buffers.
studies to bind to EBP2. We have previously shown that this
interaction can be detected by yeast two-hybrid assay and that
residues 325-376 are important for this interaction (Shire et al., 1999;
Shire et al., 2006). We have now used a more sensitive version of
this assay based on LexA fusion proteins, to evaluate whether
EBNA1 N-terminal sequences also contribute to EBP2 interactions
(Fig. 7A). Deletion of residues 34-52 in EBNA1 was found to have
a negligible effect on EBP2 binding, whereas deletion of residues
325-376 severely decreased EBP2 binding. However, some ability
to bind EBP2 was still detected with 325-376 mutants, and this
was further decreased by removal of residues 34-52 (compare 325376 to 34-52325-376 in the bottom right panel of Fig. 7A).
Differences in EBP2 binding were not due to different levels of
expression of the EBNA1 mutants, because all were expressed in
yeast at similar levels (Fig. 7B). The observed effects mirror the
results for EBNA1 attachment to mitotic chromosomes, raising the
possibility that the mitotic chromosome interactions mediated by
residues 325-376 or by residues 34-52 (as occurs when 325-376 is
deleted) might involve EBP2.
Discussion
We have examined the timing with which EBP2 and EBNA1
associate with cellular chromosomes in the cell cycle and the
involvement of EBNA1 sequences in this process. EBP2 is a
nucleolar protein (Andersen et al., 2002; Chatterjee et al., 1987)
that has an essential role in rRNA processing (Huber et al., 2000;
Tsujii et al., 2000). In mammals, the nucleolus undergoes an ordered
disassembly at the beginning of mitosis, then reforms in telophase.
The mechanisms by which this complex structure disintegrates and
reforms at each cell cycle has been the subject of several studies,
but is not well understood. The proteins that normally reside in the
nucleolus in interphase have been reported to undergo various fates
in mitosis. Proteins involved in rRNA transcription generally
remain associated with the nucleolar-organizing regions in the
condensed chromosomes. However, protein components involved
in pre-rRNA processing have been reported to associate with the
periphery of all of the chromosomes and/or form particles in the
mitotic cytoplasmic particles, including nucleolus-derived foci
(Dundr et al., 1997; Gautier et al., 1992; Hernandez-Verdun and
Gautier, 1994; Olson and Dundr, 2005; Shaw and Jordan, 1995).
Fig. 7. Interaction between EBP2 and EBNA1 mutants. (A)Yeast two-hybrid
assays showing the interaction of EBNA1 mutants with EBP2 (bottom panels)
or with the empty pACT2 plasmid (top panels) as determined by activation of
a HIS3 reporter gene (right panels). Wild-type EBNA1 or 8-67, 34-52,
325-376 or 34-52325-376 mutants were expressed as fusions to the lexA
DNA-binding domain and tested for interactions with EBP2 fused to the
GAL4 activation domain, expressed from pACT2 (bottom panels). Tenfold
serial dilutions of the cultures were grown on plates containing or lacking His,
as indicated. (B)Western blot of equal amounts of yeast cell lysates probed
with anti-EBNA1 antibody showing similar expression levels of each protein.
Our observations indicate that EBP2 is one of the nucleolar proteins
that associates with the periphery of the chromosomes in mitosis,
and this is consistent with previous observations that proteins
involved in pre-rRNA processing form part of the peripheral layer
of the chromosomes (Leung et al., 2004; Zatsepina et al., 1997).
One of the most studied nucleolar proteins in terms of its
localization in mitosis is B23, which is involved in late rRNA
processing steps and is part of the granular component of the
nucleolus. B23 leaves the nucleolus in prophase to associate with
the chromosome periphery and this relocalization has been found
to occur later in prophase than for other nucleolar proteins,
particularly those from the fibrillar center of the nucleolus (Leung
et al., 2004). Here, we have shown that EBP2 leaves the nucleolus
even later than B23. However, similarly to B23, EBP2 is largely
(but not entirely) associated with the peripheral chromosome layer.
EBNA1 is essential for the long-term maintenance of EBV
episomes in replicating cells because of contributions to both the
replication and mitotic segregation of the episomes, the latter of
which requires EBNA1 attachment to the chromosomes. However,
the timing by which EBNA1 associates with chromosomes in the
cell cycle has not been clear, owing to conflicting data. For
example, Ito and co-workers (Ito et al., 2002) reported that EBNA1
is associated with interphase chromatin that had been artificially
condensed by calyculin A treatment; however, this treatment might
also artificially induce protein-chromosome interactions. Using
biochemical fractionation, Kanda and colleagues (Kanda et al.,
2001) observed that EBNA1 from interphase cells sedimented with
the chromosomal pellet and was released by micrococcal nuclease,
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which is consistent with interaction of EBNA1 with chromosomes
in interphase. By contrast, biochemical fractionation approaches
used by Daikoku, Ritzi and colleagues (Daikoku et al., 2004; Ritzi
et al., 2003) found little EBNA1 from interphase cells in the
chromosomal pellet. Such divergent results might be due to the
different buffer conditions used in each study. Indeed, we showed
that seemingly small changes in buffer conditions can have major
effects on EBNA1 solubility, and that the association of EBNA1
with mitotic chromosomes is disrupted in some buffers used to
demonstrate chromosome interactions of other proteins (such as
CSK buffer with 0.5% Triton X-100). Therefore, one cannot draw
conclusions on EBNA1-interphase chromatin interactions under
these conditions. Using buffers that do not disrupt EBNA1-mitotic
chromosome interactions, we found that EBNA1 fractionates with
the chromosomal pellet in log-phase cells, which is consistent with
a chromatin interaction in interphase. In addition, we have clearly
shown by immunofluorescence imaging, that EBNA1 is associated
with chromosomes by prophase.
EBNA1 contains two Gly-Arg-rich regions, one between amino
acids 33-53 and a larger one spanning residues 325-376. Studies
involving excision of these sequences and fusions to other proteins
or to the EBNA1 DNA-binding domain have indicated that each
region has some ability to interact with metaphase chromosomes,
suggesting that they are important in the mitotic segregation function
of EBNA1 (Hung et al., 2001; Marechal et al., 1999; Sears et al.,
2004). However, functional analysis of EBNA1 deletion mutants
indicated that only residues 325-376 are important for segregation.
Specifically, deletion of amino acids 325-376 abrogated the ability
of EBNA1 to maintain EBV-based plasmids in replicating cells,
without affecting the DNA replication function of EBNA1, whereas
deletion of residues 34-52 had no effect on either the long-term
maintenance or the replication of EBV-based plasmids (Shire et al.,
1999; Wu et al., 2002). The fact that these deletion mutants retained
at least one functional activity of EBNA1 shows that these deletions
did not cause misfolding of EBNA1, a result that is consistent with
the fact that these regions are within an extended region of EBNA1
as opposed to being folded into a protease-resistant domain (E.
Bochkareva and L. Frappier, unpublished results).
To better understand the above observations, it was necessary to
more carefully examine the contributions of residues 33-53 and 325376 to chromosome attachment throughout mitosis. Here, we have
shown that residues 325-376 have a major role in the chromosome
interactions of EBNA1 at each stage of mitosis, whereas deletion
of residues 34-52 had no obvious effect on chromosome interactions
at any stage of mitosis. These results agree well with the functional
effects on segregation, and strongly suggest that the segregation
defect of 325-376 is due to insufficient chromosome attachment.
In addition, the ability of EBNA1 N-terminal sequences to mediate
chromosome attachment was revealed when residues 325-376 were
deleted, because the chromosome interactions that were seen with
this mutant were abrogated by removal of amino acids 34-52 or 867. This suggests that the N-terminal Gly-Arg-rich sequences can
mediate weak chromosome interactions in the absence of residues
325-376 that form the principal chromosome-binding region. It is
also interesting that8-67 mutant on its own resulted in detectable
effects on chromosome interactions at each stage of mitosis (which
was not seen with the 34-52 mutant), suggesting that sequences
adjacent to the N-terminal Gly-Arg contribute to chromosome
interactions. This observation is consistent with previous functional
data showing that the 8-67 mutant had reduced ability to maintain
oriP plasmids (Wu et al., 2002).
In our previous studies, we identified EBP2 as a protein capable
of interacting with EBNA1 through its C-terminal domain (Kapoor
et al., 2001; Shire et al., 1999). We also showed that this protein
interacts with chromosomes in metaphase-blocked cells through its
central coiled-coil domain (Kapoor and Frappier, 2003; Kapoor et
al., 2005; Wu et al., 2000). Remarkably, expression of human EBP2
in S. cerevisiae enabled EBNA1 to segregate plasmids containing
the EBV segregation element (FR) in yeast in a manner that was
dependent on both the chromosome-interacting and EBNA1interacting domains of EBP2, and segregation in this system was
shown to be sensitive to the same EBNA1 mutations as human cells
(Kapoor and Frappier, 2003; Kapoor et al., 2001). In addition,
segregation of FR-containing plasmids in yeast was also achieved
by expressing a single protein in which the EBP2 chromosomebinding domain was fused to the EBNA1 DNA-binding domain
(Kapoor and Frappier, 2003). The importance of EBP2 for EBNA1mediated segregation in human cells was suggested by the close
correlation of effects of point mutations in residues 325-376 on
plasmid-maintenance activity and EBP2 binding (Shire et al., 2006),
and further confirmed by the finding that silencing of EBP2 in four
different human cells lines (including EBV-positive cells) led to a
substantial decrease in the association of EBNA1 with metaphase
chromosomes (Kapoor et al., 2005). As expected, this loss of EBNA1
from the mitotic chromosomes was accompanied by a similar loss
of oriP plasmids from the chromosomes (Kapoor et al., 2005). The
results as a whole indicate that an interaction between EBP2 and
EBNA1 is important for proper association of EBNA1 with mitotic
chromosomes and hence for the segregation function of EBNA1.
However, it is not clear why EBP2 was required for the association
of EBNA1 with mitotic chromosomes. Two possibilities seem likely:
(1) EBP2 might be required for the initial attachment of EBNA1 to
chromosomes by mediating EBNA1 interactions with the
chromosomes; or (2) EBP2 might associate with EBNA1 after its
initial chromosome interaction and may be required to stabilize the
EBNA1-chromosome interaction. The data presented here on the
timing of the association of EBNA1 and EBP2 with chromosomes
through mitosis strongly support the second possibility.
The cumulative results on EBNA1-chromosome associations
suggest a model in which interactions between EBNA1 and
chromosomes that govern mitotic segregation involve at least two
steps. The first chromosome interaction probably occurs in
interphase and does not involve EBP2. At this stage, EBNA1 might
interact directly with the cellular DNA, as proposed by Sears and
colleagues (Sears et al., 2004), or it might involve interactions with
one or more chromatin-associated proteins. The second stage of
EBNA1-chromosome interaction occurs during the second half of
mitosis, and might require an interaction with EBP2 to keep EBNA1
tightly associated with the chromosomes, either because of
stabilization of the initial EBNA1-chromosome contact or the
addition of a second contact. This two-step scenario has interesting
parallels with the segregation of papillomavirus genomes that is
mediated by the E2 protein of bovine papillomavirus (BPV), which,
similarly to EBNA1, governs the segregation of BPV episomes by
tethering them to the cellular chromosomes in mitosis. Interactions
of E2 with mitotic chromosomes have been shown to occur through
two different host proteins, Brd4 (Brannon et al., 2005; You et al.,
2004) and ChlR1 (Parish et al., 2006), leading to a model in which
ChlR1 is required for the initial attachment of E2 to chromosomes
before mitosis and/or in early mitosis, and in which Brd4 is involved
in E2-chromosome interactions at later stages of mitosis (Parish et
al., 2006). Thus several mechanisms of chromosome interaction at
EBNA1 and EBP2 chromosome interactions
different stages of mitosis might be a common theme for viral
episomes that segregate by chromosome attachment.
1% Triton X-100. EBNA1 was detected with OT1x monoclonal antibody followed
by Alexa-Fluor-488-conjugated goat anti-mouse antibody (Invitrogen) and DAPI (25
ng/l) staining. Microscopy was performed as described above.
Materials and Methods
Biochemical fractionation
Cell culture
Biochemical fractionation was performed either on log-phase cells or on cells blocked
in mitosis with colcemid treatment (0.1 g/ml medium) followed by mitotic shakeoff, as previously described (Kapoor et al., 2005). Unless otherwise indicated, cells
from a 6-cm dish were lysed in 100 l of isotonic buffer [20 mM Tris-HCl pH 7.5,
75 mM KCl, 30 mM MgCl2, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), 0.5
mM EDTA, 1 mM PMSF, 1 mM benzamidine]. A 50 l sample of the cell lysate
was removed (sample W) and the remaining 50 l of lysate was spun for 10 minutes
at 4°C in a microcentrifuge. The supernatant (sample S) was removed and the pellet
was resuspended in 50 l isotonic buffer (sample P). Where indicated, isotonic buffer
was substituted with modified CSK buffer (mCSK: 10 mM PIPES pH 6.8, 100 mM
NaCl, 300 mM sucrose, 1 mM MgCl2, I mM EGTA, 1 mM DTT, 1 mM PMSF, 0.5%
Triton X-100) (Daikoku et al., 2004) or hypotonic sucrose buffer (HSB: 10 mM
HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM
DTT, 0.1% Triton X-100, protease inhibitors) (Kanda et al., 2001). Aliquots of 25
l of each of W, S and P samples were subjected to SDS-PAGE and western blotting
using OT1x monoclonal antibody against EBNA1 and goat anti-mouse antibody
conjugated with HRP (Santa Cruz Biotechnology). Membranes were developed with
the ECL Plus system (Amersham Biosciences).
Rat2 thymidine kinase-deficient rat fibroblasts (Reynolds et al., 1987; Topp, 1981)
were propagated in DMEM. CFPAC-1 pancreatic adenocarcinoma cells (Schoumacher
et al., 1990) were maintained in Iscove’s modified Dulbecco’s medium (Gibco). Akata
cells (Burkitt’s lymphoma cell line) were maintained in RPMI 2640 medium (Sigma).
CNE2Z EBV-negative nasopharyngeal carcinoma cells were grown in -MEM.
CNE2Z cells were derived from EBV-positive tumour cells that lose the EBV upon
growth in culture (Sun et al., 1992). In all cases, medium was supplemented with
10% fetal bovine serum, 5% penicillin and streptomycin and 5% L-glutamine.
Plasmid constructs
Journal of Cell Science
4349
To make the diHcRed-tagged EBNA1 construct, EBNA1 (lacking most of the GlyAla repeat) was PCR amplified from the pAS2.EBNA1 vector (Shire et al., 1999)
with primers that placed HindIII and BamHI sites flanking the cDNA. After digestion
with HindIII and BamHI, the cDNA was inserted between the HindIII and BamHI
sites of the pdiHcRed-C1 vector [a modification of pHcRed1-C1 from Clontech
Laboratories kindly supplied by David Bazett-Jones, The Hospital for Sick Children,
Toronto, Canada (Dellaire et al., 2006)] just after the cDNA encoding a dimeric version
of HcRed. The cDNA encoding the fusion protein was then excised with NheI and
BamHI and inserted between the NheI and BamHI sites of pcDNA3.1/Hygro(–)
(Invitrogen). For experiments involving the localization of EBNA1 mutants in human
cells, EBNA1 proteins were expressed from pc3oriP and were generated as previously
described (Wu et al., 2002). Constructs for yeast two-hybrid assays were generated
by subcloning the EBNA1 mutants into pLexA-kan (a gift from Igor Stagljar,
University of Toronto, Toronto, Canada) between the NdeI and BamH1 sites,
resulting in fusion to the LexA DNA-binding domain.
Yeast two-hybrid assays
Two-hybrid assays were performed as described (Shire et al., 2006). Briefly, S.
cerevisiae strain L40a (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) was
transformed with one of the pLexA.EBNA1 constructs and with pACTII expressing
hEBP2 (fused to the GAL4 activation domain) or with empty pACTII (negative
control). Yeast were grown overnight in medium selective for both plasmids (lacking
Trp and Leu) and tenfold serial dilutions of the cultures were spotted and grown on
plates lacking Trp and Leu with and without His.
Immunofluorescence microscopy of Rat2 and CFPAC cells
Rat2 and CFPAC cells were plated on coverslips at a density of 1-2⫻105 cells/ml at
37°C in complete medium, washed with PBS, and fixed in 3% formaldehyde in PBS.
The cells were then permeabilized with 0.1% Triton X-100 for 5-10 minutes, washed
with PBS, and blocked in 3% BSA in PBS for 30 minutes before staining with rabbit
anti-EBP2 (Wu et al., 2000), goat anti-B23 (C10; Santa Cruz Biotechnology, CA)
and mouse anti-phosphohistone H3 (Upstate Laboratories) antibodies. After washing
in PBS with 0.01% Tween 20, cells were treated with the following secondary
antibodies: fluorescein-isothiocyanate-conjugated donkey anti-rabbit antibody for
EBP2 (Santa Cruz Biotechnology), donkey anti-goat Cy3 secondary antibody for
B23 (Chemicon) and Texas-red-conjugated goat anti-mouse for phosphohistone H3
(Molecular Probes, Eugene, OR). After washing, cells were mounted on coverslips
with Prolong Antifade, containing DAPI (4⬘,6⬘-diamidino-2-phenylindole; Invitrogen).
To generate Rat2 cells expressing diHcRed-EBNA1, a 10 cm plate of Rat2 cells were
transfected with 5 g pdiHcRed-EBNA1 using lipofectamine then propagated in
medium containing 200 g/ml hygromycin (Gibco) to select for the plasmid. After
3 weeks of selection, cells were fixed and stained for EBP2, as described above.
Microscopy was performed using a Leica DM-IRE2 wide-field inverted fluorescence
microscope, a Hamamatsu ORCA-AG CCD camera, appropriate filters and Openlab
software (version 4.0.2). Exposure times for DAPI, hEBP2 and diHcRedEBNA1
imaging were kept constant within each set of experiments. Where indicated, digital
images were collected at defined intervals (0.2-0.3 m) along the Z-axis and the images
were deconvolved (60 iterations) using the 3D deconvolution software (Image Pro
software, MediaCybernetics). Digital images were imported into ImageJ (NIH,
Bethesda, MD) for line pixel analysis and calculation of the extent of colocalization
between EBNA1 and EBP2. Images were false-coloured using ImageJ software and
presented using Adobe PhotoShop 5.5 (Improvision, San Jose, CA). The Pearson product
moment correlation coefficient (r), a measure of the correlation of two variables X and
Y on the same object or organism, was calculated using ImageJ software.
Akata chromosome spreads
For chromosome spreads, Akata cells (EBV-positive Burkitt’s lymphoma) were
blocked in mitosis with colcemid (0.1 g/ml) for 16 hours, swollen in hypotonic
buffer, fixed with methanol:acetic acid (70:30) and dropped onto cooled slides as
previously described (Kapoor and Frappier, 2003). The chromosomes were then
stained for EBP2 and EBNA1 and counterstained with DAPI, as previously described
(Kapoor et al., 2005), using anti-EBP2 rabbit antibody and OT1x monoclonal antibody
against EBNA1 (kindly supplied by Jaap Middeldorp, VU University Medical Center,
Amsterdam, The Netherlands), followed by fluorescein-isothiocyanate-conjugated
donkey anti-rabbit and Texas-red-conjugated goat anti-mouse secondary antibodies.
Images were captured and processed as described above.
Immunofluorescence microscopy of EBNA1 mutants in CNE2Z cells
CNE2Z cells, grown on coverslips in a six-well dish, were transfected with 1 g
pc3oriP expressing EBNA1 or EBNA mutants using Fugene HD (Roche). Two days
later, cells were fixed in 3% formaldehyde for 30 minutes and permeabilized with
We thank Andy Wilde, David Bazett-Jones and Reagan Ching for
access to and assistance with deconvolution software and expert
microscopy advice. We also thank Igor Stagliar for pLexA and the L40a
yeast strain, and Japp Middeldorp for EBNA1 monoclonal antibody.
This work was funded by grant number 12477 from the Canadian
Institutes of Health Research. L.F. is a Tier 1 Canada Research Chair
in Molecular Virology.
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