2946
Research Article
SPOC1: a novel PHD-containing protein modulating
chromatin structure and mitotic chromosome
condensation
Sarah Kinkley1, Hannah Staege1, Gerrit Mohrmann1,*, Gabor Rohaly1, Theres Schaub1,‡, Elisabeth Kremmer2,
Andreas Winterpacht3 and Hans Will1,§
1
Heinrich-Pette Institute for Experimental Virology and Immunology, Martinistrasse 52, 20251 Hamburg, Germany
Institute of Molecular Immunology, Helmholtz Center Munich, German Center for Environmental Health (GmbH), Marchioninstrasse 25, 81377
Munich, Germany
3
Institute for Human Genetics, Schwabachanlage 10, 91054 Erlangen, Germany
2
*Present address: Labor Arndt, Lademannbogen 61, 22339 Hamburg, Germany
‡
Present address: Institute for Human Genetics, University Hospital Eppendorf (UKE), Martinistrasse 52, 20246 Hamburg, Germany
§
Author for correspondence ([email protected])
Accepted 11 May 2009
Journal of Cell Science 122, 2946-2956 Published by The Company of Biologists 2009
doi:10.1242/jcs.047365
Summary
In this study, we characterize the molecular and functional
features of a novel protein called SPOC1. SPOC1 RNA
expression was previously reported to be highest in highly
proliferating tissues and increased in a subset of ovarian
carcinoma patients, which statistically correlated with poor
prognosis and residual disease. These observations implied that
SPOC1 might play a role in cellular proliferation and
oncogenesis. Here we show that the endogenous SPOC1 protein
is labile, primarily chromatin associated and its expression as
well as localization are regulated throughout the cell cycle.
SPOC1 is dynamically regulated during mitosis with increased
expression levels and biphasic localization to mitotic
chromosomes indicating a functional role of SPOC1 in mitotic
processes. Consistent with this postulate, SPOC1 siRNA
knockdown experiments resulted in defects in mitotic
Introduction
We have recently identified a novel human gene, which contains
an open reading frame for a protein with a single PHD domain,
coined Survival time associated PHD finger protein in Ovarian
Cancer 1 (SPOC1) (Mohrmann et al., 2005). To date, nothing has
been reported on SPOC1 protein expression and there is only
information on its RNA expression and its chromosomal location.
The SPOC1 gene is located in chromosomal region 1p36.3, an area
previously identified for chromosomal instability and implicated in
tumour development and progression (Van Gele et al., 1998; Varga
et al., 2001). Consistent with this possibility, SPOC1 RNA transcript
levels were found to be significantly enhanced in a subset of ovarian
carcinomas and statistically correlated with poorer survival and
residual disease in these patients (Mohrmann et al., 2005). SPOC1
transcripts are most highly expressed in the testis and ovaries and
are ubiquitously expressed to a low level in all tissues. Furthermore,
SPOC1 RNA was found to be specifically expressed in the highly
proliferating cell types of the testis such as the spermatagonia but
not in the mature non-proliferating sperm cells (Mohrmann et al.,
2005). These data are compatible with a possible role of this protein
in cell proliferation and oncogenesis of specific cell types.
The human SPOC1 protein is 300 amino acids in length and has
a predicted molecular mass of 34 kDa. Except for the single C-
chromosome condensation, alignment and aberrant sister
chromatid segregation. Finally, we have been able to show, using
micrococcal nuclease (MNase) chromatin-digestion assays that
SPOC1 expression levels proportionally influence the degree of
chromatin compaction. Collectively, our findings show that
SPOC1 modulates chromatin structure and that tight regulation
of its expression levels and subcellular localization during
mitosis are crucial for proper chromosome condensation and
cell division.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/16/2946/DC1
Key words: PHD domain, Chromatin, Mitosis, Chromosome
condensation
terminal PHD domain and a bipartite nuclear localization sequence,
suggesting that it may be a nuclear chromatin affiliated protein, no
additional functional domains for the SPOC1 protein have so far
been identified. Therefore, based on the current limited knowledge
it can only be postulated that SPOC1 is an expressed nuclear
localized protein and that it may play a role in cell proliferation
and oncogenesis.
To understand the involvement of SPOC1 in these processes and
to gain insight into its biological function we have characterized it
at the protein level. In order to experimentally evaluate SPOC1 we
generated expression constructs, antibodies, stable cell lines, siRNA
oligos and performed a more detailed ‘in silico’ analysis. Using
these tools and various biochemical approaches we show that
SPOC1 is a tightly regulated chromatin-associated protein capable
of modulating chromatin structure and important for proper mitotic
chromosome condensation and cell division.
Results
SPOC1 is a novel protein conserved across higher eukaryotes
and contains several interesting putative functional domains
The evolutionary conservation of a protein is a good indication of
biological relevance and implies an important biological function.
Therefore we evaluated in detail the SPOC1 protein by ‘in silico’
SPOC1: a novel mitotic player
sequence alignment of all SPOC1 proteins currently available in
the GenBank. This revealed that SPOC1 is highly conserved across
higher eukaryotes and probably originated in evolution in the
phylum Chordata, subphylum Vertebrata (supplementary material
Fig. S1). The SPOC1 protein (also designated PHF13 in GenBank
annotations) is conserved in species ranging from fish to humans
and shows total sequence homologies ranging from 62% to 100%
(supplementary material Table S1). Further evaluation of the
SPOC1 sequences for putative domain structures identified a highly
conserved N-terminal region of unknown function (except in Gallus
gallus) from amino acid residues 20 to 70 (Mac Vector 7.0), a
centrally located bipartite nuclear localization sequence (Prosite
motif scan) from residues 110 to 127, a C-terminal PHD domain
from residues 232 to 280 (PFAM and SMART servers) and two high
scoring PEST domains at amino acid residues 52-88 and 141-190
(www.at.embnet.org/embnet/tools/bio/PESTfind; supplementary
material Fig. S1; Fig. 1A). Interestingly, these motifs, with the
exception of the PEST domains, show very little sequence diversity
and are almost 100% conserved from fish to humans (supplementary
material Fig. S1) arguing strongly for the functional conservation
of the SPOC1 protein across the vertebrate species in the phylum
Chordata. Furthermore, although the sequence length and amino
acid composition for the two PEST domains varies in lower
2947
Chordata species, they received similar or better PEST scores by
the PESTfind program (data not shown), indicating that these
degradation motifs are also conserved.
SPOC1 is a novel chromatin-associated protein
The putative domain structure of SPOC1 implies that it may be a
labile [PEST domains (Chevaillier, 1993; Garcia-Alai et al., 2006)]
nuclear (NLS) protein that is capable of interacting with and
modulating chromatin [PHD domain (Aasland et al., 1995; Bienz,
2006; Mellor, 2006; Zhang, 2006)]. To evaluate SPOC1 at the protein
level, rabbit polyclonal and rat monoclonal antibodies against SPOC1
were generated and tested for specificity (supplementary material Figs
S2-S4). Consistent with its conserved NLS and PHD domain,
immunofluorescence localization of both exogenous and endogenous
SPOC1 showed that it is in fact a nuclear protein (Fig. 1B). To
demonstrate the functionality of the SPOC1 NLS, a FLAG-SPOC1
construct deleted of its NLS was expressed. SPOC1 ΔNLS was found
to localize to both the cytoplasm and the nucleus, indicative of the
NLS being at least in part responsible for the nuclear localization of
SPOC1. The partial nuclear localization of SPOC1 ΔNLS may be
explained by either simple diffusion ratios and/or that it has a nuclear
binding partner that helps to retain it in the nucleus. In contrast to
the NLS, deleting SPOC1 of either of its PEST domains or its PHD
Fig. 1. Predicted domain structure and subcellular
localization of SPOC1. (A) A schematic depiction
of the predicted domain structure and putative
GSK3β phosphorylation sites (P) of the human
SPOC1 protein. (B) Immunofluorescence
localization of endogenous and exogenous
SPOC1, FLAG-SPOC1 ΔNLS, SPOC1 ΔPEST1,
SPOC1 ΔPEST2, SPOC1 ΔPHD, GFP-β-gal and
GFP-β-gal fused to the SPOC1 NLS in U2OS
cells. Endogenous SPOC1 (upper row) and
transiently expressing SPOC1 (second row) were
detected with the rabbit polyclonal CR53
antibody (green). FLAG-SPOC1 ΔNLS was
detected with a mouse anti-FLAG antibody. GFPβ-gal and GFP-β-gal SPOC1 NLS were detected
by the intrinsic fluorescence of GFP. SPOC1
ΔPEST1 was detected with rat monoclonal 7F8,
whereas SPOC1 ΔPEST2 and SPOC1 ΔPHD
were detected with the rat mAb 6F6. DNA was
stained with Hoechst (blue), and phase contrast
(PC) images were captured. Scale bars: 10 μm.
(C) Immunoblot detection of SPOC1 from U2OS
cells lysed by either a 2% SDS total cell lysis
approach (Tot.) or after a differential fractionation
procedure producing soluble (Sol.), chromatin
(Chr.) and insoluble (Ins.) fractions. SPOC1 was
detected with the rat mAb 6F6. Histone H3 (H3)
and HP1α served as chromatin fraction markers
and p21 served as a soluble fraction marker.
(D) Immunoblot detection of exogenous
expression of SPOC1, SPOC1 ΔPEST1, SPOC1
ΔPEST2 and SPOC1 ΔPHD in differentially
fractionated lysates, soluble (S), moderately
chromatin-associated (C1) and tightly chromatinassociated (C2) fractions. Histone H3, HP1α and
p21 served as fraction-specific markers. SPOC1,
SPOC1 ΔPEST2 and SPOC1 ΔPHD were
detected with the rat mAb 6F6 whereas SPOC1
ΔPEST1 was detected with the rat mAb 7F8.
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Journal of Cell Science 122 (16)
domain did not alter its nuclear localization (Fig. 1B). To investigate
the functionality of the SPOC1 NLS further, it was fused to GFP-βgal, a strictly cytoplasmic protein. This fusion resulted in the nuclear
localization of GFP-β-gal, demonstrating that the NLS of SPOC1 is
functional (Fig. 1B). Taken together these findings convincingly show
that SPOC1 is a nuclear-localized protein and that it contains a
functional NLS.
To further evaluate which subcellular fraction SPOC1 localizes
to, immunoblotting was performed with total and differentially
fractionated cellular lysates. Although SPOC1 is predicted to be a
34 kDa protein, it shows aberrant electrophoretic mobility with an
apparent molecular mass of 43 kDa. The SPOC1 protein was
primarily detected in the chromatin-associated fraction in
immunoblots along with the marker proteins histone H3 and HP1α
(Fig. 1C) arguing strongly that it is a chromatin-associated protein.
However, it is important to note that upon longer exposure SPOC1
was also detected in the soluble fraction along with p21 (soluble
nuclear protein), suggesting that a small percentage of SPOC1 is
present in the soluble nuclear fraction of asynchronous cells. The
fact that SPOC1 primarily localizes to the chromatin-associated
fraction is consistent with the prediction of a C-terminal PHD
domain, a domain which is known to directly interact with chromatin
(Aasland et al., 1995; Bienz, 2006) and implicates a chromatinrelated function for SPOC1.
The observations that deleting SPOC1 of either one of its PEST
domains or its PHD domain did not alter its nuclear localization as
determined by immunofluorescence microscopy (Fig. 1B),
prompted us to further investigate whether deleting these regions
had any effect on its subnuclear localization. To answer this
question, we expressed full length SPOC1, SPOC1 ΔPEST1,
SPOC1 ΔPEST2 and SPOC1 ΔPHD in cells, and then subjected
the cells to a differential fractionation procedure that produced a
soluble fraction (S), a moderately chromatin-associated fraction (C1)
using a chromatin buffer without nuclease, and a tightly chromatinassociated fraction (C2) using a chromatin buffer supplemented with
benzonase (Fig. 1D). Histone H3, HP1α and p21 served as fraction
specific markers for the tightly chromatin-associated (H3 and
HP1α), moderately chromatin-associated (HP1α) and soluble (p21)
fractions. In this way we were able to determine if deleting any of
these motifs resulted in an altered subcellular localization of the
protein in comparison to full length SPOC1. Our results show that
deleting the PEST1 domain results in no observable change to the
subcellular localization of SPOC1. By contrast, however, deleting
SPOC1 of its PEST2 domain or its PHD domain did result in a
substantial shift in its biochemical localization and appeared to have
contrasting effects on the solubility of the protein (Fig. 1D).
Interestingly, deleting SPOC1 of its PEST2 domain appeared to
make it more tightly associated with chromatin and very little was
detected in either the soluble or the moderately chromatin-associated
fraction. These findings imply that some region within the PEST2
domain is essential for SPOC1s ability to be released from chromatin
into the nucleoplasm. In stark contrast to this, deleting the PHD
domain of SPOC1, made the resulting protein significantly more
soluble in comparison to the full length protein. These findings
strongly support the prediction that the PHD domain of SPOC1 is
important for its association with chromatin.
SPOC1 is a labile protein that is regulated by GSK3β and by
proteasome-mediated degradation
The prediction of two highly conserved and high scoring PEST
domains in the SPOC1 protein by the PEST FIND program, suggests
that it may be a labile protein. PEST domains, which are found in
less than 10% of all mammalian proteins, can confer susceptibility
to degradation and are frequently found in proteins with short halflifes (Rechsteiner and Rogers, 1996). PEST sequences are enriched
in proline (P), glutamic acid (E), serine (S), threonine (T) and
aspartic acid (D) and have been identified in transcriptional
regulators, key metabolic enzymes and cell cycle regulating proteins.
To evaluate the significance of the PEST motifs in the SPOC1
protein we first measured the half-life of endogenous SPOC1 in
both the soluble and chromatin subcellular fractions by treating cells
in culture for varying periods of time with cycloheximide, an
inhibitor of protein synthesis (Farber and Roberts, 1971).
Endogenous soluble SPOC1 was found to have a very short halflife of approximately 10-15 minutes whereas chromatin-associated
SPOC1 was found to be stable across the time frame of our
experiment (Fig. 2A). These findings argue that soluble SPOC1 is
in fact a labile protein and susceptible to degradation, whereas
chromatin-associated SPOC1 is much more stable, further
supporting the likelihood of a chromatin-associated function of
SPOC1. With these results in mind we decided to further address
the functionality of the PEST domains in regulating SPOC1
stability. To do this we measured the half-life of exogenous SPOC1
protein as well as of SPOC1 deleted of each of its PEST domains
or of its PHD domain, in the soluble nuclear fraction (Fig. 2B-E).
Exogenous SPOC1 was found to have a half-life of approximately
10-15 minutes similar to our findings for endogenous SPOC1 (Fig.
2A,B). Interestingly, however, deleting either one of the PEST
domains (SPOC1 ΔPEST1 or SPOC1 ΔPEST2) resulted in a
markedly stabilized soluble nuclear protein (Fig. 2C,D).
Furthermore, deletion of the C-terminal PHD domain, a domain
with no known function in protein stability and of proportional size
to the PEST2 domain, resulted in no stabilization of SPOC1 and
the protein was found to have a similar half-life to that of both
endogenous and exogenous full length SPOC1 (Fig. 2E). These
findings strongly suggest that SPOC1 is a labile protein and that
the two PEST domains play a role in regulating the stability and
half-life of the SPOC1 protein.
In order to gain additional evidence that SPOC1 is a short lived
protein, we also evaluated the influence of different proteasome
inhibitors on SPOC1 stability. We treated one of our inducible
SPOC1 stable cell lines (U2OS SPOC1 clone #5) with doxycycline
for 24 hours to induce SPOC1 expression and then with either
MG132 or Velcade for varying periods of time (Fig. 2F). Consistent
with SPOC1 being a labile protein, both Velcade and MG132
increased SPOC1 expression levels in a time-dependent manner.
These results further support that SPOC1 is a short-lived protein
and that proteasome-mediated degradation also regulates SPOC1
expression levels.
The SPOC1 amino acid sequence is also predicted to contain
eight GSK3β phosphorylation motifs (SxxxS) as determined by the
Elm server (www.elm.eu.org), most of which are evolutionarily
conserved and six of which lie directly within the two predicted
PEST domains. This observation was intriguing, since GSK3β
phosphorylation has been previously reported to play a role in the
regulation of protein stability for a variety of labile proteins and in
some cases by directly cooperating with the ubiquitin proteasome
system (Alao et al., 2006a; Alao et al., 2006b; Zhou et al., 2004).
GSK3β is a serine-threonine kinase that requires its substrates to
be pre-phosphorylated by an additional priming kinase at the +4
position before it physically binds and phosphorylates its substrate
at the N-terminal serine (Meijer et al., 2004; Patel et al., 2004). As
SPOC1: a novel mitotic player
2949
To evaluate whether GSK3β is capable of regulating SPOC1
expression levels, we treated cells in culture with two independent
GSK3β inhibitors, LiCl and insulin (Meijer et al., 2004; Patel et
al., 2004). LiCl and insulin were found to both independently
stabilize SPOC1 expression levels in comparison to the induced
only control (Fig. 2H) and further implicate a role of this kinase in
the regulation of SPOC1. The observations that both GSK3β and
proteasome inhibitors alone were capable of stabilizing SPOC1
expression levels, indicates that both of the corresponding enzymes
play a role in SPOC1 degradation. Interestingly when used in
combination, LiCl and MG132 had a synergistic effect, supporting
the possibility that both GSK3β and proteasomal proteases may act
together in regulating SPOC1 stability and half-life (Fig. 2H).
Consistent with this possibility, endogenous SPOC1 protein was
also similarly stabilized in various human cell lines after treatment
with LiCl and MG132 (Fig. 2I). Taken together, these findings show
that SPOC1 is a labile protein and that it is regulated in its expression
by both GSK3β and the proteasome degradation system.
SPOC1 is dynamically regulated during the cell cycle and
mitosis
Fig. 2. SPOC1 is a labile protein regulated in part by GSK3β and proteasomal
degradation. (A) Immunoblot analysis of endogenous SPOC1 expression
levels from fractionated lysates of U2OS cells that were treated for varying
periods of time with cycloheximide (CHX). Tubulin and actin served as
loading controls for the soluble (Sol.) and chromatin (Chr.) fractions,
respectively. (B-E) Immunoblot analysis of soluble lysates from U2OS cells
exogenously expressing SPOC1, SPOC1 ΔPEST1, SPOC1 ΔPEST2 or SPOC1
ΔPHD and treated for varying periods of time with cycloheximide (CHX).
Tubulin was used as a loading control and GFP as a transfection control.
(F) Doxycycline-induced expression of exogenous SPOC1 (24 hours) in a
stable U2OS (clone #5) cell line is stabilized by two independent proteasome
inhibitors in a time-dependent manner. Tubulin and actin served as loading
controls. (G) Endogenous GSK3β co-immunoprecipitates with FLAG-SPOC1
but not from the control vector-transfected lysate when purified with FLAGM2 agarose. IN, input; S, supernatant; IP, immunoprecipitate; WB, western
blot. (H) LiCl and MG132 both independently stabilize SPOC1 expression and
act synergistically. Doxycycline-induced (24 hours) U2OS SPOC1 clone #5
was left untreated or treated for 6 hours with LiCl, insulin, MG132 or
LiCl+MG132 and then analyzed by immunoblotting for SPOC1 expression
levels. Tubulin and actin served as loading controls. (I) Endogenous SPOC1
expression is stabilized in various human cell lines (U2OS, HeLa, Ovcar 5 and
Ovcar 8) after 6 hours of LiCl+MG132 treatment. Tubulin served as a loading
control. SPOC1 was detected in A, B, D, E, G and I with the rat mAb 6F6, in
C with the rat mAb 7F8, and in F and H with the rabbit polyclonal CR53
antibody.
a result of this docking property it is often possible to coimmunoprecipitate this kinase with its substrates. Therefore to
evaluate whether or not SPOC1 may be a GSK3β substrate, we
expressed FLAG-tagged SPOC1 or empty FLAG-vector (as a
control) in cells and immunoprecipitated the lysates with antiFLAG-M2 agarose and then eluted the purified proteins using FLAG
peptide (Fig. 2G). FLAG-SPOC1 was found to specifically coimmunoprecipitate endogenous GSK3β from cell lysate, implicating
that the two proteins interact either directly or indirectly.
Triggered by the observations that SPOC1 is stable when associated
with chromatin but labile when soluble in the nucleoplasm, we
decided to investigate its cell-cycle-specific expression and
subcellular localization profile to see if these properties were evident
at specific cell cycle stages. To evaluate this, we synchronized CV1
cells in specific cell cycle stages by growing them in an isoleucinedeficient medium for 40 hours to block them in G0. The cells were
then released in normal FBS supplemented medium for 8 hours to
bring them to the G1-S phase border and were then collected every
2 hours and analyzed by both FACS profiling and immunoblotting
(Fig. 3A,B). Interestingly, these experiments showed that SPOC1
expression levels are significantly decreased both in the total cell
lysate and in the chromatin fraction at the G1-S phase transition (0
hours) and during early S phase (2 hours) as was determined by
both the FACS profile and the levels of cyclin A and geminin.
Furthermore, SPOC1 was detected at these time points in the soluble
fraction upon longer exposure (Fig. 3B). In contrast to this, SPOC1
expression levels and its chromatin association are dramatically
increased during late G2 and M phases, as determined by both the
FACS profile and the expression levels of cyclin A and cyclin B,
indicating a potential functional role of SPOC1 during both late G2
and mitosis (Fig. 3B). These findings indicate that both the
expression levels and localization of SPOC1 are regulated during
the cell cycle, further indicating a potential cell-cycle-specific
function of the SPOC1 protein.
The results of the synchronization experiments suggested that
SPOC1 may have a functional purpose during mitosis. In an effort
to analyze the relevance of this we looked at the localization of
SPOC1 to mitotic chromosomes during the distinct mitotic phases
by indirect immunofluorescence microscopy and immunoblotting
(Fig. 3C,D). Consistent with our synchronization data (Fig. 3B),
SPOC1 showed greater immunofluorescence intensity in mitotic
cells than in interphase cells (white arrow in Fig. 3C) supporting
the conclusion that SPOC1 expression is increased during mitosis.
Most notably these experiments demonstrated also that SPOC1 is
very dynamic in its chromatin association during mitosis. SPOC1
was found to be associated with chromatin during prophase, soluble
during metaphase and early anaphase and then associated with
chromatin once again during mid to late anaphase and telophase
(Fig. 3C). These findings were also confirmed biochemically by
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Fig. 3. Cell cycle regulation and mitotic
localization of SPOC1. Synchronized CV1 cells
were analyzed by FACS (A) and
immunoblotting (B) to identify the cell cycle
stage-specific expression and localization of
SPOC1. Geminin, cyclin A and cyclin B1 were
used to control the cell cycle stage and tubulin
and actin were used as loading controls. SPOC1
was detected with the rat mAb 6F6. The
asterisk denotes that the blot with the soluble
fraction was exposed 25 times longer than the
chromatin fraction blot.
(C) Immunofluorescence localization of
endogenous SPOC1 (green) during the distinct
mitotic phases was detected with the rabbit
peptide polyclonal CR53 antibody. DNA was
stained with Hoechst and phase contrast (PC)
images were captured. The white arrow
indicates an interphase cell that shows weaker
fluorescence intensity compared with the
mitotic cells. Scale bars: 10 μm.
(D) Immunoblot analysis of SPOC1 in
fractionated and total cell lysates from
nocodazole (Noc)-treated (24 hours) and
released U2OS cells. Pre-metaphase (PM),
metaphase (M), early anaphase (EA), anaphase
(A) and late anaphase-telophase (LA/T) time
points were evaluated. Cyclin B1 and histone
H3 phosphorylated at serine 10 (H3P10) served
to mark the metaphase anaphase transition and
anaphase, respectively. Actin served as a
loading control. SPOC1 was detected with the
rat mAb 6F6.
synchronizing cells in pre-metaphase with nocodazole (microtubule
inhibitor) and then releasing them for varying periods of time to
follow them through the distinct mitotic phases. SPOC1 localization
to chromatin or the cytoplasm/nucleoplasm was determined by
fractionating the cell pellets into soluble and chromatin-associated
lysates (Fig. 3D). Consistent with the immunofluorescence findings
SPOC1 was found to be both soluble and chromatin associated at
late prophase/pre-metaphase (0 minutes), primarily soluble during
metaphase and early anaphase (15-60 minutes) and then increased
again back on chromatin at mid-late anaphase and telophase (90105 minutes; Fig. 3D). Together these data indicate that SPOC1 is
very dynamically regulated in its association with chromatin during
mitosis and further supports a potential functional role of this protein
in the mitotic processes.
phospho 10 (H3P10) and analyzed by immunofluorescence
microscopy (Fig. 4). When the mitotic cells of the SPOC1 siRNA
knockdown group were analyzed at higher magnification, various
mitotic defects were observed, including chromosome
misalignments, lagging chromosomes and chromosome bridges
(Fig. 4A). Although these defects were also observed occasionally
in the control groups, it occurred with a greater frequency in the
SPOC1 siRNA treated cells. To statistically evaluate these
consequences, 500 mitotic cells from each group were screened
and showed that these chromosome abnormalities occurred with a
frequency of ~23% in SPOC1 siRNA-treated cells compared with
only 4.7% and 7.3% in the control groups (Fig. 4B). Taken together
these findings further substantiate the functional importance of
SPOC1 in mitotic processes.
SPOC1 siRNA-mediated reduction results in pleiotropic mitotic
defects
SPOC1 modulates chromatin structure and is important for
mitotic chromosome condensation
Based on our aforementioned results we speculated that SPOC1
may play an important mitotic role. To evaluate this possibility we
examined the efficacy of mitosis in U2OS cells after SPOC1 siRNA
mediated knockdown, using two independent oligos. To this end,
asynchronous U2OS cells were either left untreated, or transfected
with a control or SPOC1-specific siRNA oligos (768 or 941) for
72 hours. Mitotic cells were then stained with histone H3 serine
With the observations that SPOC1 associates with mitotic
chromosomes during prophase and anaphase (Fig. 3C,D), the two
stages of the mitotic cell cycle where chromosome condensation
occurs, and since defects in mitotic chromosome condensation have
been previously reported to lead to the pleiotropic mitotic defects
(Cimini et al., 2003; Inoue et al., 2008; Mora-Bermudez et al., 2007;
Nasmyth, 2002; Yong-Gonzalez et al., 2007) that we have observed
SPOC1: a novel mitotic player
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Fig. 4. SiRNA-mediated reduction of SPOC1 results
in various mitotic chromosome defects.
(A) Immunofluorescence detection of histone H3
phosphophorylated at serine 10 (H3P10) in wild-type
U2OS cells, control or SPOC1 siRNA-768 or -941transfected cells (green). DNA (blue) was stained
with Hoechst. Scale bars: 5 μm. (B) Quantification of
chromosome misalignments, lagging chromosomes
and chromosome bridges (aberrant mitosis) of wildtype U2OS cells (Wt), control (si-Cont.) and SPOC1
siRNA (si-768, si-941)-transfected cells. 500 mitotic
cells were counted for each sample.
(C) Immunoblotting of total cell lysates from wildtype U2OS cells (Wt) or U2OS cells treated with
either a control siRNA (C) or with SPOC1-specific
siRNAs (768, 941). SPOC1 was detected with the rat
mAb 6F6 and tubulin was used as a loading control.
in our SPOC1 siRNA knockdown cells, we further questioned
whether reducing SPOC1 expression levels interfered with proper
mitotic chromosome condensation. In order to investigate this
possibility, we designed a functional assay in which SPOC1
expression was knocked down in cells for 48 hours by 768 siRNA
transfection and then treated with nocodazole for 12 hours to
evaluate the efficiency and ability of the cells to form typical premetaphase condensed structures. These cells were evaluated by
comparative immunofluorescence microscopy against control cells
treated only with nocodazole for 12 hours or that were first
transfected with a control siRNA for 48 hours before the addition
of nocodazole for 12 hours (Fig. 5A-C). To identify the mitotic
nuclei we stained the cells for Ki-67, a proliferation marker which
is localized to the nucleolus during interphase and labels mitotic
chromosomes during mitosis (Endl and Gerdes, 2000; Scholzen and
Gerdes, 2000). These experiments revealed that the majority of
mitotic cells from the wild-type and control siRNA cohorts had the
typical rounded-up pre-metaphase morphology that is expected after
nocodazole treatment. By contrast, however, the mitotic cells of the
SPOC1 siRNA cohort showed a more prophase-like morphology
after nocodazole treatment (Fig. 5B). In order to quantify the
significance of this, 500 mitotic cells were counted from each group
and classified as either prophase-like or pre-metaphase-like (Table
1). Cells were scored into these two categories based on the size
and shape of their mitotic nuclei and on the degree of visible
chromosome condensation. We observed that unlike the control cells
that were primarily blocked at pre-metaphase (72.4-74.4%), SPOC1
768 siRNA-transfected cells appeared to a much greater extent to
block in prophase (59.4%) showing significantly less condensed
mitotic chromosomes (Fig. 5B, Table 1). Similar results were also
obtained using a second SPOC1 siRNA (941) oligo (supplementary
material Fig. S5A,B). These findings suggest that reducing SPOC1
expression levels may result in defective prophase chromosome
condensation. To analyze whether or not the observed downstream
chromosome abnormalities occur as a secondary consequence to
defective chromosome condensation we used a high throughput
modular microscope (ScanR, Olympus). Using this imaging system
we were able to measure and capture all cells on a coverslip and
divide them into different categories based on DNA content (cell
cycle profile), Ki-67 staining intensities (mitotic cells) and cell size
(prophase versus pre-metaphase cells). We were therefore able to
demonstrate that the pre-metaphase cells of the SPOC1 siRNA
cohort (40.6%) were significantly larger and less condensed than
the pre-metaphase cells in the control cohorts (Fig. 5C).
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Journal of Cell Science 122 (16)
Fig. 5. SPOC1 protein expression levels modulate chromatin structure and condensation. (A) Immunoblotting of total cell lysates from wild-type (Wt) U2OS cells
or cells transfected for 48 hours with control siRNA (Cont.) or SPOC1 siRNA 768. SPOC1 was detected with the rat mAb 6F6 and actin was used as a loading
control. (B) Immunofluorescence analysis of wild-type (Wt) U2OS cells or U2OS cells transfected with control or SPOC1-specific 768 siRNA for 36 hours and
then treated with nocodazole for 12 hours. Ki67 (green), DNA (blue) and phase contrast (PC) imaging was used to detect mitotic cells and pre-metaphase cells.
Scale bar: 10 μm. (C) Pre-metaphase cells from each experimental group were identified using a scanning microscope and SCAN-R software. (D,E) U2OS cells
were either left untransfected (Wt) or transfected with a SPOC1 siRNA (768 or 941), full length SPOC1 or SPOC1 ΔPHD constructs for 48 hours. SPOC1 levels
were determined by immunoblotting (upper panels). SPOC1 was detected with rat mAb 6F6 and tubulin served as a loading control. Chromatin sensitivity assays
(lower panels) were performed either using an RNase treatment before digestion with 40 U of MNase for the indicated periods of time (D) or digestion with 80 U
of MNase for the indicated periods of time followed by an RNase treatment (E). DNA was separated on a 1% agarose gel. Mono-, di- and tri-nucleosomes are
denoted.
Furthermore, using both SPOC1 siRNAs we observed a significant
increase in the percentage of pre-metaphase cells with
misalignments (~35% compared with the ~10% in the control cells;
supplementary material Fig. S5C,D). These findings demonstrate
that SPOC1 is important for mitotic chromosome architecture and
for proper mitotic chromosome condensation. Furthermore, it is
conceivable that condensation defects can result in downstream
mitotic chromosomal abnormalities and therefore it is tempting to
speculate that a disruption to proper prophase condensation may
induce the observed alignment and segregation defects in SPOC1
siRNA-treated cells.
In order to gain some mechanistic insight into the ability of
SPOC1 to modulate mitotic chromosome architecture and
condensation we performed MNase chromatin sensitivity assays to
evaluate directly the consequence of SPOC1-mediated reduction
and SPOC1 transient overexpression on chromatin architecture and
condensation. These experiments were performed using chromatin
templates that were first treated with RNase (Fig. 5D) followed by
the MNase treatment or vice versa (Fig. 5E) to preserve chromatin
structure. The expression levels of SPOC1 were determined by
immunoblotting (Fig. 5D,E). In these experiments we analyzed the
MNase sensitivity of chromatin from wild-type U2OS cells in
SPOC1: a novel mitotic player
Table 1. Percentage of prophase and pre-metaphase cells
% of cells
Cell type
U2OS
Control siRNA
SPOC1 siRNA
Prophase
Pre-metaphase
27.6
25.6
59.4
72.4
74.4
40.6
The percentage of prophase and pre-metaphase cells were counted in the
U2OS, control siRNA and SPOC1 siRNA cohorts after 12 hours of
nocodazole treatment.
comparison to chromatin from U2OS cells transfected with a
SPOC1-specific siRNA (768 or 941) or chromatin from U2OS cells
transfected with either a SPOC1 or SPOC1 ΔPHD expression vector
for 48 hours (Fig. 5D,E). We found, regardless of the experimental
approach, that SPOC1 siRNA-mediated reduction resulted in a
greater sensitivity of the chromatin to MNase digestion compared
with the wild-type U2OS cells (Fig. 5D,E). This is evident from
the increase in mono-, di- and tri-nucleosomes after equivalent
lengths of MNase treatment (Fig. 5D,E) and by the reduced amount
of undigested chromatin in SPOC1 siRNA-treated samples
compared with the wild type control (Fig. 5E). In contrast to this,
overexpression of SPOC1 led to a reduced sensitivity of the
chromatin to MNase digestion compared with the wild-type U2OS
cells, as is indicated by the absence mononucleosomes (Fig. 5D)
or the substantial increase in undigested chromatin (Fig. 5E) after
equivalent lengths of MNase treatment. Furthermore as a negative
control, over expression of SPOC1 deleted of its PHD domain
showed equivalent sensitivity to MNase digestion as the wild-type
control U2OS cells (Fig. 5D). Therefore, we have been able to show
here that over expression of wild-type SPOC1 results in a more
condensed and less accessible chromatin architecture which is
dependent on its PHD domain whereas siRNA-mediated reduction
of SPOC1 results in less condensed and a more accessible chromatin
architecture than in wild-type U2OS cells (Fig. 5D,E). These
findings demonstrate that SPOC1 expression levels are important
for chromatin architecture and show that depletion of SPOC1 results
in a less condensed chromatin state, whereas its over expression
increases the condensation state of chromatin.
Discussion
In this study we have described the expression and biological
function of a novel protein called SPOC1. Using the predicted and
conserved domain motifs of SPOC1 as a platform for our
experimental evaluation, we have systematically dissected SPOC1
at the protein level. Based on previous work that suggested a
relationship of enhanced SPOC1 RNA levels with tumourigenesis,
we deemed it important to try and elucidate the physiological
function of the SPOC1 protein. Lending credence to the biological
importance of this protein is the fact that it is highly conserved
across higher eukaryotes, it is tightly regulated in both its expression
and localization during the cell cycle and because both its enhanced
and reduced expression has been associated with proliferation,
genomic instability and/or cancer.
Our findings support, as is predicted by its C-terminal PHD
domain, that SPOC1 is a chromatin-associated protein with an
important role in the regulation of chromatin structure and function.
PHD domains are found in less than 150 mammalian proteins with
functions ranging from transcriptional activators, repressors to
chromatin remodelling enzymes (Aasland et al., 1995; Bienz, 2006;
2953
Eberharter et al., 2004; Nourani et al., 2001; Papait et al., 2008),
for example HATs (Nourani et al., 2001; Ullah et al., 2008), HDACs
(Li et al., 2007; Yochum and Ayer, 2001), demethylases (Iwase et
al., 2007) and methyltransferases (Tanaka et al., 2008). Recently,
PHD domains have been identified as a molecular reader of the
epigenetic modifications H3K4(me3) and H3K36(me3) both of
which normally denote actively transcribed euchromatin (Li et al.,
2006; Mellor, 2006; Ramon-Maiques et al., 2007; Shi et al., 2007;
Zhang, 2006). PHD-containing proteins therefore participate in the
epigenetic regulation of chromatin by dictating its structure and
function, and how chromatin is interpreted by other molecular
players.
Based on SPOC1’s increased expression and dynamic biphasic
chromatin localization during mitosis, we speculated that the
protein may have a functional role in the regulation of chromatin
structure and function during mitosis. Furthermore, since SPOC1
was specifically localized to chromatin during prophase and then
later during anaphase, the stages of the mitotic cell cycle where
chromosome condensation occurs, we further speculated that it
plays a role in the regulation of mitotic chromosome condensation.
Our results support these postulates. Functional assays employing
siRNA-mediated reduction of SPOC1 were designed to measure
the impact of SPOC1 knockdown on mitotic chromatin structure
and function. Reducing SPOC1 protein levels resulted in various
mitotic defects such as hypocondensed mitotic chromosomes,
misalignment and segregation defects. Similar mitotic defects have
been previously described after the disruption of other important
factors involved in mitotic chromosome condensation (Cimini et
al., 2003; Nasmyth, 2002; Somma et al., 2003) such as,
components of the condensin complex (Yong-Gonzalez et al.,
2007), chromokinesins (Mazumdar and Misteli, 2005; Mazumdar
et al., 2004) topoisomerase IIα (Dawlaty et al., 2008; Downes et
al., 1991), aurora kinase (Mora-Bermudez et al., 2007) and HP1α
(Inoue et al., 2008). This can be explained by the fact that the
failure of a mitotic cell to properly condense it chromosomes can
result in unresolved sister chromatids, which can preclude
successful metaphase alignment, often resulting in mitotic delays
owing to activated checkpoints and ultimately leading to various
segregation defects, such as lagging chromosomes, chromosome
bridges and aneuploidy or polyploidy (Arlot-Bonnemains et al.,
2001; Cortes and Pastor, 2003; Fu et al., 2007; Giet and Glover,
2001; Hodges et al., 2005; Storchova et al., 2006). We found that
the mitotic chromosome structure was significantly altered after
SPOC1 siRNA-mediated reduction, being larger and less
condensed than normal mitotic chromosomes. These findings
strongly indicate that SPOC1 does play a role in chromatin
organization during mitosis and that reducing its expression levels
can have deleterious consequences to cellular proliferation and
genomic stability. Further evidence supporting a functional role
of SPOC1 in modulating chromatin structure and condensation,
was obtained from chromatin MNase sensitivity assays which
demonstrated that SPOC1 overexpression results in a more
compacted chromatin architecture in a PHD-domain-dependent
manner. Therefore, based on all of these data we believe that
SPOC1 plays a role in the condensation of mitotic chromosomes
by directly altering the chromatin structure and by favouring a
more condensed state. We postulate that SPOC1 directly tethers
itself to specific histone residues in chromatin via its PHD domain
and that when it is concentrated on the chromatin, as it is during
early mitosis and later during anaphase, that this aids in condensing
the chromatin architecture.
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Journal of Cell Science 122 (16)
Owing to their important biological roles, PHD-containing
proteins have often either tumour suppressor functions or oncogenic
potentials (Coles and Jones, 2009; Mohrmann et al., 2005; Shi et
al., 2006; Ythier et al., 2008). As a consequence of their chromatin
regulatory functions, disruptive aberrations to PHD-fingercontaining proteins can have severe genomic consequences, and
therefore have been found to be involved in various human
pathological conditions, ranging from mental retardation to
malignancy (Badens et al., 2006; Barrett et al., 2007; Bronner et
al., 2007; Campos et al., 2004; Chen et al., 2008; Coles and Jones,
2009; Ythier et al., 2008). The PHF23 protein is the most closely
related protein to SPOC1, containing a predicted N-terminal
domain, two predicted PEST motifs, a NLS and a PHD domain
with strong sequence homologies (supplementary material Fig. S6).
Although the function of PHF23 is not yet known, chromosomal
translocation of the PHD finger of PHF23 to nucleoporin protein
98 has been recently identified in acute myeloid leukaemia and the
corresponding predicted fusion protein was proposed to act as an
aberrant transcription factor (Reader et al., 2007).
Since SPOC1 appears to be important for cell division and
proliferation it is conceivable that aberrant SPOC1 expression levels
or localization may also favour oncogenic transformation which
may explain its tight cellular regulation. Consistent with this
assumption, we have shown here that reducing SPOC1 expression
levels led to mitotic chromosome alignment and segregation defects.
When left unresolved these mitotic defects lead to genomic
instability and aneuploid phenotypes, which is the hallmark of
almost all known malignancies. Furthermore, it was previously
shown that increased SPOC1 RNA expression levels were found
in a subset of ovarian carcinomas and resulted in a significantly
poorer prognosis (Mohrmann et al., 2005). It will be important in
the future to evaluate SPOC1 protein expression levels not only in
ovarian carcinomas but also in other malignancies, to study the
biological consequences of SPOC1 overexpression to genomic
stability. Lastly, it will also be important to perform specific
epigenetic studies to evaluate which chromatin modifications
SPOC1 recognizes, in what context and if it is part of a greater
molecular chromatin remodelling complex. In summation, we
show here that the SPOC1 protein is an expressed chromatinassociated protein that is tightly regulated and is important for proper
mitotic chromosome structure and function and therefore cell
division processes.
GST-SPOC1. Lou/c rats were immunized twice intraperitoneally and subcutaneously
with approximately 50 μg of affinity-purified GST-SPOC1 protein. After fusion of
the myeloma cell line P3X63 Ag 8.653 with immune rat spleen cells, positive clones
were identified with a solid phase enzyme linked immunosorbent assay (ELISA)
using cell extracts of E. coli expressing the GST-SPOC1 fusion protein. An irrelevant
GST fusion protein was used as a negative control. Positive clones were then further
confirmed by immunoblotting, siRNA knockdown and transient expression
experiments (see supplementary material Figs S2-S4). The rat monoclonal 6F6 and
7F8 antibodies were used in a 1:50 dilution for immunoblotting and undiluted for
immunofluorescence staining of cells.
SPOC1 stable inducible cell lines were generated using the Tet-On system
commercially available from Invitrogen. Briefly a stable tetracycline (Tet) repressorexpressing cell line under blasticidine selection (Invivogen) was generated using the
pcDNA6/TR expression vector. pCDNA-4TO-SPOC1 was then co-transfected into
a Tet-repressor-expressing cell line and placed under the selection markers zeocin
(0.5 mg/ml) and blasticidine (1 μg/ml). Surviving colonies were then isolated, grown
and tested for doxycycline (Sigma)-inducible SPOC1 expression. Doxycycline was
used at a concentration of 1 μg/ml and was added to the culture medium for 16-24
hours.
Cell lines and antibodies
Human osteosarcoma cells (U2OS), cervical carcinoma cells (HeLa), two ovarian
cancer cell lines (OVCAR 5 and OVCAR 8) and an African green monkey kidney
cell line (CV1) were used in our experiments. All cell lines were cultured under
standard conditions and grown in DMEM medium supplemented with 10% FCS, 1⫻
penicillin-streptomycin (PAA) and 10 mM Hepes (Gibco). Rabbit monoclonal antihistone H3 (Epitomics), rabbit polyclonal anti-geminin (Santa Cruz Biotechnology),
anti-cyclin A (Santa Cruz Biotechnology) and anti-histone H3 serine phospho 10
(Upstate) and mouse monoclonal anti-HP1α (Upstate), anti-p21 (Santa Cruz
Biotechnology), anti-tubulin (Sigma), anti-β-actin (Sigma), anti-GFP (Roche), anticyclin B1 (Santa Cruz Biotechnology), anti-FLAG (Sigma) and anti-Ki67 (Dako)
antibodies were purchased from the respective companies and used according to
manufacturer’s instructions. All HRPO-conjugated and fluorescently labelled
secondary antibodies were purchased from Dianova and Invitrogen and used
according to manufacturer’s instructions.
Extraction protocol and immunoblotting
SPOC1 protein was extracted from cell pellets using either (1) a SDS total cell lysate
extraction buffer (50 mM Tris, pH 6.8, 2% SDS and 10% glycerol) or (2) a fractionated
lysate protocol. (1) In brief, the cell pellet (5⫻106 cells) was resuspended in 150 μl
of SDS total cell lysis buffer supplemented with 1⫻ protease inhibitor cocktail
(Roche), 1 mM NaF (Sigma), 1 mM Na3VO4 (Sigma) and 1 mM PMSF (Calbiochem),
heated at 99°C for 5 minutes, supplemented with Laemmli buffer and then reheated
for an additional 5 minutes at 99°C before loading on a SDS polyacrylamide gel. (2)
The fractionated lysate protocol was performed as follows: soluble cellular proteins
from approximately 5⫻106 cells were extracted with 150 μl of a standard NP40 CSK
buffer (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.1%
NP40) supplemented with 1⫻ protease inhibitor cocktail (Roche), 10 μM MG132
proteasome inhibitor and 1 mM PMSF (Calbiochem) for 10 minutes on ice.
Chromatin-associated proteins were extracted from the remaining pellet in 150 μl of
a chromatin extraction buffer (20 mM Tris-HCl pH 7.5, 350 mM NaCl, 1% Triton
X-100, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF) supplemented with
1⫻ protease cocktail inhibitor, 1 mM PMSF, 2.5 mM MgCl2 and 25 units of Benzonase
(Novagen) for 30 minutes on ice. SDS-PAGE and immunoblotting were performed
using standard protocols.
Materials and Methods
Plasmids
SiRNA knockdown
Full length human SPOC1 constructs were generated in the pCDNA4-TO (Invitrogen),
pFLAG-CMV-4 (Sigma) and GST pGEX4-T1 (Amersham) expression vectors.
Human SPOC1 constructs deleted of their PEST1 domain, PEST2 domain or PHD
domain were also generated in the pCDNA4-TO expression vector, and SPOC1 deleted
of its NLS in the pFLAG-CMV4 vector. The GFP–β-gal (β-galactosidase) fusion
construct (pHM830) was kindly provided by Frank Fackelmayer (Biochemical
Research Institute, Ioannina, Greece) and was used to generate the GFP–β-galSPOC1NLS construct. All constructs were generated using standard PCR and cloning
approaches and were verified by sequencing. Unless otherwise stated, all transfections
were performed using Fugene 6 (Roche) according to the manufacturer’s instructions.
SPOC1 protein was knocked down using the 768 siRNA oligo 5⬘-UCACCUGUCCUGUGCGAAA-3⬘ or the 941 siRNA oligo 5⬘-GAUCAGGUCAAAGAAAUAAA3⬘. Briefly, cells were allowed to grow to 80% confluence and then transfected with
100 nM end concentration of SPOC1 siRNA using Lipofectamine 2000 (Invitrogen)
according to manufacturer’s instructions. Cells were then grown at 37°C for an
additional 24-72 hours before harvesting (total cell lysate) for protein detection. A
control siRNA 5⬘-AGGUAGUGUAAUCGCCUUG-3⬘ was purchased from
Dharmacon.
Generation of SPOC1 antibodies and stable inducible cell lines
SPOC1 antibodies were generated using standard approaches. In brief, the SPOC1
rabbit peptide polyclonal CR53 antibody was generated by immunizing rabbits three
times with 100 μg of a KLH-coupled C-terminal peptide (CRDSKFDIRRSNRSRT)
in complete (1⫻) and incomplete (2⫻) Freund’s adjuvant (Sigma). The antibody was
affinity purified by chromatography using HiTrap NHS activated HP resin coupled
to the peptide (Amersham Biosciences). The CR53 antibody was used at a 1:3000
and 1:500 dilution for immunoblotting and immunofluorescence experiments,
respectively. Rat monoclonal antibodies were generated using recombinant full length
Immunofluorescence microscopy
Immunofluorescence analysis of SPOC1 was performed according to standard
protocols. In brief, cells were grown on coverslips, fixed with 4% paraformaldehyde
in PBS, permeabilized with 0.5% Triton X-100, washed three times in PBS and then
blocked overnight with 3% BSA in PBS. Coverslips were then incubated with primary
antibodies according to the manufacturer’s described dilution for 1 hour, washed three
times with PBS and then incubated with the appropriate secondary antibodies for 1
hour. The coverslips were then washed three more times in PBS and mounted on
slides using Mowiol (Calbiochem). The cells were analyzed by indirect
immunofluorescence microscopy on a Zeiss Axiophot or a high content screening
cytometry microscope (ScanR Olympus).
SPOC1: a novel mitotic player
Scan^R analysis
Cells were seeded in six-well dishes, left untransfected or were transfected with either
a control siRNA or a SPOC1-specific siRNA (768) for 36 hours. Following this, the
cells were treated for 12 hours with 100 ng/ml nocodazole. Cells were analyzed on
a high throughput scanning Olympus microscope (www.olympus.de/microscopy/
22_scan_R.htm) for comparative analysis of mitotic nuclei. In brief, this combined
microscope and computer platform scans and captures images from all cells in all
wells for comparative analysis. Then, using an integrated FACS profiling function it
is capable of generating a cell cycle profile from all cells based on a DNA stain
(Hoechst). Using this FACS profile, cells in the G2-M phase were selected for in the
first gate. From this G2-M population a second gate selecting for mitotic cells was
generated based on Ki-67-Alexa Fluor 488 staining intensity. Lastly, based on the
combined differences in DNA and nuclear volume, prophase cells were differentiated
from pre-metaphase cells to generate our final gate of pre-metaphase only cells. All
gates were constant for all experimental conditions and generated sub-galleries of
cells that were then directly comparable with the same gate from other treatment
conditions.
Cell synchronization
The African green monkey kidney cells CV-1 were synchronized as previously
described (Rohaly et al., 2005). In brief, the cells were starved of isoleucine for 3640 hours to block the cells in G0. The cells were then released back into medium
containing normal serum for 8 hours before collecting at the first time point (0 hours)
at the G1-S transition point. Cells were collected at 2-hour intervals up to 14 hours
for FACS and immunoblot analysis. The DNA was stained with propidium iodide
for FACS analysis.
Cells were synchronized in mitosis at premetaphase by treating the cells in culture
with a 100 ng/ml nocodazole (Sigma) for 12-24 hours. A mitotic shake-off was then
performed and the cells were collected and replated.
In culture drug treatments
Cycloheximide experiments were performed by incubating cells in culture with 10
μg/ml of cycloheximide (Sigma) for varying lengths of time. Inhibition of proteasomal
activity was performed by incubating cells in culture for up to 6 hours with 10 μM
MG132 (Calbiochem) or 1 μM Velcade (kindly provided by Sven Behrens, University
of Halle-Wittenberg, Germany). GSK3β was inhibited by incubating cells in culture
for 6 hours with either 40 mM LiCl (Merck) or with 100 nM insulin (Sigma).
MNase digestion assay
In brief, cell pellets were lysed in a hypotonic buffer (10 mM Tris-HCl pH 7.4, 10
mM KCl, 15 mM MgCl2) on ice for 10 minutes. Nuclei were pelleted by centrifugation
and resuspended in MNase digestion buffer (50 mM Tris-HCl pH 7.9, 5 mM CaCl2)
supplemented with RNase and incubated at 37°C for 30 minutes. The DNA was then
pelleted once again by centrifugation and resuspended in MNase digestion buffer
supplemented with 100 μg/ml BSA and 40 IU MNase and incubated at room
temperature for varying periods of time. The MNase reaction was stopped by the
addition of 10 mM EDTA. Alternatively, nuclei were first treated with 80 IU of MNase
at 37°C for varying periods of time. The MNase reaction was stopped by the addition
of 10 mM EDTA followed by centrifugation. The pellet was then resuspensed in
MNase digestion buffer supplemented with 10 mM EDTA and RNase and incubated
at 37°C for 30 minutes. The DNA was extracted and purified by standard procedures.
This work was supported by a grant from the Deutsche Krebshilfe
#10-2243-Wi4 and the Müggenburg Foundation. The Heinrich-Pette
Institute is supported by the Freie und Hansestadt Hamburg and the
German Federal Ministry of Health and Social Security. We would also
like to thank Nicole Lohrengel, Urte Matschl and Kerstin Reumann for
their excellent technical support.
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