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Cyclin E2 is the predominant E-cyclin associated
with NPAT in breast cancer cells
Rogers et al.
Rogers et al. Cell Division (2015) 10:1
DOI 10.1186/s13008-015-0007-9
S H O R T R E P O R T Open Access
Cyclin E2 is the predominant E-cyclin associated
with NPAT in breast cancer cells
Samuel Rogers
1
, Brian S Gloss
1,2
, Christine S Lee
1
, Claudio Marcelo Sergio
1
, Marcel E Dinger
1,2
,
Elizabeth A Musgrove
3
, Andrew Burgess
1,2
and Catherine Elizabeth Caldon
1,2*
Abstract
Background: The cyclin E oncogene activates CDK2 to drive cells from G
1
to S phase of the cell cycle to commence
DNA replication. It coordinates essential cellular functions with the cell cycle including histone biogenesis,
splicing, centrosome duplication and origin firing for DNA replication. The two E-cyclins, E1 and E2, are assumed
to act interchangeably in these functions. However recent reports have identified unique functions for cyclins E1
and E2 in different tissues, and particularly in breast cancer.
Findings: Cyclins E1 and E2 localise to distinct foci in breast cancer cells as well as co-localising within the cell. Both
E-cyclins are found in complex with CDK2, at centrosomes and with the splicing machinery in nuclear speckles. However
cyclin E2 uniquely co-localises with NPAT, the main activator of cell-cycle regulated histone transcription. Increased cyclin
E2, but not cyclin E1, expression is associated with high expression of replication-dependent histones in breast cancers.
Conclusions: The preferential localisation of cyclin E1 or cyclin E2 to distinct foci indicates that each E-cyclin has
unique roles. Cyclin E2 uniquely interacts with NPAT in breast cancer cells, and is associated with higher levels of
histones in breast cancer. This could explain the unique correlations of high cyclin E2 expression with poor outcome
and genomic instability in breast cancer.
Keywords: Cyclin E1, Cyclin E2, CDK2, Centrosome, Cajal bodies, Histone Locus bodies (HLB), Spliceosomes, NPAT,
Histones, Breast cancer
Findings
The canonical function of cyclin E is the activation of
CDK2 (cyclin dependent kinase 2) to phosphorylate Rb,
hence promoting the release of E2F transcription factors
and progression of the cell cycle from G
1
to S phase [1].
However there are other functions for cyclin E that may
be CDK2 dependent or independent, including tran-
scriptional processing, origin firing, and centrosome du-
plication [2]. The wide range of cyclin E functions may
explain the necessity for two cyclin E proteins: E1 and
E2. Both these proteins activate CDK2, but are encoded
by genes on different chromosomes (cyclin E1: CCNE1
at 19q12; cyclin E2: CCNE2 at 8q22.1). Cyclin E1 and E2
have differences in tissue expression, transcription and
post-transcriptional regulation, and have distinct affinities
for other proteins, e.g. p107 [1,3]. In this study we exam-
ined the localisation of cyclin E1 and E2 and report unique
sites of localisation in breast cancer cells.
We previously identified that cyclin E1 and E2 are
expressed in different cell line subpopulations due to
distinct cell cycle regulation [4]. Close examination re-
vealed that cyclin E1 and E2 localise to unique foci
within the nucleus of T-47D and MCF-7 breast cancer
cells (Figure 1A and Additional file 1). Several large
bright foci exclusively localised with either cyclin E1 or
E2, while some foci showed co-localisation (Figure 1A,
inset, and Additional file 1, inset).
Cyclins E1 and E2 have cytoplasmic, nuclear and chro-
matin associated functions [1,2]. Cell fractionation
showed that both cyclin E1 and E2 were predominantly
nuclear and a large proportion was extracted with chro-
matin (Figure 1B). However a significant proportion of
cyclin E1 was nucleolar and not chromatin associated
(18.5%) compared to a smaller proportion of cyclin E2
(4.2%), and both proteins occurred at only very low
* Correspondence: l.caldon@garvan.org.au
1
The Kinghorn Cancer Centre and Cancer Research Program, Garvan Institute
of Medical Research, Sydney, NSW, Australia
2
St Vincent’s Clinical School, Faculty of Medicine UNSW, Sydney, Australia
Full list of author information is available at the end of the article
© 2015 Rogers et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Rogers et al. Cell Division (2015) 10:1
DOI 10.1186/s13008-015-0007-9
levels in the cytoplasm (Figure 1B). Thus the majority of
cyclin E1 and E2 is located on chromatin, but there is a
small but significant proportion of cyclin E1 that is lo-
calized to non-chromatin foci.
We next examined a range of cyclin E functions to de-
termine if unique localisation of cyclin E1 or E2 was as-
sociated with a unique function. Cyclin E binds and
activates CDK2, and this activity is inhibited by CDK
inhibitors p21
Waf1/Cip1
and p27
Kip1
. Both cyclin E1 and
E2 form cyclin/CDK2/CDK inhibitor complexes, although
these complexes are mutually exclusive (Figure 2A). Cyc-
lin E/CDK2 phosphorylates splicing complexes which may
coordinate pre-mRNA splicing with the G
1
/S transition
[5]. These functional complexes appear common to cyc-
lin E1 and cyclin E2, as in T-47D cells both proteins co-
immunoprecipitate a major component of the spliceosome,
cdc6
cyclin E1
cyclin E2
CDK2
% protein
0
20
40
60
80
100
cytoplasmic
soluble nuclear
chromatin-bound
cyclin E2
cyclin E1
cytoplasmic
soluble nuclear
chromatin-bound
cytoplasmic
soluble nuclear
chromatin-bound
total cell extract
cytoplasmic
nuclear
chromatin
extraction
nuclear/
cytoplasmic
extraction
A
BC
cyclin E2cyclin E1 overlayToPro3
cyclin E1 /
cyclin E2
overlay
Figure 1 Cyclins E1 and E2 localise to unique foci, and have distinct subcellular distribution. A. Confocal images of T-47D breast cancer cells
immunoprobed with cyclin E1 (red) or cyclin E2 (green), and counterstained with ToPro3 (blue, nuclei). Inset at higher magnification. Scale bars = 5
μm. Experiments are performed in triplicate. Similar data obtained in MCF-7 cells are shown in Additional file 1. B. T-47D cells were lysed to extract total
cell proteins (lane 1), total nuclear (lane 2) and total cytoplasmic (lane 3) lysates. In parallel, cell lysates were purified to extract soluble cytoplasmic
proteins, soluble nuclear proteins, and chromatin bound proteins. PAGE separated proteins were western blotted for Cdc6 (predominantly chromatin
bound), CDK2 (cytoplasmic, nuclear and chromatin bound), cyclin E1 and cyclin E2. C. Cyclins E1 and E2 were quantitated from duplicate experiments
using densitometry (ImageJ), and soluble cytoplasmic, soluble nuclear, and chromatin-bound fractions graphed as a percentage of total extracted
protein. Error bars show range.
Rogers et al. Cell Division (2015) 10:1 Page 2 of 9
lysate
7123456 8
sucrose gradient fractions
910
cyclin E1
centrin-2
γ-tubulin
ER
cyclin E2
B
SAP145
cyclin E1
cyclin E2
CDK2
IB
CDK2 cyclin E1
IP
IgG
lysate +
cyclin E2 IgG
+++++
antibody
only control +++
C
A
IB
CDK2 cyclin E1
IP
lysate +
cyclin E2IgG
++++
antibody
only control +++
cyclin E1
cyclin E2
CDK2
IgG
p27
p21
IgG
Figure 2 (See legend on next page.)
Rogers et al. Cell Division (2015) 10:1 Page 3 of 9
SAP 145 (Figure 2B). Centrosomes are major cytoplasmic
bodies located at the nuclear periphery. We identified that
both cyclins E1 and E2 were localised to the centrosome
complexes of T-47D and MCF-7 breast cancer cells using
sucrose gradient fractionation of centrosomes and western
blotting (Figure 2C, and Additional file 2), consistent with
previous data showing specific localisation of cyclin E1 to
centrosomes by immunofluorescence [6].
Cyclin E directly coordinates histone gene transcrip-
tion with G
1
to S phase transition via the phosphoryl-
ation of histone transcription factor NPAT in the
Histone Locus Bodies (HLB) which localise to histone
gene clusters on chromosomes 1 and 6 [7-9]. We found
by immunofluorescence that cyclin E2 co-localised with
the major HLB protein, NPAT, in T-47D (Figure 3A)
and MCF-7 breast cancer cells (Additional file 3), but
NPAT rarely co-localised with cyclin E1. The strong as-
sociation between cyclin E2 and NPAT may be due to
the relatively high levels of cyclin E2 observed in breast
cancer cell lines [4]. However we observe that cyclin E1
does not relocalise to NPAT foci upon cyclin E2 siRNA
treatment (Figure 3B and C). This suggests that the spe-
cific cyclin E2-NPAT interaction is due to intrinsic fea-
tures of cyclin E2 rather than excess cyclin E2 preventing
an interaction between cyclin E1 and NPAT.
We confirmed our findings using the in situ Proximity
Ligation Assay (PLA), which detects the co-localisation
of two antibodies within 40nm on fixed cells by PCR
amplification of a linker probe. PLA analysis identified
an average of 22 nuclear NPAT-E2 foci per cell, consist-
ent with the multiple HLBs which are detected in aneu-
ploid cancer cell lines [10] (Figure 4A). NPAT-cyclin E2
interactions were 4-fold higher than the number of cyc-
lin E1-NPAT interactions (P < 0.0001; Figure 4B). Cyclin
E1-NPAT interactions did not exceed background levels
of the αGST/NPAT negative control, and hence are un-
likely to represent true HLBs (Figure 4B). Together the
immunofluorescence and PLA data indicate that cyclin
E2 is the major E-cyclin within HLBs in breast cancer
cells and is likely to have a particular role in coordinat-
ing the cell cycle with histone transcription.
As a positive control for PLA analysis we examined
cyclin E1-CDK2 and cyclin E2-CDK2 interactions. We
observed that both cyclin E1 and cyclin E2 had
predominantly nuclear interactions with CDK2 (Figure 4C
and D). A proportion of both cyclin E1-CDK2 and cyclin
E2-CDK2 foci were cytoplasmic (Figure 4C and D) which
is consistent with nuclear-cytoplasmic shuttling of these
complexes [11]. Cyclin E1-CDK2 interactions were 2-fold
more abundant than cyclin E2-CDK2 (Figure 4E), which
again suggests that it is unlikely that excess cyclin E2 pre-
vents cyclin E1 from interacting with other binding part-
ners such as NPAT.
Previous publications describe binding of “cyclin E”to
NPAT, whereas we here identify that cyclin E2 is the
major E-cyclin within HLBs in breast cancer cells. The
previous studies were performed prior to the develop-
ment of specific cyclin E1 and E2 antibodies, and relied
upon the cyclin E HE67 (cyclin E1 aa366-381) and HE11
(full-length protein) antibodies which are raised using
epitopes that may not effectively discriminate cyclin E1
and cyclin E2 [8,9]. While cyclin E1 may not influence
histone transcription in breast cells via NPAT it could
influence it via other pathways. Cyclin E/CDK2 indir-
ectly controls histone transcription via E2F-mediated
transcription of NPAT [12], and by phosphorylation of
the HIRA protein which is a repressor of histone tran-
scription that operates outside S phase [13].
Our observation of a specific NPAT-cyclin E2 inter-
action in breast cancer cell lines was supported by our
findings of high expression of replication-dependent his-
tones in breast cancers that have high expression of cyc-
lin E2. We examined the transcript profiles of breast
cancers from The Cancer Genome Atlas (TCGA) for
cyclin E and histone expression. In 526 breast cancers,
high CCNE2 expression is associated with high levels of
replication-dependent histones that are under the con-
trol of NPAT (Figure 5A). However this pattern is not
observed for CCNE1 (Figure 5A), nor with non-
replication dependent histones (Figure 5B).
Cyclin E1 has been recognised as an important onco-
gene for 20 years [14]. The high degree of sequence
homology between cyclin E1 and E2 suggests that many
of their functions may be interchangeable, but recent
publications in cancer and liver biology show that these
proteins have unique regulation and function [15,16].
Our re-examination of cyclin E function has identified
that cyclin E2 is likely to have particular role in histone
(See figure on previous page.)
Figure 2 Common functional complexes of cyclin E1 and E2. A. Cyclin E1 and E2 both co-immunoprecipitate CDK2/CDK2 inhibitor complexes.
Lysates of T-47D cells were immunoprecipitated and then western blotted using the indicated antibodies. Data are representative of duplicate experiments.
Similar data from MCF7 cells are shown in [19]. IB: immunoblot; IP: immunoprecipitation B. Cyclin E1 and E2 both co-immunoprecipitate SAP145. Lysates of
T-47D cells were immunoprecipitated and then western blotted using the indicated antibodies. Data are representative of triplicate experiments. InA.and
B. arrows indicate protein of interest; IgG is non-specific immunoglobulin G staining. C. Cyclins E1 and E2 both co-purify with centrosomes. T-47D cells were
arrested and synchronised at G
0
with anti-estrogen ICI 182780 followed by estrogen stimulation for 16h. Lysates were separated by ultracentrifugation on
sucrose gradients, fractionated, then pelleted and resuspended in sample buffer for western blotting with the indicated antibodies. γ-tubulin and centrin-2
are centrosome components, and estrogen receptor α(ER) is a non-centrosomal negative control. Data are representative of duplicate experiments. Similar
data obtained in MCF-7 cells are shown in Additional file 2.
Rogers et al. Cell Division (2015) 10:1 Page 4 of 9
cyclin E1
cyclin E2
NPAT
NPAT
overlay
overlay
A
overlay (enlarged)
overlay (enlarged)
cyclin E2 NPAT overlay overlay (enlarged)
cyclin E1 NPAT overlay overlay (enlarged)
B
C
NPAT/cyclin E2 foci
NPAT/cyclin E1 foci
NPAT/cyclin E1 foci
+ cyclin E2 siRNA
cyclin E2 siRNA
co-localisation (PCC)
N.S.
**
**
0 0.2 0.4 0.6
Figure 3 Cyclin E2, but not cyclin E1, co-localises with NPAT by immunofluorescence in breast cancer cells. A. Cyclin E2 localises to NPAT
foci. Confocal images of T-47D cells immunoprobed with cyclin E1 or cyclin E2 (red) and NPAT (green). Experiments performed in triplicate.
Example of lack of co-localisation of cyclin E1 (antibody: HE12) and NPAT (antibody: C-19) is shown, and is representative of similar data with
cyclin E1 (antibody: Epitomics) and NPAT (antibody: 27) co-staining (not shown). Scale bars = 5μm. Similar data obtained in MCF-7 cells are
shown in Additional file 3. B. Confocal images of T-47D cells treated with 20nM cyclin E2 siRNA for 48h, and then immunoprobed with cyclin
E1 or cyclin E2 (red) and NPAT (green). Scale bars =10μm. C. Quantitation of co-localisation using Pearson's correlation coefficient (PCC) which
quantifies positional relationship from confocal images on a scale of -1 to +1. Statistical significance was calculated with one-way ANOVA and
Tukey’s multiple comparisons, where N.S. indicates not significant and ** indicates P < 0.01. Data pooled from duplicate experiments. Similar
data obtained in MCF-7 cells are shown in Additional file 3.
Rogers et al. Cell Division (2015) 10:1 Page 5 of 9
regulation in breast cancer via its unique interaction with
NPAT. Cyclin E2 has a strong prognostic role in breast
cancer [15], and induces genomic instability that is associ-
ated with defects in chromosome condensation [3]. This
could be in part due to excessive histone production, as
disruption of histone equilibrium is a predicted cause of
genomic instability [17].
Our identification of multiple foci that contained only
cyclin E1 or E2 indicates that there are other unique in-
teractions. This is not surprising given that the low
C
B
αGST / NPAT
NPAT / cyclin E1
NPAT / cyclin E2
0
10
20
30
antibody
pair
aGST / NPAT
NPAT / cyclin E1
NPAT / cyclin E2
number of foci
****
N.S.
****
A
CDK2 / cyclin E1
CDK2 / cyclin E2
CDK2 / cyclin E1
CDK2 / cyclin E2
0
20
40
60
80
D
0
25
50
75
100
cytoplasmic
nuclear
CDK2 / cyclin E1
CDK2 / cyclin E2
E
number of foci
nuclear/cytoplasmic foci (%)
Figure 4 Cyclin E2, but not cyclin E1, co-localises with NPAT in T-47D cells by PLA. A. Proximity Ligation Assay (PLA) for cyclin E1/NPAT
(antibodies: cyclin E1 –Epitomics; NPAT –27) and cyclin E2/NPAT (antibodies: cyclin E2 –Epitomics; NPAT –27). Images are 3-D rendered serially
stacked confocal images assembled with Imaris software. NPAT/αGST staining was performed as a negative control (antibodies: NPAT –27,
αGST –[23]). Representative cells are shown, scale bars = 10μm. B. Quantitation of A. where number of foci were quantitated from 10-15 cells
per antibody pair. Statistical significance was calculated with one-way ANOVA and Tukey’s multiple comparisons, where N.S. indicates not significant and
**** indicates P < 0.0001. Data pooled from duplicate experiments. C. Cyclin E1/CDK2 (cyclin E1- HE12, CDK2 –M2) and cyclin E2/CDK2 (cyclin E2 –
Epitomics, CDK2 –D12) PLA were performed as positive controls. Representative cells are shown with nuclear foci pseudocoloured in red, and cytoplasmic
foci pseudocoloured in white. Scale bars = 10μm. D./E. Quantitation of C. including relative nuclear/cytoplasmic foci (D.) and total foci (E.).Datapooled
from duplicate experiments.
Rogers et al. Cell Division (2015) 10:1 Page 6 of 9
molecular weight derivatives of cyclin E1 also has unique
binding and function in cancer cells compared to the full
length protein [18]. Future studies should carefully dif-
ferentiate cyclin E1 and E2 and their isoforms, especially
since each protein has unique expression patterns and
their expression has distinct correlation with patient out-
come in cancer [1].
Methods
Cell lines
Cell lines were authenticated by STR profiling (CellBank
Australia, Westmead, NSW, Australia) and cultured for
<6 months after authentication. Cyclin E1 and E2 siRNA
treatment was performed and validated by western blot-
ting as described in [19].
Immunoblotting and immunoprecipitation
Collection of whole cell lysates [20], chromatin [21] and
sucrose gradient fractions of centrosomes [22] were per-
formed as described. Lysates were separated using
NuPage polyacrylamide gels (Invitrogen) prior to transfer
to PVDF membranes. Western blotting, immunofluores-
cence and PLA antibodies are: Cdc6 (180.2), CDK2 (M2,
D12), centrin-2 (S-19), cyclin E1 (HE12), estrogen recep-
tor α(HC20), NPAT (C-19, 27), SAP145 (A-20), γ-tubulin
(C-11) (Santa Cruz Biotechnology); cyclin E2 (Epitomics);
N.S. N.S. N.S. ****
**
Replication-Dependent Histones
0 1 2 3456
High expression of “n” histones
−1
1
2
3
4
0
Cyclin expression
0 1 2 3456
High expression of “n” histones
1
2
3
4
0
N.S. ** ** ** N.S. N.S. N.S.
−1
−2
−2
CCNE1
CCNE2 N.S.: Non-significant
* p <0.01
** p <0.001
Non-Replication-Dependent Histones
Cyclin expression
A
B
Figure 5 Increased Cyclin E2 expression is associated with higher levels of replication-dependent histones in breast cancers. Box plots
illustrate the change in mRNA expression levels of CCNE2 compared to CCNE1 as replication-dependent (A.) and non-replication-dependent (B.) histone
expression increases in 526 breast cancer samples. Breast cancer samples were grouped according to the number of replication dependent
and independent histones displaying above median expression. Gene expression was normalized to the median expression of group 0 for
each sample. p-values were calculated using a Mann-Whitney-U test. Boxes represent the normalized median expression and the 1
st
and 3
rd
quartiles and whiskers extend 1.5x the IQR from median.
Rogers et al. Cell Division (2015) 10:1 Page 7 of 9
p21 (610234) and p27 (610242) (BD Biosciences); αGST
[23]. Immunoprecipitation antibodies are: CDK2 (C-19),
cyclin E1 (C-19), NPAT (C-19, 27), non-immune IgG
(Santa Cruz Biotechnology), and cyclin E2 (Epitomics).
Specificity of cyclin E1 and E2 antibodies was demon-
strated in [15,19]. Additionally, we show specific loss of
cyclin E1 and cyclin E2 immunofluorescence signal with
siRNA treatment to cyclin E1 (Additional file 4) and cyclin
E2 (Figure 3).
Immunofluorescence and microscopy
Cells were fixed with 4% PFA/PBS for 20 min at room
temperature, with or without methanol post-fixation ( -20°
C for 20 min). Samples were blocked with 1% BSA/PBS,
stained with the indicated antibodies and counterstained
with ToPro3/DAPI (Jackson ImmunoResearch Labora-
tories). Co-localisation was quantitated by detecting
overlapping pixels with Imaris v8.0 (Bitplane) and ana-
lysed with Pearson’s Correlation Coefficient [24]. For
PLA, PFA fixed cells were subjected to the Duolink
Proximity Ligation Assay (Sigma) as described by the
manufacturer. Confocal microscopy was performed on
Leica DMRBE/DMIRE2. Images were analysed with
Imaris where individual spots were defined with a variable
and initial size estimate of 0.5 μm. Images were processed
with Adobe Photoshop, and adjusted for optimal bright-
ness/contrast. Minimal gamma changes were made to en-
able visualisation of overlaid signals.
Bioinformatics
Expression values in 526 breast cancer samples of CCNE1,
CCNE2 and representative replication-dependent and
-independent histones (Additional file 5) were accessed
from the cBioPortal [25] using the CGDSR package [26]
in R [27]. For each sample the number of histones with
high expression (> median across patients) was established
for histone subsets. Samples were grouped according to
the number of histones having above median expression.
For each group, the expression level of CCNE1 and
CCNE2 was normalised to 100% of the median expression
in the patient group with zero highly expressed histones.
Additional files
Additional file 1: Cyclins E1 and E2 localise to distinct nuclear foci in
MCF-7 cells. A. Confocal images of MCF-7 breast cancer cells immunoprobed
with cyclin E1 (red) or cyclin E2 (green), and counterstained with ToPro3 (blue,
nuclei). Inset at higher magnification. Scale bars = 5 μm. Experiments
are performed in triplicate.
Additional file 2: Cyclins E1 and E2 co-sediment with centrosome
components in MCF-7 cells. A. Cyclins E1 and E2 both co-purify with
centrosomes. MCF-7 cells were arrested and synchronised at G
0
with
anti-estrogen ICI 182780 followed by estrogen stimulation for 16h. Lysates
were separated by ultracentrifugation on sucrose gradients, fractionated,
then pelleted and resuspended in sample buffer for western blotting
with the indicated antibodies. γ-tubulin and centrin-2 are centrosome
components, and Grb2 and estrogen receptor α(ER) are non-centrosomal
negative controls. Data are representative of duplicate experiments.
Additional file 3: Cyclin E2, but not cyclin E1, co-localises with NPAT
by immunofluorescence in MCF-7 cells. A. Confocal images of MCF-7 cells
immunoprobed with cyclin E1 or cyclin E2 (red) and NPAT (green).
Experiments performed in duplicate. Scale bars = 10μm. B. MCF-7 cells were
treated with 20nM cyclin E2 siRNA for 48h as described in [19]. Co-localisation
of cyclin E1 or cyclin E2 with NPAT using Pearson's correlation coefficient
(PCC) which quantifies positional relationship from confocal images on a scale
of -1 to +1. Statistical significance was calculated with one-way ANOVA and
Tukey’s multiple comparisons, where N.S. indicates not significant and **
indicates P < 0.01. Data pooled from du plicate experiments.
Additional file 4: Specific immunostaining for cyclin E2 in the
presence of cyclin E1 siRNA. Breast cancer cells were transfected with
20nM cyclin E1 siRNA for 48h as described in [19]. Confocal images of MCF-
7 cells (A.) and T-47D cells (B.) immunoprobed with cyclin E1 (red), cyclin E2
(green) and DAPI (blue). Inset at higher magnification. Experiments
performed in duplicate. Scale bars = 10μm.
Additional file 5: Representative subsets of replication-dependent
and -independent histones. Table of replication-dependent and replication
independent histones used in this study. Table includes histone gene name
and chromosomal location.
Abbreviations
CDK2: Cyclin dependent kinase 2; HLB: Histone Locus Body; PLA: Proximity
Ligation Assay; TCGA: The Cancer Genome Atlas.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
SR performed immunofluorescence, PLA staining and analysis. BSG designed
and performed the bioinformatic analyses and MED provided bioinformatics
expertise. CSL performed siRNA and immunoprecipitation experiments. CMS
assisted in centrosome purification by sucrose gradients. EAM helped
conceive the study. AB participated in experimental design, provided
microscopy expertise and advised on the manuscript. CEC conceived the
study, designed and performed experiments and drafted the manuscript. All
authors read and approved the final manuscript.
Acknowledgements
We gratefully acknowledge the assistance of Dr Marco Nousch in the collection
of centrosome fractions derived from the sucrose gradients. BSG and CEC are
supported by Cancer Institute NSW Fellowships and AB is a Cancer Institute
NSW Future Leader Fellow. EAM was suppported by a Cancer Institute NSW
Fellowship and is now supported by Cancer Research UK (C596/A18076).
Author details
1
The Kinghorn Cancer Centre and Cancer Research Program, Garvan Institute
of Medical Research, Sydney, NSW, Australia.
2
St Vincent’s Clinical School,
Faculty of Medicine UNSW, Sydney, Australia.
3
Wolfson Wohl Cancer
Research Centre, University of Glasgow, Garscube Estate, Glasgow G61 1QH,
UK.
Received: 24 September 2014 Accepted: 2 February 2015
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