From the Department of Biology, University of Konstanz, 78457 Konstanz, Germany
Received for publication, January 9, 2001, and in revised form, April 26, 2001
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ABSTRACT |
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Recent data revealed that DEK associates with
splicing complexes through interactions mediated by
serine/arginine-repeat proteins. However, the DEK protein has
also been shown to change the topology of DNA in chromatin in
vitro. This could indicate that the DEK protein resides on
cellular chromatin. To investigate the in vivo localization
of DEK, we performed cell fractionation studies, immunolabeling, and
micrococcal nuclease digestion analysis. Most of the DEK protein was
found to be released by DNase treatment of nuclei, and only a small
amount by treatment with RNase. Furthermore, micrococcal nuclease
digestion of nuclei followed by glycerol gradient sedimentation
revealed that DEK co-sedimentates with oligonucleosomes, clearly
demonstrating that DEK is associated with chromatin in
vivo. Additional chromatin fractionation studies, based on the
different accessibilities to micrococcal nuclease, showed that DEK is
associated both with extended, genetically active and more densely
organized, inactive chromatin. We found no significant change in the
amount and localization of DEK in cells that synchronously traversed
the cell cycle. In summary these data demonstrate that the major
portion of DEK is associated with chromatin in vivo and
suggest that it might play a role in chromatin architecture.
DNA in the nucleus is organized into a hierarchy of structures
with the nucleosome as the basic building block. It has become widely
accepted that modification of nucleosome structure is an important
mechanism that regulates the accessibility of chromatin to DNA binding
factors (1, 2).
In the search for factors that change the structure of chromatin and
the replicational activity of chromatin templates, we recently
identified the proto-oncogene protein DEK as a candidate protein that
changes the topology of DNA in chromatin in vitro (3). DEK
is a 43-kDa phosphoprotein that was first isolated as part of a fusion
protein expressed in a subtype of acute myeloid leukemias with (t6;9)
chromosomal translocations (4). DEK was later identified as an
autoimmune antigen in patients with pauciarticular onset juvenile
rheumatoid arthritis, systemic lupus erythematosus, and other
autoimmune diseases (5-7). In addition, DEK has been reported to be a
site-specific DNA binding factor, which recognizes a specific DNA
element in the human immunodeficiency virus enhancer (8).
In a recent study, it was demonstrated that DEK associates with
splicing complexes through interactions promoted by
SR1 proteins. It was shown
that DEK associates with mRNA in a splicing-dependent manner, indicating that it could function to coordinate splicing with
subsequent steps in gene expression (9). In addition DEK was found in a
~335-kDa five-component complex at a conserved position 20-24
nucleotides upstream of exon-exon junctions (10).
Our recent experiments have identified DEK as a protein that induces
alterations in the superhelical density of DNA in chromatin (3). The
change in topology was only observed with chromatin but not with naked
DNA and depends on the presence of histone H2A/H2B dimers. In addition
we could show that DEK inhibits the replication efficiency of chromatin
templates but not of naked DNA in vitro, demonstrating that
DEK acts in a chromatin-specific manner. Association with chromatin has
already been reported by Fornerod et al. (11), who
demonstrated that DEK is associated with condensed chromosomes during metaphase.
Thus, DEK seems to be a factor with dual RNA and DNA binding
properties. In order to elucidate the localization of DEK in the cell,
we performed fractionation studies. We found that DEK is an abundant
protein in the cell and is eluted from the nuclei with 250 mM salt. Treatment of nuclei with RNase released only ~10% of the DEK protein, whereas DNase treatment released most of
the DEK protein, indicating that most of DEK is associated with
chromatin in vivo. Treatment of nuclei with micrococcal
nuclease followed by glycerol gradient sedimentation revealed that DEK is associated with oligonucleosomes. Chromatin fractionation studies demonstrated that DEK is more or less equally distributed on
transcriptional active and inactive chromatin regions. The amount and
localization of DEK does not change during the cell cycle.
Cell Culture and Cell Cycle Synchronization--
Human HeLa S3
cells were grown on plastic dishes in Dulbecco's modified Eagle's
medium with 5% fetal calf serum. Cells were synchronized by a double
thymidine block at the beginning of S phase and released into
thymidine-free medium (12). S phase was determined by cell counting and
by pulse-labeling with [3H]thymidine and mitosis by
mitotic indexes exactly as described recently (Fig. 1) (12).
Immunoblotting and Antibodies--
Immunoblotting was carried
out according to standard procedures. Proteins were separated by
SDS-PAGE (13) and transferred to nitrocellulose. The membrane was
blocked in Rotiblock solution (Roth) and incubated with different
antibodies. Enhanced chemiluminescence reagents (ECL, Amersham
Pharmacia Biotech) were used for detection. The polyclonal DEK
antibodies (raised against His-DEK) were kindly provided by Gerald
Grosveld (St. Jude Hospital, Memphis, TN) and the anti-SR antibodies
mAb NM4 were a gift from Benjamin Blencowe (University of Toronto,
Toronto, Ontario, Canada). The MCM5 antibodies have been described
(12).
Cell Fractionation--
Cells were washed three times on the
plate with ice cold hypotonic buffer A (20 mM HEPES pH 7.4, 20 mM NaCl, 5 mM MgCl2, 1 mM ATP) and lysed by Dounce homogenization. After 15 min on
ice, the cytosolic supernatant was separated from the nuclear pellet by
centrifugation (5 min, 600 × g). Nuclei were
resuspended in buffer A supplemented with 0.5% Nonident P40 (Nonidet
P-40) and kept on ice for 15 min to lyse the nuclear envelope.
Centrifugation separated the free nucleosolic proteins from the
pelleted nuclei (5 min, 1000 × g). The pellet was
extracted for 15 min on ice in buffer B (20 mM HEPES, pH
7.4, 0.5 mM MgCl2, 1 mM ATP, 0.3 M sucrose) plus NaCl in concentrations from 0.1 to 0.45 M to release structure-bound proteins. The final pellet was
extracted in RIPA (50 mM Tris-HCl, pH 8, 150 mM
NaCl, 1% Nonidet P-40, 0.5% sodium desoxycholate). Proteins of each
fraction were precipitated according to Wessel and Flügge (14)
and analyzed by SDS-PAGE and Western blotting. Histone H1 was isolated
from the individual fractions by perchloric acid extraction as
described previously (15) and visualized by silver staining.
Micrococcal Nuclease Digestion, DNase I, and RNase
Treatment--
Nuclei were prepared as described above and finally
washed in elution buffer (20 mM HEPES, pH 7.4, 0.5 mM MgCl2, 1 mM ATP, 0.3 M sucrose) containing 100 mM NaCl. Nuclei (500 µg of DNA/ml) were incubated for 30 min at 37 °C with 17 units of
DNase I/100 µl of cell nuclei or 40 units of RNase/100 µl of cell
nuclei; enzymes (Roche) were RNase- and DNase-free, respectively. The reaction was stopped on ice with 8 mM EDTA. Supernatant and
pellet were separated by one centrifugation step. For micrococcal
nuclease digestion, nuclei (500 µg of DNA/ml) were adjusted to 2 mM CaCl2 and digested with micrococcal nuclease
with the concentration and time indicated under "Results." The
reaction was stopped on ice with 8 mM EDTA. Released
chromatin fragments were separated from insoluble material by
centrifugation (10 min, 12,000 × g, 4 °C). Proteins
were removed from the DNA for 30 min at 50 °C in 2% SDS, extracted
as described by Wessel and Flügge (14), and analyzed by SDS-PAGE
and Western blotting. DNA was visualized on agarose gels by ethidium
bromide staining.
Glycerol Gradient Sedimentation--
The supernatant from the
micrococcal nuclease digestion was separated for 14 h (40,000 rpm,
SW40) on 5-40% glycerol gradients (20 mM HEPES, pH 7.4, 0.5 mM MgCl2, 100 mM NaCl, 1 mM ATP). Proteins and DNA from the individual fractions
were analyzed as described above.
Chromatin Fractionation--
The isolation of S1, S2, and P
chromatin fractions was based on the procedure described by Rose and
Garrard (16) with minor modifications. 107 nuclei were
isolated in HNB buffer (0.5 M sucrose, 15 mM
Tris-HCl, pH 7.5, 60 mM KCl, 0.25 mM EDTA, pH
8, 0.125 mM EGTA, 0.5 mM spermidine, 0.15 µM spermine, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, supplemented with a
protease inhibitor mixture (Complete, Roche)) and finally resuspended
in 200 µl of nuclear buffer (20 mM Tris-HCl, pH 7.5, 70 mM NaCl, 20 mM KCl, 5 mM
MgCl2, 3 mM CaCl2, supplemented
with protease inhibitors). Nuclei suspension was incubated with 300 units of micrococcal nuclease at 22 °C. The reaction was stopped on
ice after 1, 2, and 4 min of digestion, and samples were centrifuged
(12 min, 12,000 rpm, 4 °C). The first supernatant was designated the
S1 fraction. The pellet was resuspended in 200 µl of 2 mM
EDTA, incubated for 10 min on ice, and centrifuged again. The
supernatant and the pellet were designated the S2 and P fractions,
respectively. Equal aliquots of the fractions were deproteinized, and
the DNA was investigated by agarose gel electrophoresis (0.8%) and
ethidium bromide staining. Proteins were extracted according to Wessel
and Flügge (14) and analyzed by SDS-PAGE and Western blotting
with DEK-specific antibodies.
Immunofluorescence Analysis--
HeLa S3 cells grown on
coverslips were fixed for 15 min at room temperature in
phosphate-buffered saline (PBS) containing 3.5% paraformaldehyde,
followed by a 3-min permeabilization step in PBS containing 0.3%
Triton X-100. Cells were then blocked for 60 min at room temperature in
a humid chamber in PBS containing 3% BSA, followed by the incubation
with the primary antibodies for 1 h at 37 °C. After three
washing steps in PBS, cells were incubated for 30 min at 37 °C with
species-specific secondary antibodies. These antibodies were either
Texas Red-labeled goat anti-rabbit or Oregon Green-labeled goat
anti-mouse. After washing in PBS, the DNA was labeled with Hoechst
33258 in PBS containing 40% glycerol. For cell fractionation, cells
were first permeabilized and then extracted with CSK (10 mM
Pipes, pH 6.8, 0.3 M sucrose, 3 mM
MgCl2, 1 mM EGTA, 0, 5% Triton X-100)
containing different NaCl concentrations.
Mitotic cells, obtained after double thymidine block, were
permeabilized for 5 min in PHEM (60 mM Pipes, 25 mM HEPES, pH 7.4, 10 mM EGTA, 4 mM
MgSO4, 0.5% Triton X-100) and then fixed in 3.5% paraformaldehyde followed by blocking in PBS with 3% BSA and
incubation with the first and secondary antibodies.
Immunofluorescence Labeling of Spread Chromosomes--
Metaphase
chromosome spreads were obtained from HeLa S3 cells treated for 14 h with 40 ng/ml nocodazole. Blocked cells were collected by
centrifugation at 250 × g for 5 min, resuspended in
hypotonic buffer (10 mM Tris-HCl, pH 7.4, 10 mM
NaCl, 5 mM MgCl2), and kept for 5 min at room
temperature. Hypotonically swollen cells were centrifuged at 250 × g for 5 min, resuspended in fixation buffer
(methanol:acetic acid 3:1), and incubated for 30 min at room
temperature. After another centrifugation step, the pellet was
resuspended in a few droplets fixation buffer and kept on ice. The
solution was pipetted on coverslips and dried at 37 °C. Coverslips
were washed for rehydration three times for 2 min in TEEN (1 mM triethanolamine, pH 8.5, 0.2 mM EDTA, 25 mM NaCl, 0, 1% BSA, 0.5% Triton X-100), then incubated
with the primary antibodies for 1 h in TEEN at 37 °C. After
washing in KB+ (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0, 1% BSA, 0, 1% Triton X-100), chromosomes were
covered for 30 min at 37 °C with the secondary antibodies, diluted
in KB+. DNA was stained with Hoechst 33258.
DEK Is an Abundant Protein That Elutes at 250 mM Salt
from Cell Nuclei--
The DEK protein changes the topology of
chromatin in vitro at a ratio of two to three molecules of
DEK/nucleosome by specifically interacting with histone H2A/H2B dimers
(3). It was of interest to investigate the subcellular distribution of
the protein. For that purpose, we performed cell fractionation experiments.
HeLa cells were disrupted in hypotonic buffer including 5 mM MgCl2 (17) to prepare a supernatant
containing cytosolic proteins. The isolated nuclei were then lysed in
0.5% Nonidet P-40 and centrifuged again to obtain nucleosolic
proteins. The remaining nuclear structure including chromatin and the
nuclear matrix was successively extracted with a buffer containing
increasing NaCl concentrations and finally with RIPA. Most of the DEK
protein dissociated at 0.25 M NaCl, and only small amounts
were also present in the nucleosol or extracted with 100 or 450 mM salt (Fig. 1A).
The salt extractability of the DEK protein was compared with that of
other nuclear proteins. We found that the DEK protein behaved much like
typical chromatin-bound proteins, namely hCdc6 and hOrc2 protein (18,
19). The transcription factor SP1 was, however, found predominately in
the nucleoplasm and only traces in the 250 mM and 450 mM extract. As already known, most of the linker histone H1
eluted from the nuclei with 450 mM salt (see,
e.g., Ref. 20), a minor portion was also detected after
extraction of the nuclei with RIPA. The distribution of nuclear
proteins found by this cell fractionation technique reflects only the
steady state level of a protein; however, proteins seem to move in the
nucleus and to exchange continuously between chromatin regions (see
"Discussion").
Immunofluorescence studies revealed a strictly nuclear signal, whereby
the nucleoli were excluded from staining (Fig. 1B, control). The signal disappeared upon extraction of nuclei
with increasing salt concentrations, and most of the DEK protein was released between 250 and 400 mM salt (Fig. 1B),
in agreement with the biochemical fractionation studies of Fig.
1A.
DEK could be linked to nuclear structures via RNA or DNA. To address
this point, we treated nuclei with DNase I or RNase. The mobilization
of DEK was determined by Western blotting (Fig. 2A) and by immunofluorescence
(Fig. 2B). Treatment of nuclei with DNase I released a large
fraction of DEK, whereas RNase released only a small portion of DEK
(Fig. 2A). This was supported by immunofluorescence analysis: A bright nuclear signal remained after RNase treatment of
nuclei, which was substantially reduced after DNase digestion (Fig.
2B). Residual DEK staining was detected after DNase
treatment and could be due to the association of some DEK with splicing complexes (9). We found indeed that the DEK staining, remaining after
DNase digestion, overlapped with regions stained by SR-specific antibodies (21), indicating that this fraction of DEK might be
associated with splicing complexes in vivo.
The cell fractionation experiment and the immunofluorescence data both
implied that DEK is an abundant protein in the nucleus. To estimate the
number of DEK molecules and the possible ratio of the DEK protein to
nucleosomes in vivo, we prepared whole cell extracts of HeLa
cells. The amount of DEK in a given cell number was determined by
Western analysis with DEK-specific antibodies in comparison with known
amounts of recombinant glutathione S-transferase-DEK protein
(data not shown). The calculation revealed that 4-6 × 106 molecules of DEK/cell are present, which corresponds to
a theoretical ratio of 0.25-0.4 molecules of DEK/nucleosome. Thus, DEK
is an abundant protein in the cell.
DEK Is Associated with Chromatin in Vivo--
To further
characterize the subnuclear distribution of the DEK protein, we treated
HeLa nuclei with increasing amounts of micrococcal nuclease (Fig.
3), an endonuclease that preferentially attacks the linker DNA between adjacent nucleosomes. We thus produced DNA fragments of the size of mono- and oligonucleosomes (Fig. 3A). Proteins were extracted from the soluble and insoluble
chromatin and investigated by Western blotting (Fig. 3B,
S and P). With increasing amounts of micrococcal
nuclease, up to 40% of DNA in chromatin became solubilized. Higher
enzyme concentrations degraded the released oligonucleosomal fragments
to mononucleosomes, but failed to mobilize additional DNA from the
chromatin pellet (Fig. 3A). This is consistent with earlier
studies, which have reported that a fraction of chromatin is refractory
to nuclease attack even at high enzyme concentrations (22-24).
Approximately 50% of the DEK protein were released already at rather
low enzyme concentrations (2 units, Fig. 3B). However, the
second half of DEK remained associated with an insoluble nuclear
structure, even at very high micrococcal nuclease concentrations (Fig.
3B) and after prolonged incubation times (data not shown).
To quantify the immunoblots, the stained bands of Fig. 3B
were investigated by NIH imager and plotted together with the fraction
of solubilized DNA as a function of the enzyme concentration used (Fig.
3B). The quantitation shows again that a fraction of DEK was
already released with low micrococcal nuclease concentrations,
indicating that a fraction of DEK resides in chromatin regions that are
more accessible to nucleases than bulk chromatin.
To obtain further information on the DEK-carrying chromatin, the
products of micrococcal nuclease digestion were separated on glycerol
gradients. Equal aliquots of the individual fractions were
deproteinized and investigated by agarose gel electrophoresis, to
determine the position of chromatin fragments in the gradient (Fig.
4, upper part).
Other aliquots of the gradient fractions were used to localize the DEK
protein (Fig. 4, untreated). We found that all of the DEK
protein released after treatment of nuclei with micrococcal nuclease
co-sedimentated with chromatin fragments of various lengths, suggesting
that DEK remained associated with chromatin after nuclease digestion.
We considered the possibility that at least some DEK may be bound to
ribonucleoprotein, and treated the chromatin fragments with RNase
before separation on glycerol gradients. Indeed, a small portion of DEK
was released from fast sedimenting material and migrated after RNase
treatment just like isolated soluble DEK (Fig. 4, RNase and
DEK
However, the majority of DEK was clearly bound to chromatin because
DNase I digestion of chromatin fragments resulted in their complete
degradation (not shown) and the release of the DEK protein (Fig. 4,
DNase). Our conclusion is that a major portion of DEK is
associated with chromatin.
Rose and Garrard (16) have shown that differential extraction of
micrococcal nuclease-treated chromatin allows a separation of active
and inactive chromatin. We have used their procedure to investigate the
partition of DEK to functionally different chromatin regions. Briefly,
nuclei were digested for 1, 2, and 4 min with micrococcal nuclease and
centrifuged to prepare the supernatant fraction S1. This fraction has
been described to consist of transcriptionally active chromatin that is
deficient in histone H1 and enriched in HMG proteins (16). The pellet
was resuspended in an EDTA-containing buffer and centrifuged to obtain
supernatant fraction S2, which has been shown to be depleted of
transcribed sequences (16). The remaining insoluble fraction P includes the nuclear matrix with actively transcribed genes. The resistance to
nucleases may be due to associated protein complexes as RNA polymerase
(16) or the SWI/SNF complex, which has also been detected in fraction P
(25). As shown by DNA analysis on an agarose gel, the fractions
represent differences in nuclease accessibility of chromatin (Fig.
5). Fraction S1, containing between 5%
and 10% of the cellular DNA, is mainly composed of
mononucleosomal-sized DNA, whereas fraction S2, containing ~50% of
cellular DNA, showed a typical nucleosomal ladder of DNA fragments. The
P fraction, with ~40% of cellular DNA, consisted of heterogeneously
sized DNA (Fig. 5A). Equal aliquots of fractions S1, S2, and
P were subjected to SDS-PAGE, and the DEK protein was detected by
immunoblotting with DEK-specific antibodies (Fig. 5B). We
found that the DEK protein is present in all three fractions. However,
taking into account that fraction S2 contains the major part of the
DNA, the DEK protein is enriched approximately 5-fold in fraction S1
with respect to fraction S2. This indicates that the DEK protein is more abundant in transcriptionally active chromatin.
Localization of DEK during the Cell Cycle--
In order to
investigate the localization of DEK during the cell cycle, HeLa cells
were arrested at the transition between G1 and S phase by a
double thymidine block (26). Synthesis of DNA, as determined by the
incorporation of labeled DNA precursors, started between 1 and 2 h
after thymidine removal and continued for ~7 h (data not shown).
Equal cell aliquots were taken at the indicated times after thymidine
release and fractionated as described above (Fig. 1). Proteins were
separated by SDS-PAGE, and the DEK protein was detected after
immunoblotting with DEK-specific antibodies. As a control Western blots
were also stained with MCM5-specific antibodies (Fig.
6). Minichromosome maintenance (MCM)
proteins are known to dissociate from chromatin during S phase (27), but rapidly bind to chromatin again at the end of mitosis (12). As
shown in Fig. 6, DEK remained on chromatin during all stages of the
cell cycle. Furthermore, DEK in all cases could be eluted at 250 mM salt from chromatin, suggesting that the mode of
DEK-chromatin interaction does not change during S phase when chromatin
is newly assembled. The internal control here is the MCM5 protein,
which was found on G1 phase chromatin (Fig. 6, 3 h), but not on chromatin from S phase and post-S phase cells (Fig.
6, 6-11 h). Similar results were obtained after release of
HeLa cells from a nocodazole block, where no change in the amount and
localization of the DEK protein occurred during the following
G1 phase (data not shown).
Immunofluorescence was used to investigate the association of DEK with
chromatin during mitosis. HeLa cells were released from a thymidine
block and 9 h later permeabilized and fixed with formaldehyde. The
localization of DEK was detected with DEK-specific antibodies and Texas
Red-conjugated secondary antibodies (Fig. 7A). We found that DEK is
associated with chromatin during all phases of mitosis, starting in
pro-phase to metaphase, anaphase, and telophase (data not shown). In
addition, we prepared metaphase chromosomes from nocodazole-treated
cells (Fig. 7B). After treatment with DEK-specific
antibodies, we found a homogenous staining of the metaphase
chromosomes. Thus, DEK remains bound to mitotic chromatin.
Our recent experiments have shown that DEK changes the topology of
DNA in chromatin in vitro (3). It was therefore of interest to know whether DEK is in contact with chromatin in vivo. In
this work we present evidence that the DEK protein is associated with chromatin in vivo. We found that ~50% of the DEK protein
can be released from nuclei with low micrococcal nuclease
concentrations. The resistant fraction remains in the nucleus even
after prolonged incubation with high amounts of micrococcal nuclease.
Analysis of released chromatin fragments on glycerol gradients revealed that DEK co-sediments with oligonucleosome-sized DNA fragments. After
treatment of these preparations with RNase, a small fraction of DEK was
released and appeared at the position of free DEK protein. This
indicates that a minor fraction of DEK is associated with RNA-containing complexes in vivo, as has been shown
previously (9). A co-localization of a fraction of DEK protein with
splicing complexes was also confirmed by immunostaining with anti-SR
antibodies. In addition to the fractions of DEK, which are solubilized
by short treatment with micrococcal nuclease and include chromatin- and
RNP-bound DEK, a third fraction exists, which could not be released
from nuclei by micrococcal nuclease or DNase treatment but is eluted
with 250 mM salt and might be associated with
heterochromatic regions. Whether the distribution of DEK in the nucleus
reflects different forms of the protein has yet to be shown. In fact,
DEK can be phosphorylated (11), but whether this or other
post-translational modifications direct the protein to different
chromatin compartments or to ribonucleoprotein is not known.
DEK is not unique among nuclear proteins that bind to chromatin and to
RNA containing structures. Interestingly, several examples of factors
with dual RNA and DNA binding properties are known. Like the DEK
protein, some of these factors are also involved in human malignancies.
To this class belongs the Wilm's tumor protein (WT-1), which binds to
DNA but also concentrates within splicing factor domains in the nucleus
(28, 29). In addition the oncoprotein TLS/FUS binds to DNA and also
interacts with SR protein splicing factors (30). Furthermore, the
Ets-related transcription factor Spi-1/PU.1, a hematopoietic specific
transcription factor has been shown to influence splicing activity (31,
32). An intriguing question is whether the fusion of the DEK protein with CAN, expressed in acute myelogenous leukemia (4), alters the
location of DEK on chromatin and thus contributes to the transforming potential of the protein or whether alterations in RNA processing are
responsible for the oncogenic processes.
DEK is an abundant protein with more than a million copies per nucleus.
A comparison of the DEK amino acid sequence with sequences in data
bases identified homologous sequences within predicted open reading
frames of zebrafish, fly, and plant expressed sequence tag cDNAs,
but did not reveal a significant homology with any characterized
protein. Interestingly, amino acids 149-183 of the DEK protein show
strong homology to the SAF (scaffold attachment factor) box (33, 34), also termed SAP domain (after
SAF-A/B, Acinus, and PIAS) (35), a
DNA-binding motif involved in chromosomal organization. This 31-amino
acid motif reveals a bipartite distribution of strongly conserved
hydrophobic, polar, and bulky amino acids separated by a region that
contains an invariant glycine (34). The positions enriched in
positively charged amino acids might make contacts with the DNA
backbone. SAF boxes have been found in many different proteins as, for
example, polyADP-ribose polymerase, the Ku autoantigen, and the RAD18
protein, involved in DNA repair. Furthermore, the SAF box is associated
with different proteins involved in the assembly of RNA-processing
complexes (35) and might tether a diverse set of domains involved in
pre-mRNA processing to transcriptionally active chromatin. It has
also been shown that the SAF box organizes interphase chromosomes by
binding to SAR regions, and its release by caspases causes the
chromosomes to collapse during apoptotic cell death and facilitates
chromatin degradation (33, 36). Thus, the SAF box targets a variety of
proteins to specific chromosomal locations. In the case of the DEK
protein, the association with chromatin might be mediated by the
binding of the SAF box to DNA and by additional protein-protein interactions with histone H2A-H2B dimers (3).
In accordance with previous experiments (11), our immunofluorescence
data show a co-localization of the DEK protein with chromatin during
mitosis. In addition we could demonstrate that there is no change in
the amount and localization of the DEK protein during the cell cycle,
indicating that DEK is associated with chromatin during the whole cell
cycle. This suggests that the DEK protein might be involved in
maintaining architectural features of chromatin.
Recent experiments using photobleaching techniques have demonstrated
that different proteins as the nucleosomal binding protein HMG-17, the
pre-mRNA splicing factor SF2/ASF, and the rRNA processing protein
fibrillarin, which are involved in diverse nuclear processes move
rapidly throughout the entire nucleus (37). In addition it was found
that almost the entire population of histone H1-green fluorescence
protein is bound to chromatin at any one time, but is exchanged
continuously between chromatin regions (20, 38). Thus, H1-green
fluorescence protein molecules reside on chromatin in living cells for
~220 s before dissociating and rapidly rebinding to an available
binding site (20).
Our studies on the association of the DEK protein with nuclear
structures have analyzed the steady-state level of the DEK protein. In
light of the new data that most proteins constantly move in the
nucleus, it seems, however, quite likely that the DEK protein may also
move between chromatin regions or even between chromatin and RNA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The main fraction of DEK is eluted from HeLa
nuclei at 250 mM NaCl. A, 107
HeLa cells were fractionated in cytoplasmatic proteins
(cytosol), nucleosolic proteins (nucleoplasm), and a Nonidet
P-40-resistant structure, which was eluted with 100, 250, and 450 mM NaCl and RIPA as indicated. Equal aliquots of the
supernatants and the final pellet were analyzed by SDS-PAGE and Western
blotting using DEK-specific antibodies. The same blot was reprobed
using Cdc6-, Orc2-, and Sp1-specific antibodies. Histone H1 was
extracted from the individual fractions with 5% perchloric acid and
visualized by silver staining. The positions of molecular size markers
(kDa) are indicated. B, immunofluorescence studies. HeLa
cells grown on coverslips were permeabilized and extracted with buffer
without salt (control) or containing 100, 250, or 450 mM NaCl. After fixation in formaldehyde cells were
incubated first with DEK-specific antibodies and then with Texas
Red-conjugated secondary antibodies. Cellular DNA was stained with
Hoechst 33258 (bar, 5 µm).
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Fig. 2.
The main portion of DEK is resistant to RNase
treatment but is extracted from nuclei with DNase I. A,
cell nuclei (500 µg of DNA/ml) were incubated for 30 min at 37 °C
in the absence (control) or presence of DNase I or RNase.
Supernatant (S) and pellet (P) were separated by
one centrifugation step. Proteins were analyzed by SDS-PAGE and Western
blotting with DEK-specific antibodies. B, immunolocalization
of DEK protein in control nuclei and after DNase and RNase treatment.
Cells grown on coverslips were treated with DNase I or RNase for 40 min
at 37 °C after permeabilization in PBS, 0.5% Triton X-100 and
hypotonic extraction for 10 min. Cells were either labeled with
DEK-specific antibodies or with anti-SR antibodies as described in Fig.
1 (bar, 5 µm).
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Fig. 3.
Mobilization of DEK protein by micrococcal
nuclease. A, cell nuclei (500 µg of DNA/ml)
were incubated for 15 min at 14 °C with increasing amounts of
micrococcal nuclease. Reactions were stopped with 8 mM
EDTA, and insoluble material was removed by centrifugation. Purified
DNA was analyzed by agarose gel electrophoresis and ethidium bromide
staining. The positions of mono-, di-, tri-, tetra-, and
pentanucleosomes are indicated. Proteins were extracted from soluble
chromatin (S) and the insoluble pellet (P),
separated by SDS-PAGE, and analyzed by Western blotting with
DEK-specific antibodies. B, DNA amounts of nuclease-treated
chromatin were determined by fluorimetry and expressed as percentage of
total DNA. The immunologically stained bands in A were
scanned and given as percentage of the control value (lane
0 in the pellet in A).
chromatin).
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Fig. 4.
DEK co-sedimentates with oligonucleosomes on
glycerol gradients. HeLa cell nuclei (500 µg of DNA/ml) were
digested for 10 min at 14 °C with micrococcal nuclease (5 units/100
µl). Reactions were stopped, and insoluble material was removed by
centrifugation. The supernatant was either left untreated or was
digested for 30 min at 37 °C with RNase or DNase I. Samples were
separated on 5-40% glycerol gradients. Purified DNA from the
individual fractions was analyzed by agarose gel electrophoresis and
ethidium bromide staining (upper panel).
Positions of DNA fragments corresponding to mono-, di-, tri-, etc.
nucleosomes are indicated. Proteins were separated by SDS-PAGE and
analyzed by Western blotting with DEK-specific antibodies
(lower panels). As a control the DEK protein was
centrifuged on a parallel gradient in the absence of chromatin
(DEK, chromatin).
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[in a new window]
Fig. 5.
DEK is found on active, inactive, and
matrix-bound chromatin. A, chromatin fractionation.
HeLa cell nuclei were treated for the indicated time with 300 units of
micrococcal nuclease at 14 °C. The reaction was stopped on ice. Low
speed centrifugation yielded fraction S1. The pellet was washed with 8 mM EDTA to yield the supernatant fraction S2 and the pellet
fraction P. Percentage of released DNA is indicated. DNA was purified
and analyzed by agarose gel electrophoresis. B, proteins
were extracted from the individual fractions, and equal aliquots were
separated by SDS-PAGE and analyzed by Western blotting with
DEK-specific antibodies.
View larger version (28K):
[in a new window]
Fig. 6.
The amount and localization of DEK does not
change during the cell cycle. HeLa cells were arrested in early S
phase using double thymidine block. Samples were taken at the indicated
times, and cell number was determined by a Coulter counter.
107 cells were fractionated as described in Fig. 1. Equal
aliquots of the fractions were analyzed by SDS-PAGE and Western
blotting using DEK- or MCM5-specific antibodies.
View larger version (26K):
[in a new window]
Fig. 7.
DEK is bound to chromatin during
mitosis. A, HeLa cells grown on coverslips, were
synchronized with thymidine. 7.5 h after release cells were
permeabilized, fixed in 3.5% formaldehyde, and incubated with
DEK-specific antibodies and then with Texas Red-conjugated secondary
antibodies (bar, 5 µm). B, preparation of
metaphase chromosomes. Mitotic cells were collected after nocodazole
block, swollen in hypotonic buffer, washed once in methanol:acetic acid
(3:1), and then dried on coverslips. After rehydration, chromosomes
were incubated with DEK-specific antibodies and then with Texas
Red-conjugated secondary antibodies. Cellular DNA was stained with
Hoechst 33258 (bar, 2 µ m).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Rolf Knippers, Vassilios Alexiadis, and Jim Kadonaga for stimulating discussions and critical reading of the manuscript. The glutathione S-transferase-DEK expression vector and the DEK antibodies were a kind gift from Gerald Grosveld. The mAb NM4 antibodies were kindly provided by Benjamin Blencowe.
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FOOTNOTES |
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* This work was supported by grants from the Deutscheforschungsgemeinschaft (to C. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Molecular Cell Biology,
Heinrich-Pette-Institut, 20251 Hamburg, Germany.
§ To whom correspondence should be addressed. Tel.: 49-7531-882125; Fax: 49-7531-884036; E-mail: claudia.gruss@uni-konstanz.de.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M100162200
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ABBREVIATIONS |
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The abbreviations used are: SR, serine/arginine-repeat; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; RIPA, radioimmune precipitation buffer; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Pipes, 1,4-piperazinediethanesulfonic acid; MCM, minichromosome maintenance.
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