| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 28, 24988-24994, July 12, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Biology, University of Konstanz, Konstanz 78457, Federal Republic of Germany
Received for publication, April 25, 2002, and in revised form, May 6, 2002
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
We have investigated the molecular mechanism by
which the proto-oncogene protein DEK, an abundant chromatin-associated
protein, changes the topology of DNA in chromatin in vitro.
Band-shift assays and electron microscopy revealed that DEK induces
both intra- and intermolecular interactions between DNA molecules. Binding of the DEK protein introduces constrained positive supercoils both into protein-free DNA and into DNA in chromatin. The induced change in topology is reversible after removal of the DEK protein. As
shown by sedimentation analysis and electron microscopy, the DEK-induced positive supercoiling causes distinct structural changes of
DNA and chromatin. The observed direct effects of DEK on chromatin folding help to understand the function that this major chromatin protein performs in the nucleus.
DNA in the eukaryotic nucleus is highly organized in a complex
chromatin structure (reviewed in Ref. 1). The coiling of DNA by
histones in nucleosomes is a barrier to the entry of proteins involved
in transcription, replication, and other DNA transactions. Changes in
chromatin structure as triggered by the combined action of histone
acetyltransferases and histone deacetylases as well as chromatin
remodeling complexes have a significant influence on the processes
occurring in DNA (reviewed in Refs. 2-5). In search for factors that
change the structure of chromatin and the replicative activity of
chromatin templates, we have recently identified the proto-oncogene
protein DEK as a candidate protein that changes the topology of DNA in
chromatin in vitro (6).
The DEK protein was initially identified in a fusion with the CAN
nucleoporin in a subtype of acute myeloid leukemias involving chromosomal translocations (7). DEK was later identified as an
autoantigen in several autoimmune diseases such as juvenile rheumatoid
arthritis (8) or systemic lupus erythematosus (9). Despite these
disease associations the function of the DEK protein in the cell
remains elusive. There are two recent reports demonstrating that DEK
could be involved in RNA metabolism. It was shown that DEK associates
with splicing complexes through interactions mediated by SR proteins.
DEK associates with the SRm160 splicing coactivator in vitro
and remains bound to the exon product RNA after splicing. This
association requires the prior formation of a spliceosome (10). In
addition, DEK has been found in a five-component complex of ~335 kDa
at a conserved position 20-24 nucleotides upstream of exon-exon
junctions in mRNAs (11).
However, DEK also binds to DNA (12-14) and to metaphase chromosomes
(15). In support of this, we determined that DEK is a constituent of
oligonucleosomes, generated by micrococcal nuclease digestion of
chromatin in isolated nuclei (16). Most of the DEK protein was released
from nuclei after DNase treatment, whereas only around 10% was
released after RNase treatment, indicating that the major fraction of
DEK is associated with chromatin in vivo (16).
Isolated DEK changes the topology of DNA in viral minichromosomes and
reduces the accessibility of chromatin to binding factors, including
components of the replication machinery (6). However, the mechanism of
the DEK-induced change in topology has not been investigated. We
describe here that DEK changes not only the topology of nucleosomal DNA
but of protein-free DNA as well.
Glycerol gradients and electron microscopy showed that DEK induces
distinct alterations of the DNA structure due to an introduction of
constrained positive supercoils. We believe that these findings have
profound implications for the current understanding of chromatin functions.
Purification of GST-DEK--
The DEK gene was
cloned in the pGEX3 vector (Amersham Biosciences) to generate a
GST1 fusion protein. The
GST-DEK fusion protein was expressed in BL21(DE3)pLysS. A standard
preparation of GST-DEK was prepared from 1 liter of liquid culture in
2× YT media. Further purification steps were done according to the
manufacturer's protocol (Amersham Biosciences).
Purification of His-DEK--
Full-length DEK cDNA was cloned
into pBlueBac His2A and cotransfected with linearized Autographa
californica nuclear polyhedrosis virus DNA (Bac-N-Blue DNA)
into SF9 cells as described in the manufacturer's protocol
(Invitrogen). Passage level three virus stocks were used to infect
HighFive cells for protein expression. After 2 days post-infection the
cells were washed twice with phosphate-buffered saline and then lysed
with lysis buffer (100 mM Tris, pH 7.5; 150 mM
NaCl; 5 mM KCl; 0.5 mM MgCl2; 1%
Nonidet P-40; and 5 mM imidazole). Soluble
His-tagged DEK was purified by nickel-nitrilotriacetic acid-Agarose
(Qiagen) chromatography according to the manufacturer's protocol.
DEK Assay--
Purified GST-DEK protein was dialyzed on Whatman
filters (Type VS, pore size, 0.025 µm) against buffer A-100 (20 mM HEPES at pH 7.6, 100 mM NaCl, 10 mM sodium bisulfite, 1 mM EDTA) in the presence
of 1 µg/µl bovine serum albumin (New England BioLabs) for 90 min at 4 °C. Salt-treated SV40 minichromosomes or SV40 DNA (17) were
incubated with the dialyzed GST-DEK protein for 1 h at 37 °C
(molar ratios of DEK to DNA ranged from 10 to 234 mol of DEK/mol of
DNA) in the presence of 12 ng of human topoisomerase I. The reactions
were performed in buffer A-100, containing 0.2 µg/µl bovine serum
albumin in a total volume of 90 µl. Supercoiling reactions with
Escherichia coli topoisomerase I were done in the presence
of 6 mM MgCl2. After Proteinase K digestion,
DNA was precipitated and analyzed on 0.8% agarose gels in 0.5×
TBE (45 mM Tris-borate, 1 mM EDTA) at 2 V/cm for 16 h.
Sedimentation Analysis--
1.5 µg of salt-treated SV40
minichromosomes or SV40 DNA was incubated as described above, except
that the reaction volumes were 500 µl. The samples were loaded on
10-35% or 5-60% glycerol gradients (for chromatin and DNA,
respectively) (20 mM HEPES at pH 7.6, 10 mM
sodium bisulfite, 1 mM EDTA, 100 mM or 700 mM NaCl as indicated) and run in a SW40 rotor (Beckman) for
4.5 h (minichromosomes) or 4 h (DNA) at 40,000 rpm at
4 °C. The gradients were fractionated in 600-µl fractions;
proteins were precipitated according to the Wessel-Flügge method
(18) and analyzed by 10% SDS-PAGE followed by Western blot analysis
and immunostaining with a DEK-specific antibody. The DNA was purified
and analyzed on a 0.8% agarose gel in 0.5× TBE at 2 V/cm for 16 h.
Chromatin Reconstitution--
Nucleosomal cores were
reconstituted onto a 1000-bp fragment of SV40 DNA by using a
modification of the salt dilution method (19). In a standard
reconstitution reaction, histone octamers were mixed in an initial
volume of 10 µl with 6 µg of the EcoRI/EcoRV fragment of SV40 DNA (1000 bp) in 2 M NaCl, 10 mM Tris-HCl, pH 7.4, 0.5 mM EDTA. Samples were
diluted at successive intervals of 60 min to contain 1.12, 0.8, 0.6, 0.4, and finally 0.12 M NaCl in the same buffer at room
temperature. Samples were centrifuged at 12,000 × g for 10 min to remove aggregated material and stored on ice
until use.
Electron Microscopy--
SV40 DNA and SV40 minichromosomes were
incubated with increasing amounts of DEK and human topoisomerase I. Unbound protein was removed by gel filtration through a Biogel A-5
column, and samples were fixed with glutaraldehyde (0.1% v/v) for 15 min at 37 °C. Protein-DNA complexes were spread by the alkyl benzyl
dimethyl ammonium chloride technique of Vollenweider et al.
(20). Electron micrographs were taken with a Zeiss EM 900 electron
microscope. The enclosed area of nucleoprotein complexes was determined
with the IMAGE program (National Institutes of Health), and length measurements were made with the AutoCAD program.
Gel Mobility Shift Assays--
500 ng of SV40 DNA (forms I, II,
and III) or SV40 minichromosomes was incubated with increasing amounts
of GST-DEK for 1 h at 37 °C. Standard reactions contained
buffer A-100 in a total volume of 30 µl. Binding of GST-DEK was
analyzed on 0.6% agarose gels in 0.5× TBE at 3.5 V/cm for 4 h.
Two-dimensional Gel Electrophoresis--
Supercoiling reactions
were performed as described above and run in the first dimension on a
0.8% agarose gel in 0.5× TBE at 2 V/cm for 16 h. The second
dimension was run in the presence of 0.25 µg/µl chloroquine on a
0.8% agarose gel in 0.5× TBE plus 0.25 µg/µl chloroquine at 2 V/cm for 16 h. DNA was analyzed by Southern blot and hybridization
with [32P]dATP-labeled HinfI-digested SV40
DNA, followed by autoradiography.
Purification of E. coli Topoisomerase I--
XL-1 blue cells
were transformed with pJW 312Sal. Purification of topoisomerase I was
done from a 500-ml liquid culture exactly as described previously
(21).
DEK Binds to Chromatin and DNA--
Alexiadis et al.
(6) have shown that incubation of SV40 minichromosomes with DEK,
purified from human HeLa cells in the presence of topoisomerase I,
causes a drastic change in superhelical density. However, in our
initial experiments, we failed to detect DEK-induced changes of the
topology of relaxed protein-free DNA and concluded that DEK acts in a
chromatin-specific manner (6). This was clearly in contradiction to the
experiments from the Markovitz group (12-14), who had demonstrated
that DEK binds to DNA in a site-specific manner. To reinvestigate this
point, we used mobility shift assays to compare the binding of DEK to
protein-free DNA and to chromatin (Fig.
1A). DNA or chromatin was
incubated with increasing amounts of the DEK protein, and the resulting nucleoprotein complexes were separated by agarose gel electrophoresis (Fig. 1A). We found that DEK binds to DNA and chromatin
substrates with similar affinities producing multiple complexes whose
mobility decreased with increasing protein concentration until
saturation was reached. High amounts of DEK on protein-free DNA
resulted in nucleoprotein complexes, which did not enter the gel. This indicates that DEK directs the formation of multimeric DNA complexes (compare Figs. 6A, panel e, and Fig. 7, see
below).
To determine whether DEK is able to bind different topological forms of
DNA, we compared the binding of the DEK protein to supercoiled form I
DNA, relaxed form II DNA, and linear form III DNA (Fig. 1B).
In three independent experiments, we found that DEK binds with similar
affinities to the three DNA structures tested.
We next asked whether DEK is able to change the topology of
protein-free DNA. For this purpose, we used increasing amounts of DEK,
comparing protein-free SV40 DNA and SV40 minichromosomes as substrates
in the presence of topoisomerase I. Deproteinized DNA was investigated
by agarose gel electrophoresis and ethidium bromide staining (Fig.
1C). As in our previous experiments, we found no significant
change in the topology of protein-free DNA at low DEK/DNA ratios. At
these ratios a topological alteration of DEK-treated SV40
minichromosomes was observed. However, topological changes of
protein-free DNA occurred at higher DEK/DNA ratios, reaching a maximal
value at ratios between 52 and 78 mol of DEK/mol of DNA. Thus, about
2-fold higher amounts of DEK are necessary to change the topology of
protein-free DNA compared with minichromosomes. Titration of
topoisomerase I revealed that we used saturating amounts of
topoisomerase I in our assays; a further increase did not affect the
topoisomer ladder (data not shown). In kinetic experiments, we found
that rates at which linking number changes were introduced into DNA
were similar for protein-free DNA and SV40 minichromosomes (data not shown).
The experiments shown here were performed with GST-tagged DEK protein
purified from bacteria. To exclude an effect of the GST tag alone, we
repeated the assays with recombinant His-tagged DEK expressed and
purified from insect cells (Fig. 1D). We found that the
His-tagged DEK protein exhibited the same activity as the GST-tagged
DEK protein, both in mobility shift assays (Fig. 1D,
left panel) and in topology assays (Fig. 1D,
right panels). Together with the experiments performed with
untagged human DEK protein (6), these data clearly demonstrate that the
observed effects are solely due to the DEK protein itself.
The DEK-induced Change in DNA Topology Is Reversible and Caused by
the Introduction of Positive Supercoils--
We addressed the question
of whether the DEK-induced change in DNA topology is reversible after
removal of the DEK protein. For this purpose, we incubated SV40
minichromosomes with DEK and topoisomerase I and separated the
chromatin on glycerol gradients containing either 100 or 700 mM NaCl (Fig. 2). Histones
were still present in stoichiometric amounts on chromatin purified on
700 mM gradients (data not shown). As determined by Western
blot analysis, DEK was associated with chromatin at 100 mM
NaCl but not at 700 mM NaCl. The interesting point here is
that the topology of DNA did not change after removal of DEK protein,
as shown by a similar distribution of DNA topoisomers in the 100 mM NaCl and the 700 mM NaCl gradient. Thus,
once introduced, the change in topology is maintained.
An explanation for this effect could be the introduction of constrained
positive supercoils by the DEK protein as outlined in the model in Fig.
3A. The hypothetical
minichromosome in Fig. 3A contains eight nucleosomes,
corresponding to eight negative supercoils after removal of the
nucleosomes by Proteinase K. The five positive supercoils, introduced
by DEK, would neutralize the same number of negative supercoils. This
model is consistent with the data of Fig. 2, because sedimentation
through 700 mM NaCl removes not only DEK but topoisomerase
as well (data not shown) with the consequence that the DEK-induced
topoisomer ladder remains after removal of the DEK protein. In this
case, a re-addition of topoisomerase to DEK-depleted chromatin
templates should revert the topoisomer ladder to form I DNA. To test
this hypothesis we isolated DEK-associated chromatin from 100 mM (Fig. 3B, +DEK) and 700 mM gradients (Fig. 3B,
We have repeated the experiment with protein-free DNA as substrate
(Fig. 3C) except that the DEK protein was removed from the
DNA by Proteinase K and not by salt wash as in Fig. 3B.
DEK-associated (Fig. 3C, +DEK) and DEK-depleted
DNA (Fig. 3C,
To directly demonstrate the presence of positively supercoiled DNA, the
reaction products were treated with E. coli topoisomerase I
(Fig. 4A) and analyzed by
two-dimensional gel electrophoresis (Fig. 4B). Bacterial
topoisomerase, in contrast to eukaryotic topoisomerase I, which removes
both negative and positive supercoils, can only remove negative but not
positive supercoils (22). SV40 DNA was incubated with DEK and
topoisomerase I for 1 h under DEK assay conditions. The DNA was
deproteinized with Proteinase K, precipitated, used as substrate for
the topoisomerase assay, and incubated either with human topoisomerase
I or E. coli topoisomerase I. DNA was again deproteinized,
precipitated, and analyzed by agarose gel electrophoresis (Fig.
4A). The activity of the topoisomerases was checked with
protein-free DNA (Fig. 4A, control). Although human topoisomerase I reverted the topoisomer ladder to form II (see
Fig. 3C), E. coli topoisomerase did not change
the topoisomer ladder, indicating that DEK introduces constrained
positive supercoils into the DNA.
We then analyzed the supercoiling state of the reaction products by
two-dimensional gel electrophoresis with the second dimension in the
presence of chloroquine (Fig. 4B). Chloroquine changes the
topology of closed circular DNA molecules by introducing positive superhelical turns. For orientation, we mark the position of fully relaxed closed circular DNA as a reference point ( The Introduction of Positive Supercoils by the DEK Protein Causes
an Alteration of DNA and Chromatin Structure--
To determine the
effects of DEK-induced positive supercoils on DNA and chromatin
structure, we performed sedimentation analysis (Fig.
5) and electron microscopy (Fig.
6). DNA or chromatin were incubated with
increasing amounts of DEK (plus human topoisomerase I) and then
analyzed by glycerol gradient centrifugation. The position and topology
of the DNA were investigated on agarose gels, and the position of the
DEK protein was determined by Western blotting (Fig. 5). DEK influenced
the sedimentation properties of both protein-free DNA and chromatin.
However, although DEK at ratios of 45 mol of DEK/mol of DNA caused a
precipitation of free DNA, high DEK/chromatin ratios increased the
sedimentation rate but did not precipitate chromatin. Furthermore, at a
DEK/DNA ratio of ~90, corresponding to a ratio of 3.6 molecules of
DEK per nucleosome, free DEK protein appeared at the top of the
gradients, indicating that the substrates were saturated with DEK. The
sedimentation behavior in the presence of DEK indicated that DEK
protein induced structural alterations of DNA and chromatin.
To further investigate the structural changes induced by DEK, we
examined SV40 DNA or SV40 minichromosomes by electron microscopy (Fig.
6). With increasing amounts of DEK we observed different types of
structures. At low DEK/DNA ratios, most DNA molecules possessed one
single protein dot. The size of the dot was similar to that of
nucleosomes, indicating that the DEK protein multimerizes when
bound to DNA (Fig. 6A, panel b). At increasing
DEK/DNA ratios, individual protein dots fused to form loops of variable
sizes, indicating that DEK molecules on different sites on the DNA
interact with each other (Fig. 6A, panel c) until
large portions of DNA are covered by the DEK protein (Fig.
6A, panel d). In addition, DNA networks
containing several SV40 DNA molecules connected by the DEK protein
through intermolecular interactions were formed (Fig. 6A,
panel e). Statistical evaluation revealed a heterogeneous population of molecules at all DEK concentrations used, which were
never completely covered by the DEK protein (Fig. 6B). This supports biochemical data showing that saturating DEK concentrations lead to a spectrum of DNA topoisomers but never to fully superhelical DNA. A likely explanation is that DEK induces topological strain in the
molecule, which prevents further loading of DEK protein.
The DEK-induced alterations of SV40 chromatin were analyzed in the same
way (Fig. 6C). Without DEK, the chromatin carried about
23-25 regularly spaced nucleosomes (Fig. 6C, panel
a). At increasing DEK/chromatin ratios, the structure became more
compact until the area of the nucleoprotein complexes measured 10-30% of the DEK-free control (Fig. 6C, panels b-e).
Nucleosomes on DEK-treated chromatin appeared in clusters, indicating
that DEK induced internucleosomal interactions. To exclude artifacts
due to spreading problems, protein-free DNA was included in all samples as a control and was found in an extended configuration (Fig. 6C, panel f).
To gain further insight into the mode of DEK-DNA interaction, a
linear DNA fragment of 1000 bp was incubated in the presence of
increasing amounts of the DEK protein and topoisomerase I and analyzed by electron microscopy (Fig. 7).
One possibility is that right-handed wrapping of the DNA around the DEK
protein causes the introduction of positive supercoils. Thus at
moderate DEK concentrations we expected that wrapping of the DNA around
the DEK protein should cause a shortening of the molecules. We found, however, that molecules were around 10% longer in the presence of
moderate DEK amounts (Fig. 7, upper panel). At higher DEK
concentrations, different types of molecules were observed (Fig. 7,
middle panel). These consisted of molecules arising by
multiple intra- and intermolecular DEK-DEK interactions, which result
in different forms of compacted molecules. In a control experiment, we
used purified histone octamers and salt gradient dialysis and
reconstituted the 1000-bp fragment into chromatin (Fig. 7, lower
panel). Wrapping of the DNA around nucleosomes resulted in a
significant shortening of the molecules. Taken together, these data
clearly demonstrate that the DEK-induced change in topology is not
caused by wrapping of the DNA around the DEK protein.
DEK is an ubiquitous and abundant nuclear protein with ~5 × 106 copies/nucleus. A fraction of DEK is probably bound
to nuclear RNA, but the majority (80-90%) of DEK is a constituent of
chromatin (16). Despite its abundance and wide distribution,
surprisingly little is known about the functions that DEK may perform
in chromatin. We show here that DEK in vitro does not only
bind to chromatin as previously thought (6) but to protein-free DNA as
well. However, about three times more DEK is necessary to reach
saturation in the mobility shift assay compared with the amount
necessary to completely change the topology of DNA or chromatin. In
either case, the initial binding of DEK introduces positive supercoils up to a point where no further supercoils can be introduced due to
sterical hindrance, resulting in the observed topoisomer ladder. In
addition, DNA-bound DEK proteins are able to interact with each
other (Figs. 6 and 7). Therefore, additional DEK molecules can bind to
the DNA by protein-protein interactions and thus cause a further change
in the mobility shift assay.
DEK introduces positive supercoils into topologically fixed circular
DNA substrates. This reaction is specific, because other DNA binding
proteins such as the SV40 T-Ag or histone binding proteins such as NAP1
do not change the topology of DNA at the same molar ratios (data not
shown). The conclusion that DEK introduces positive supercoils into the
DNA is based on the following observations. First, we found that the
DEK-induced change in topology is reversible after removal of DEK by
eukaryotic but not bacterial topoisomerase I. Second, analysis by
two-dimensional gel electrophoresis directly showed the presence of
positive supercoils.
How could DEK introduce positive supercoils into DNA? So-called helix
tracking proteins, which translocate on the DNA, generate local
positive supercoils ahead of the moving protein and negative supercoils
behind them (24). When the substrate for DEK is protein-free DNA, any
positive and negative superhelicity generated by tracking would
probably be self-canceling or relaxed by topoisomerase. This appears
not to be the case in the presence of the DEK protein. Another
possibility is that DEK wraps DNA in a right-handed direction, generating positive supercoils. The torsional stress would lead to an
accumulation of unconstrained negative supercoils. Topoisomerase I is
able to remove negative but not positive supercoils that are
constrained by DEK binding. Subsequent deproteinization would result in
the formation of positively supercoiled DNA. However, electron
microscopic analysis with linear DNA clearly demonstrated that DEK
caused no shortening of the molecules, excluding a wrapping of the DNA
around DEK. Another possibility is that protein-protein interactions
between DNA-bound DEK molecules cause a change in helical twist by
bending the DNA and thus introducing positive supercoils. Compensatory
negative supercoils are relaxed by topoisomerase I; therefore,
positive supercoils might be stabilized by a framework of DEK molecules
(see Fig. 6). Further studies are needed to clarify the exact mechanism
of positive supercoil induction by the DEK protein.
DNA supercoiling is ubiquitous in living cells and is known to
participate in many DNA transactions (25). Previous examples of
right-handed DNA wrapping proteins include DNA gyrase (26) and the
archaeal histone HMf (27). DNA wrapping by these two proteins does not
require ATP. Both positive and negative supercoiling activity have also
been associated with the yeast recombinase complex Rad 51·Rad
54 during recombination (28). In addition, a novel nonspecific
DNA binding protein Smj 12 has recently been identified in
Sulfolobussol fataricus, which stabilizes the DNA double helix and induces positive supercoiling (29). Positive supercoiling activity has also been shown for condensins (30), multiprotein complexes, which contain the evolutionarily conserved SMC
(structural maintenance of chromosomes) proteins (31). The eukaryotic
SMC proteins form two kinds of heterodimers, which are involved in
chromatin and DNA dynamics. The two most prominent and
best-characterized complexes are cohesin and condensin, necessary for
sister chromatid cohesion and mitotic chromosome condensation (32). SMC
proteins show high affinity for cruciform DNA (33), a property that is
shared with a group of architectural proteins (34). Interestingly, we
also found binding of the DEK protein to four-way junction
DNA,2 suggesting a possible
function of the DEK protein in chromatin architecture. This possibility
is supported by our sedimentation studies and electron microscopic
analysis, which showed that DEK protein induces distinct structural
alterations in the structure of both chromatin and DNA substrates.
The amount and localization of the DEK protein does not change during
the cell cycle (16). The calculated amount of the DEK protein in the
nucleus equals one molecule of DEK per three to four nucleosomes, and
this would be sufficient to exert a general effect on chromatin
structure. Protein-protein interactions of DEK molecules might enhance
and stabilize the compaction
process.3 Because many
proteins are highly mobile in the cell nucleus (35-37), it seems quite
likely that the DEK protein exchanges between chromatin regions and
thus might influence the chromatin structure at different loci. The
mechanisms by which these reactions are regulated remain an interesting
topic for further research.
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
View larger version (66K):
[in a new window]
Fig. 1.
Gel mobility shift assay with protein-free
DNA and SV40 minichromosomes. Equal amounts of form I DNA or SV40
minichromosomes (A) or form I, II, and III DNA
(B) were incubated in the absence ( 
) or presence of
increasing amounts of DEK and loaded directly on a 0.6% agarose gel.
The molar ratios of DEK to DNA present in the reaction mixtures were:
In A, lanes 1-10: 0, 8.5, 17, 25, 50, 78, 100, 130, 180, and 234 mol of DEK/mol of DNA; in B, lanes
1-5: 0, 50, 100, 130, and 180 mol of DEK/mol of DNA. DEK assay:
C, form I SV40 DNA (175 ng) or SV40 minichromosomes (175 ng)
were incubated for 1 h in the absence (DEK) or
presence of increasing amounts of DEK protein and human topoisomerase
I. The topology of deproteinized DNA was analyzed by agarose gel
electrophoresis and ethidium bromide staining. The molar ratios of DEK
to DNA were: lanes 1-11: 0, 4.5, 8.5, 17, 25, 50, 78, 100, 130, 182, and 234 mol of DEK/mol of DNA. D, gel mobility
shift assay with His-tagged DEK. Equal amounts of DNA (form I, II, and
III) were incubated in the absence () or presence (+) of His-tagged
DEK and loaded directly on a 0.6% agarose gel (first
panel). The DEK assay is shown with protein free DNA (second
panel) and SV40 minichromosomes (third panel) in the
absence () or presence (+) of His-tagged DEK. The molar ratios of DEK
to DNA are 0 () and 182 (+). Sample analysis was as in
C; I, supercoiled; II, relaxed, closed
circular and nicked DNA.
View larger version (31K):
[in a new window]
Fig. 2.
DNA topology after removal of the DEK
protein. SV40 chromatin was incubated in the absence (mc
without DEK) or presence of DEK (mc with DEK) and human
topoisomerase I and separated on glycerol gradients containing 100 or
700 mM salt. The position and topology of the purified DNA
was analyzed by agarose gel electrophoresis, and the position of the
DEK protein was determined by Western analysis and compared with the
sedimentation behavior of free DEK protein (free DEK). Note
that free DEK protein shows a faster sedimentation in 100 mM gradients compared with 700 mM, indicating
that DEK forms multimers at low salt. The positions of DNA forms I
(supercoiled) and II (relaxed, closed circular and nicked DNA) and of
molecular mass makers (kilodaltons) are given in the
middle.
DEK) and
treated the molecules with human topoisomerase I. As predicted, the
topoisomers of DEK-depleted chromatin were completely converted into
form I DNA after treatment with human topoisomerase I, whereas no
effect was observed on the topoisomer ladder of DEK-associated
chromatin. Thus the DEK-induced change in DNA topology is reversible
after removal of the DEK protein in the presence of human topoisomerase
I.
View larger version (27K):
[in a new window]
Fig. 3.
Treatment of DEK-depleted DNA and chromatin
with human topoisomerase I. A, model for DEK action on
chromatin (see text for further explanation). B, chromatin
separated on 100 mM (+DEK) or 700 mM
( DEK) salt gradients (see Fig. 2) was incubated in the
absence () or presence (+) of human topoisomerase I. As a control for
topoisomerase activity protein-free SV40 DNA was incubated under
identical conditions (control). Deproteinized DNA was
investigated by agarose gel electrophoresis. C,
DEK-associated DNA was first incubated in the absence (+DEK)
or presence (DEK) of Proteinase K and then in the absence
() or presence (+) of human topoisomerase I. As a control
protein-free DNA was incubated under identical conditions
(control) (input-DEK, SV40 DNA was incubated
under the same conditions in the absence of DEK). Purified DNA was
separated by agarose gel electrophoresis. The positions of DNA forms I
(supercoiled) and II (relaxed, closed circular and nicked DNA) are
indicated.
DEK) was then treated with human
topoisomerase I. We found that the topology of DEK-associated DNA was
not changed by topoisomerase I, whereas the topoisomer ladder of
DEK-depleted DNA was reverted to form II DNA.
View larger version (37K):
[in a new window]
Fig. 4.
Analysis of the supercoiling state.
A, DEK-associated DNA was treated with Proteinase K
( DEK) and then incubated in the absence () or presence
(+) of either human topoisomerase I or E. coli
topoisomerase. The activity of the topoisomerases was tested with
protein-free DNA (control). Purified DNA was analyzed by
agarose gel electrophoresis. The positions of DNA forms I (supercoiled)
and II (relaxed, closed circular and nicked DNA) are indicated.
B, SV40 minichromosomes (a, b) and
SV40 DNA (c, d) were incubated in the absence
(a, c) or presence (b, d)
of DEK (at a molar ratio of DEK to DNA of 50) and topoisomerase I. The
purified DNA was separated by two-dimensional gel electrophoresis with
the second dimension in the presence of 0.25 µg/µl chloroquine. DNA
was investigated by Southern blotting with 32P-labeled
HinfI-digested SV40 DNA. The most relaxed topoisomer is
defined as 
Lk = 0, and the positions of
positive (Lk > 0) and negative
(Lk < 0) topoisomers are indicated.
Lk = 0). DNA topoisomers on the arc extending leftward from
Lk = 0 have negative supercoils, and DNA
topoisomers on the right arc have positive supercoils.
We found that DNA in chromatin possessed around 25 negative supercoils,
in accordance with the known number of 25 nucleosomes of SV40
minichromosomes (23). In contrast, DEK-treated DNA in chromatin had
only an average of 12 to 13 negative supercoils, demonstrating that
DEK together with topoisomerase I changes the topology of DNA in
chromatin. This change is most likely due to an introduction of
positive supercoils, which compensate for the negative supercoils. This
could be clearly demonstrated by studying the topology of protein-free
DNA by two-dimensional gel electrophoresis. We found that the linking
number of protein-free DNA centers around 0, with a few positive and a
few negative supercoils resulting from thermal fluctuation. After
incubation with DEK protein, there was a significant increase in
positive supercoiling. By counting the number of topoisomers we
determined that an average of one positive supercoil is introduced per
six to eight DEK molecules. We conclude that the DEK-induced change in
DNA topology both in protein-free DNA and in chromatin is caused by the
introduction of constrained positive supercoils.
View larger version (40K):
[in a new window]
Fig. 5.
Sedimentation behavior of DNA and chromatin
in the presence of increasing amounts of DEK. 1.5 µg of SV40 DNA
(A) or SV40 minichromosomes (B) was incubated in
the presence of increasing amounts of the DEK protein and topoisomerase
I. Nucleoprotein complexes were separated on glycerol gradients.
Proteins were extracted from individual fractions and analyzed on 10%
SDS-PAGE followed by Western blot analysis. The DNA was purified and
analyzed on a 0.8% agarose gel. Control gradients were run in the
absence of DEK (DEK/DNA ratio = 0) or in the absence of a DNA
template (free DEK). The molar ratios of DEK to DNA are 15, 45, 75, and
90. The positions of sedimentation markers (S), molecular
mass markers (kilodaltons), and DNA forms I and II are indicated.
View larger version (78K):
[in a new window]
Fig. 6.
Electron microscopic analysis. SV40 DNA
(A) and SV40 minichromosomes (C) were incubated
in the absence (A and C, panel
a) or presence of increasing amounts of DEK and visualized by
electron microscopy (compare DEK/DNA ratios with Fig. 5).
Bar, 100 nm. Note that the graph in C,
panel f, represents a different magnification to visualize
compacted chromatin together with protein-free DNA as spreading
control. B, subdivision of the types of DNA molecules
observed at a given DEK/DNA ratio are given as the percentage of the
total number of molecules per ratio (around 100).
View larger version (101K):
[in a new window]
Fig. 7.
Interaction of DEK with linear DNA. A
1000-bp DNA fragment was incubated in the absence ( ) or presence (+)
of DEK (molar ratio: 27 pmol of DEK/mol of DNA) and topoisomerase I and
visualized by electron microscopy. The length of molecules
(given in scan units) without DEK was measured (= 153.03 ± 7.81 scan units; 29 molecules) and compared with DNA molecules uniformly
covered by DEK (= 169.68 ± 31.52 scan units; 41 molecules)
(upper panel). At a molar ratio of 54 mol of
DEK/mol of DNA a heterogeneous population of molecules was observed due
to intra- and intermolecular interactions induced by the DEK protein
(middle panel). The 1000-bp fragment was
reconstituted into chromatin by salt gradient dialysis, and the length
of the resulting DNA fragments was measured (= 100.84 ± 20.9 scan
units; 34 molecules) (lower panel). Bar, 100 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Rolf Knippers, Vassilios Alexiadis, Eric Carstens, Ferdinand Kappes, and Ingo Scholten for stimulating discussions and critical reading of the manuscript. The GST-DEK expression vector and the DEK antibodies were a kind gift from Gerald Grosveld. Human topoisomerase I was kindly provided by Fritz Boege, and the expression vector for E. coli topoisomerase I was kindly provided by James Wang.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (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.
To whom correspondence should be addressed. Tel.: 49-7531-882-125;
Fax: 49-7531-884-036; E-mail: Claudia.Gruss@uni-konstanz.de.
Published, JBC Papers in Press, May 7, 2002, DOI 10.1074/jbc.M204045200
2 T. Waldmann, manuscript in preparation.
3 I. Scholten, manuscript in preparation.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GST, glutathione S-transferase; SMC, structural maintenance of chromosomes.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Woodcock, C. L., and Dimitrov, S. (2001) Curr. Opin. Genet. Dev. 11, 130-135[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Armstrong, J. A., and Emerson, B. M. (1998) Curr. Opin. Genet. Dev. 8, 165-172[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Flaus, A., and Owen-Hughes, T. (2001) Curr. Opin. Genet. Dev. 11, 148-154[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Peterson, C. L., and Logie, C. (2000) J. Cell. Biochem. 78, 179-185[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Pollard, K. J., and Peterson, C. L. (1998) Bioessays 20, 771-780[CrossRef][Medline] [Order article via Infotrieve] |
| 6. |
Alexiadis, V.,
Waldmann, T.,
Andersen, J.,
Mann, M.,
Knippers, R.,
and Gruss, C.
(2000)
Genes Dev.
14,
1308-1312 |
| 7. |
von Lindern, M.,
Fornerod, M.,
van Baal, S.,
Jaegle, M.,
de Wit, T.,
Buijs, A.,
and Grosveld, G.
(1992)
Mol. Cell. Biol.
12,
1687-1697 |
| 8. | Sierakowska, H., Williams, K. R., Szer, I. S., and Szer, W. (1993) Clin. Exp. Immunol. 94, 435-439[Medline] [Order article via Infotrieve] |
| 9. | Wichmann, I., Respaldiza, N., Garcia-Lozano, J. R., Montes, M., Sanchez-Roman, J., and Nunez-Roldan, A. (2000) Clin. Exp. Immunol. 119, 530-532[CrossRef][Medline] [Order article via Infotrieve] |
| 10. |
McGarvey, T.,
Rosonina, E.,
McCracken, S., Li, Q.,
Arnaout, R.,
Mientjes, E.,
Nickerson, J. A.,
Awrey, D.,
Greenblatt, J.,
Grosveld, G.,
and Blencowe, B. J.
(2000)
J. Cell Biol.
150,
309-320 |
| 11. | Le Hir, H., Izaurralde, E., Maquat, L. E., and Moore, M. J. (2000) EMBO J. 19, 6860-6869[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Faulkner, N. E.,
Hilfinger, J. M.,
and Markovitz, D. M.
(2001)
J. Biol. Chem.
276,
25804-25812 |
| 13. |
Fu, G. K.,
and Markovitz, D. M.
(1996)
J. Biol. Chem.
271,
19599-19605 |
| 14. |
Fu, G. K.,
Grosveld, G.,
and Markovitz, D. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1811-1815 |
| 15. | Fornerod, M., Boer, J., van Baal, S., Jaegle, M., von Lindern, M., Murti, K. G., Davis, D., Bonten, J., Buijs, A., and Grosveld, G. (1995) Oncogene 10, 1739-1748[Medline] [Order article via Infotrieve] |
| 16. | Kappes, F., Burger, K., Baack, M., Fackelmayer, F. O., and Gruss, C. (2001) J. Biol. Chem. 276, 26217-26323 |
| 17. | Gruss, C., and Knippers, R. (1995) Methods Mol. Genet. 7, 101-113 |
| 18. | Wessel, D., and Flügge, U. I. (1984) Anal. Biochem. 138, 141-143[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Germond, J. E.,
Hirt, B. E.,
Oudet, P.,
Gross-Bellard, M.,
and Chambon, P.
(1975)
Proc. Natl. Acad. Sci. U. S. A.
72,
1843-1847 |
| 20. |
Vollenweider, H. J.,
Sogo, J.,
and Koller, T.
(1975)
Proc. Natl. Acad. Sci. U. S. A.
72,
83-87 |
| 21. | Lynn, R. M., and Wang, J. C. (1989) Proteins 6, 231-239[CrossRef][Medline] [Order article via Infotrieve] |
| 22. | Champoux, J. J. (2001) Annu. Rev. Biochem. 70, 369-413[CrossRef][Medline] [Order article via Infotrieve] |
| 23. | Sogo, J. M., Stahl, H., Koller, T., and Knippers, R. (1986) J. Mol. Biol. 189, 189-204[CrossRef][Medline] [Order article via Infotrieve] |
| 24. | Dröge, P. (1994) Bioessays 16, 91-99[CrossRef][Medline] [Order article via Infotrieve] |
| 25. | Kanaar, R., and Cozzarelli, N. R. (1992) Curr. Opin. Struct. Biol. 2, 369-379[CrossRef] |
| 26. |
Liu, L. F.,
and Wang, J. C.
(1978)
Proc. Natl. Acad. Sci. U. S. A.
75,
2098-2102 |
| 27. |
Musgrave, D. R.,
Sandman, K. M.,
and Reeve, J. N.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
10397-10401 |
| 28. | Van Komen, S., Petukhova, G., Sigurdsson, S., Stratton, S., and Sung, P. (2000) Mol. Cell 6, 563-572[CrossRef][Medline] [Order article via Infotrieve] |
| 29. |
Napoli, A.,
Kvaratskelia, M.,
White, M. F.,
Rossi, M.,
and Ciaramella, M.
(2001)
J. Biol. Chem.
276,
10745-10752 |
| 30. | Kimura, K., and Hirano, T. (1997) Cell 90, 625-634[CrossRef][Medline] [Order article via Infotrieve] |
| 31. | Strunnikov, A. V., and Jessberger, R. (1999) Eur. J. Biochem. 263, 6-13[Medline] [Order article via Infotrieve] |
| 32. |
Hirano, T.
(1999)
Genes Dev.
13,
11-19 |
| 33. |
Akhmedov, A. T.,
Frei, C.,
Tsai-Pflugfelder, M.,
Kemper, B.,
Gasser, S. M.,
and Jessberger, R.
(1998)
J. Biol. Chem.
273,
24088-24094 |
| 34. |
Zlatanova, J.,
and van Holde, K.
(1998)
FASEB J.
12,
421-431 |
| 35. | Lever, M. A., Th'ng, J. P., Sun, X., and Hendzel, M. J. (2000) Nature 408, 873-876[CrossRef][Medline] [Order article via Infotrieve] |
| 36. | Misteli, T., Gunjan, A., Hock, R., Bustin, M., and Brown, D. T. (2000) Nature 408, 877-881[CrossRef][Medline] [Order article via Infotrieve] |
| 37. | Phair, R. D., and Misteli, T. (2000) Nature 404, 604-609[CrossRef][Medline] [Order article via Infotrieve] |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  F. Kappes, J. Fahrer, M. S. Khodadoust, A. Tabbert, C. Strasser, N. Mor-Vaknin, M. Moreno-Villanueva, A. Burkle, D. M. Markovitz, and E. Ferrando-May DEK Is a Poly(ADP-Ribose) Acceptor in Apoptosis and Mediates Resistance to Genotoxic Stress Mol. Cell. Biol., May 15, 2008; 28(10): 3245 - 3257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Devany, F. Kappes, K.-M. Chen, D. M. Markovitz, and H. Matsuo Solution NMR structure of the N-terminal domain of the human DEK protein Protein Sci., February 1, 2008; 17(2): 205 - 215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mor-Vaknin, A. Punturieri, K. Sitwala, N. Faulkner, M. Legendre, M. S. Khodadoust, F. Kappes, J. H. Ruth, A. Koch, D. Glass, et al. The DEK Nuclear Autoantigen Is a Secreted Chemotactic Factor Mol. Cell. Biol., December 15, 2006; 26(24): 9484 - 9496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Wise-Draper, H. V. Allen, E. E. Jones, K. B. Habash, H. Matsuo, and S. I. Wells Apoptosis Inhibition by the Human DEK Oncoprotein Involves Interference with p53 Functions Mol. Cell. Biol., October 15, 2006; 26(20): 7506 - 7519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Wise-Draper, H. V. Allen, M. N. Thobe, E. E. Jones, K. B. Habash, K. Munger, and S. I. Wells The Human DEK Proto-Oncogene Is a Senescence Inhibitor and an Upregulated Target of High-Risk Human Papillomavirus E7 J. Virol., November 15, 2005; 79(22): 14309 - 14317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Cleary, K. V. Sitwala, M. S. Khodadoust, R. P. S. Kwok, N. Mor-Vaknin, M. Cebrat, P. A. Cole, and D. M. Markovitz p300/CBP-associated Factor Drives DEK into Interchromatin Granule Clusters J. Biol. Chem., September 9, 2005; 280(36): 31760 - 31767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Böhm, F. Kappes, I. Scholten, N. Richter, H. Matsuo, R. Knippers, and T. Waldmann The SAF-box domain of chromatin protein DEK Nucleic Acids Res., February 18, 2005; 33(3): 1101 - 1110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Kappes, I. Scholten, N. Richter, C. Gruss, and T. Waldmann Functional Domains of the Ubiquitous Chromatin Protein DEK Mol. Cell. Biol., July 1, 2004; 24(13): 6000 - 6010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Kappes, C. Damoc, R. Knippers, M. Przybylski, L. A. Pinna, and C. Gruss Phosphorylation by Protein Kinase CK2 Changes the DNA Binding Properties of the Human Chromatin Protein DEK Mol. Cell. Biol., July 1, 2004; 24(13): 6011 - 6020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Waldmann, M. Baack, N. Richter, and C. Gruss Structure-specific binding of the proto-oncogene protein DEK to DNA Nucleic Acids Res., December 1, 2003; 31(23): 7003 - 7010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. McCracken, D. Longman, I. L. Johnstone, J. F. Caceres, and B. J. Blencowe An Evolutionarily Conserved Role for SRm160 in 3'-End Processing That Functions Independently of Exon Junction Complex Formation J. Biol. Chem., November 7, 2003; 278(45): 44153 - 44160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hoek and B. Stillman Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo PNAS, October 14, 2003; 100(21): 12183 - 12188. [Abstract] [Full Text] [PDF]
|
|
| ||
