From the Departments of Biochemistry and
§ Pathology, University of Ulsan College of Medicine,
Seoul 138-736, Korea
Received for publication, September 26, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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Ku is involved in the metabolism of DNA ends, DNA
repair, and the maintenance of telomeres. It consists of a heterodimer
of 70- and 80-kDa subunits. Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein.
Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1 Ku was originally identified as a major autoantigen in patients
with autoimmune diseases such as scleroderma-polymyositis (1). Ku is a
heterodimer of 70- and 80-kDa subunits (2, 3) and binds to DNA ends,
nicks, or single- to double-strand transition (2, 3). Ku binds to the
DNA-dependent protein kinase catalytic subunit to form a
DNA-protein kinase complex, which has been shown to be crucial for
nonhomologous double-strand break DNA repair and V(D)J recombination
(4-10).
It has been shown that Ku played an important role in the regulation
and maintenance of telomeres as well. In Saccharomyces cerevisiae, mutants of either human Ku70 or Ku80 homologues,
YKU70/HDF1 and YKU80/HDF2, were shown to have abnormally short
telomeres (11, 12) and disrupted subnuclear organization of telomeres (13). Mutations in either Ku subunit led to enhanced instability of
telomeres by increasing their sensitivity to either degradation or
recombination reactions (14). Ku has been shown to be an integral
component of yeast telomeres (15) and to bind human telomeric DNA
in vitro (16). Ku was also shown to be associated with
mammalian telomeres (17).
Ku is a relatively abundant protein and is present much more than the
DNA-dependent protein kinase catalytic subunit in the nuclei (5, 18). Recently, we showed that Ku was present in the nuclear
matrix as well as in the soluble fractions of human nucleus (19). In
the nuclear matrix, Ku70 appeared to be present in a greater amount
than Ku80 (19). Thus, it appeared that Ku70 might bind other proteins
to perform diverse biological functions at least in certain cellular
compartments. Such a possibility prompted us to look for additional
interacting proteins of Ku70.
A number of attempts have been made to identify proteins that interact
with Ku70 using the yeast two-hybrid system. Human Ku70 was identified
to be an interacting protein of GCN5/CBP/TAFII250 acetyltransferase (20), oncoprotein p95vav (21), and
apolipoprotein J (22). However, the search has been limited by the
predominant interaction with Ku80 in the yeast two-hybrid system (23,
24). To avoid such a problem, we screened a HeLa cell library using a
partial Ku70 cDNA bait, which did not overlap significantly with
the proposed binding sites to Ku80 (23-25). Using the system, we have
shown that Ku70 interacts with TRF2, a telomere-binding protein
(26).
Here, we show that Ku70 interacts with
HP1 Plasmid Construction for Yeast Two-hybrid Assay--
All
constructions were done according to standard methods and verified by
sequencing. For the yeast two-hybrid analysis, we used the system
developed by the Brent laboratory (34) that used LexA as the
DNA-binding protein, B42 as a transcriptional activator, and lacZ and
LEU2 as reporters. The full-length human Ku70 and Ku80 cDNA were
kind gifts from Dr. W. H. Reeves (35). A region corresponding to
the amino acid residues 200-385 (186 aa) of Ku70 was fused to the LexA
DNA binding domain (LexA-Ku70/186) and used as bait in the yeast
two-hybrid screening. For control baits, LexA-Ku70/139, LexA-Ku80/185,
LexA-DDX3, and LexA-thyroid receptor were generated. LexA-Ku70/139
contained residues 471-609 (139 aa) of Ku70 (35). LexA-Ku80/185
contained the N-terminal 185 amino acid residues of Ku80 (36).
LexA-DDX3 contained the full-length human DDX3 (37).
LexA-thyroid receptor was described previously (38). DNA fragments were
amplified by polymerase chain reaction and fused to the LexA DNA
binding domain in pEG202. Plasmids were constructed similarly for the
bait-prey interchange experiments.
To further delineate the interaction domains of Ku70, two truncated
versions of the original bait, LexA-Ku70/44 and LexA-Ku70/119, were
constructed. LexA-ku70/44 included amino acid residues 200-243 (44 aa), which represented the Leu-Ser repeat (35). LexA-Ku70/119 included
amino acid residues 267-385 (119 aa), which corresponded to the
flanking anionic domain. To delineate interacting domains of the
selected HP1 Yeast Two-hybrid Screen--
The baits were introduced into
yeast S. cerevisiae strain EGY48 (MATa, his3, trp1,
ura3-52, leu2::pLeu2LexAop6/pSH18-34
(LexAop-lacZ reporter)) by a modified lithium acetate
method. The transformants were selected on the yeast synthetic media
(Ura Production of Recombinant Ku70 Proteins in Escherichia
coli--
Three in-frame fusions of Ku70 were constructed with the
pET28 vector (Novagen) as N-terminal (His)6-tagged proteins
for bacterial expression. In-frame fusion of the LexA-Ku70/186 bait was
constructed by cloning the EcoRI fragment of 575 bp from
pEG202/Ku70/186 into the EcoRI site of pET28a. After
transformation to E. coli strain DE3, the recombinant
protein turned out to be insoluble.
In-frame fusion of full-length Ku70 was constructed by ligating first
the BamHI-HindIII fragment of 1476 bp from
pET28a/Ku70/1-482 and then the HindIII-XhoI
fragment of 468 bp from pET28a/Ku70/176-609, followed by cloning into
the BamHI-XhoI sites of pET28a. When the
recombinant full-length Ku70 was expressed in bacteria, its N terminus
was cleaved. Thus, it was necessary to cleave off the N-terminal part
of Ku70.
To construct a plasmid expressing the N-terminal deleted Ku70 gene
product, Ku70 Production of Recombinant HP1 GST Pull-down Assay--
GST-HP1 Coimmunoprecipitation of HP1 Immunofluorescence Microscopy of Transfected Cells--
COS
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin,
and 100 units/ml streptomycin at 37 °C in 5% CO2. The
HP1
At 48 h after transfection, coverslips were rinsed with PBS and
fixed in cold methanol ( Transfections and Reporter Assay--
pM1 and pM1-HP1
One µg of plasmids was transfected to HeLa cells, 3 × 105/30-mm dish, with proper combinations of pM1-HP1 Identification of HP1
Using the LexA-Ku70/186 bait, a HeLa cell cDNA library was screened
as described under "Experimental Procedures." The bait did not have
any intrinsic activity of transcriptional activation for the reporters.
Approximately 3 × 105 independent transformants were
pooled and respread on the selection media (Ura
DNA sequencing and data base searches revealed that the nucleotide
sequence of nine clones encoded human HP1
To confirm the specificity of the interaction, three additional baits
containing similar leucine zipper-like domains were tested as well;
they included LexA-Ku70/139, LexA-Ku80/185, and LexA-DDX3/260 (Fig.
1). LexA-Ku70/139 encompassed amino acid
residues 471-609 of Ku70, which included the second leucine
zipper-like domain. LexA-Ku80/185 and LexA-DDX3/260 included the
leucine zipper-like domains of Ku80 and the putative RNA helicase DDX3,
respectively. As shown in Fig. 1, HP1 Interaction Sites of Ku70 and HP1
To delineate the Ku70-interacting domain of HP1 GST Pull-down Analysis--
To validate the results obtained with
the yeast two-hybrid system, the interaction between recombinant Ku70
and HP1 Enhanced Interaction at Acidic pH--
HP1 In Vivo Interaction between Ku70 and HP1 Ku70 Colocalized with HP1
Transfected HP1 Analysis of Transcriptional Repressor Activity--
It has been
shown that HP1 Interaction of Ku70 and HP1 Chromo Shadow Domain of HP1
The LexA-Ku70/186 bait contained the Leu-Ser repeat and flanking
anionic domain. Other leucine zipper-like domains of control proteins
and that of Ku70 (aa 483-518) failed to interact with HP1 Transfection and Reporter Assay--
In our transfection system,
HP1 pH Dependence of the Interaction May Represent a Control
Mechanism--
We have reported that Ku interacts with another
telomere-binding protein, TRF2 (26), which has been shown to prevent
end-to-end fusion of telomeres (58). Because Ku, TRF2, and HP1 HP1
The specific interaction of HP1 (HP1
), a protein known to be associated with telomeres as well as heterochromatin. HP1
is a suppressor of the position effect variegation in
Drosophila and acts as a transcriptional suppressor in
mammalian cells. The interaction with Ku70 in the two-hybrid system was
confirmed by a glutathione S-transferase pull-down study
using bacterial recombinant proteins in vitro. The
interaction was also reproduced in vivo in HeLa cells,
where endogenous Ku70 coimmunoprecipitated with HP1
. This
interaction was more effective in acidic pH and weakened considerably
as the pH of the reaction buffer was elevated up to 7.5. Ku80 did not
interact with HP1
directly. The interaction domains of Ku70 and
HP1
included the Leu-Ser repeat (amino acids 200-385) and the
chromo shadow domain, respectively. Ku70 was largely colocalized with
transfected HP1
but not with a C-terminal deletion mutant,
HP1
C. In contrast to HP1
, Ku70 did not
repress transcriptional activity of the reporter gene when tethered to
DNA after transfection to mammalian cells. The implication of this
interaction is discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,1 which is one of the
three mammalian HP1 family proteins. HP1 homologues have been found in
many other organisms from Schizosacchromyces pombe (27) to
mammals (28, 29). HP1 is a nonhistone chromosomal protein suppressor of
position effect variegation in Drosophila (30, 31). It is
associated with the heterochromatin region (30, 31) and telomeres (32),
and prevents telomere fusion (32). It is of interest that the yeast
Ku70 homologue Hdf1 has been shown to interact with a telomere-binding
protein, Sir4, which is also a suppressor of the telomere position
effect, in S. cerevisiae (33).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/E11, three truncated versions of HP1
were cloned
into the prey vector pJG4-5: HP1
/N (containing aa 1-58), HP1
/M
(57), and HP1
/C (111). Deletion mutants were created by using convenient restriction sites and/or by PCR with appropriate primer pair combinations and verified by sequencing. The plasmids were
introduced into EGY48-expressing LexA-Ku70/186 and/or LexA-Ku70/119 hybrid proteins.
and His
) with 2% glucose and used as
a host for transformation with HeLa cDNA library as described
previously (38). The cDNA fragments were cloned in pJG4-5 using
EcoRI and XhoI to generate B42 fusion proteins,
and the expression of the fusion protein was designed to be induced by
the presence of galactose. The cDNA was introduced into the
competent yeast cells, and transformants were selected for tryptophan
prototrophy (plasmid marker) on the synthetic medium (Ura
, His
, and Trp
)
containing 2% glucose. All transformants were pooled and respread on
the synthetic medium (Ura
, His
,
Trp
, and Leu
) containing 2% galactose to
induce the introduced cDNA. Cells growing on the selection media
were retested on the synthetic medium (Ura
,
His
, Trp
, and Leu
) containing
2% galactose (inducing condition) and 2% glucose (noninducing
condition) to confirm the dependence of their growth on the presence of
galactose. Cells growing only on the galactose media were selected and
streaked on the synthetic medium (Ura
, His
,
and Trp
) containing 2% galactose or 2% glucose with
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside to test
the
-galactosidase activity. The cells expressing both reporter
genes only in the presence of galactose were finally chosen to isolate
the plasmids. After identification of the interaction between Ku70 and
library members, the reciprocal experiments between the bait and prey
vectors were carried out.
N, pET28a/Ku70/200-609, a
fragment of 1234 bp encompassing amino acids 200-609, was obtained by
PCR using a sense primer (5'-CCTTGACTTGATGCACCTGA-3') and an antisense
primer (5'-TCAGTCCTGGAAGTGCTTGG-3'). The PCR product was first cloned
into a vector, pCR2.1 (Invitrogen), and then a 1250-bp EcoRI
fragment from pCR2.1-Ku70/410 was cloned into the EcoRI site
of pET28a. The Ku70
N fusion protein, which
had amino acid residues 200-609 and 38 additional amino acids, was
partly soluble. The fusion proteins were purified using a His-bind
resin column according to the manufacturer's instructions (Novagen).
Proteins in E. coli--
In-frame fusions of full-length HP1
(aa 1-191),
HP1
N (aa 111-191), and
HP1
C (aa 1-137) were similarly cloned in
pET28 as well as pGEX-4T-1 vectors (Amersham Pharmacia Biotech) for GST
tagging. To construct a plasmid expressing intact HP1
, a full-length
cDNA was generated by EcoRI and XhoI
liberation from the library prey vector E11, followed by cloning into
the EcoRI-XhoI sites of pET28 as well as
pGEX-4T-1 vectors. HP1
N and
HP1
C were constructed by a similar
procedure, but the inserts were obtained by PCR with a full-length
cDNA clone, E11, using appropriate primer combinations. The PCR
products were digested with EcoRI and XhoI
followed by cloning into pET28 and pGEX-4T-1 vector that had been
predigested with the same enzymes.
,
GST-HP1
N, and
GST-HP1
C fusion proteins were expressed
in E. coli and purified on a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). For a negative control, GST without fusion was expressed and purified similarly. GST fusion proteins were
eluted from the beads using the elution buffer (10 mM
glutathione, 50 mM Tris, pH 8.0) according to the
manufacturer's instructions. To 10 µg of GST-HP1
and comparable
amounts of other GST fusion proteins, equimolar purified His-tagged
Ku70
N was added in the reaction buffer (10 mM 1,4-piperazinediethanesulfonic acid, pH 6.5, 0.5%
Nonidet P-40, 83 mM KCl, 17 mM NaCl, 1 mM MgCl2, 0.5 mM
phenylmethylsulfonyl fluoride, 5 mM EDTA, 1 mM
dithiothreitol) containing 3 M urea. After 1 h
incubation, the mixtures were dialyzed overnight at 4 °C in the
reaction buffer without urea. Then glutathione-Sepharose 4B beads were
added and incubated for 4 h. Beads were washed five times in the
reaction buffer and resuspended in Laemmli buffer, and the proteins
were separated on 10% SDS-polyacrylamide gel electrophoresis. Proteins
were blotted onto a nitrocellulose membrane and probed with monoclonal
anti-T7.tag antibody (Novagen), followed by goat anti-mouse second
antibody (Sigma), and visualized using a nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color reaction
(Roche Molecular Biochemicals). To figure out the influence of pH on
the interaction, GST pull-down experiments were repeated in the
reaction buffer at pH 6.2, 6.5, 6.8, 7.2, and 7.5, respectively.
and Ku70 in Vivo--
For
whole-cell extracts, 5 × 105 HeLa cells were washed
with cold phosphate-buffered saline, harvested by scraping, and
centrifuged for 2 min in an Eppendorf microfuge at 4000 × g. The cells were resuspended in 200 µl of reaction buffer
and Dounced on ice thoroughly. Immunoprecipitation was performed with
anti-HP1
antibody. The affinity-purified anti-HP1
antibody was
obtained from antisera of a rabbit, which was immunized using the
purified HP1
-GST fusion protein as described previously (19). For a
negative control, nonimmune rabbit serum was used at the same
concentration. Immune complexes were precipitated with protein
A-Sepharose beads. The beads were washed three times in PBS, resolved
in the SDS-polyacrylamide gel electrophoresis sample buffer, and
electrotransferred to a nitrocellulose membrane. Coimmunoprecipitation
of Ku proteins was analyzed by immunoblotting with monoclonal anti-Ku70
and anti-Ku80 antibodies, respectively (19). Immune complexes were
visualized using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate color reaction and/or the enhanced chemiluminescence method
(ECL detection system; Amersham Pharmacia Biotech).
and HP1
C cDNAs with T7.tag
were cloned into pcDNA3 (Invitrogen), a mammalian expression
plasmid that contains a cytomegalovirus promoter. COS cells were
transfected with 10 µg of plasmid DNA/30-mm dish using lipofectAMINE
reagent (Life Technologies, Inc.) according to the manufacturer's
instructions. Transfected COS cells were grown on coverslips.
20 °C) for 10 min. Then primary antibodies were incubated with cells for 2 h at room temperature. Rabbit polyclonal anti-HP1
and monoclonal anti-Ku70 antibodies were described above. Mouse polyclonal anti-HP1
antibody was a generous gift from Dr. William C. Earnshaw (University of Edinburgh, Edinburgh, United Kingdom). Monoclonal anti-T7.tag antibody (Novagen) and goat
anti-Ku70 antisera (Santa Cruz Biotechnology) were purchased. For
double immunofluorescence microscopy, proper combinations of monoclonal
and polyclonal antibodies were applied simultaneously. After washing
with PBS, Texas red-labeled anti-mouse and fluorescein isothiocyanate-labeled anti-goat secondary antibodies (Sigma) were
applied for 1 h. After washing with PBS three times, DNA staining
was done with 4',6-diamidino-2-phenylindole (1 µg/ml PBS) for 15 s (Sigma). Cells were viewed in a confocal laser microscope (Zeiss
LSM510). As controls, cells were stained with primary or secondary
antibody alone. Control slides did not display significant fluorescence
in any case
effector plasmids and G5-SV40-CAT reporter plasmid were generous gifts
from Dr. Nobert Lehming (Max Delbruck Laboratorium, Cologne, Germany).
The GAL4 (1) fusion was made using pM1, a mammalian expression
vector containing the DNA binding domain of GAL4 under the control of
the early SV40 enhancer (39). pM1/Ku70 was constructed by cloning the
full-length Ku70 obtained by PCR into the BamHI-linearized
and blunted pM1. pM1/Ku70
N (aa 200-609) was
similarly constructed using the PCR primers as described above. The
pG5-SV40-CAT reporter had five GAL4 sites fused to the SV40 promoter
upstream of the CAT reporter gene (40, 41).
,
pM1-Ku70, or pM1 effector plasmid DNA and pG5-SV40-CAT reporter plasmid
using lipofectAMINE reagent (Life Technologies) according to the
manufacturer's instructions. At 48 h after transfection, cell
lysates were analyzed for CAT activity using assay kits from Promega.
CAT reporter activity was normalized for transfection efficiency to an
internal
-galactosidase control and expressed as a percentage of the
activity obtained with the vector alone. Every construct was tested in
four different transfection experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as a Binding Partner of
Ku70--
cDNA corresponding to the amino acid residues 200-385
(186 aa) of Ku70 was fused to the LexA DNA binding domain
(LexA-Ku70/186) and used as bait in the yeast two-hybrid screening.
This region included the periodic repeat of leucine and serine (aa
215-243) in every seventh position and an anionic domain (35). It was chosen as bait because (i) it does not overlap significantly with the
published Ku80 binding sites, and (ii) it contains a domain that is
reminiscent of a leucine zipper (42) and therefore likely to interact
with other molecules.
,
His
, Trp
, and Leu
) containing
2% galactose to induce the expression of cDNAs. In the selection
media, a total of 72 colonies showed galactose dependence. The plasmids
were extracted by yeast miniprep, and the cDNAs were PCR-amplified
with primers derived from the vector pJG4-5, followed by sequence determination.
(32). Although 8 clones
encoded the full-length HP1
, one contained a 5'-truncated cDNA
encoding amino acids 32-191 of HP1
. Thus, the binding site to Ku70
did not appear to involve the N terminus.
did not interact with any one
of the control baits, indicating that the association of Ku70/HP1
was not mediated by nonspecific interaction of the leucine zipper-like
-helix domains
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Fig. 1.
Yeast two-hybrid analysis. Domains of
Ku70 are shown at the top of the BAIT section.
Bars below the lines designate inserts of three
baits used for the yeast two-hybrid analysis. Inserts of the Ku80 and
DDX3 control baits are shown below. Domains of HP1 are shown at the
top of the PREY section. C11 was one of the prey
clones containing full-length HP1
. The full-length HP1
and
HP1
/C (aa 111-191) interacted with the bait Lex-Ku70/186,
confirming that the interaction site was in the chromo shadow domain.
The interactions were confirmed by reciprocal experiments.
CD, chromo domain; CSD, chromo shadow
domain.
--
To map the interaction
domains of the Ku70 with HP1
, we did a deletion analysis of Ku70 in
the yeast two-hybrid system. The truncated LexA-Ku70/44 (aa 200-243),
which corresponded to the Leu-Ser repeat, could not be used because it
showed intrinsic transcriptional activity. The second deletion,
LexA-Ku70/119 (aa 267-385), completely abolished the interaction with
HP1
(Fig. 1). Therefore, the Leu-Ser repeat appeared to be essential
for the interaction with HP1
. The results were confirmed by
reciprocal yeast-two hybrid experiments.
, three deletion
derivatives of HP1
were generated: HP1
/N (containing aa 1-58),
HP1
/H (aa 57-137), and HP1
/C (aa 111-191), representing the
chromo domain (43), "hinge" region (44), and chromo shadow domain
(45) of HP1
, respectively. Mutant forms of HP1
were tested for
their ability to bind to the LexA-Ku70/186 bait by yeast two-hybrid
assay. As shown in Fig. 1, HP1
/C was solely responsible for the
interaction with Ku70. Thus, it appeared that the chromo shadow domain,
which spanned amino acid residues 123-175, was the Ku70 binding site
of HP1
.
proteins was studied in vitro. For the GST
pull-down experiments, GST-HP1
, GST-HP1
N, and
GST-HP1
C fusion proteins were expressed
in E. coli and purified on a glutathione-Sepharose 4B
column. As a binding partner, recombinant His-tagged
Ku70
N was expressed and purified on His
binding resin (see "Experimental Procedures"). To increase the
solubility of recombinant His-tagged Ku70
N,
3 M urea was added to the reaction buffer. After the
incubation, the mixtures were dialyzed in the reaction buffer without
urea at 4 °C overnight and were pulled down by glutathione-Sepharose beads. As shown in Fig. 2A,
Ku70
N was pulled down by the interaction
with GST-HP1
and GST-HP1
N fusion
proteins, whereas no interaction was noted with
GST-HP1
C or GST alone. The result
coincided with the yeast two-hybrid data indicating that the binding
site of HP1
to Ku70 was at the C-terminal part.
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Fig. 2.
GST pull-down experiment using recombinant
Ku70 and HP1 . A, ten µg of
purified His-tagged recombinant Ku70
N was
incubated with equimolar GST-HP1
,
GST-HP1
N, or
GST-HP1
C fusion proteins in the reaction
buffer (10 mM 1,4-piperazinediethanesulfonic acid, pH 6.5, 0.5% Nonidet P-40, 83 mM KCl, 17 mM NaCl, 1 mM MgCl2, 0.5 mM
phenylmethylsulfonyl fluoride, 5 mM EDTA, 1 mM
dithiothreitol) containing 3 M urea to increase the
solubility of Ku70
N. Then the mixtures were
dialyzed overnight at 4 °C in the reaction buffer without urea.
Recombinant HP1
proteins were pulled down by glutathione-Sepharose
4B beads, and the proteins were resolved on 10% SDS-polyacrylamide gel
electrophoresis. The immunoblotting using anti-T7.tag antibody showed
that Ku70
N was coprecipitated with HP1
and HP1
N but not with
HP1
C or GST alone. This result confirmed
that the binding site was in the C-terminal region of HP1
, the
chromo shadow domain. B, pH effect on the interaction
in vitro. GST pull-down experiments were done in the
reaction buffer at various pH: 6.2, 6.5, 6.8, 7.2, and 7.5, respectively. Ten µg of purified Ku70
N was
incubated with 5 µg of purified GST-HP1
in every experiment. By
immunoblotting using anti-T7.tag antibody,
Ku70
N was shown to be most effectively
pulled down at pH 6.2 and 6.5 through the interaction with GST-HP1
.
The pulled down Ku70
N decreased gradually as
the pH was elevated until it was detected barely at pH 7.5. MW, molecular weight. WB, Western blot.
is an acidic protein
the expected isoeletric point of which is ~5.6. To study the
influence of pH on the interaction, we repeated the GST pull-down
experiments at various pH. After the incubation of same amounts of
purified recombinant His-tagged Ku70
N and
GST-HP1
, Ku70
N was most efficiently
pulled down by GST-HP1
at pH 6.2, and then the interaction weakened
gradually as the pH was elevated to 7.5 (Fig. 2B).
--
The
protein-protein interaction between Ku70 and HP1
in vivo
was confirmed by immunoprecipitation using anti-HP1
antibody. When
HP1
was immunoprecipitated from the whole HeLa cell extracts, a
protein of ~70 kDa coprecipitated (Fig.
3A). By immunoblotting, it was
confirmed to be Ku70 (Fig. 3B). As the in vitro
binding study demonstrated, Ku70 was most efficiently coprecipitated at acidic pH. In the same precipitate, Ku80 was not detected by
immunoblotting using an anti-Ku80 monoclonal antibody (data not
shown).
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Fig. 3.
In vivo interaction of Ku70 and
HP1 . The HeLa cell extract was incubated
with affinity-purified anti-HP1
antibody. For a negative control,
preimmune rabbit serum was applied at the same concentration. Immune
complexes were precipitated with protein A-Sepharose beads, and the
proteins were resolved in SDS-polyacrylamide gel electrophoresis. By
Coomassie blue staining, a band of ~70-kDa protein was shown to
coprecipitate with HP1
(A). By immunoblotting using
monoclonal anti-Ku70 antibody, it was confirmed to be Ku70
(B). MW, molecular weight; Ig,
immunoglubulin heavy chain; WB, Western blot.
but Not with
HP1
C--
To confirm the interaction between Ku70
and HP1
in vivo, we examined the subcellular localization
of wild-type HP1
and its deletion derivative,
HP1
C, after transfection into COS cells.
On immunostaining using anti-T7.Tag antibody, ~5% of cells displayed
the expression of transfected HP1
as a punctated pattern throughout
the nuclei (Fig. 4A). No cytoplasmic staining was present. Rabbit and mouse anti-HP1
antisera displayed a similar distribution and immunostaining pattern of positive
cells. By double immunofluorescence microscopy, the immunostaining pattern of Ku70 did not appear to change considerably in those cells
(Ref. 19 and Fig. 4B). Transfected HP1
and native Ku70 largely colocalized with minor exceptions (Fig. 4,
A-C).
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Fig. 4.
Immunofluorescence microscopy of transfected
COS cells. COS cells were transfected with either
pcDNA3/HP1 or pcDNA3/HP1
C
using lipofectAMINE reagent (Life Technologies). After 24 h, cells
were fixed in cold methanol, and then double immunostaining was done
using monoclonal anti-T7.Tag antibody and polyclonal anti-Ku70
antibody. Transfected HP1
was expressed as a punctated pattern in
~5% of cells (A; rhodamine-labeled). Native Ku70
distributed similarly in the cells (B; fluorescein
isothiocyanate-labeled). A merged image showed that Ku70 and
transfected HP1
largely colocalized except for a few spots
(C). Transfected HP1
C was
detected in <1% of cells. HP1
C was
mostly detected as nodular aggregates (D), which tended to
be much larger than the spots of full-length HP1
(A). By
double immunostaining, HP1
C and Ku70 were
present in a mutually exclusive manner (E and F).
Slides were examined using a Zeiss LSM510 confocal microscope.
C was detected in <1% of
total cells. Although the nuclei were immunostained primarily, it was
also detected as small granules in the perinuclear cytoplasm in some
cells. In the nuclei, HP1
C was mostly
detected as irregular nodules, which tended to be much larger than the
spots of wild-type HP1
(Fig. 4D). By double immunofluorescence staining, HP1
C and
Ku70 were present in a mutually exclusive manner (Fig. 4, D-F). The result was consistent with the yeast two-hybrid
and biochemical data indicating that the binding site of HP1
to Ku70 was at the C-terminal region.
had transcriptional repressor activity in mammalian
cells (40, 41). Thus, we decided to determine whether Ku70 had a
transcriptional repressor activity. A fusion construct of Ku70 with the
GAL4-DBD was generated for transfection into COS cells together with a
CAT reporter plasmid bearing five GAL4 binding sites upstream of the
SV40 promoter. As reported previously (40, 41), the expression of
HP1
-GAL4-DBD fusions resulted in a 25-50-fold decrease in the
activity from the SV40 reporter, compared with the control obtained
with the GAL4-DBD alone (Fig. 5). However,
the expression of Ku70-GAL4-DBD or
Ku70
N-GAL4-DBD fusions did not show any
evidence of transcriptional repression but a slight increase of the
activity from the SV40 reporter (Fig. 5).
View larger version (34K):
[in a new window]
Fig. 5.
Transcriptional regulator activity of
HP1 and Ku70 in transfected mammalian
cells. The diagram at the top shows the
reporter plasmid, which has five GAL4 binding sites. The mammalian
expression vector pM1 contains the DNA binding domain of GAL4 under the
control of the early SV40 enhancer. One µg of pM1/HP1
, pM1/Ku70,
or pM1/Ku70
N (aa 200-609) plasmid was
transfected to HeLa cells together with the G5-SV40-CAT reporter
plasmid using lipofectAMINE reagent (Life Technologies). At 48 h
after transfection, the cell lysates were analyzed for CAT activity
(Promega). The CAT reporter activity was normalized for transfection
efficiency to an internal
-galactosidase control and expressed as a
percentage of the activity obtained with the vector alone. Every
construct was tested in four different transfection experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The growing list of
interacting proteins of Ku and HP1 indicates that they are involved in
diverse biological functions. HP1 interacts with a number of proteins
involved in gene silencing: suppressors of variegation 3-7 (46),
suppressors of variegation 3-9 (47), and transcriptional intermediary
factor
and -
(48, 49). It also interacts with other
nuclear proteins, including lamin B receptor (50), inner centromere
protein (44), chromatin assembly factor 1 (51), SP100, a major
component of promyelocytic leukemia protein nuclear bodies (40,
41), origin recognition complexes 1 and 2 (52), and ATR-X
syndrome, a putative helicase and transcriptional regulator
(53). So far, no protein has been shown to interact with both Ku and
HP1
. Ku and HP1
may perform diverse biological functions
depending on which binding partner they interact with in the given
nuclear microenvironment. Because Ku80 did not interact with HP1
, it
is suggested that subunits of Ku might function independently from each
other depending on binding partners.
as Interaction Site--
HP1
has
a chromo domain, chromo shadow domain, and hinge region (43-45). Among
the interacting proteins, origin recognition complex and inner
centromere protein bind to the chromo domain (52) and hinge region (44)
of HP1
, respectively. Except for the two, all others interact with
HP1
through the chromo shadow domain. Our data show that Ku70 also
binds to the chromo shadow domain of HP1
. Taken together, it appears
that the chromo shadow domain is a major site for interaction with
other nuclear proteins, which would be critical for the function of
HP1
.
in our
yeast two-hybrid system, confirming the specificity of the interaction.
Further direct delineation of the interaction site was not possible,
because the Leu-Ser repeat alone showed an intrinsic transcriptional
activity, and the anionic domain alone did not interact with HP1
.
Recently, a pentamer sequence, PXVXL, has been
proposed to be a consensus sequence of the HP1
binding site (54,
55). Intriguingly, a compatible pentamer sequence, PLVLL (aa 352-356),
was included in LexA-Ku70/119, representing the anionic domain. It
is possible that the truncated version of Ku70 in LexA-Ku70/119 is
not in proper configuration for the interaction in the yeast two-hybrid system.
suppressed the transcriptional activity of the reporter as much
as was reported previously (40, 41). However, Ku70 did not show any
transcriptional suppressor activity when tethered to DNA in either
full-length or N terminus-deleted form. Rather, the reporter
transcriptional activity was increased slightly. This result suggests
that certain transcriptional activators compete with HP1
for binding
with Ku70 in the system. Because Ku itself has also been shown to
repress glucocorticoid-induced mouse mammary tumor virus transcription
(56) and glycophorin B transcription mildly (57), the slight but
consistent increase of the reporter transcriptional activity is in
favor of the possibility of a competition.
share
the implicated function of telomere maintenance, it appears that they perform the function in close collaboration with each other through direct interactions. Because TRF2 and HP1
interact with Ku70 through
the same binding area, they may not be able to bind Ku70 simultaneously. In contrast to the interaction between Ku and TRF2,
which works effectively in both acidic and basic environments (26),
HP1
interacts with Ku70 in acidic conditions primarily. Thus, it is
possible that the complex interaction may be controlled in
vivo in such a way that the pH of the microenvironment in the nucleus would play an important role. Further study would be necessary to address the possibility.
as a Mammalian Counterpart of Yeast Sir Protein--
Yeast
genetic studies have shown that Ku is a key player in the replication
and protection of telomeres (11-15). In addition, Mre11, Rad50, Xrs2,
Rap1, Sir2, Sir3, Sir4, Rif1, and Rif2 are also required to
maintain the telomeres normally (for review, see Ref. 59). Mutants of
Rad50, Mre11, and Xrs2, which are known to form a protein complex, all
display a telomere-shortening phenotype similar to that of Ku mutants
(60, 61); however, in contrast to Sir proteins, the mutants are not
defective in telomere silencing (60). Thus, the yeast telomere proteins
may be divided into two groups, namely proteins with or without a
silencing effect.
with Ku70 suggests that HP1
is a
mammalian counterpart of Sir4, which has been sought for. If HP1
represented a telomere protein of the silencing group, other telomere
proteins such as TRF1, TRF2, TRF1-interacting nuclear protein 2 (62), and hRap1 (63) could be regarded as mammalian proteins that
regulate the telomere length without a silencing effect. Another
telomere protein, tankyrase, may regulate other proteins by
ADP-ribosylation (64). Through the direct interaction with TRF2 and
HP1
, Ku appears to play a key role in the maintenance and regulation
of telomeres in mammalian cells as in yeast cells. The role of Ku70 in
heterochromatin formation and maintenance remains to be elucidated.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. W. H. Reeves for providing the Ku70 and Ku80 cDNA clones, Dr. W. C. Earnshaw for anti-HP1 antibody, and Dr. N. Lehming for the vectors for the transcriptional repression study and valuable discussion. We also thank Dr. Jaeseob Kim for the confocal microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by Korea Research Foundation Grant 1998-021-F00130 (to I. L. and K. S.) and by the Asan Foundation.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 Pathology, Ghil Medical Center, Gachon Medical College, 1198 Guwol-Dong, Nam-Gu, Inchon 405-706, Korea.
To whom correspondence should be addressed: Dept. of
Pathology, University of Ulsan College of Medicine, 388-1 Poongnap-Dong, Songpa-Gu, Seoul 138-736, Korea. Tel.: 822-2224-4551;
Fax: 822-472-7898; E-mail: iclee@www.amc.seoul.kr.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M008779200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HP1, heterochromatin protein 1
;
aa, amino acids;
PCR, polymerase chain
reaction;
bp, base pair;
GST, glutathione S-transferase;
PBS, phosphate-buffered saline;
CAT, chloramphenicol acetyltransferase;
DBD, DNA binding domain;
chromo, chromatin modifier organization;
DDX3, DEAD box 3.
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