Human Ku70 Interacts with Heterochromatin Protein 1alpha *

Kyuyoung SongDagger , Yusun Jung§, Donghae Jung§, and Inchul Lee§||

From the Departments of Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 1alpha (HP1alpha ), 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 HP1alpha . 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 HP1alpha directly. The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively. Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha Delta C. In contrast to HP1alpha , 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

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 HP1alpha ,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

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 HP1alpha /E11, three truncated versions of HP1alpha were cloned into the prey vector pJG4-5: HP1alpha /N (containing aa 1-58), HP1alpha /M (57), and HP1alpha /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.

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- 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 beta -D-galactopyranoside to test the beta -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.

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, Ku70Delta 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 Ku70Delta 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).

Production of Recombinant HP1alpha Proteins in E. coli-- In-frame fusions of full-length HP1alpha (aa 1-191), HP1alpha Delta N (aa 111-191), and HP1alpha Delta 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 HP1alpha , 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. HP1alpha Delta N and HP1alpha Delta 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 Pull-down Assay-- GST-HP1alpha , GST-HP1alpha Delta N, and GST-HP1alpha Delta 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-HP1alpha and comparable amounts of other GST fusion proteins, equimolar purified His-tagged Ku70Delta 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.

Coimmunoprecipitation of HP1alpha 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-HP1alpha antibody. The affinity-purified anti-HP1alpha antibody was obtained from antisera of a rabbit, which was immunized using the purified HP1alpha -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).

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 HP1alpha and HP1alpha Delta 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.

At 48 h after transfection, coverslips were rinsed with PBS and fixed in cold methanol (-20 °C) for 10 min. Then primary antibodies were incubated with cells for 2 h at room temperature. Rabbit polyclonal anti-HP1alpha and monoclonal anti-Ku70 antibodies were described above. Mouse polyclonal anti-HP1alpha 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

Transfections and Reporter Assay-- pM1 and pM1-HP1alpha 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/Ku70Delta 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).

One µg of plasmids was transfected to HeLa cells, 3 × 105/30-mm dish, with proper combinations of pM1-HP1alpha , 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 beta -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

Identification of HP1alpha 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.

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-, 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.

DNA sequencing and data base searches revealed that the nucleotide sequence of nine clones encoded human HP1alpha (32). Although 8 clones encoded the full-length HP1alpha , one contained a 5'-truncated cDNA encoding amino acids 32-191 of HP1alpha . Thus, the binding site to Ku70 did not appear to involve the N terminus.

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, HP1alpha did not interact with any one of the control baits, indicating that the association of Ku70/HP1alpha was not mediated by nonspecific interaction of the leucine zipper-like alpha -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 HP1alpha are shown at the top of the PREY section. C11 was one of the prey clones containing full-length HP1alpha . The full-length HP1alpha and HP1alpha /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.

Interaction Sites of Ku70 and HP1alpha -- To map the interaction domains of the Ku70 with HP1alpha , 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 HP1alpha (Fig. 1). Therefore, the Leu-Ser repeat appeared to be essential for the interaction with HP1alpha . The results were confirmed by reciprocal yeast-two hybrid experiments.

To delineate the Ku70-interacting domain of HP1alpha , three deletion derivatives of HP1alpha were generated: HP1alpha /N (containing aa 1-58), HP1alpha /H (aa 57-137), and HP1alpha /C (aa 111-191), representing the chromo domain (43), "hinge" region (44), and chromo shadow domain (45) of HP1alpha , respectively. Mutant forms of HP1alpha were tested for their ability to bind to the LexA-Ku70/186 bait by yeast two-hybrid assay. As shown in Fig. 1, HP1alpha /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 HP1alpha .

GST Pull-down Analysis-- To validate the results obtained with the yeast two-hybrid system, the interaction between recombinant Ku70 and HP1alpha proteins was studied in vitro. For the GST pull-down experiments, GST-HP1alpha , GST-HP1alpha Delta N, and GST-HP1alpha Delta C fusion proteins were expressed in E. coli and purified on a glutathione-Sepharose 4B column. As a binding partner, recombinant His-tagged Ku70Delta N was expressed and purified on His binding resin (see "Experimental Procedures"). To increase the solubility of recombinant His-tagged Ku70Delta 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, Ku70Delta N was pulled down by the interaction with GST-HP1alpha and GST-HP1alpha Delta N fusion proteins, whereas no interaction was noted with GST-HP1alpha Delta C or GST alone. The result coincided with the yeast two-hybrid data indicating that the binding site of HP1alpha to Ku70 was at the C-terminal part.



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Fig. 2.   GST pull-down experiment using recombinant Ku70 and HP1alpha . A, ten µg of purified His-tagged recombinant Ku70Delta N was incubated with equimolar GST-HP1alpha , GST-HP1alpha Delta N, or GST-HP1alpha Delta 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 Ku70Delta N. Then the mixtures were dialyzed overnight at 4 °C in the reaction buffer without urea. Recombinant HP1alpha 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 Ku70Delta N was coprecipitated with HP1alpha and HP1alpha Delta N but not with HP1alpha Delta C or GST alone. This result confirmed that the binding site was in the C-terminal region of HP1alpha , 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 Ku70Delta N was incubated with 5 µg of purified GST-HP1alpha in every experiment. By immunoblotting using anti-T7.tag antibody, Ku70Delta N was shown to be most effectively pulled down at pH 6.2 and 6.5 through the interaction with GST-HP1alpha . The pulled down Ku70Delta N decreased gradually as the pH was elevated until it was detected barely at pH 7.5. MW, molecular weight. WB, Western blot.

Enhanced Interaction at Acidic pH-- HP1alpha 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 Ku70Delta N and GST-HP1alpha , Ku70Delta N was most efficiently pulled down by GST-HP1alpha at pH 6.2, and then the interaction weakened gradually as the pH was elevated to 7.5 (Fig. 2B).

In Vivo Interaction between Ku70 and HP1alpha -- The protein-protein interaction between Ku70 and HP1alpha in vivo was confirmed by immunoprecipitation using anti-HP1alpha antibody. When HP1alpha 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 HP1alpha . The HeLa cell extract was incubated with affinity-purified anti-HP1alpha 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 HP1alpha (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.

Ku70 Colocalized with HP1alpha but Not with HP1alpha Delta C-- To confirm the interaction between Ku70 and HP1alpha in vivo, we examined the subcellular localization of wild-type HP1alpha and its deletion derivative, HP1alpha Delta C, after transfection into COS cells. On immunostaining using anti-T7.Tag antibody, ~5% of cells displayed the expression of transfected HP1alpha as a punctated pattern throughout the nuclei (Fig. 4A). No cytoplasmic staining was present. Rabbit and mouse anti-HP1alpha 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 HP1alpha 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/HP1alpha or pcDNA3/HP1alpha Delta 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 HP1alpha 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 HP1alpha largely colocalized except for a few spots (C). Transfected HP1alpha Delta C was detected in <1% of cells. HP1alpha Delta C was mostly detected as nodular aggregates (D), which tended to be much larger than the spots of full-length HP1alpha (A). By double immunostaining, HP1alpha Delta C and Ku70 were present in a mutually exclusive manner (E and F). Slides were examined using a Zeiss LSM510 confocal microscope.

Transfected HP1alpha Delta 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, HP1alpha Delta C was mostly detected as irregular nodules, which tended to be much larger than the spots of wild-type HP1alpha (Fig. 4D). By double immunofluorescence staining, HP1alpha Delta 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 HP1alpha to Ku70 was at the C-terminal region.

Analysis of Transcriptional Repressor Activity-- It has been shown that HP1alpha 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 HP1alpha -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 Ku70Delta 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).



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Fig. 5.   Transcriptional regulator activity of HP1alpha 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/HP1alpha , pM1/Ku70, or pM1/Ku70Delta 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 beta -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

Interaction of Ku70 and HP1alpha -- 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 alpha  and -beta (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 HP1alpha . Ku and HP1alpha may perform diverse biological functions depending on which binding partner they interact with in the given nuclear microenvironment. Because Ku80 did not interact with HP1alpha , it is suggested that subunits of Ku might function independently from each other depending on binding partners.

Chromo Shadow Domain of HP1alpha as Interaction Site-- HP1alpha 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 HP1alpha , respectively. Except for the two, all others interact with HP1alpha through the chromo shadow domain. Our data show that Ku70 also binds to the chromo shadow domain of HP1alpha . 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 HP1alpha .

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 HP1alpha 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 HP1alpha . Recently, a pentamer sequence, PXVXL, has been proposed to be a consensus sequence of the HP1alpha 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.

Transfection and Reporter Assay-- In our transfection system, HP1alpha 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 HP1alpha 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.

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 HP1alpha 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 HP1alpha 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), HP1alpha 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.

HP1alpha 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.

The specific interaction of HP1alpha with Ku70 suggests that HP1alpha is a mammalian counterpart of Sir4, which has been sought for. If HP1alpha 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 HP1alpha , 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.


    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: HP1alpha , heterochromatin protein 1alpha ; 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Mimori, T., Akizuki, M., Yamagata, H., Inaba, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620[Medline] [Order article via Infotrieve]
2. Mimori, T., and Hardin, J. A. (1986) J. Biol. Chem. 261, 10375-10379[Abstract/Free Full Text]
3. Rathmell, W. K., and Chu, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7623-7627[Abstract]
4. Finnie, N. J., Gottlieb, T. M., Blunt, T., Jeggo, P. A., and Jackson, S. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 320-324[Abstract]
5. Peterson, S. R., Kurimasa, A., Oshimura, M., Dynan, W. S., Bradbury, E. M., and Chen, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3171-3174[Abstract]
6. Gu, Y., Jin, S., Gao, Y., Weaver, D. T., and Alt, F. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8076-8081[Abstract/Free Full Text]
7. Nussenzweig, A. K., Sokol, P., Burgman, L., and Li, G. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13588-13593[Abstract/Free Full Text]
8. Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestley, A., Demengeot, J., Mizuta, R., Lehmann, A. R., Alt, F. W., Jackson, S. P., and Jeggo, P. A. (1994) Science 265, 1442-1445[Medline] [Order article via Infotrieve]
9. Smider, V., Rathmell, W. K., Lieber, M. R., and Chu, G. (1994) Science 266, 288-292[Medline] [Order article via Infotrieve]
10. Lees-Miller, S. P., Godbout, R., Chan, D. W., Weinfeld, M., Day, R. S., III, Barron, G. M., and Allaunits-Turner, J. (1995) Science 267, 1183-1185[Medline] [Order article via Infotrieve]
11. Boulton, S. J., and Jackson, S. P. (1996) Nucleic Acids Res. 24, 4639-4648[Abstract/Free Full Text]
12. Porter, S. E., Greenwell, P. W., Ritchie, K. B., and Petes, T. D. (1996) Nucleic Acids Res. 24, 582-585[Abstract/Free Full Text]
13. Laroche, T., Martin, S. G., Gotta, M., Gorham, H. C., Pryde, F. E., Louis, E. J., and Gasser, S. M. (1988) Curr. Biol. 8, 653-656
14. Polotnianka, R. M., Li, J., and Lustig, A. J. (1998) Curr. Biol. 8, 831-834[Medline] [Order article via Infotrieve]
15. Gravel, S., Larrivee, M., Labrecque, P., and Wellinger, R. J. (1998) Science 280, 741-744[Abstract/Free Full Text]
16. Bianchi, A., and de Lange, T. (1999) J. Biol. Chem. 274, 21223-21227[Abstract/Free Full Text]
17. Hsu, H. L., Gilley, D., Blackburn, E. H., and Chen, D. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12454-12458[Abstract/Free Full Text]
18. Anderson, C. W., and Carter, T. H. (1996) in Molecular Analysis of DNA Rearrangements in the Immune System (Jessberger, R. , and Lieber, M. R., eds) , pp. 91-112, Springer-Verlag, Heidelberg
19. Yu, E., Song, K., Moon, H., Maul, G. G., and Lee, I. (1998) Hybridoma 17, 413-419[Medline] [Order article via Infotrieve]
20. Romero, F., Dargemont, C., Pozo, F., Reeves, W. H., Camonis, J., Gisselbrecht, S., and Fischer, S. (1996) Mol. Cell. Biol. 16, 37-44[Abstract]
21. Barlev, N. A., Poltoratsky, V., Owen-Hughes, T., Ying, C., Liu, L., Workman, J. L., and Berger, S. L. (1998) Mol. Cell. Biol. 18, 1349-1358[Abstract/Free Full Text]
22. Yang, C.-R., Yeh, S., Leskov, K., Odegaard, E., Hsu, H.-L., Chang, C., Kinsella, T. J., Chen, D. J., and Boothman, D. A. (1999) Nucleic Acids Res. 27, 2165-2174[Abstract/Free Full Text]
23. Wu, X., and Lieber, M. R. (1996) Mol. Cell. Biol. 16, 5186-5192[Abstract]
24. Osipovich, O., Durum, S. K., and Muegge, K. (1997) J. Biol. Chem. 272, 27259-27265[Abstract/Free Full Text]
25. Wang, J., Dong, X., Myung, K., Hendrickson, E. A., and Reeves, W. H. (1998) J. Biol. Chem. 273, 842-848[Abstract/Free Full Text]
26. Song, K., Jung, D., Jung, Y., Lee, S. G., and Lee, I. (2000) FEBS Lett. 481, 81-85[CrossRef][Medline] [Order article via Infotrieve]
27. Lorentz, A., Ostermann, K., Fleck, O., and Schmidt, H. (1994) Gene (Amst.) 243, 139-143[CrossRef]
28. Singh, P. B., Miller, J. R., Pearce, J., Kotharty, R., Burton, R. D., Paro, R., James, T. C., and Gaunt, S. J. (1991) Nucleic Acids Res. 19, 789-794[Abstract]
29. Saunders, W. S., Chue, C., Goebl, M., Craig, C., Clark, R. F., Powers, J. A., Eissenberg, J. C., Elgin, S. C. R., Rothfield, N. F., and Earnshaw, W. C. (1993) J. Cell Sci. 104, 573-582[Abstract/Free Full Text]
30. James, T. C., and Elgin, S. C. (1986) Mol. Cell. Biol. 6, 3862-3872[Medline] [Order article via Infotrieve]
31. Eissenberg, J. C., James, T. C., Foster-Harnett, D. M., Harnett, T., Ngan, V., and Elgin, S. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9923-9927[Abstract]
32. Fanti, L., Giovinazzo, G., Berloco, M., and Pimpinelli, S. (1998) Mol. Cell 2, 527-538[Medline] [Order article via Infotrieve]
33. Tsukamoto, Y., Kato, J.-I., and Ikeda, H. (1997) Nature 388, 900-903[CrossRef][Medline] [Order article via Infotrieve]
34. Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cell 75, 791-803[Medline] [Order article via Infotrieve]
35. Reeves, W. H., and Sthoeger, Z. M. (1989) J. Biol. Chem. 264, 5047-5052[Abstract/Free Full Text]
36. Yaneva, M., Wen, J., Ayala, A., and Cook, R. (1989) J. Biol. Chem. 264, 13407-13411[Abstract/Free Full Text]
37. Park, S. H., Lee, S. G., Kim, Y., and Song, K. (1998) Cytogenet. Cell Genet. 81, 178-179[Medline] [Order article via Infotrieve]
38. Rho, S. B., Lee, K. H., Kim, J. W., Shiba, K., Jo, Y. J., and Kim, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10128-10133[Abstract/Free Full Text]
39. Sadowski, I., Bell, B., Broad, P., and Hollis, M. (1992) Gene (Amst.) 118, 137-141[CrossRef][Medline] [Order article via Infotrieve]
40. Seeler, J. S., Marchio, A., Sitterlin, D., Transy, C., and Dejean, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7316-7321[Abstract/Free Full Text]
41. Lehming, N., Le, S. A., Schueller, J., and Ptashne, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7322-7326[Abstract/Free Full Text]
42. Landschultz, W. H., Johnson, P. F., and McKnight, S. L. (1988) Science 240, 1759-1764[Medline] [Order article via Infotrieve]
43. Paro, R., and Hogness, D. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 263-267[Abstract]
44. Ainsztein, A. M., Kandels-Lewis, S. E., Mackay, A. M., and Earnshaw, W. C. (1998) J. Cell Biol. 143, 1763-1774[Abstract/Free Full Text]
45. Aasland, R., and Stewart, A. F. (1995) Nucleic Acids Res. 23, 3168-3173[Abstract]
46. Cleard, F., Delattre, M., and Spierer, P. (1997) EMBO J. 16, 5280-5288[Abstract/Free Full Text]
47. Aagaard, L., Laible, G., Selenko, P., Schmid, M., Dom, R., Schotta, G., Kuhfittig, S., Wolf, A., Lebersorger, A., Singh, P. B., Reuter, G., and Jenuwein, T. (1999) EMBO J. 18, 1923-1938[Abstract/Free Full Text]
48. Le Dourain, B., Nielsen, A. L., Garnier, J. M., Ichinose, H., Jeanmougin, F., Losson, R., and Chambon, P. (1996) EMBO J. 15, 6701-6715[Abstract]
49. Nielson, A. L., Ortiz, J. A., You, J., Oulad-Abdelghani, M., Khechumian, R., Gansmuller, A., Chambon, P., and Losson, R. (1996) EMBO J. 18, 6385-6395[Abstract/Free Full Text]
50. Ye, Q., and Worman, H. J. (1996) J. Biol. Chem. 271, 14653-14656[Abstract/Free Full Text]
51. Murzina, N., Verreault, A., Laue, E., and Stillman, B. (1999) Mol. Cell 4, 529-540[Medline] [Order article via Infotrieve]
52. Pak, D. T. S., Pflumm, M., Chesnokov, I., Huang, D. W., Kellum, R., Marr, J., Romanowski, P., and Botschan, M. R. (1997) Mol. Cell. Biol. 91, 311-323
53. McDowell, T. S., Gibbons, R. J., Sutherland, H., O'Rourke, D. M., Bickmore, W. A., Pombo, A., Turley, H., Gatter, K., Picketts, D. J., Buckle, V. J., Chapman, L., Rhodes, D., and Higgs, D. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13983-13988[Abstract/Free Full Text]
54. Smothers, J. F., and Henikoff, S. (2000) Curr. Biol. 10, 27-30[CrossRef][Medline] [Order article via Infotrieve]
55. Lechner, M. S., Begg, G. E., Speicher, D. W., and Rauscher, F. J., III. (2000) Mol. Cell. Biol. 20, 6449-6465[Abstract/Free Full Text]
56. Giffin, W., Torrance, H., Rodda, D. J., Prefontaine, G. G., Pope, L., and Hache, R. J. (1996) Nature 380, 265-268[CrossRef][Medline] [Order article via Infotrieve]
57. Camara-Clayette, V., Thomas, D., Rahuel, C., Barbey, R., Cartron, J.-P., and Bertrand, O. (1999) Nucleic Acids Res. 27, 1656-1663[Abstract/Free Full Text]
58. van Steensel, B., Smogorzewska, A., and de Lange, T. (1998) Cell 92, 401-413[Medline] [Order article via Infotrieve]
59. Shore, D. (1998) Science 281, 1818-1819[Free Full Text]
60. Boulton, S. J., and Jackson, S. P. (1998) EMBO J. 17, 1819-1828[Abstract/Free Full Text]
61. Nugent, C. I., Bosco, G., Ross, L. O., Evans, S. K., Sallinger, A. P., Moore, J. K., Haber, J. E., and Lundblad, V. (1998) Curr. Biol. 8, 657-660[Medline] [Order article via Infotrieve]
62. Kim, S. H., Kaminker, P., and Campisi, J. (1999) Nat. Genet. 23, 405-412[CrossRef][Medline] [Order article via Infotrieve]
63. Li, B., Oestreich, S., and de Lange, T. (2000) Cell 101, 471-483[Medline] [Order article via Infotrieve]
64. Smith, S., Giriat, I., Schmitt, A., and de Lange, T. (1998) Science 282, 1484-1487[Abstract/Free Full Text]


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