©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Constitutive Heat Shock Element-binding Factor Is Immunologically Identical to the Ku Autoantigen (*)

Dooha Kim , Honghai Ouyang , Shao-Hua Yang , Andre Nussenzweig , Paul Burgman , Gloria C. Li (§)

From the (1)Departments of Medical Physics and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1. We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column. The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa. Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen. The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen. In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro. Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct. These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo.


INTRODUCTION

In eukaryotic cells, heat shock-induced transcriptional activation of the heat shock genes involves a highly conserved, positive, cis-acting element termed the heat shock element (HSE).()HSE, defined as a repetitive sequence of a 5-nucleotide NGAAN module arranged in an alternating orientation, is present in multiple copies upstream of the transcriptional start site of all heat shock genes. It is well established that HSE is the binding site of heat shock transcription factor (HSF), and that the heat-induced binding of HSF to HSE is a major regulatory step in heat shock gene activation(1, 2) . Extensive studies in Drosophila, yeast, mouse, and human cells have provided strong evidence that protein modification and oligomerization are important steps that convert inactive HSF to an active transcription activator upon stress(3, 4, 5, 6) . In plants and vertebrates, more than a single HSF has been identified. For example, in mouse and human cells, there exist two HSFs, termed HSF1 and HSF2(7, 8) . Utilizing specific antisera, Sarge et al.(6) demonstrated that HSF1 is the mediator of stress-induced heat shock gene transcriptional activation. Under normal growth conditions, mammalian HSF1 is present in a monomeric non-DNA-binding form. Upon heat shock, HSF1 displays stress-induced DNA-binding competence, oligomerization, nuclear localization, and phosphorylation(6) .

Previously, two distinct HSE binding activities have been detected in HeLa cells by the gel mobility shift assay(9, 10) . The electrophoretic migrating patterns of HSE-binding proteins in a nondenaturing gel show that there are two distinct HSE-protein complexes. Unstressed cells contain a constitutive HSE binding activity that appears as a faster migrating complex, while a distinct HSE binding activity (HSF1), evidenced as a slower migrating complex, is present only in the heat-shocked cells. Our recent studies on the response of rodent cells to heat shock, sodium arsenite, or sodium salicylate indicated that a high level of HSF1-HSE binding activity by itself is neither sufficient nor necessary for the induction of hsp70 mRNA transcription(11) . Analysis of the protein factors capable of binding to HSE in extracts of control and heat-shocked rodent cells indicate that, similar to HeLa cells, rodent cells also contain two HSE-binding factors: one constitutively present (termed the constitutive HSE-binding factor, CHBF), and the other heat shock-induced (HSF1)(11) . Upon heat shock, the heat-induced decrease of CHBF-HSE binding activity correlates well with the increase of HSF1-HSE binding activity. During post-heat shock recovery, HSF1-HSE binding activity decreases with time, while CHBF-HSE binding activity recovers gradually. The relationship between CHBF and HSF1 and the significance of the inverse correlation between their levels of HSE binding activity during heat shock and subsequent recovery are currently unknown. However, the tight temporal inverse correlation between HSE-binding ability of HSF1 and HSE-binding ability of CHBF suggests that this correlation may be functionally significant. The molecular and biochemical basis of this observation is also unclear. We have previously suggested that the inverse correlation between the HSE binding activity of HSF1 and CHBF may reflect the involvement of both in the regulation of heat shock gene expression, the former as a positive and the latter as a negative regulator.

In order to study the role of CHBF in the regulation of heat shock response, we have purified and partially characterized this protein. The purified CHBF is composed of two polypeptides of approximately 70 and 86 kDa. Immunoblot and gel mobility shift analysis show that both components of CHBF are recognized by monoclonal antibodies specific to human Ku autoantigen (p70/p80), suggesting that CHBF is identical or closely related to the Ku autoantigen.

Ku protein is an abundant DNA-binding protein composed of a heterodimer of 70 kDa (p70) and 86 kDa (p80) polypeptides(12, 13, 14) , and is well characterized as a transcription factor for tRNA synthesis (15, 16, 17) and as a regulatory subunit of a DNA-dependent protein kinase (18), which phosphorylates a variety of transcription factors in vitro(19, 20, 21, 22) . We have performed gel mobility shift analysis using purified CHBF and purified recombinant human HSF1, and found that purified CHBF competes with purified recombinant human HSF1 for the binding of DNA sequences containing HSEs in vitro. We have also performed in vivo experiments to assess whether the Ku autoantigen complex may play a role in the modulation/regulation of heat shock response. The Ku-70 and Ku-80 cDNA-containing expression vectors were co-transfected into Rat-1 cells with the N Luc hsp70-luciferase plasmid, which contains the mouse hsp70 promoter upstream of the firefly luciferase gene. Co-transfection of Ku-70 plus Ku-80 expression vectors with hsp70-luciferase reporter gene resulted in an average of 4-fold lower thermal induction of luciferase activity relative to cells transfected with only the hsp70-luciferase. These results demonstrate that heat-induced transcriptional activation of hsp70-luciferase can be modified in vivo by the overexpression of Ku protein. Taken together our findings suggest a role of Ku protein in the modulation of heat shock response in vivo. Further studies with this protein should lead to an understanding of the role of CHBF (or Ku protein) in the regulation of heat shock gene expression.


MATERIALS AND METHODS

Purification of the Constitutive Heat Shock Element-binding Factor (CHBF)

Taking advantage of the known CHBF-HSE binding activity established previously(11) , we have purified CHBF from HeLa cells using the following successive column fractionations: (i) DEAE-ion exchange, (ii) gel filtration, (iii) heparin-agarose, and (iv) oligomeric HSE-agarose column chromatography.

HeLa cells (1 10 cells) grown under low serum conditions and harvested in exponential growth phase were purchased from Life Technologies, Inc.; the frozen cells were thawed, and pelleted by centrifugation. The cell pellet was extracted with extraction buffer (10 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, and 0.5 mM PMSF, 5% glycerol) as described previously(11) . The whole cell extract was dialyzed against dialysis buffer DB1 (20 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1.5 mM MgCl, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 20% glycerol), and was loaded onto a DEAE-agarose column pre-equilibrated with equilibration buffer EB1 (DB1 with 10% glycerol). The column was eluted with a NaCl gradient (0.01-0.4 M in EB1). The HSE binding activity of each fraction was analyzed by dot-blot analysis and gel mobility shift assays. The fractions containing relatively high HSE binding activities were pooled and concentrated using an Amicon concentrator with a YM-10 ultrafiltration membrane. Concentrated proteins were dialyzed against buffer DB2 (20 mM HEPES, pH 7.9, 200 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF, 10% glycerol). The dialyzed sample was loaded onto a Bio-Gel A-0.5m (Bio-Rad) column, and was eluted with buffer DB2. Again, fractions containing a high HSE binding activity were pooled, concentrated, and subsequently loaded onto a heparin-agarose (Bio-Rad) column, and eluted with a NaCl gradient (0.2-1.5 M in DB2). Fractions eluted between 0.85 and 1.5 M NaCl contained high HSE binding activities. These active heparin-agarose fractions were combined, dialyzed against the equilibration buffer EB2 (20 mM HEPES, pH 7.9, 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF, 10% glycerol), and applied to a 1-ml HSE oligonucleotide-agarose affinity column. The loaded column was washed with 50 column volumes of a washing buffer (300 mM NaCl in the equilibration buffer EB2) to remove unbound proteins. Proteins bound to the HSE oligonucleotide-agarose were then eluted with a step gradient of NaCl (0.1 M increment of NaCl in the same buffer). Protein fractions eluted between 0.3 and 0.8 M NaCl contained the highest HSE binding activity and were pooled, dialyzed, and stored at -80 °C for further characterization.

The purification scheme yielded 3.8 µg of purified protein. The overall recovery of activity was estimated to be approximately 7% as analyzed by gel mobility shift assay described below. The specific activity of the purified protein was increased approximately 8000-fold over that from the starting material.

Preparation of the HSE Oligonucleotide-Agarose Affinity Column

A 49-mer double-stranded DNA oligonucleotide containing six repeats of HSE sequence, NGAAN, was prepared by annealing the two complementary oligonucleotides: upper strand, 5`-TCTAACAGACCCGAAACTGCTGGAAGATTCCCGAAACTTCTGGTTCGGG-3`, and lower strand, 3`-AGATTGTCTGGGCTTTGACGACCTTCTAAGGGCTTTGAAGACC-5`. The 6-nucleotides gap at the 5`-end of the lower strand was filled in by Klenow enzyme(23) . The annealed oligonucleotide (20 nmol) was incubated at 37 °C for 1 h, with 100 units of Klenow enzyme in the reaction mixture containing 20 nmol of biotin-11-dCTP, 1.0 mM each of dATP and dGTP in 50 mM Tris-HCl, pH 7.5, 7 mM MgCl, 1 mM DTT, 50 mM NaCl. The reaction was completed by further incubation for 1 h following addition of dCTP to 1.0 mM. The biotin-labeled HSE oligonucleotide was subsequently incubated overnight at 4 °C with 1 ml (29 nmol of biotin equivalent) of streptavidin-agarose (Pierce) in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and packed into a 1 ml Econo-Column (Bio-Rad).

Gel Mobility Shift Assay and Preparation of Probes

Preparation of the cell extracts and the gel mobility shift assay were performed as described previously (11) with minor modifications. The double-stranded oligonucleotide containing the HSE (lower strand, 5`-GGGCCAAGAATCTTCCAGCAGTTTCGGG-3`; upper strand, 3 bases shorter than lower strand from 3` end) was labeled with Klenow enzyme (23) and [-P]dCTP in 50 mM Tris-HCl, pH 7.5, 7 mM MgCl, 1 mM DTT, 50 mM NaCl. Equal amounts of cellular proteins (50 µg) were added to a binding mixture containing 10 µg of yeast tRNA, 1 µg of Escherichia coli DNA, 0.25 µg of poly[dI-dC]poly[dI-dC], 50 µg of BSA in 15 mM Tris-HCl, pH 7.4, 75 mM NaCl, 0.1 mM EGTA, 5% glycerol, 0.5 mM DTT, and incubated with 1 ng of P-labeled probe for 25 min at 20 °C. The protein-bound and free oligonucleotides were electrophoretically separated on 4% native polyacrylamide gels in 0.5 TBE buffer (44.5 mM Tris, 1 mM EDTA, and 44.5 mM boric acid, pH 8.0) for 2 h at 140 V. E. coli DNA and poly[dI-dC]poly[dI-dC] were omitted for the sample of purified CHBF or other column fractions. Competition assays were performed by co-incubating the cell extracts from control or heat-shocked cells with unlabeled double-stranded (ds)- or single-stranded (ss)- HSE oligonucleotides. The gels were dried, autoradiographed, and quantified by a radio analytical imaging system (AMBIS).

For supershift analysis, purified CHBF or extracts from non-heat-shocked cells were preincubated with various amounts of monoclonal antibodies (anti-Ku-70 and anti-Ku-80) for 1 h on ice, and followed by incubation with P-labeled oligonucleotide for 20 min at 25 °C before being subjected to gel mobility shift analysis. In some experiments, purified CHBF or cell extracts were incubated with P-labeled oligonucleotide first, then incubated with monoclonal antibodies specific to Ku protein before being subjected to a gel mobility shift analysis.

Dot-blot Analysis of CHBF-HSE Binding Activity

Aliquots of proteins from each column fraction were mixed with P-labeled HSE oligonucleotide (1 ng) in a filter binding buffer (15 mM Tris-HCl, pH 7.4, 3 mM MgCl, 100 mM NaCl, 0.5 mM DTT, and 5% glycerol). After a 20-min incubation at 20 °C, the reaction mixtures were loaded on the membrane in a preassembled dot-blot apparatus (Bio-Rad, 3 mm diameter), washed three times with the filter binding buffer, and dried. The radioactivity of each sample was counted using AMBIS.

UV Cross-linking between CHBF and HSE

P-Labeled HSE oligonucleotide, the same one as used in the gel mobility shift assay, was used for the UV cross-linking experiments. Purified CHBF (4 ng) or cell extract (30 µg) was mixed with two volumes of the binding buffer (15 mM HEPES, pH 7.4, 75 mM NaCl, 0.1 mM EGTA, 0.5 mM DTT, 5% glycerol) and 1 ng of P-labeled HSE oligonucleotide. After 20 min of incubation at 20 °C, the reaction mixtures were irradiated in a UV cross-linker (Stratagene) for 1-5 min with the power setting of 3 milliwatts/cm.

Western Blot Analysis

Proteins from the purified CHBF fraction or cell extract were separated by one-dimensional SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a mixture of two monoclonal antibodies raised against the 70-kDa (Ku-70 or p70) or the 86-kDa (Ku-80 or p80) subunit of human Ku autoantigen (provided by Dr. J. Wang, Dr. W. H. Reeves, and Dr. N. Thompson). Monoclonal antibodies, anti-Ku-70 (class IgG) and anti-Ku-80 (class IgG), were mixed and used with a final dilution of 1:500. Alkaline phosphatase-conjugated anti-mouse IgG was employed as a second antibody to visualize the immunocomplexes. Purified human Ku autoantigen (provided by Dr. A. Dvir) was simultaneously analyzed as a reference. HSF1 was detected with an enhanced chemiluminescence system (Amersham) by employing a 1:5,000 diluted first antibody specific to HSF1, and a 1:10,000 diluted second antibody conjugated to horseradish peroxidase.

Expression and Purification of Recombinant Human HSF1 in E. coli

The human HSF1 gene in a plasmid pSKHSF1 was obtained from Dr. C. Wu(24) . This plasmid contains the entire coding sequence, the 5`- and 3`-untranslated regions, and poly(A) tail cloned into the EcoRI site of pBluescript SK(-) (Stratagene). The 529-amino acid open reading frame was subcloned into an expression vector pETMod (obtained from Dr. W. Lee). pETMod, a derivative of pETME, contains a sequence of 6 histidine residues at the NH terminus of the target protein. The expressed fusion protein thus contains 6 histidine residues on its amino terminus.

To purify recombinant human HSF1, E. coli cells (strain BL21(DE3)pLysS) were transformed with the expression plasmid, grown in LB broth with ampicillin (200 µg/ml), and induced with a 1 mM isopropyl-1-thio--D-galactopyranoside. These cells were harvested, washed with cold phosphate-buffered saline, and sonicated in 10 ml of 10 mM Tris-HCl, pH 7.9. Cytosolic proteins were extracted, applied onto, and eluted from a nickel-agarose affinity column (Novogen) according to the manufacturer's protocol. The eluted fractions containing high HSF1-HSE binding activity as assayed by gel mobility shift assay were pooled, dialyzed, and applied on an Econo-PackQ cartridge column (Bio-Rad). The bound proteins were eluted with a 0-800 mM NaCl gradient in 20 mM Tris-HCl, pH 7.9. The fractions containing high HSE binding activity were applied onto an HSE oligonucleotide-agarose affinity column prepared with biotinylated-HSE oligonucleotide and avidin cross-linked agarose. The bound HSF1 was then eluted with 600 mM NaCl in Tris-HCl, pH 7.9, dialyzed, and concentrated using an Amicon ultrafiltration membrane YM10. The purified protein was analyzed by SDS-PAGE and silver staining, as well as Western blotting using anti-human HSF1 antibody (provided by Dr. C. Wu) and an enhanced chemiluminescence system (Amersham).

Transfection of Rat-1 Cells with Human Ku-70, Human Ku-80 Expression Vectors, and Assays for Transient Expression of Reporter Gene

For transient expression of human Ku-70 and human Ku-80 in Rat-1 cells, the full-length cDNA of each was cloned into pcDNA1-NEO (Invitrogen). The plasmid NLuc containing the mouse hsp70 promoter-driven luciferase reporter gene was a generous gift from Dr. O. Bensaude (Ecole Normale Superieur, Paris, France)(25) . DNA transfection and assay for transient expression of reporter gene were done as described previously(26) . Briefly, monolayers of Rat-1 fibroblasts were either co-transfected with pcDNA-Ku70, pcDNA-Ku80, and NLuc (DNA ratio 1:1:1 or 5:5:1) or transfected with NLuc only. pBluescript SK(-) (Stratagene) was added to make up equal amounts of DNA in the different transfections. To test for heat-inducible expression of the luciferase gene, cells were replated into 35-mm Petri dishes 24 h after the transfection. Forty-eight hours after the transfection, cells were heat-shocked at 45 °C for 15 min and returned to 37 °C for 8 h, after which cell extracts were prepared. Luciferase activity present in cell extracts was assayed as described previously(26) . Experiments were always performed in duplicate dishes. The results are averaged and expressed relative to the luciferase activity in the unheated control cells.


RESULTS

Inverse Correlation between CHBF and HSF1 during Heat Shock Response

Protein factors in cell extracts from Rat-1, CHO HA-1 and HeLa cells that interact with HSE were analyzed by the gel mobility shift assay. Fig. 1A shows the electrophoretic migration patterns of the HSE-binding proteins in a nondenaturing gel. As previously reported, there are two distinct HSE-binding complexes: a faster migrating complex in extracts of unshocked cells (CHBF) and a slower migrating complex in extracts of heat-shocked cells (HSF1). The induction of HSF1-HSE-binding is rapid, reaching a maximal level by 5 min at 45 °C. In contrast to the HSF1-HSE binding activity, the CHBF-HSE binding activity decreases upon heat shock. The heat-induced decrease of CHBF-HSE binding activity correlates with the increase of HSF1-HSE binding activity during heat shock. During post-heat shock recovery at 37 °C, HSF1-HSE binding activity decreases, while CHBF-HSE binding activity gradually recovers at 37 °C. There is a tight temporal correlation between the disappearance of HSF1-HSE binding activity and the recovery of CHBF-HSE binding activity in Rat-1, Chinese hamster HA-1 (Fig. 1B), and HeLa cells (data not shown).


Figure 1: Analysis of HSE binding activities in control and heat-shocked mammalian cells. A, gel mobility shift analysis of whole cell extracts from heat-shocked Rat-1, HA-1, and HeLa cells. The HSE binding activity was analyzed with samples prepared from monolayers of exponentially growing Rat-1 cells heated at 45 °C for 5-15 min (lanes 1-3), HA-1 cells heated at 45 °C for 5-15 min (lanes 5-7), HeLa cells heated at 45 °C for 15-60 min (lanes 9-11), HeLa cells heated at 46 °C for 15-30 min (lanes 12 and 13), and HeLa cells at 47 °C for 15-30 min (lanes 14 and 15). B, the level of CHBF and HSF during recovery at 37 °C. Whole cell extracts (50 µg) were analyzed by gel mobility shift analysis with samples prepared from HA-1 cells (lanes 2-6) and Rat-1 cells (lanes8-12) incubated at 37 °C for 0-8 h following a 15-min heat shock at 45 °C. The constitutive HSE-binding complex (CHBF) and the heat shock-induced HSF1-HSE complex (HSF) are indicated by an openarrow and a closedarrow, respectively.



We have also tested a variety of heat shock conditions, including various heating temperatures and heating times, on the induction of hsp70 and found that hsp70 is induced only under the condition when HSF1 was activated, and the CHBF-HSE binding activity was significantly reduced(11) .() Taken together, these data suggest that CHBF may also be involved in the regulation of heat shock response.

Purification of CHBF

Following protocols similar to those used for the purification of HSF(27) , the CHBF was purified by successive chromatography on (i) DEAE-ion exchange column, (ii) gel filtration column, (iii) heparin-agarose column, and (iv) HSE oligonucleotide-agarose column. HSE binding activity from column fractions was determined by gel mobility shift assay or dot-blot analysis using a P-labeled double-stranded oligonucleotide containing repetitive HSE sequences. In Fig. 2A, the relative HSE binding activities of various DEAE-ion exchange column fractions show that these fractions were resolved into two activity peaks: fractions 10-37 (eluted with 0.05-0.2 M NaCl) and fractions 40-74 (eluted with 0.2-0.3 M NaCl). The first peak, containing both HSF1-HSE binding activity and CHBF-HSE binding activity, was discarded. The second peak, containing high CHBF-HSE binding activity, was pooled and applied onto the gel filtration column. A major activity peak, eluting close to the void volume of the column, was pooled and subsequently applied onto the heparin-agarose column. As shown in Fig. 2B, the heparin column fractions were resolved into two activity peaks; one eluted with 0.2-0.8 M NaCl and the other eluted with 0.85-1.5 M NaCl. The latter peak, showing a higher specific activity of HSE binding activity, was pooled and used for further purification with an HSE oligonucleotide affinity column. Proteins bound to the HSE oligonucleotide-agarose column were eluted by a step gradient of NaCl with 0.1 M increments (Fig. 2C). The recovered fractions with the highest HSE binding activity (0.3-0.8 M) showed significant levels of impurities when analyzed by SDS-PAGE and silver staining. However, washing of the loaded column with 50 column volumes of the elution buffer containing 0.3 M NaCl eliminated these impurities. The 0.3-0.8 M fractions were pooled, dialyzed, stored at -80 °C, and used for further analysis and characterization. The overall recovery of activity, as estimated from the gel mobility shift assays, was approximately 7%. The specific activity of the purified protein was increased about 8000-fold over that from the starting material. Fig. 3A shows the protein profiles of fractions obtained from various stages of chromatographic purification. The final HSE oligonucleotide-agarose column fraction contains only two major polypeptides, with apparent molecular masses of 70 and 86 kDa, and present in equimolar amounts.


Figure 2: Column chromatographic purification of the constitutive heat shock element-binding factor. A, soluble proteins of HeLa cells prepared as described under ``Materials and Methods'' were applied onto a DEAE-agarose column (5.5 10 cm) and eluted with a NaCl gradient (10-400 mM) following column wash. Equal volumes of protein samples were taken from every third fraction and subjected to gel mobility shift analysis. A P-labeled oligonucleotide containing the HSE sequence (5`-GGGCCAAGAATCTTCCAGCAGTTTCGGG-3`) was employed for the gel mobility shift assay. The CHBF- and HSF-DNA complexes are indicated as CHBF and HSF, respectively. Fractions 40-74 (indicated by &cjs0822;-&cjs0822;) were pooled and subjected to the next purification step. B, heparin-agarose column chromatography. Active fractions from Bio-Gel A-0.5m following DEAE-agarose column were pooled, applied onto a heparin-agarose column (1.5 14 cm), and eluted with a NaCl gradient (0.2-1.5 M). Protein content and HSE binding activity were analyzed by Bradford and dot-blot assay, respectively. Solidcircle, radioactivity; opencircle, protein concentration. Fractions showing high specific activity of HSE-binding were pooled (indicated by &cjs0822;-&cjs0822;), and subjected to the next purification step. C, HSE oligonucleotide-agarose column chromatography of pooled fractions collected from heparin-agarose column. Pooled fractions from the later peak of the heparin column were applied onto an oligonucleotide-agarose column (1 ml) prepared from biotin-labeled oligonucleotides containing the HSE sequence and streptavidin-agarose. The column was eluted with a NaCl step gradient. The DNA binding activity of each fraction was determined by gel mobility shift assay and scanning of the dried gel by using a radio analytic imaging system (AMBIS). The fractions (0.3-0.8 M, indicated by &cjs0822;-&cjs0822;) were pooled and used for further characterization of CHBF.




Figure 3: Analysis of protein profiles from each purification step and immunological identification of the purified protein. A, equal amount of proteins (1 µg each) from the starting material, eluted from DEAE, gel filtration, heparin column, and purified proteins (20 ng) from the HSE-agarose column were analyzed using a SDS-polyacrylamide gel electrophoresis and silver staining. B, antigenic cross-reactivity of P70 and P80 of Ku autoantigen. HeLa cell lysate, HSE affinity-purified proteins (10 ng), and Ku autoantigen (2 and 10 ng) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with monoclonal antibodies specific to the human Ku autoantigen. Human Ku proteins were used as a reference. The arrowheads indicate 70- and 86-kDa polypeptides.



UV Cross-linking between CHBF and HSE

We have confirmed that the purified polypeptides are the constitutive HSE-binding factor using UV cross-linking technique. P-Labeled oligonucleotide containing HSE identical to the natural regulatory sequence of the rat hsp70 gene was mixed with the purified proteins, and the reaction mixture was cross-linked under ultraviolet light. SDS-sample buffer was added to the protein-DNA complex mixture, which was subsequently boiled and analyzed by one-dimensional SDS-PAGE and autoradiography (Fig. 4). A P-labeled doublet with apparent molecular masses of 77 and 86 kDa was observed, suggesting that both subunits of the 70/86-kDa protein complex can be cross-linked to DNA. The UV cross-linking is due to CHBF binding to HSE, because in a competition experiment, addition of excess unlabeled HSE abolishes the signal completely.


Figure 4: UV cross-linking of the purified CHBF to HSE probe. The purified CHBF (4 ng) was incubated with P-labeled HSE oligonucleotide, the same as used in the gel mobility shift assay, was incubated for 20 min at 25 °C, and followed by exposure to UV light for 1-5 min (3 milliwatts/cm). The unlabeled oligonucleotide (200-fold excess) was added to one of the samples as a competitor. Similar results were obtained when lower levels (50-fold excess) of competitor were used. Cell extracts (30 µg) from Rat-1, HA-1, and HeLa cells were treated under the same condition as the purified protein and subjected to UV irradiation. The cross-linked DNA-protein complexes were analyzed by a 10% polyacrylamide gel and followed by autoradiography. The 77- and 86-kDa doublet is indicated by arrows.



We have also performed UV cross-linking experiments using crude cell extracts from HeLa, Rat-1, and Chinese hamster HA-1 cells. Cell lysates were mixed with P-labeled HSE and UV cross-linked. The protein-DNA complex was analyzed by one-dimensional SDS-PAGE. Specific P labeling of the 70/86-kDa protein complex in crude extracts prepared from these different cell lines (HeLa, Rat-1, HA-1) under normal growth, unheated conditions (37 °C) is clearly observed (Fig. 4).

CHBF Is Closely Related or Identical to Ku Autoantigen

The polypeptide composition and molecular masses of CHBF are very similar to that of a well characterized protein named Ku autoantigen(13, 28, 29, 30) . The cloned subunits of Ku protein have predicted molecular weights of 69,851 and 82,713(30, 31) , which are in good agreement with the observed molecular weights of CHBF. To verify immunologically that CHBF is similar to Ku protein, purified CHBF was analyzed by Western blot using monoclonal antibodies specific to human Ku autoantigen. It is clearly shown in Fig. 3B that both subunits of CHBF reacted with the antibodies specific to Ku autoantigen. The 70- and 86-kDa polypeptides of purified CHBF not only showed similar electrophoretic mobility in the SDS-PAGE as the purified Ku protein, but were also equally well recognized by antibodies specific to Ku protein (Fig. 3B). Since the anti-Ku monoclonal antibodies are highly specific and recognize only the p70 and p80 subunits of Ku protein, respectively, our purified CHBF seems to be identical to the Ku protein.

To further examine whether CHBF is closely related or identical to Ku protein, we tested the ability of monoclonal antibodies against Ku protein to selectively deplete or modify the DNA-binding ability of CHBF. Gel mobility shift assays were performed with P-labeled HSE oligonucleotide and purified CHBF that were preincubated with a mixture of monoclonal antibodies against Ku protein. Addition of anti-Ku antibodies to HeLa cell extract caused a supershift of the protein-DNA complex (Fig. 5A, lanes 1-3), while it completely abolished the DNA binding activity of purified CHBF (Fig. 5A, lanes4-6) and purified Ku autoantigen (Fig. 5A, lanes 7-9). In a separate set of experiments, anti-Ku antibodies when added after the formation of CHBF-HSE complex, caused a supershift in the mobility of the HSE-binding complexes of HeLa cell extract, purified CHBF, and purified Ku autoantigen, respectively (Fig. 5A, lanes 10-12, respectively). Our observation is consistent with the results of Mimori and Hardin(14) , who first reported that Ku antibodies prevent the Ku DNA binding. Compared to HeLa cells (Fig. 5B, lanes1 and 2), addition of anti-Ku antibodies specific to human Ku protein had less effect on the CHBF-HSE binding activity in extracts prepared from Rat-1 cells (Fig. 5B, lanes3 and 4) or HA-1 cells (data not shown). The CHBF-DNA binding activity was inhibited by preincubation with either anti-p70 or anti-p80 (data not shown), suggesting that the overall integrity of the p70/p80 heterodimeric structure is essential for the DNA binding competence of the protein.


Figure 5: Recognition of nondenatured CHBF by monoclonal antibodies specific to human Ku autoantigen. A, whole cell extract from non-heat-shocked HeLa cells containing 30 µg of proteins (lanes 1-3), 20 ng of purified CHBF (lanes 4-6), or purified human Ku autoantigen (lanes7-9) were preincubated with various amounts of monoclonal antibodies specific to human Ku autoantigen, and followed by incubation with P-labeled oligonucleotide for 20 min at 25 °C before being subjected to gel mobility shift analysis. In lanes10-12, HeLa cell extract (lane10), purified CHBF (lane11), and purified Ku autoantigen (lane12) were incubated with P-labeled HSE first, then incubated with the monoclonal antibodies before being subjected to gel mobility shift analysis. B, as a control, cell extract from Rat-1 cells (lanes3 and 4) was analyzed as in A in parallel to that of HeLa cells (lanes1 and 2) with (lanes2 and 4) or without (lanes1 and 3) preincubation with the monoclonal antibodies specific to human Ku autoantigen. Open arrow indicates CHBF.



We also analyzed the molecular size of the protein-DNA complex using size exclusion gradient nondenaturing acrylamide gel electrophoresis. The molecular size of CHBF in its native oligomeric form, as evidenced by its binding to the P-labeled HSE oligonucleotide probe, was estimated to be approximately 140 kDa (data not shown), which is in good agreement with the estimated value (approximately 156 kDa) of Ku protein.

CHBF Binds to Both ss- and dsDNA in Vitro

The ssDNA and dsDNA binding activities of the purified CHBF were examined by competition experiments using the gel mobility shift assay. P-Labeled double-stranded-DNA probe was incubated with the purified CHBF in the absence or presence of various amounts of single- or double-stranded unlabeled oligonucleotides, and the CHBF-DNA-binding complexes were analyzed by gel mobility shift analysis. As shown in Fig. 6, unlabeled dsHSE competed more efficiently with P-labeled double-stranded probe than the unlabeled ssHSE for its binding to the purified CHBF (compare Fig. 6and Fig. 4). These data suggest that CHBF has a higher binding affinity to double-stranded DNA than to single-stranded-DNA. The results shown in Fig. 6are consistent with previous reports by Falzon et al.(12) , Blier et al.(32) , and Mimori and Hardin(14) , who have similarly tested ssDNA binding characteristics of Ku protein by competition experiments using P-labeled dsDNA probe and unlabeled ssDNA.


Figure 6: Binding characteristics of CHBF to dsDNA and ssDNA. Purified CHBF (10 ng) was incubated with P-labeled dsHSE probe (1 ng, the same as used in Fig. 1) with various amounts of unlabeled dsHSE (lanes 1-3) or unlabeled ssHSE (lanes 4-7) for 25 min at 20 °C in the reaction mixture. The reaction mixture contains 10 µg of yeast tRNA, 1 µg of E. coli DNA, 0.25 µg of poly[dI-dC]poly[dI-dC], 50 µg of BSA in the binding buffer (15 mM Tris-HCl, pH 7.4, 75 mM NaCl, 0.1 mM EGTA, 5% glycerol, 0.5 mM DTT). In lanes1-3, 0, 10, and 100-fold excess amounts of unlabeled dsHSE relative to the amount of the P-labeled probe was added as a competitor. In lanes 4-7, 100, 200, 500, and 1000-fold excess amounts of ssHSE were added as competitor. The arrowhead indicates CHBF-DNA complex. Note that the binding of purified CHBF to dsHSE probe was only partially decreased by 200-fold excess of unlabeled ssHSE, while in Fig. 4, the CHBF-HSE binding was completely eliminated at 200-fold excess of dsHSE. This difference is due to the use of single-stranded versus double-stranded competitors.



Competition in the DNA Binding Activity between HSF1 and CHBF

The biological significance of the inverse correlation between the levels of CHBF-HSE and HSF1-HSE binding activity during heat shock and cells' subsequent recovery is not known at present. However, these data suggest a possible involvement of CHBF in the regulation of heat shock response. We have performed three sets of experiments to examine the competition between HSF1 and CHBF for the binding of DNA sequences containing HSE in vitro. Recombinant human HSF1 produced in E. coli constitutively binds to HSE without requiring heat activation and, therefore, was used in our experiment. As shown in Fig. 7, histidine-tagged human HSF1 purified through a nickel-agarose column followed by the HSE oligonucleotide-agarose affinity column was apparently homogeneous and was recognized by an anti-HSF1 antibody.


Figure 7: Column chromatographic purification of a recombinant human HSF1. A, E. coli cells transformed with the expression plasmid containing the human HSF1 sequence were grown and induced as described under ``Materials and Methods.'' Cytosolic proteins were applied to and eluted from a nickel-agarose affinity column. Starting cytosolic proteins, flow-through of the column, and Ni-binding proteins are shown in lanes 2-4, respectively. Molecular size markers (lane1) and the sizes in kilodalton are shown to left of the gel lane. B, the eluted positive fractions were pooled, dialyzed, and applied to an Econo-PackQ cartridge column (from Bio-Rad). The bound proteins eluted with 0-800 mM NaCl gradient were applied to an HSE oligonucleotide-agarose affinity column prepared as described under ``Materials and Methods.'' The bound HSF1 were then eluted, dialyzed, concentrated, and analyzed by SDS-PAGE/silver staining. Lanes5 and 6 represent 10 and 20 ng of HSF1, respectively. C, the purified HSF1 (2 ng) was analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and probed by a polyclonal antibody against human HSF1 by employing an enhanced chemiluminescence detection system (lane7). Arrows indicate the purified recombinant human HSF1.



In the first set of experiments, when the purified CHBF and the purified recombinant HSF1 were mixed simultaneously with P-labeled DNA probe and protein-DNA complexes were analyzed by gel mobility shift assay, there was clearly competition between HSF1 and CHBF for DNA binding in vitro (Fig. 8A). When the CHBF to HSF1 ratio is increased, the formation of HSF1-DNA complex decreases, whereas the formation of CHBF-DNA complex increases, indicating that CHBF competes with HSF1 for its binding to DNA. In a second set of experiments, CHBF was incubated first with the P-labeled DNA probe (allowing the formation of CHBF-DNA-binding complex), and then mixed with graded concentrations of purified recombinant human HSF1. As shown in Fig. 8B, in the presence of preformed CHBF-DNA-binding complex, the binding of HSF1 to the same DNA is significantly inhibited (comparing Fig. 8A, lane1 with Fig. 8B, lane4). In the third set of experiments, HSF1 was incubated first with theP-labeled DNA probe (allowing the formation of HSF1-DNA complex), and then mixed with graded concentrations of CHBF. As shown in Fig. 8C, the presence of increasing amounts of CHBF appears to facilitate the dissociation of preformed HSF1-DNA-binding complex. Similar results were reproducibly observed using crude cell extracts prepared from control and heated Rat-1, HA-1, and HeLa cells (data not shown).


Figure 8: In vitro competition between HSF and CHBF in DNA binding. The DNA binding activities of CHBF and HSF were analyzed by gel mobility shift assay employing purified CHBF and purified recombinant human HSF1. A, fixed amounts of HSF1 (10 ng) and various amounts of CHBF (2, 4, 10, and 20 ng for lanes 1-4, respectively) were incubated with P-labeled HSE probe, followed by gel mobility shift analysis. B, fixed amount of CHBF (10 ng) was preincubated with P-labeled HSE (0.1 ng) for 30 min, subsequently various amounts of HSF1 (2, 4, 10, and 20 ng for lanes1-4, respectively) were added to the reaction mixture, incubated for an additional 30 min, and followed by gel mobility shift analysis. C, fixed amount of HSF1 (10 ng) was preincubated with P-labeled HSE (0.1 ng) for 30 min, after which various amounts of CHBF (2, 4, 10, and 20 ng for lanes 1-4, respectively) were added and incubated for an additional 30 min before being subjected to gel shift analysis.



Overexpression of Human Ku Protein in Rat-1 Cells Suppresses the Heat-induced Expression of Firefly Luciferase in Vivo

To assess whether there is an in vivo role of CHBF/Ku in the regulation of heat shock response, we have performed experiments using an hsp70 promoter-driven firefly luciferase reporter gene. The human Ku-70 and human Ku-80 expression vectors were co-transfected into Rat-1 cells with hsp70-luciferase reporter gene, which contains the mouse hsp70 promoter upstream of the firefly luciferase gene. The relative abilities of overexpressed human Ku protein to suppress heat-induced transcription were determined by comparing the heat induction of luciferase activities with that from Rat-1 cells transfected with only the hsp70-luciferase construct. Our data (Fig. 9) show that co-transfection of human Ku-70 and Ku-80 expression vector with hsp70-luciferase resulted in an average of 4-fold reduction in heat-induced luciferase activity relative to that from transfection with only the hsp70-luciferase construct. These results demonstrate that heat-induced transcriptional activation of hsp70-luciferase can be modified in vivo by the overexpression of Ku protein.


Figure 9: Overexpression of human Ku protein in Rat-1 cells suppresses the heat induction of luciferase expression in vivo. Monolayers of Rat-1 cells were either co-transfected with pcDNA-Ku-70, pcDNA-Ku-80, and NLuc (hsp70 promoter-driven luciferase reporter gene construct) with 1:1:1 or 5:5:1 DNA ratio) or transfected with NLuc construct only. To test for heat-inducible expression of the luciferase gene, cells were replated into 35-mm Petri dishes 24 h after the transfection. Forty-eight hours after the transfection, cells were heat-shocked at 45 °C for 15 min and returned to 37 °C for 8 h, cell extracts were prepared, and luciferase activities present in the extracts of control unshocked (CON) and heat-shocked cells (HS) were determined. Experiments were always performed in duplicate dishes. The results are averaged and expressed relative to the luciferase activity in the unheated control cells (normalized as 1). Solidbar, cells were transfected with only NLuc; openbar, cells were co-transfected with Ku-70, Ku-80, and Luc with DNA ratio 1:1:1; hatchedbar, Ku-70, Ku-80, and Luc with DNA ratio 5:5:1.




DISCUSSION

Mammalian heat shock transcription factor HSF1 has been the target of extensive studies relating to heat shock gene activation in recent years. On the other hand, few studies have focused on the constitutive HSE binding activity of CHBF. Recently, we have performed detailed analyses on the binding of protein factors to HSE during heat shock at different temperatures (41-47 °C), as well as cells' subsequent recovery at 37 °C(11, 33) . In all cell lines studied (e.g. HeLa, HA-1, Rat-1), we found that, upon heat shock, the heat-induced decrease of CHBF-HSE binding activity correlates well with the rapid increase in HSF1-HSE binding activity. Furthermore, during post-heat shock recovery, kinetically there is a good correlation between the decrease of HSF1-HSE binding activity and the recovery of the CHBF-HSE binding activity. The biological significance of this inverse correlation in the levels of binding activity of CHBF and HSF1 is not known. However, these data suggest a possible involvement of CHBF in the regulation of heat shock response. To further investigate the possible role of CHBF in the regulation of heat shock response, we proceeded to purify this protein.

In the present study, we report the purification of CHBF from HeLa cells. The purified protein contains two subunits with molecular masses of 70 and 86 kDa, respectively. The polypeptide composition and sizes resemble those of the Ku autoantigen. The Ku protein is known to contain equimolar amounts of 70- and 86-kDa polypeptides and to form heterodimers or tetramers in solution(14, 15, 16, 19) . A close similarity between CHBF and Ku protein is confirmed by their antigen cross-reactivity. Monoclonal antibodies raised specifically against human Ku-70 (p70) and Ku-80 (p80) were used in Western blot analysis and gel retardation assay. As clearly shown in the immunoblot analysis (Fig. 3B), both subunits of CHBF reacted with the monoclonal antibodies specific to Ku autoantigen. Furthermore, preincubation of anti-Ku antibodies with either purified CHBF or HeLa cell extract abolished or modified the DNA-binding ability of CHBF, as was observed with purified Ku autoantigen (Fig. 5). Addition of anti-Ku antibodies to already formed CHBF-HSE complex leads to a super-shift in the mobility of the DNA-protein complex in the nondenaturing gel. On the other hand, addition of anti-Ku antibodies (highly specific against human Ku protein, but not showing significant cross-reactivity with rodent Ku protein) to extracts of Chinese hamster HA-1 or Rat-1 cells has little effect on the CHBF-DNA-binding ability of these cells. Taken together, Fig. 3and Fig. 5show that the anti-Ku antibodies specifically recognize human CHBF in its denatured, native, or native DNA-bound form. In addition, we have shown that the DNA binding characteristics of CHBF are very similar to that of Ku protein, i.e. it prefers dsDNA to ssDNA; the binding is competed by either form of DNA, but much more efficiently by dsDNA. Judging by the composition, molecular size, DNA binding activity, and cross-reactivity with antibodies specific to Ku protein, the CHBF appears to be identical or closely related to the Ku autoantigen.

Many known characteristics of Ku autoantigen suggest a possibility that Ku protein may play some regulatory role(s) in transcription. For example, studies on the DNA binding characteristics of Ku protein showed that Ku protein first binds single-stranded DNA in a single/double transition region and subsequently slides on to the double-stranded DNA in an energy and sequence-independent manner(12) . Yaneva and Busch (34) found that Ku protein appeared to be associated with DNase I-sensitive nucleosomes lacking H1 histone, and Ku protein binds chromosomal DNA in vivo. The binding of transcription factors to nucleosome regions close to the promoter is a prerequisite to transcriptional activation(35) . The possibility of a role for Ku protein in transcriptional regulation is further supported by the observation that Ku autoantigen is specifically localized on certain transcriptionally active loci of chromosomal DNA(36, 37) . Ku autoantigen has been shown to be one component of a DNA-dependent protein kinase (the other component is a 350-kDa polypeptide), which phosphorylates many different transcription factors such as Sp1(19) , c-Jun(20) , p53(22) , c-Myc, Oct-1, and Oct-2 (21), or RNA POL-II (18) in vitro. These studies indicate that Ku protein may exert certain regulatory roles in transcription through phosphorylation of DNA-binding factors. Finally, many previously known DNA-binding factors such as NF-IV (38) and transcription factors such as PSE1(17) , HTFR(17) , and EBP-80 (12) have been shown to be similar or identical to the Ku autoantigen, implying a role of Ku protein as a transcription factor. Several studies (15, 16, 17) have demonstrated that Ku autoantigen directly modulates the RNA POL-I-mediated transcription. However, it is not clear whether Ku plays any regulatory role in RNA POL-II-mediated transcription. It is possible that Ku protein may exert certain regulatory roles in the RNA POL-II-mediated transcription through phosphorylation of the carboxyl-terminal domain of the RNA POL-II as described by Dvir et al.(18) . However, a role for Ku protein in the heat shock-related transcriptional regulation remains to be shown.

In order to investigate a possible role of CHBF in the transcriptional regulation of heat shock gene expression, we first examined whether the inverse correlation of DNA binding activities between HSF1 and CHBF could be reproduced in vitro with purified proteins. As shown in Fig. 8, CHBF competed with HSF1 for the binding of DNA sequences containing HSE in vitro. Interestingly, the preformed CHBF-DNA complex was replaced less efficiently by HSF1-HSE complex (Fig. 8B). It is plausible that heat-induced dissociation of CHBF from HSE may be a prerequisite for the formation of HSF1-HSE-binding complex. Conversely, the dissociation of HSF1-HSE complex may be facilitated by the presence of DNA-binding competent CHBF (Fig. 8C).

Ku autoantigen, which preferentially binds double-stranded DNA ends, has a K value of 15-20 10M(12) . Compared to K of 8.3 10M of HSF1(39) , the DNA-binding affinity of CHBF is approximately 2 orders of magnitude higher than that of HSF1. Our in vitro observations with the gel mobility shift assay also imply that the DNA binding affinity of CHBF is higher than that of HSF1. Replacement of a DNA-binding factor with a higher DNA-binding affinity known as ``squelching'' is a common phenomenon, as shown by steroid hormone receptor-DNA binding (40) and enhancer-binding protein/Adh promoter interaction(41) . As suggested by Hoff et al.(42) , Ku protein might play either positive or negative regulatory roles in transcription depending on parameters such as the abundance of Ku protein, or the presence of other DNA-binding factors with higher affinity to the DNA sequence. It is plausible that CHBF (or Ku protein) has similar roles in the regulation of heat shock gene expression.

We have performed additional in vivo experiments to assess whether the appearance of the Ku autoantigen complex may play a role in the regulation of heat shock response. The Ku-70 and Ku-80 cDNA-containing expression vectors were co-transfected into Rat-1 cells with the NLuc hsp70-luciferase plasmid, which contains the mouse hsp70 promoter upstream of the firefly luciferase gene. The relative ability of overexpressed Ku protein to suppress heat-induced transcription was determined by comparing the heat induction of luciferase activities to Rat-1 cells transfected with only the hsp70-luciferase construct. Co-transfection of Ku-70 plus Ku-80 expression vectors with hsp70-luciferase resulted in an average of 4-fold lower induction of luciferase activity relative to cells transfected with only the hsp70-luciferase. These results demonstrate that heat-induced transcriptional activation of hsp70-luciferase can be modified in vivo by the overexpression of Ku protein. These findings provide support for a role of Ku protein in the regulation of heat shock response in vivo.

The mechanism for a role of CHBF/Ku in the regulation of heat shock response in vivo may be much more complicated; because there are many unknown DNA-binding factors, the chromosomal structures are enormously complex, and the mechanisms and components involved in eukaryotic transcription are still obscure. However, it is plausible that HSF1 and CHBF are both involved in a dual control mechanism of the heat shock response. Consistent with the transient co-transfection experiments with human Ku gene and hsp70-luciferase reporter gene constructs, we have also shown that the heat-induced expression of hsp70 gene is significantly repressed in Rat-1 cells stably and constitutively overexpressing the 70-kDa subunit of human Ku autoantigen. Taken together, these data suggest that CHBF (or Ku protein) plays a role in the modulation of the heat shock response in vivo(43) . However, it is not known whether CHBF (or Ku protein) exerts roles by simply competing for DNA sequences in the hsp70 promoter region, by itself as a regulatory transcription factor, or through phosphorylation of other DNA-binding factors such as Sp1, HSF1, or RNA POL-II that are involved in heat shock gene expression.


FOOTNOTES

*
This work was supported in part by Grants CA 31397 and CA56909 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Depts. of Medical Physics and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-6028; Fax: 212-639-2611.

The abbreviations used are: HSE, heat shock element; hsp, heat shock protein; HSF, heat shock factor; HSF1, heat shock factor 1; CHBF, constitutive heat shock element-binding factor; ss, single-stranded; ds, double-stranded; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride.

G. C. Li, unpublished results.


ACKNOWLEDGEMENTS

We greatly appreciate Dr. Nancy Thompson (University of Wisconsin, Madison), Dr. J. Wang and Dr. W. H. Reeves (University of North Carolina) for providing the anti-Ku monoclonal antibodies, Dr. Carl Wu (National Institutes of Health) for providing the DNA and the antisera for human HSF1, Dr. Arik Dvir (University of Colorado at Boulder) for providing the purified human Ku autoantigen, and Dr. William Lee (University of Pennsylvania) for providing the expression vector with histidine tag. We especially thank Pat Krechmer for preparation of this manuscript and Patricia Park for excellent technical assistance.


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