From the
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.
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).
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
HeLa cells (1
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.
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
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-
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
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
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 N
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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) .
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.
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.
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.
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).
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.
-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
N
Luc (DNA ratio 1:1:1 or 5:5:1) or transfected with
N
Luc 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.
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.
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 the
P-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 N
Luc 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 N
Luc; 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.
value of 15-20
10
M(12) . Compared to K
of 8.3
10
M 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.
Luc 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.