![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 12, No. 5, pp. 654-666, March 1, 1998
1 Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, Illinois 60208 USA; 2 Biotechnology Research Institute, National Research Council, Montreal, Canada
 |
Abstract |
|---|
Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
|
|---|
The rapid yet transient transcriptional activation of heat shock genes is mediated by the reversible conversion of HSF1 from an inert negatively regulated monomer to a transcriptionally active DNA-binding trimer. During attenuation of the heat shock response, transcription of heat shock genes returns to basal levels and HSF1 reverts to an inert monomer. These events coincide with elevated levels of Hsp70 and other heat shock proteins (molecular chaperones). Here, we show that the molecular chaperone Hsp70 and the cochaperone Hdj1 interact directly with the transactivation domain of HSF1 and repress heat shock gene transcription. Overexpression of either chaperone represses the transcriptional activity of a transfected GAL4-HSF1 activation domain fusion protein and endogenous HSF1. As neither the activation of HSF1 DNA binding nor inducible phosphorylation of HSF1 was affected, the primary autoregulatory role of Hsp70 is to negatively regulate HSF1 transcriptional activity. These results reveal that the repression of heat shock gene transcription, which occurs during attenuation, is due to the association of Hsp70 with the HSF1 transactivation domain, thus providing a plausible explanation for the role of molecular chaperones in at least one key step in the autoregulation of the heat shock response.
[Key Words: Transcriptional control; autoregulation; heat shock proteins; Hsp70; activation domain]
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
|
|---|
The cellular response to diverse forms of environmental and
physiological stress, including heat shock, heavy metals, oxidants, UV,
cytokines, or hormones, involves the rapid transcriptional induction of
target genes whose activity is regulated by different stress-specific
transactivators (Baeuerle 1995
). The molecular response to each of these stresses serves to protect the cell against
lethal exposures to the same and other potentially deleterious forms of
stress. The transcription of heat shock genes, for example, is rapidly
induced yet persists at maximal levels transiently and attenuates
either proportionally to the intensity of the stress or upon return to
control conditions (DiDomenico et al. 1982a,b; Mosser et al. 1988;
Straus et al. 1990; Abravaya et al. 1991). In higher eukaryotes, the
stress-induced component(s) of the heat shock transcriptional response
are principally heat shock factors (HSFs), which are ubiquitously
expressed and maintained in unstressed cells in an inert
non-DNA-binding state. Upon exposure of cells to seemingly diverse
stress conditions, HSFs become activated to a DNA-binding,
transcriptionally active state, which results in the preferential
transcription of heat shock genes (Mosser et al. 1988; Morimoto et al.
1990; Lis and Wu 1993; Morimoto 1993; Rabindran et al. 1993; Sarge et
al. 1993; Westwood and Wu 1993; Wu 1995).
The heat shock response provides the cell with a mechanism to
reestablish protein homeostasis, the balance between protein synthesis,
protein folding and assembly, and protein degradation. Whereas exposure
to elevated temperatures results in a response that attenuates upon
prolonged heat shock or recovery, other inducers of the heat shock
response, such as amino acid analogs, activate heat shock gene
expression when the analogs are incorporated into nascent polypeptides.
Under the latter conditions, heat shock genes are constitutively
up-regulated and attenuation does not occur (DiDomenico et al. 1982b;
Mosser et al. 1988). These observations, among others, have led to a
widely held proposal that the heat shock response is autoregulated by
heat shock proteins. This is additionally supported by genetic evidence
that mutations in yeast Hsp70 result in the overexpression of heat
shock genes (Craig and Jakobsen 1984; Craig and Gross 1991) and are
dependent on the HSF binding site to DNA (Boorstein and Craig 1990),
and biochemical evidence that Hsp70 interacts with HSFs (Abravaya et
al. 1992; Baler et al. 1992; Mosser et al. 1993; Rabindran et al.
1994). It has been unclear, however, which of the many events in HSF regulation
including trimerization, DNA binding, inducible
phosphorylation, or stress-induced transcriptional activationwere
affected by Hsp70. As yeast HSF is constitutively trimeric and does not
convert to non-DNA-binding monomer during attenuation, the principal
form of regulation is likely to be at the level of transcriptional activation (Sorger et al. 1987; Jakobsen and Pelham 1988; Sorger and
Nelson 1989; Boorstein and Craig 1990). A role for molecular chaperones
in the regulation of the heat shock response has also been shown in
Escherichia coli. The E. coli DnaK chaperone machine comprised of DnaK, DnaJ, and GrpE negatively regulates the
transcription of heat shock genes by direct interaction with
32 (Tilly et al. 1983; Grossman et al. 1987; Straus et
al. 1990; Liberek et al. 1992; Blaszczak et al. 1995; Gamer et al. 1996).
In this study we demonstrate that Hsp70 stably associates in vivo and in vitro with the transactivation domain of HSF1. The consequence is to negatively regulate the transcriptional activity of HSF1 with little effect on the DNA-binding or inducibly phosphorylated state of HSF1, thus indicating that molecular chaperone Hsp70 functions as a repressor of transcriptional activity of the heat shock-specific transactivator.
| |
Results |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
|
|---|
Hsp70 associates with the HSF1 transactivation domain
Attenuation of the heat shock transcriptional response occurs
during continuous exposure to intermediate heat shock conditions or
upon recovery from stress (Abravaya et al. 1991). A characteristic feature of attenuation is the rapid repression of heat shock gene transcription, which precedes the conversion of HSF1 trimers to monomers, and the loss of HSF1 DNA-binding activity (Abravaya et al.
1991; Kline and Morimoto 1997). The kinetics of these complex events
have suggested a role for other proteins that could act directly on the
HSF1 activation domain, perhaps to repress its transcriptional
activity. Therefore, we screened for potential regulatory proteins that
interact with the HSF1 transactivation domain (amino acids 395-503)
using a direct protein-protein interaction assay. From an extract of
HeLa 35S-labeled proteins, four proteins ranging in size from
30 to 70 kD bound specifically with the wild-type HSF1 activation
domain fused to GST and not with GST alone or with a transcriptionally inert HSF1 activation domain deletion mutant (amino acids 451-503) (Fig. 1A). Hsp70 was identified as one of the
proteins that associated with the HSF1 activation domain (Fig. 1B). The
interaction with Hsp70 appears selective, as Hsp90 was not detected in
the collection of activation domain-binding proteins (data not shown).
|
To further examine the interaction of the HSF1 transactivation domain
with Hsp70, cells transiently overexpressing GAL4-HSF1 and Hsp70 were
assayed by immunoprecipitation analysis using antibodies specific to
Hsp70. The Hsp70-containing immunocomplexes were analyzed by Western
blot analysis for the presence of HSF1. Both the transfected GAL4-HSF1
activation domain fusion protein and endogenous HSF1 were among the
Hsp70-associated proteins (Fig. 1C). This confirms previous results
that Hsp70 interacts with HSF1 (Abravaya et al. 1992; Baler et al.
1992; Rabindran et al. 1994) and demonstrates that a specific site for
Hsp70 interaction is the HSF1 activation domain. The association of
Hsp70 with HSF1 was also detected by coimmunoprecipitation with
HSF1-specific antibody during a 4-hr heat shock time course. During
attenuation and recovery from heat shock, increased levels of Hsp70
were associated in complexes with HSF1 (Fig. 1D).
HSF1 activation domain interacts directly with Hsp70 and Hdj1
To examine whether the interaction of Hsp70 with the HSF1
activation domain is direct or involves other proteins, purified recombinant human Hsp70 was incubated with the GST-HSF1 activation domain and examined for complex formation. Hsp70, in the absence of
other proteins, interacts directly with the HSF1 activation domain
(Fig. 2A). The HSF1 activation domain also binds to
the Hsp70 homologs, Hsc70 (Fig. 2A) and DnaK (data not shown), but not
with an Hsp70 AAAA mutant (Fig. 2A) that has the last four amino acids
of Hsp70 mutated from EEVD to AAAA and is deficient in chaperone
function (Freeman et al. 1995). To further examine the specificity of
the interaction, other chaperones including Hdj1 and Hsp90 were studied
in the binding assay. Hdj1 also interacts directly with the HSF1
activation domain, but Hsp90 does not (Fig. 2B). A characteristic
feature of Hsp70-substrate binding is ATP-mediated substrate release
(Pelham 1986; Clarke et al. 1988; Flynn et al. 1989; Kost et al. 1989;
Beckmann et al. 1990; Palleros et al. 1991). To examine the effect of
ATP on Hsp70-HSF1 activation domain interaction, a similar binding
assay was performed in the absence or presence of ATP, or its
nonhydrolyzable analog ATP-
s. The HSF1-Hsp70 complexes were
dissociated upon addition of ATP and unaffected by ATP-s (Fig.
2C), similar to the Hsp70-substrate interaction. The HSF1 activation
domain also interacts with Hdj-1, however the HSF1-Hdj1 complexes were
insensitive to nucleotide (Fig. 2C), consistent with other observations
on the ATP-insensitivity of DnaJ-substrate interaction (Wawrzynow and
Zylicz 1995).
|
The domain of Hsp70 that interacts with HSF1 was subsequently
identified in binding assays with purified full-length Hsp70, the
amino-terminal ATPase domain, or the carboxyl-terminal substrate binding domain (Fig. 3A). Only full-length Hsp70 and
the substrate binding domain interact with the HSF1 activation domain;
no binding of the Hsp70 ATPase domain to the HSF1 activation domain was
detected (Fig. 3B). These results, together with the ATP sensitivity of the HSF1-Hsp70 complexes (Fig. 2C), indicate that the interaction between HSF1 activation domain and Hsp70 has the features of a substrate-chaperone complex and is distinct from cochaperone-Hsp70 interactions that predominantly occur through the ATPase domain of
Hsp70 (Hohfeld et al. 1995; Tsai and Douglas 1996).
|
The region of the HSF1 activation domain that interacts with Hsp70 was identified using in vitro binding assays. A collection of GST-HSF1 activation domain fusion proteins, including the wild-type activation domain I (amino acids 395-503) and deletion mutants II (amino acids 411-503), III (amino acids 425-503), IV (amino acids 439-503), and V (amino acids 451-503) (Fig. 4A), were purified as recombinant proteins (Fig. 4C) and incubated with HeLa whole cell extracts containing Hsp70 or separately with recombinant Hsp70. The wild-type activation domain I and deletion mutants II and III were associated with Hsp70, whereas deletion mutants IV and V did not bind to Hsp70 (Fig. 4B; data not shown). These results delimit the Hsp70-binding site to amino acid residues 425-439 of the HSF1 activation domain.
|
Hsp70 and Hdj1 negatively regulate the transcriptional activation property of HSF1
Having demonstrated that Hsp70 and Hdj1 interact directly with HSF1, we addressed whether these interactions affect HSF1 transcriptional activity. Overexpression of either chaperone by transfection was employed to examine the effect of elevated expression of the chaperone independent of other consequences of the heat shock response. The transcriptional activity of the HSF1 activation domain fused to the GAL4 DNA-binding domain, as measured by CAT activity from the G5BCAT reporter, was repressed four- to fivefold when coexpressed with a vector that expresses high levels of Hsp70 (Fig. 5A, i and iii). As neither the level of GAL4-HSF1 fusion protein (data not shown) nor its DNA-binding activity (Fig. 5A, ii) was affected, we conclude that Hsp70 functions principally as a negative regulator of the transactivation domain of HSF1.
|
The negative effect of Hsp70 on HSF1 transcriptional activity requires
that the chaperone is functional. The Hsp70 AAAA mutant, which lacks
chaperone activity, does not bind to the HSF1 activation domain (Fig.
2A) and consequently does not repress HSF1 transcriptional activity
(Fig. 5B). Overexpression of Hsp70 neither represses the expression of
a cotransfected RSV-
-gal control (data not shown) nor the
transcriptional activity of the GAL4-VP16 activation domain (Fig. 5C)
and other GAL4-activation domains (data not shown). These results
indicate that the negative effects of Hsp70 are specific to the HSF1
transactivation domain.
In parallel experiments, overexpression of Hdj1 also resulted in the negative regulation of HSF1 transcriptional activity. Coexpression of Hdj1 with GAL4-HSF1 activation domain resulted in a fourfold repression of the transcriptional activity of GAL4-HSF1 (Fig. 6A). As observed for Hsp70, Hdj1 did not repress transcriptional activity of the GAL4-VP16 activation domain (Fig. 6B).
|
Autoregulation of hsp90 and hsp70 gene transcription in cells conditionally expressing Hsp70
If Hsp70 negatively regulates HSF1 transcriptional activity, it
should be possible to control the levels of Hsp70 and examine the
effects of increased levels of Hsp70 on the transcriptional activity of
HSF1. To accomplish this, we used a stably transfected cell line
(PETA70) conditionally expressing human Hsp70 under the control of a
tetracycline-regulated system (Mosser et al. 1997). PETA70 cells were
cotransfected with the GAL4-HSF1 activation domain, G5BCAT reporter,
and RSV--gal internal control. Upon induction and accumulation of
Hsp70, GAL4-HSF1 activity, measured by the expression of a CAT
reporter, was repressed fourfold (Fig. 7A,C). No
detectable changes in the levels of GAL4-HSF1 DNA-binding activity
were observed (Fig. 7B); these results demonstrate that the effect of
Hsp70 is on the transactivation activity of HSF1.
|
To address whether the effects of elevated levels of chaperone on HSF1
transcriptional activity were physiologically relevant to that achieved
during heat shock, we examined the effects of increased levels of Hsp70
on inducible transcription of the endogenous heat shock genes. In
PETA70 cells expressing elevated levels of Hsp70 (Fig. 8E), the
transcription of endogenous heat shock genes was not induced upon heat
shock; this result was in striking contrast to the typical pattern of
heat shock-induced transcription of the hsp70 and
hsp90
genes in uninduced
cells. For example, in Hsp70-overexpressing cells,
transcription of the hsp90 gene was not induced
following heat shock, whereas in a parallel experiment using cells
uninduced for Hsp70, there was a dramatic heat shock induction of
hsp90 gene transcription (Fig. 8A). These
results clearly establish that Hsp70 negatively regulates heat shock
gene transcription. Assessing this for the hsp70 gene was not
possible as the PETA70 cells contain multiple copies of the
hsp70 gene under the tetracycline-inducible promoter, thus
accounting for high basal hsp70 gene transcription. However,
consistent with the results obtained for the
hsp90 gene, heat shock did not enhance the
transcription of the hsp70 gene and noticeably reduced the signal, presumbly because of the general repressive effects of heat
shock on transcription (Fig. 8A). Nevertheless, in the absence or
presence of Hsp70 overexpression, HSF1 acquired high DNA-binding activity and was inducibly phosphorylated (Fig. 8B,D), suggesting that
Hsp70 primarily affects HSF1 transcriptional activity.
|
Previously, we had established that Hsp70 was detected in a complex with HSF1 (Fig. 1). To determine whether Hsp70 overexpressed in the PETA70 cells was associated with HSF1, we performed gel mobility-shift assays using whole cell extracts. HSF-HSE (heat shock element) complexes in extracts from heat-shocked cells expressing high levels of Hsp70 exhibited a slower electrophoretic mobility compared to that of heat-shocked cells without Hsp70 overexpression (Fig. 8B). Antibody supershift of HSF-HSE complexes from heat-shocked cells overexpressing Hsp70 detected a supershifted ternary complex (Hsp70:HSF1-HSE) with Hsp70-specific antibody, whereas no such complex was detected in extracts from heat-shocked cells without Hsp70 overexpression (Fig. 8C). The HSF1-Hsp70 complexes were disrupted upon addition of ATP and resulted in a faster migrating HSF-HSE complex, which could not be supershifted by Hsp70-specific antibody (Fig. 8C), corroborating the in vitro data that HSF-Hsp70 complexes are ATP sensitive (Fig. 2C).
To further establish that these chaperone-HSF1 interactions and
subsequent effects on HSF1 activity are specific, similar experiments
were performed using a stably transfected human cell line
(PERTA70-AAAA) conditionally overexpressing a nonfunctional Hsp70 AAAA
mutant (Fig. 9A). Upon overexpression of the Hsp70 AAAA mutant to a level similar to that obtained for wild-type Hsp70
(Fig. 9E), neither heat-shock induced transcription of the hsp90 gene (Fig. 9B) nor HSF1 DNA-binding
activity (Fig. 9C) was affected. Antibody supershift of HSF-HSE
complexes with Hsp70-specific antibody did not detect any HSF-HSP70
complexes in either uninduced or AAAA-expressing cells (Fig. 9D), in
contrast to that from wild-type Hsp70-expressing cells (Fig. 8C). These
results further indicate that the transcriptional regulation by Hsp70
requires its chaperone function for direct binding to HSF1.
|
Is the transcriptional repression by Hsp70 dependent on a specific
level of Hsp70? To assess the levels of Hsp70 necessary to block heat
shock gene transcription, the PETA70 cells were induced over a period
of 48 hr to accumulate Hsp70. During this period, aliquots of cells
were removed at various time points, exposed to heat shock, and
examined for the levels of Hsp70, transcription of endogenous heat
shock genes, and HSF1 DNA-binding activity. Relative to the typical
pattern of heat shock transcription observed in the nonoverexpressing
cells (maintained in the presence of tetracycline), the transcription
of the endogenous hsp90 gene was reduced 6-fold
after 12 hr of Hsp70 induction, an 11-fold repression was observed
after 24 hr, and a 25-fold repression after 48 hr (Fig.
10A). During this period, the levels of HSF1 DNA
binding were initially unaffected and reduced by 25% at later time
points (Fig. 10A). The levels of Hsp70 induced over this period were
measured by Western blot analysis and compared to the levels of Hsp70
accumulated during attenuation of the heat shock response (4-hr heat
shock). The level of Hsp70 achieved during attenuation was equivalent
to that achieved upon 12-hr tetracycline induction (Fig. 10B) and
corresponds to a 300-fold excess of Hsp70 to HSF1. These results reveal
that Hsp70 primarily affects the transcriptional activity of HSF1 with
only modest effects on HSF1 DNA-binding activity.
|
In summary, the results presented here indicate that the attenuation of the heat shock transcriptional response is a multistep event in which the elevated synthesis and accumulation of Hsp70 lead directly to the binding of Hsp70 to the HSF1 activation domain, and result in the repression of heat shock-induced transcription. Subsequent to transcriptional arrest is the conversion of HSF1 trimers to monomers and the loss of HSF1 DNA binding.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
|
|---|
The transcription of heat shock genes is induced transiently upon continuous exposure to elevated temperatures. Attenuation of the inducible response reveals that even in the presence of the stress signal, the elevated expression of heat shock genes either dampens the signal or negatively regulates components of the activation pathway. Conditional overexpression of Hsp70 in human cells is by itself sufficient to repress heat shock-induced transcription. These results, together with the in vivo and in vitro demonstration that chaperones associate directly with the HSF1 transactivation domain, establish a role for Hsp70 (and other chaperones) as transcriptional repressors.
A role for molecular chaperones in the autoregulation of the heat shock
response is consistent with observations in Drosophila and
other organisms in which exposure to amino acid analogs leads to
continuous activation of heat shock gene expression (DiDomenico et al.
1982b; Mosser et al. 1988). The incorporation of amino acid analogs
into nascent polypeptides that consequently do not fold to the native
state sequesters the preexisting Hsp70 into futile cycles of
chaperone-substrate interactions rather than the transient
interactions between early folding intermediates and Hsp70 (Beckmann et
al. 1990). Furthermore, as the Hsp70 synthesized during amino acid
analog-induced stress is itself misfolded due to incorporation of amino
acid analogs, the newly synthesized chaperone is nonfunctional and
cannot repress HSF activity. Consequently, HSF, which is a stable
protein and not dependent on continuous protein synthesis, remains in a
transcriptionally active state (Zimarino and Wu 1987; Amici et al. 1992).
The consequence of elevated levels of Hsp70 appears specific to
transcriptional repression of heat shock genes with little or no
immediate effect on other features of HSF1 such as DNA-binding or
stress-induced phosphorylation. This reveals that the attenuation phase
of the heat shock response is comprised of distinct events in which the
rate of heat shock gene transcription can be uncoupled from the
DNA-binding and inducibly phosphorylated state of HSF1. These
conclusions are independently supported by a detailed kinetic analysis
of heat shock gene transcription, HSF1 DNA binding, and inducible
phosphorylation, which showed that the rate of heat shock gene
transcription declined prior to the loss of HSF1 DNA-binding activity
(Kline and Morimoto 1997). Likewise, upon exposure to transient heat
shock (42°C) and recovery at 37°C, heat shock gene transcription
was rapidly repressed, yet HSF1 DNA-binding activity was sustained as
measured by in vitro gel mobility shift assay and by in vivo
footprinting assay (Abravaya et al. 1991). The uncoupling of HSF1 DNA
binding from transcriptional activity noted here during the attenuation
of the heat shock response has also been observed during activation of
HSF1 by anti-inflammatory drugs. Under these conditions activation of
HSF1 leads to a DNA-binding-competent form that lacks transcriptional
activity (Jurivich et al. 1992; Giardina and Lis 1995; Cotto et al. 1996).
Although our data are consistent with a role for Hsp70 as a
transcriptional repressor, we are not proposing a novel activity for
Hsp70 that is distinct from its properties in other
chaperone-substrate complexes. The demonstration that overexpression
of Hsp70 does not directly affect the acquisition of HSF1 DNA-binding
activity is consistent with previous studies in rat M21 and TTRat1
cells and in Drosophila TTSL2 cells in which the constitutive
overexpression of Hsp70 did not interfere with the activation of HSF1
DNA-binding activity (Rabindran et al. 1994). Increased levels of Hsp70
had a slight effect on the kinetics of deactivation of HSF1 DNA-binding activity (Mosser et al. 1993; Rabindran et al. 1994). The regulation of
heat shock genes in the yeast Saccharomyces cerevisiae
involves repression of the transcriptional activity of HSFs independent of effects on the constitutively trimeric form of HSFs (Boorstein and
Craig 1990). The convergence of genetic and biochemical observations, from yeast to humans, reveals that the transcriptional activity of HSF
is intimately linked to autoregulatory aspects of Hsp70.
Although much of the emphasis on the role of heat shock proteins in the
regulation of the heat shock response has centered on Hsp70, our data
also reveal a role for other molecular chaperones such as Hdj1. The
interaction of Hdj1 with HSF1 in higher eukaryotes and its repressive
effect on HSF1 transcriptional activity is supported in yeast by the
observation that the S. cerevisiae DnaJ homolog SIS1
negatively regulates its own expression. However, SIS1 autoregulation
requires the HSE and other sequences, suggesting that additional
regulatory molecules might be involved (Zhong et al. 1996). HSF may
also associate with Hsp90; however this result seems variable as such
associations have been detected with yeast, rat, and rabbit Hsp90
(Nadeau et al. 1993; Nair et al. 1996) but not with human Hsp90 (this
study; Baler et al. 1992; Rabindran et al. 1994). A related issue is
whether autoregulation requires elevated levels of specific chaperones
or that the concentration of one or more chaperones increases
substantially over a relatively short period of time to signal
attenuation. Although the data presented here provide a clear
demonstration that transcriptional repression occurs when a certain
level of Hsp70 is attained, the constitutively expressed member of the
Hsp70 family, Hsc70, is typically expressed at high levels in human
cells. However, because Hsc70 is normally engaged in a variety of
cellular activities, including protein synthesis, protein assembly and
translocation, and protein degradation, presumably it is not available
to negatively regulate HSF1.
Regardless of how chaperones lead to inaccessibility of the HSF1
activation domain, Hsp70 and other molecular chaperones protect the
cell against the deleterious effects of protein aggregates by
stabilizing and refolding stress-induced folding intermediates. This
suggests that an equilibrium must exist between protecting proteins
against stress-induced damage and the activation and attenuation of the
heat shock transcriptional response. Although it is unclear which
component(s) of the basal transcriptional machinery contacts HSF1 to
effect the high level of inducible transcription, these interactions
are also likely to be transient. As the levels of Hsp70 increase in the
nuclear compartment during heat shock (Velazquez et al. 1980; Welch and
Feramisco 1984) or by conditional overexpression (data not shown), HSF1
is provided with an alternative interaction leading to the
Hsp70-dependent repression of heat shock gene transcription. A model of
the regulation of HSF1 transcriptional activity by molecular chaperones
is presented in Figure 11. It is striking that the
effects of overexpression of Hsp70 are specific to heat shock
transcription and that the formation of stress-induced phosphorylated
HSF1 trimers are unaffected. Presumably other stress-induced events,
perhaps working in conjunction with Hsp70, are important for
dephosphorylation and conversion of HSF1 trimers to monomers.
|
Although our data do not reveal how the interaction between a chaperone
and the activation domain of HSF1 leads to transcriptional repression,
the presence of a chaperone-binding site within the activation domain
could either competitively sequester the contact site(s) between HSF1
and the transcriptional apparatus or lead to a chaperone-dependent
change in the conformation of HSF1. In either case, the activation
domain is rendered inaccessible to the transcriptional machinery. This
shares certain parallels with the role of the DnaK and DnaJ chaperones
in the regulation of RepA (Wickner et al. 1991) and in phage 
replication (Georgopoulos 1990). In the former case, chaperones
function as a conformational activator of RepA, and in the latter
case, the chaperones interfere with the interaction of P protein
to dnaB, thus allowing unwinding of the phage chromosome. A
role for chaperones as regulators of transcriptional activators has
also been described for the family of steroid aporeceptors, although in
this case multiple chaperones are recruited to maintain the activator
in a repressed state by formation of a stable chaperone-substrate
complex (Bohen and Yamamoto 1994). It is tempting to consider that the
interactions observed between p53 and Hsp70 (Hsc70) could also reflect
a form of chaperone-dependent regulation of a transcriptional activator
(Hupp et al. 1992).
| |
Materials and methods |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
|
|---|
Plasmid constructions and protein purification
The GST-HSF1 activation domain (amino acids 395-503) fusion
construct was created by EcoRI digestion of the corresponding GAL4-HSF1 fusion construct X (Shi et al. 1995), which has two EcoRI sitesone located in the polylinker region between the
GAL4 DNA-binding domain and the HSF1 activation domain coding sequence, and the other located beyond the HSF1 stop codonsto generate a 640-bp
fragment containing the HSF1 activation domain-coding region. The
640-bp EcoRI fragment was ligated into EcoRI-digested pGEX-5X-1 vector (Pharmacia-LKB). Constructs of the HSF1 activation domain-GST fusion deletion mutants II, III, IV, and V were created similarly from corresponding GAL4-HSF1 fusion constructs XI, XII, XIII, and XIV (Shi et al. 1995). All constructs were sequenced across
the junction between the GST and HSF1 sequences to ensure that the HSF1
reading frame was maintained and to confirm the boundaries of the
deletion mutants. -Actin hsp70 AAAA was constructed by
PCR mutating the last 4 amino acids of Hsp70 from EEVD to AAAA with
plasmid pH2.3 (Wu et al. 1985) as a PCR template and creating a
BamHI site at the 5
end and an EcoRI site,
followed by a BamHI site at the 3 end of the PCR product.
The PCR product was digested with BamHI and ligated into
BamHI-digested pH-actin-1-neo vector (Gunning
et al. 1987) so that the amplified hsp70 AAAA was under the
control of the human -actin promoter. Mutation of the residues was
confirmed by sequencing. -Actin hdj1 was created by PCR
with pGEX-hdj1 (B.C. Freeman, unpubl.) as a template to
create a BamHI site at each end of the amplified human
hdj1 DNA fragment, and the BamHI-digested PCR product
was ligated into BamHI-digested pH-actin-1-neo
vector to be downstream of the human -actin promoter. All DNA
fragments that were amplified by PCR were sequenced in their entirety
to ensure that mutations had not occurred randomly. pTR-DC/hsp70-gfp was created as described
(Mosser et al. 1997). pTR-DC/hsp70 AAAA-gfp was
created by ClaI and EcoRI digestion of -actin
hsp70 AAAA, and the 490-bp ClaI-EcoRI
fragment containing the AAAA mutation was ligated into ClaI
and EcoRI partially digested pTR-DC/hsp70-gfp to replace the corresponding
wild-type fragment of hsp70. The purification of GST fusion proteins,
wild-type or mutant Hsp70, Hsc70, Hdj1, and Hsp90, has been described
(Freeman et al. 1995; Freeman and Morimoto 1996).
GST in vitro binding assays
HeLa whole cell extracts or purified recombinant proteins were
incubated with purified GST or GST-HSF1 fusions fixed on
glutathione-agarose beads. The beads were washed four times in 100 bed
volumes of NETN buffer (0.5% NP-40, 0.1 mM EDTA, 20 mM Tris at pH 7.4, 300 mM NaCl) for HeLa whole cell
extracts, and TEN buffer (20 mM Tris at pH 7.4, 0.1 mM EDTA, 100 mM NaCl) for purified proteins. The bound proteins were eluted by boiling in SDS-sample buffer and analyzed by SDS-PAGE and Coomassie blue staining or Western blot analysis. To examine the nucleotide effect on the interaction, purified
recombinant Hsp70/Hdj1 proteins were incubated with
GST-HSF1 activation domain in the absence or presence of 1 mM ATP, or 1 mM ATP-s in TEK-Mg buffer (20 mM Tris at pH 7.4, 0.1 mM EDTA, 50 mM
KCl, 5 mM MgCl2).
Cell culture, metabolic labeling, and transient and stable transfection
HeLa cells were grown in Dulbecco's modified Eagle medium (DMEM)
supplemented with 5% calf serum (GIBCO BRL) in a humidified 5%
CO2 incubator at 37°C. For labeling, cells were washed
with methionine-deficient DMEM and labeled for 12 hr in 1 ml of the same medium supplemented with 100 µCi of Tran35S-label
(ICN), then washed with 1× PBS and harvested. COS-7 cells were grown
in DMEM supplemented with 10% calf serum in a 37°C incubator with
5% CO2. Cells at a density of 40%-50% confluence were
transfected by the calcium phosphate precipitation method (Shi et al.
1995). In all of the transient transfection experiments, cells were
cotransfected with 1 µg of GAL4-HSF1 activation domain plasmid, 2 µg of G5BCAT reporter plasmid, 1 µg of RSV--gal internal control plasmid, and various amounts of Hsp70- or Hdj1-expressing plasmid as indicated in the legends to Figures 5 and 6. PETA70 cells
(Mosser et al. 1997) were grown in RPMI 1640 supplemented with 10%
fetal calf serum (Sigma), 2 mM L-glutamine (GIBCO
BRL), 200 µg/ml of G418 (GIBCO BRL), 100 µg/ml of hygromycin (Sigma), and 10 ng/ml of anhydrotetracycline (Acros Organics).
Anhydrotetracycline was withdrawn from the media to induce Hsp70
expression. PETA70 cells were transfected by electroporation (Mosser et
al. 1997). The PERTA70-AAAA cell line was generated by cotransfection
of pTR-DC/hsp70 AAAA-gfp plasmid with the
plasmid ptk/hygro into PEER cells expressing the
reverse tetracycline-controlled transactivator (rtTA) (Gossen et al.
1995). Hygromycin-resistant cells (200 µg/ml) were
selected in batch culture, and cells with tetracycline-regulatable expression of the Hsp70 AAAA mutant protein were selected by adding the
tetracycline derivative doxycycline (Sigma) into media followed by flow
cytometric cell sorting of the GFP-positive cells as described (Mosser
et al. 1997). PERTA70-AAAA cells were grown in RPMI 1640 supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 200 µg/ml of G418, and 100 µg/ml of
hygromycin. Doxycycline (1 µg/ml) was added to the
media to induce the expression of the Hsp70 AAAA mutant.
CAT, gel mobility shift, Western blot, and immunoprecipitation assays
Conditions for the CAT assay and GAL4 gel mobility shift assay
were as described previously (Shi et al. 1995). The HSF1 gel mobility
shift assay was as described (Mosser et al. 1988). The antibody
supershift assay was performed with Hsp70-specific antibody C92
(Amersham) as described (Abravaya et al. 1992). Western blot analysis
for HSF1 was performed with HSF1-specific polyclonal antibody (Sarge et
al. 1993) or monoclonal antibody 4B4 (Cotto et al. 1997). Western blot
analysis for Hsp70 was performed with Hsp70-specific monoclonal
antibody 3A3, 5A5, 4G4 (S.P. Murphy, unpubl.), or C92 (Amersham).
Western blot analysis for Hdj1 was performed with Hsp40-specific
polyclonal antibody (Hattori et al. 1992). The condition for Western
blot analysis was as described (Sarge et al. 1993). Monoclonal
antibodies 3A3 and 5A5 recognize both Hsp70 and Hsc70. Monoclonal
antibodies 4G4 and C92 specifically recognize Hsp70 but not the other
members of the 70-kD heat shock proteins. Immunoprecipitations were
carried out with either monoclonal antibody specific to Hsp70 (C92) or
polyclonal antibody specific to HSF1 as described (Kline and Morimoto 1997).
Transcriptional run-on analysis
Run-on transcription reactions were performed with isolated cell
nuclei in the presence of 50 µCi of [-32P]UTP
(Amersham) as described previously (Banerji et al. 1984). Radioactive
RNA was hybridized to DNA probes for the human hsp70 gene (pH
2.3) (Wu et al. 1985), the human hsp90 gene
(pUC801) (Hickey et al. 1989), pBR322 (Promega) as a control for
nonspecific hybridization, and the rat gapdh gene (Fort et al. 1985)
as a normalization control for transcription. The intensities of radioactive signals were quantitated by using a PhosphorImager (Molecular Dynamics).
| |
Acknowledgments |
|---|
We thank Brian Freeman and David Bimston for providing purified chaperone proteins and valuable discussion, Sue Fox for excellent technical assistance and comments, Jose Cotto for advice, and Sameer Mathur and Anu Mathew for comments. Antoine Caron provided assistance in the selection of stable cell lines, Steven Triezenberg (Michigan State University) provided the GAL4-VP16 (pSGVP) construct, James Douglas Engel (Northwestern University) provided the GAL4-GATA3 construct, and Kenzo Ohtsuka (Aichi Cancer Research Institute, Nagoya, Japan) provided the Hsp40 specific antibody. These studies were supported by a grant to R.M. from the National Institutes of Health. Y.S. is supported in part by a Gramm Travel Fellowship Award from the Lurie Cancer Center of Northwestern University.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
| |
Footnotes |
|---|
3 Corresponding author.
E-MAIL r-morimoto{at}nwu.edu; FAX (847) 491-4461.
| |
References |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials & Methods
References
|
|---|
70 and 32 factors assembled with RNA polymerase.
EMBO J.
14:
5085-5093[Medline]. 32.
EMBO J.
15:
607-617[Medline]. growth.
In Stress proteins in biology and medicine (ed. R.I. Morimoto,
A. Tissieres and
C. Georgopoulos), pp. 191-221. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli.
Genes & Dev.
1:
179-184-actin expression vector system directs high-level accumulation of antisense transcripts.
Proc. Natl. Acad. Sci.
84:
4831-483532 transcription factor.
Proc. Natl. Acad. Sci.
89:
3516-3520
AD) were incubated with 35S-labeled HeLa whole
cell extracts. Bound proteins were analyzed by SDS-PAGE and
fluorography. The sizes of the molecular weight markers (MW) are
indicated. (B) Hsp70 is among the HSF1 activation domain
interactive proteins. Proteins bound to GST, GST-AD, or GST-
) or together with the GAL4-HSF1 activation domain (GAL4-AD) construct (+). Cell lysates were immunoprecipitated with
Hsp70-specific antibody 3A3 and immunoblotted with HSF1-specific polyclonal antibody. Endogenous HSF1 and transfected GAL4-AD are indicated (right). An aliquot of the cell lysates (WCE) was
analyzed directly by Western blot as a control. (D) Increased
Hsp70 is associated with HSF1 during attenuation or recovery from heat shock. HeLa cells were heat-shocked at 42°C for 0, 15, 60, and 240 min, or at 42°C for 120 min and then allowed to recover at 37°C
for 240 min (R). Cell lysates were immunoprecipitated with HSF1-specific polyclonal antibody and immunoblotted with Hsp70-specific monoclonal antibody C92 or with HSF1-specific monoclonal antibody 4B4.
