From the Dana Farber Cancer Institute, Harvard Medical School, and
the Department of Medicine, Molecular and Cellular
Biology Laboratory, Beth Israel and Deaconess Medical Center, Harvard
Medical School, Boston, Massachusetts 02115
Received for publication, October 4, 2002, and in revised form, November 26, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heat shock factor 1 (HSF1), in addition to its
pivotal role as a regulator of the heat shock response, functions as a
versatile gene repressor. We have investigated the structural domains
involved in gene repression using mutational analysis of the
hsf1 gene. Our studies indicate that HSF1 contains two
adjacent sequences located within the N-terminal half of the protein
that mediate the repression of c-fos and c-fms.
One region (NF) appears to be involved in quenching transcriptional
activation factors on target promoters and binds to the basic zipper
transcription factor NF-IL6 required for activation of c-fms and
IL-1 Heat shock factor 1 (HSF1)1 is the regulator of
heat shock protein (hsp) gene transcription and controls the
response to protein stress conserved in eukaryotic cells (1-7). HSF1
senses exposure to stresses such as heat shock at least partially by
monitoring the presence of denatured and aggregated proteins in cells
(8, 9). Upon activation, HSF1 trimerizes and binds to the promoters of
hsp genes in a hyper-phosphorylated form competent to
activate transcription (2, 5, 10-14).
We have found that, in addition to activating the transcription of
hsp genes, HSF1 acts as a repressor of non-heat shock genes (15-21). Heat shock inhibits the transcription of many inducible genes
involved in macrophage activation and the acute phase response, including interleukin 1 In the work presented here we have examined the mechanisms of gene
repression by HSF1 using mutational analysis to map repression domains.
We have found that gene repression is conferred by sequences in the
N-terminal half of the protein and that the C terminus, which contains
the major trans-activation domains, does not play a major
role in repression. We have identified two regions in the N terminus of
HSF1 essential in the repression of c-fos and c-fms, including a region (amino acids 229-279) needed for
repression but not required for NF-IL6 binding and a sequence (amino
acids 106-205) essential for NF-IL6 binding. We next developed a
dominant interfering negative construct (HSF1-(1-205)) that
competitively inhibits c-fos repression by HSF1. This
construct was also found to inhibit repression of the
IL-1 Cells and Constructs--
Chinese hamster ovaricytes, CHO K1,
were obtained from the American Type Tissue Culture Collection and
maintained in Ham's F-12 medium supplemented with 10% fetal bovine
serum and 2.0 mM L-glutamine.
The c-fms promoter reporter gene, pGLfms,
contains the 500-bp promoter sequence of the murine c-fms
gene (21). The IL-1
The HSF1 expression plasmid, pcDNA3.1( Transfection Methods and Assays for Luciferase and
Western Analysis--
Nuclear or whole cell extracts were
prepared and subjected to SDS-PAGE by using standard methodology.
Proteins were then transferred electrophoretically onto polyvinylidene
difluoride (Immobilon) membranes (Millipore) as described (33). The
membranes were then blocked by incubation in 1× phosphate-buffered
saline containing 5% nonfat dried milk and incubated at 4 °C with a
specific antibody against the N terminus of HSF1 (for C-terminal
deletion mutants), against the C terminus of HSF1 (for chimeras and
mutants containing complete C terminus), or against His6
tag (for mutants with both N- and C-terminal deletions). The membranes
were then washed and incubated with a second antibody coupled to
horseradish peroxidase (Vector Laboratories). Antigen-antibody
complexes were detected by chemiluminescence (ECL, Amersham Biosciences).
In Vitro Transcription and Translation of HSF1 and
NF-IL6--
HSF1 and NF-IL6 were produced in vitro from
pcDNA3.1( EMSA--
Nuclear extracts were prepared from cells using
nuclear and cytoplasmic extraction reagents (Pierce). Briefly,
cells were incubated for 10 min on ice in 200 µl of CERI solution
containing 0.75 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml aprotinin and leupeptin, 20 mM NaF, and 2.0 mM Na3VO4. 11 µl of CERII
solution was than added, and cytoplasmic extracts were collected by
centrifugation at 12,000 × g for 5 min. Nuclear
pellets were lysed in 100 µl of nuclear extraction reagent
solution containing 2 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml aprotinin and leupeptin. Extracts were then aliquoted and
stored at
The oligonucleotide probes were synthesized and labeled by end
filling with 32P. Consensus HSE from human
hsp70A gene, 5'-CACCTCGGCTGGAATATTCCCGACCTGGCAGCCGA-3', was used in EMSA.
Each binding mixture (12 µl) for EMSA contained 2.0 µl of nuclear
extract or 10 µl of in vitro translated protein, 2.0 µg of bovine serum albumin, 2.0 µg of poly(dI-dC), 0.5-1.0 ng of labeled double-stranded oligonucleotide probe, 12 mM HEPES,
12% glycerol, 0.12 mM EDTA, 0.9 mM
MgCl2, 0.6 mM dithiothreitol, 0.6 mM phenylmethylsulfonyl fluoride, and 2.0 µg/ml aprotinin
and leupeptin (pH 7.9). Final concentrations of KCl in the binding mixture were defined for optimal binding of each oligonucleotide. Samples were incubated at room temperature for 15 min, and complexes were then analyzed by electrophoresis on 4.5% non-denaturing
polyacrylamide gels. The protein-DNA complexes were visualized by autoradiography.
In Vitro Protein Interaction Assay--
To produce GST fusion
proteins and control GST protein, 250-ml cultures of Escherichia
coli DH5 Functional Domains within HSF1 That Mediate Repression--
As our
previous studies showed that heat shock represses the transcription of
multiple genes, including c-fos, uPA,
IL-1
Our previous studies showed that heat shock also blocks the
transcription of the endogenous c-fms gene, and both heat
shock and HSF1 overexpression inhibit the activity of transfected
c-fms promoter reporter constructs (20, 21). We have
therefore carried out similar experiments on the c-fms
promoter to those on the c-fos promoter described above,
using experimental conditions defined in our earlier publication (21).
The results of the deletion experiments were essentially similar, in
that HSF1 strongly represses c-fms activity (Fig. 1C,
lanes 1 and 2), and sequences from HSF1 could be
deleted from the C terminus spanning codon 529 to codon 279 without
significant loss of ability to repress c-fms Fig. 1C
(lanes 2-7). Our previous experiments indicate that HSF1
does not affect expression of NF-IL6 and that repression is due to
interaction of HSF1 and NF-IL6 on the c-fms promoter (14).
c-fms repression was, however, partially reversed in some of
the C-terminal deletion mutants that contain a fragment of the
C-terminal trans-activation domain (Fig. 1C, lanes
3-6). We have shown that HSF1 physically binds to NF-IL6 and
antagonizes the trans-activation of IL-1
To test the role of the HSF1 REP domain as an essential repression
domain, we next investigated the effect of an HSF1 mutant with an
internal deletion of the majority of this domain (amino acids 215-278)
(Fig. 2). Our experiments show that
excision of this region results in a partial reversal of repression of
c-fos (by 70%) and c-fms (by 60%) promoter
activity, whereas the activation of hsp70B promoter was not
affected in this deletion mutant, which activates hsp70B
efficiently (lane 5, Fig. 2, B-D). (Compare
lane 1, which shows promoter activity in the absence of
HSF1, with lane 2, in the presence of wild-type HSF1, and
lane 5, in the presence of internally deleted
HSF1-(1-214)/-(279-529).) These experiments indicate that the amino
acid 215-278 region of HSF1 is important for
trans-repression but is dispensable in hsp70B trans-activation by HSF1 overexpression. We then tested the
potency of the REP region as a portable repression domain that could
function independently from the remainder of the HSF1 molecule. This
was tested by examining the ability of HSF2/HSF1 chimeras to repress c-fos and c-fms (Fig. 2). HSF2 was deemed a
suitable protein for these studies because it is a member of the
hsf family that lacks the ability to repress
c-fos or c-fms (compare lanes 2 and
3 in Fig. 2, B and C) (16, 21). We
have prepared chimeric constructs between the region of the
hsf2 gene encoding the N-terminal core (DNA binding
domain/trimerization domain, amino acids 1-222) and the test domains
derived from the hsf1 gene. HSF1 and HSF2 are most similar
in sequence in the N-terminal core domain, which contains the highly
conserved DNA binding and trimerization domains (6). As HSF1
trimerization is required for gene repression, we also chose the HSF2 N
terminus for its potential to supply an essential
trimerization/DNA binding domain that might be needed for gene
repression. However, chimeras containing HSF2-(1-222) fused to either
the REP domain (HSF2-(1-222)/HSF1-(225-281)) or the entire C terminus
of HSF1 (HSF2-(1-222)/HSF1-(225-529)) were equally ineffective in
repressing the c-fos and c-fms promoters. (Compare the effects of wild-type HSF1 (lane 2) with
HSF2-(1-222)/HSF1-(225-281) (lane 6) and
HSF2-(1-222)/HSF1-(225-529) (lane 7).) Similarly, HSF2-(1-222)/HSF1-(279-529) was also ineffective in repressing both
promoters (lane 8 in Fig. 2, B and C).
However, the mutants containing the complete
trans-activation domain of HSF1 (225-529 or 279-529) fused
to the N terminus of HSF2 activate the hsp70B promoter as
efficiently as the wild-type HSF1, indicating that the mutants were
appropriated folded in vivo and functioned effectively in
the context of the hsp70B promoter (Fig. 2D).
Conversely, replacing the entire HSF1 trans-activation
domain with the C-terminal 192 amino acid residues of HSF2,
HSF1-(1-327)/HSF2-(344-536), does not decrease the repression of
c-fos and c-fms but completely abolishes the
ability to activate hsp70B (Fig. 2). This result is
consistent with the C-terminal deletion experiments shown in Fig. 1,
when deletion of the trans-activation domain abolished activation of hsp70B but retained repression of
c-fos and c-fms. These findings therefore suggest
an essential role for residues within the N-terminal core domain of
HSF1 that are not conserved in HSF2 as well as the essential REP domain
(between amino acids 205 and 279) in c-fos and
c-fms repression but not in hsp70B activation (Fig. 2, B-D). Analysis of protein levels in the
transfectants indicates that HSF1 wild type, HSF1-(1-214)/-(279-529),
HSF2-(1-222)/HSF1-(225-529), and HSF2-(1-222)/HSF1-(279-529) are
expressed efficiently in the cells as determined by Western analysis
(Fig. 2E). However, we were unable to examine expression
levels of HSF2-(1-222)/HSF1-(225-281) as it contains neither the HSF1
N nor C terminus, and our efforts to make antibodies to domains within
the N terminus of HSF2 have proven unsuccessful. In addition, HSF2/HSF1
chimeric constructs prepared as His tag or HA fusions were likewise not
detected by Western analysis (data not shown). We were, however, able
to detect efficient expression (and HSE binding) of
HSF2-(1-222)/HSF1-(225-281) using EMSA analysis of nuclear extracts
from cells transfected with this construct as well as wild-type HSF1,
HSF2, and the other mutants (Fig. 2F). These proteins
containing the N terminus of HSF2 bind to HSE, and the intensity of the
HSF-HSE bands was similar to the intensity of the HSF-HSE band seen in
heat-shocked control cells (Fig. 2, F and G). The
HSE binding activity in cells expressing the chimera containing the
HSF1 N terminus and the HSF2 C terminus (HSF1-(1-327)/HSF2-(344-536))
was relatively strong and far exceeded HSF-HSE binding both in
heat-shocked cells and in the other transfected constructs (Fig.
1F). The mechanism behind this is unclear but does not
involve enhanced protein expression of this construct as may be
observed in Fig. 2E.
N-terminal Sequences in HSF1 Are Required for Repression--
We
next attempted to use simple N-terminal truncation from codon 1 in the
open reading frame of hsf1, in a similar approach to the
C-terminal deletion experiments, to characterize N-terminal residues
essential for repression. However, these experiments were complicated
by the fact that all the N-terminal truncation mutants we prepared
non-specifically repressed the c-fos promoter as well as
control promoters, probably by a squelching mechanism (38). Because
squelching is likely to be mediated by the trans-activation domains of HSF1 competing for downstream transcriptional co-activators, we tried the alternative approach of using an HSF1 construct lacking the activation domains as a starting template for making N-terminal deletion mutants of HSF1. As shown previously (Fig. 1, B and
C), the HSF1-(1-279) construct lacks the
trans-activation domains but is entirely proficient in
c-fos and c-fms repression, and this construct
was therefore used as a starting template to prepare the N-terminal
deletion mutants. Indeed, HSF1-(1-279) repressed the c-fos
promoter, but the repression was diminished by deletion of amino acids
1-105 from this sequence (HSF1-(106-279)) and abolished by further
deletion of amino acids 1-145 (HSF1-(146-279)) (Fig. 3). Control experiments indicated that
these constructs based on HSF1-(1-279) do not non-specifically inhibit
the activity of control promoters such as the CMV immediate early and
Role of HSF1 Binding to NF-IL6 in Gene Repression--
Our
previous studies (20, 21) on repression of the IL-1 Development of Dominant Interfering Negative Constructs That
Inhibit Repression by HSF1--
To test further the mechanism of gene
repression by HSF1, we examined the ability of mutants null for
repression to inhibit repression by wild-type HSF1. We have discovered
two HSF1 fragments with null activity for c-fos repression
when transfected into cells but which competitively inhibit gene
repression by wild-type HSF1. These are HSF1-(1-205) and
HSF1-(146-279). We chose these two fragments because, although
HSF1-(1-205) contains the NF domain but not REP, HSF1-(146-279)
encompasses the REP domain but only part of NF. Intracellular
expression of either fragment competitively inhibited the
c-fos repression mediated by HSF1 (Fig.
5, A and B). These
HSF1 fragments may block repression by interacting with HSF1 itself or
with proteins on the target promoter to overcome HSF1-mediated
repression. These reagents allowed us to test the generality of the
mechanisms of gene repression between different target genes, and we
next examined the ability of HSF1-(1-205) to prevent HSF1 repression
of either the IL-1
Our experiments indicate that HSF1 contains at least two closely
apposed (if not contiguous) domains that together are sufficient to
mediate gene repression. There is the fairly well defined REP domain
(represented by amino acids 229-279) that is essential for gene
repression (Figs. 1 and 2). Removal of this domain by internal deletion
or C-terminal truncation leads to loss of capacity to repress
c-fos and c-fms (Figs. 1 and 2). However, this
region does not function as a portable repression domain independently of the N-terminal trimerization domain of HSF1, and chimeras
constructed by fusing the HSF2 N terminus and the REP domain did not
repress target promoters (Fig. 2). The REP domain thus functions in
cooperation with other regions in the N terminus of HSF1. N-terminal
truncation experiments show that amino acids between 106 and 145 are
also essential for repression (Fig. 3). As our previous experiments have suggested a mechanism for HSF1 repression of IL-1
Although our deletion studies suggest the presence of common sequences
within HSF1 that are required for repression of a number of genes, some
differences were also found. One marked difference between
c-fos and c-fms was noted in the study of
C-terminal truncations in Fig. 1, B and C.
Deletion through the trans-activation domains of HSF1 led to
an inhibition of c-fms repression most marked in mutants
HSF1-(1-429) and -(1-379) (Fig. 1C). Further truncation to amino acid 279, producing a construct with the "core repression sequence" 1-279, completely restored the repression capacity (Fig. 1C). These experiments suggest that, in some contexts,
sequences in the C terminus can inhibit gene repression. The exact
nature of these interactions is unclear, although one possible
mechanism might involve the binding of molecular chaperones
to C-terminal sequences in HSF1 that can promote repression. It has
been shown previously (45) that hsp70 can bind to sequences in the C
terminus of HSF1, including amino acids 425-439, and block
trans-activation of HSP promoters. Our previous studies (46)
have shown that hsp70 acts as a transcriptional co-repressor with HSF1
and that intracellular expression of hsp70 antisense oligonucleotides
can block gene repression by HSF1, whereas hsp70 overexpression
promotes repression. We suggest that hsp70-containing molecular
chaperone complexes that bind to the C terminus of HSF1 override the
C-terminal inhibitory sequences and promote gene repression; hsp70
binding is disrupted by C-terminal truncation (45), and this could
account for the inhibition of repression observed in some of the
C-terminal deletion mutants. This effect appears to be very pronounced
in c-fms while less obvious in c-fos (Fig. 1,
C and D). It is not clear whether these
differences are due to the design of the model systems used to map
repression domains or reflect intrinsic differences between the way
HSF1 interacts with c-fos and c-fms.
Our experiments therefore suggest a model in which HSF1 represses
inducible genes in part by a mechanism involving the quenching of
active transcription factors on target promoters (20). In an earlier
study of IL-1
In summary, we have found two regions in the HSF1 sequence that are
essential in repressing transcription of multiple target genes. These
sequences appear to carry out unique functions in gene repression over
and above their established roles in HSF1 uncoiling, trimerization, and
transcriptional activation in response to stress.
. The NF domain encompasses the leucine zipper 1 and 2 sequences as well as the linker domain between the DNA binding and
leucine zipper regions. The function of this domain in gene repression
is highly specific for HSF1, and the homologous region from conserved
family member HSF2 does not restore repressive function in HSF2/HSF1
chimeras. In addition, HSF2 is not capable of binding to NF-IL6. The NF domain, although necessary for repression, is not sufficient, and a
second region (REP) occupying a portion of the regulatory domain is
required for repression. Neither domain functions independently, and
both are required for repression. Furthermore, we constructed dominant
inhibitors of c-fos repression by HSF1, which also blocked the repression of c-fms and IL-1
, suggesting
a shared mechanism for repression of these genes by HSF1. Our studies
suggest a complex mechanism for gene repression by HSF1 involving the
binding to and quenching of activating factors on target promoters.
Mapping the structural domains involved in this process should permit further characterization of molecular mechanisms that mediate repression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(IL-1
), tumor necrosis factor
(TNF
), and c-fms through the mediation of
HSF1 (15, 17, 18, 20-22). In addition, other inducible genes not
involved in the specialized function of macrophages, including the
immediate early genes c-fos and urokinase plasminogen
activator (uPA) are repressed by heat shock and HSF1 (16).
Indeed repression by HSF1 may be a conserved property in eukaryotes as
evidenced by the finding that developmental loci in
Drosophila, which become repressed during heat shock, are
associated with the Drosophila HSF homologue (23). This capacity for gene repression is specific for HSF1 within the
hsf family in mammalian cells and is not a property of HSF2
(16, 20). We have examined in most detail the mechanism of gene
repression by HSF1 in monocytes responding to bacterial endotoxin
exposure. We find that the pro-inflammatory IL-1
gene is
repressed by HSF1 and that this response is mediated by HSF1 binding to
and quenching the activating effect of an essential factor on the
IL-1
promoter, nuclear factor of interleukin 6 (NF-IL6/C/EBP
), which regulates the transcription of many genes in
myeloid cells (15, 20, 21, 24). Our previous studies (24) show that
HSF1 binds to NF-IL6 both in vitro and in vivo
through the basic zipper (bZIP) region. The bZIP region contains the
leucine zipper dimerization domain and DNA binding region common to
many bZIP families of transcription factors (25). The bZIP region
mediates cooperative interactions with a number of other essential
transcription factors including, in the case of the IL-1
promoter, Spi1/PU.1 (20, 24, 26-28). HSF1 appears to
repress the IL-1
promoter by a mechanism that involves
HSF1 binding to a functional heat shock element (HSE) and interaction
with NF-IL6; HSF1 then blocks essential cooperative interactions
between NF-IL6 and PU.1 required for IL-1
promoter
activation (15, 20). A similar mechanism appears to be involved in HSF1
repression of the c-fms gene (20). The HSF1-NF-IL6
interaction may constitute a molecular mechanism involved in the
multiple levels of cross-talk between the heat shock response and the
innate immune/acute phase response in myeloid cells (20, 29,
30). Indeed, NF-IL6 activation caused either by overexpression of the
protein from transfected expression plasmid or by bacterial endotoxin
stimulation of endogenous NF-IL6 leads to the repression of the
hsp70b promoter through a mechanism that appears to involve directly HSF1-NF-IL6 binding (21). It is not known whether other members of the bZIP family including C/EBP, AP-1 binding, or
activating transcription factor/cAMP-response element-binding
protein family proteins can interact with HSF1 (31). Such an
interaction could potentially be involved in the repression of genes
such as c-fos or TNF
in which NF-IL6 does not
play a major role in transcriptional activation.
promoter and the c-fms promoter by HSF1
suggesting the presence of a common repression domain within HSF1 that
acts on a range of target promoters.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
core promoter reporter gene,
pGL3il-1
, contains the sequence of
59 to +12 of the
human IL-1
gene (20). Both reporter genes were
constructed using the commercially available pGL3.Basic plasmid (Promega, Madison, WI). The c-fos reporter gene,
pGLfos, and the hsp70B promoter reporter gene,
pGLhsp70B, were constructed as described previously (16).
The transfection efficiency control vector, pCMV-
Gal, contains the
-galactosidase-coding sequence controlled by the cytomegalovirus
(CMV) promoter.
)/HSF1, contains the human
hsf1 coding sequence driven by the CMV promoter in mammalian expression vector pcDNA3.1(
) (Invitrogen). A series of C-terminal truncation mutants derived from HSF1, which result in deletions of
amino acid residues from the C terminus (amino acid 529) to amino acids
479, 429, 379, 329, 279, 264, 249, 229, 205, and 179 were generated by
PCR-based mutagenesis using pcDNA3.1(
)/HSF1 as the template. N-
and C-terminal deletion mutants, HSF1-(106-279) and HSF1-(146-279),
and the internal deletion mutant, HSF1-(
215-278), were
generated similarly. The N- and C-terminal deletion mutants were cloned
into a plasmid encoding the His6 tag. The HSF1/HSF2 chimeras, HSF1-(1-327)/HSF2-(344-536), HSF2-(1-222)/HSF1-(225-281), HSF2-(1-222)/HSF1-(225-529), and HSF2-(1-222)/HSF1-(279-529), were
constructed by fusing PCR-amplified fragments from HSF1 and HSF2
cDNA templates using standard techniques. The expression plasmid
for the full-length NF-IL6, pcDNA3.1(
)/NF-IL6, was derived by
cloning the entire NF-IL6 cDNA into pcDNA3.1(
). A truncated form of NF-IL6, pcDNA3.1(
)/NF-IL6bZIP, was prepared from an
internal SplI deletion (amino acids 41-205) of the
trans-activation domain from the full-length NF-IL6 cDNA
that retained the intact basic zipper (b-ZIP) region (32). The
pcDNA3.1 (+)/HSF-2A, which contains the coding sequence of HSF-2A,
was used in in vitro protein interaction assays as control.
The expression plasmid for the GST/HSF1 fusion protein contains the
full-length HSF1 coding sequence inserted in-frame downstream of the
coding sequence for glutathione S-transferase in the pGEX
vector (Amersham Biosciences). The expression plasmid for the
GST-NF-IL6b-ZIP fusion protein contains the truncated NF-IL6 cDNA
inserted in pGEX vector and is designated as GST/NF-IL6b-ZIP. The
control plasmid, pGEX-2T, was used to produce GST control protein.
-Galactosidase--
For promoter activity analysis, transient
transfection was carried out using the liposomal transfection reagent
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (Roche Molecular Biochemicals). Unless specified in the figure
legends, cells were plated in 24-well tissue culture plates at 4 × 104/well and cultured for 18 h before being
transfected with 0.4 µg/well of promoter reporter construct. As the
control for transfection efficiency, 0.2 µg/well of pCMV-
Gal
expression vector was simultaneously transfected. For co-expression
assays, a total of 0.4 µg/well expression vector for transcription
factors was used. Cells were harvested 18-24 h after transfection, and
the luciferase activity and
-galactosidase expression levels were
assayed according to the manufacturer's protocols (Promega). The
promoter activities were normalized in relative light units per
milliunits of
-galactosidase activity.
)/HSF1, pcDNA3.1 (
)/NF-IL6, and
pcDNA3.1(
)/NF-IL6bZIP using a TNT Quick T7
Transcription/Translation kit according to manufacturer's protocol
(Promega). HSF1 constructs generated by PCR as described above and
cloned into the eukaryotic expression vectors (which contain promoters
for in vitro transcription) were used as the templates for
in vitro production of truncated proteins for
"pull-down" analysis. The in vitro translated proteins
were checked for size and integrity by SDS-PAGE analysis and for
function by assaying the binding properties to oligonucleotides
containing specific binding motifs for HSF1 and NF-IL6 using
electrophoretic mobility shift assay (EMSA).
80 °C.
cells expressing GST/NF-IL6b-ZIP fusion protein,
GST/HSF1 fusion protein, or GST control protein were incubated by
shaking at 37 °C until the A600
reached 0.4-0.6. Isopropyl-
-D-thiogalactopyranoside was
then added to the bacterial culture to a final concentration of 0.5 mM in order to induce GST fusion protein expression. GST
proteins were prepared as described previously (34). For each in
vitro protein binding reaction, 50 pmol of GST fusion protein or
GST control protein was immobilized on glutathione-Sepharose beads and
then incubated with 20 µl of in vitro translated,
35S-labeled proteins in 500 µl of binding buffer
containing 20 mM Tris-HCl (pH 8.0), 100 mM
NaCl, 10 mM EDTA, 2.5% Nonidet P-40, 1.0 mM
dithiothreitol, 2.0 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml aprotinin, and 5.0 µg/ml leupeptin. The binding reaction was
carried out at 4 °C for 30 min with gentle rocking. The protein-GST beads were then washed five times with binding buffer and analyzed on
10% SDS-PAGE gel. As input controls, 1 µl each of in
vitro translation samples was run in parallel with relevant
binding reactions.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, TNF
, and c-fms through a
mechanism involving interactions between HSF1 and the target promoters
of these genes, we have investigated potential structural domains
within HSF1 that mediate repression. We first examined a series of
deletion mutants generated from the human hsf1 gene as well
as chimeras between hsf1 and the structurally related
hsf2 gene. We generated C-terminal truncation mutants by depleting successive 50 codon segments from the 3' terminus of the
hsf1 coding region and expressed the fragments in CHO-K1 cells in vivo using a eukaryotic expression vector (Fig.
1A). After expression in
vivo, the mutants were then tested for their ability to repress
the activity of co-transfected target promoter-reporter constructs. For
this purpose, cells were first transfected with a luciferase reporter
construct driven by the human c-fos core promoter and an
expression vector for c-fos activator, H-Ras (16). The
c-fos core promoter has fairly low basal activity, and the activity is induced by exposure of cells to growth factors such as
epidermal growth factor (35, 36). H-ras transfection substitutes for
growth factor activation by mimicking the signaling effects of cellular
Ras activated by ligand-bound growth factor receptors, a system
described by us previously (16). We showed previously (16) that heat
shock inhibits serum-induced c-fos expression. We have
observed that wild-type HSF1 is a very effective repressor of the
c-fos promoter previously activated either by H-Ras
expression or serum stimulation and reduces activity of the reporter
luciferase by over 90% (16) (Fig. 1). We used the Ras-activated
c-fos promoter as a well defined model system of choice in
these studies due to this susceptibility of repression by HSF1. In
addition, as c-fos repression by HSF1 does not involve
binding to HSE, we were able to concentrate on domains required for
functional repression without the complication of taking into account
the already characterized DNA binding domain (16). Our control studies
indicated that neither heat shock nor HSF1 overexpression affect Ras
synthesis and that the effects of heat and HSF1 are on factors proximal to the c-fos promoter (16). These effects appear to be
downstream of extracellular signal-regulated protein
kinase as indicated by our studies showing that extracellular
signal-regulated kinase activity is actually stimulated rather than
inhibited by heat shock in CHO
cells.2 When the C-terminal
deletion mutants of HSF1 were co-expressed with the c-fos
promoter, we found that sequences from HSF1 could be deleted from the C
terminus spanning codon 529 to codon 279 without significant loss of
ability to repress Ras-induced activity (Fig. 1B). Wild-type
HSF1 repressed c-fos by over 90% (Fig. 1B, compare lane 2 with lane 1), and this repression
was similar in each mutant up to HSF1-(1-279) (lanes 3-7).
Although the 1-279 fragment efficiently represses the c-fos
promoter (Fig. 1B, lane 7), this mutation has lost all
ability to activate the hsp70B promoter as indicated in our
control studies (Fig. 1D, compare lanes 2 and
7), largely due to removal of the C-terminal
trans-activation domains (amino acids 379-529). By
contrast, overexpression of wild-type HSF1 activates the
hsp70B promoter (Fig. 1D). Thus, the
transcriptional activation domains of HSF1 do not play a major role in
trans-repression of target genes. Further deletion from the
C terminus led to the loss of ability to repress the c-fos promoter (Fig. 1B). Much of the potency of HSF1 to repress
c-fos was lost by deletion from codon 279 to 264, and
repression was abolished by deletion to codon 205 (Fig. 1B).
We also carried out similar experiments with the c-fms
promoter activated by co-transfection with the factor NF-IL6 (Fig.
1C).
View larger version (41K):
[in a new window]
Fig. 1.
The HSF1-(1-279) domain is sufficient for
transcriptional repression. A, serial C-terminal deletion
mutants of HSF1 were constructed as described under "Experimental
Procedures" and used in transient transfection assays to determine
domain(s) that are responsible for gene repression. B, we
first examined repression of Ras-activated c-fos activity by
HSF1 co-transfection. Cells were co-transfected with Ras expression
plasmid, pGLfos and pCMV-bGal transfection efficiency control either
alone (lane 1) or with HSF1 constructs as described under
"Experimental Procedures." Incubations were carried out in
triplicate, and luciferase activities were corrected for transfection
efficiency. The whole experiment was repeated (reproducibly) 3 times,
and the mean values of relative luciferase activity are plotted ± 1 S.D. The columns showing c-fos activity in HSF1
co-transfection experiments (lanes 2-12 in B)
are plotted beneath the figures in A representing the
structure of the HSF1 constructs transfected in each incubation.
C, we have examined the effect of the HSF1 deletion
constructs on NF-IL6-activated c-fms transcription. Cells
were transfected with pGLfms reporter gene and pCMV-bGal
plus NF-IL6 expression vector together alone (lane 1) or
with the various HSF1 C-terminal deletion mutants (lanes 2-12) in the same
order as in B. The complete experiment was repeated
(reproducibly) 3 times, and mean values of relative luciferase activity
are plotted ± 1 S.D. D, to control for the effects of
the various HSF1 constructs on transcriptional activation of
hsp promoters, the same mutant constructs were also tested
by co-transfection with the stress-inducible hsp70B reporter
construct pGLhsp70B, in the same order as in lanes
1-12 in B. As before, luciferase values were
normalized to transfection efficiency, and the experiment was repeated
(reproducibly) 3 times, and mean values of relative luciferase activity
are plotted ± 1 S.D. In all experiments presented here, plasmid
DNA from empty expression vector was added to achieve equal amounts of
total DNA in each transfection. E, the expression levels of
the HSF1 mutants in transfectants in an experiment carried out parallel
to B were determined by Western analysis of the whole cell
lysates with antibody against the N terminus of HSF1 (52). Control
cultures and transfectants were washed 3 times in ice-cold
phosphate-buffered saline and lysed in SDS-PAGE sample buffer, and
equal amounts of protein were fractionated by 10% SDS-PAGE prior to
electrophoretic transfer and Western analysis as described under
"Experimental Procedures." Similar results were obtained in
duplicate Western analyses carried out in parallel with the incubations
in C. F, in addition the ability of selected
mutants to bind HSE elements from the hsp70B promoter was
determined. Wild-type HSF1 and the various deletion mutants were
in vitro translated, and the proteins synthesized in this
way were incubated with 32P-labeled HSE and DNA-protein
interaction determined by EMSA as described under "Experimental
Procedures." A representative autoradiograph showing the relative
positions of complexes between HSF1 and HSE is included. For
comparison, the 1st lane shows a HSF1-HSE complex (marked
with HSF-1) formed in an incubation of 32P-labeled HSE with
nuclear extract from heat-shocked CHO cells. G, we have
compared the relative quantities of the HSE-HSF complexes by
densitometric analysis of the x-ray film autoradiographs. As many of
the bands on the EMSA were not singlets, we quantitated the most
intense band on the autoradiograph.
and
c-fms genes by NF-IL6 (20, 21). The partial loss of
c-fms repression in HSF1 mutants with removal of a portion
of the trans-activation domain may be due to the
interference of HSF1/NF-IL6 physical and functional interaction by the
remaining tail of the trans-activation domain (this is discussed in more detail later). Indeed, when this region was completely removed from the mutants, as in HSF1-(1-279), the
repression of NF-IL6-activated c-fms transcription by
HSF1-(1-279) was equally effective as wild-type HSF1 (Fig.
1C). Further deletion of HSF1 to amino acid 229 effectively
destroyed the capacity to repress c-fms (Fig.
1C). Examination of the relative expression of the deletion
mutants by Western analysis with an antibody to the HSF1 N terminus
shows that the HSF1 mutants were expressed efficiently and at
equivalent levels (Fig. 1E). Endogenous HSF1 can be seen in
each lane at an approximate mass of 69 kDa (Fig. 1E).
The HSF1 deletion mutants contain an N-terminal His tag and therefore
the transfected, untruncated HSF1 (lane marked 1-529)
migrates more slowly than the endogenous HSF1 (Fig. 1E). In
addition the HSF1 deletion mutants were functionally active and capable
of binding to heat shock elements (HSE) in the EMSA assay, indicating
that loss of repression in certain of the mutants was not due to the production of unstable or dysfunctional peptide fragments (Fig. 1F). Deletion mutant 1-229, which we show above has lost
all ability to repress either c-fos or c-fms,
binds avidly to HSE; deletion mutant HSF1-(1-179) loses all DNA
binding capacity, while still being expressed to high level in cells
(Fig. 1, E and F). Overall, these experiments
suggest that the amino acid sequence between amino acids 229 and 279, which we have designated the REP domain and which includes a portion of
the transcriptional regulatory domain (amino acid 221-310), is
essential for gene repression by HSF1. (In the case of the
c-fos promoter, the N-terminal boundary of the REP sequence
may be slightly different involving amino acids 205-279; Fig.
1B.) HSE binding activity was much greater in HSF1
truncation mutants with a deletion of 150 or more amino acids (Fig. 1,
F and G). This is due to the deletion in these constructs of leucine zipper 4, a motif located between amino acids 379 and 429, that functions as an intramolecular inhibitor of trimerization
and DNA binding (37). Wild-type HSF1, and the 1-479 and 1-429
mutants, demonstrate decreased HSF-HSE binding activity (Fig.
1G) due to the inhibitory presence of leucine zipper 4 in
these constructs.
View larger version (43K):
[in a new window]
Fig. 2.
HSF1 residues in the N-terminal core domain
and the 215-279 region are essential for repression. A,
HSF1 with an internal deletion and HSF1/HSF2 chimeras were constructed
as described under "Experimental Procedures" and used in transient
transfection assays to determine domain(s) that are responsible for
gene repression. A, solid bars represent
sequences from HSF1 and gray bars are from HSF2. We next
examined the effects of transfection of these constructs on Ras
activated c-fos (B) and NF-IL6 activated
c-fms transcription (C) essentially as described in Fig. 1 and "Experimental Procedures."
Incubations were carried out in triplicate, and luciferase activities
were corrected for transfection efficiency. Similarly, each experiment
was repeated (reproducibly) 3 times, and mean values of relative
luciferase activity are plotted ± 1 S.D. The columns showing
c-fos and c-fms activity in HSF co-transfection
experiments (lanes 2-8 in B and C)
are plotted beneath the figures in A representing the
structure of the HSF1/HSF2 constructs transfected in each incubation.
D, we tested for the effects of the HSF constructs on the
hsp70B reporter, with experimental replication and data
analysis as in B and C. E, the
expression levels of the HSF1-containing constructs were next examined
by Western analysis using an antibody directed against the C terminus
of HSF1 (52). F, expression of the HSF2-containing
constructs could be observed using EMSA. Wild-type HSF1 and the various
mutants were transiently transfected into cells, and nuclear extracts
from the cells were incubated with 32P-labeled HSE and
DNA-protein interaction determined by EMSA as described under
"Experimental Procedures." A representative autoradiograph showing
the relative positions of complexes between the HSF and HSE is
included. For comparison, the 2nd lane shows an HSF-HSE
complex (lane HS) formed in an incubation of
32P-labeled HSE with nuclear extract from heat-shocked CHO
cells, and the 3rd lane shows a similar incubation that has
also been incubated with anti-HSF1 antibody. A number of nonspecific
complexes of lower electrophoretic mobility were also observed in the
gels. These seemed to be more abundant in the transfected cells for
unknown reasons (4th to 8th lanes). All
experiments were performed three times in triplicate with highly
reproducible findings. G, we have compared the relative
quantities of the HSE-HSF complexes by densitometric analysis of the
x-ray film autoradiographs. We have shown the relative density values
for untreated controls, heat-shocked cells, and cells transfected with
the HSF2-HSF1 chimeric constructs HSF2-(1-222)/HSF1-(225-281),
HSF2-(1-222)/HSF1-(225-529), and HSF2-(1-222)/HSF1-(279-529)
(designated H2/225-281, H2/225-529, and
H2/279-529 in the figure). The anti-HSF1 antibody
supershifted band and the HSF1-(1-327)/HSF2-(344-536) band were not
included in the analysis.
-actin promoters.3 Amino
acid residues 1-145 of HSF1 encompass the complete sequence of the DNA
binding domain (amino acids 10-81), the linker region, and part of the
trimerization domain (amino acids 137-203), which contains two leucine
zipper structures (6, 39-42). We have shown previously that HSF1
binding to the c-fos promoter and amino acids 1-50 are not
required for c-fos repression by HSF1 (16). Our results
therefore indicate an essential role in gene repression for amino acids
106-146, which compose a proportion of linker domain and read into the
first leucine zipper of the trimerization domain (Fig. 3). In a recent
study, we have also demonstrated that serine residue 195 plays an
important role in maintaining transcriptional repression by
HSF1.4 Serine 195 locates
adjacent to the hydrophobic residues of the second leucine zipper.
These data suggest that the leucine zippers in the trimerization domain
are required for the repression.
View larger version (7K):
[in a new window]
Fig. 3.
N-terminal sequences are required for
transcriptional repression by HSF1. The sequences of HSF1 mutant
constructs based on the HSF1-(1-279) template, containing further
N-terminal deletions, are shown in diagrammatic form in A.
We examined the effects of transiently transfecting wild-type HSF1
(lane 1), HSF1-(1-279), and mutants 106-279 and 146-279
to determine domain(s) essential for repression of Ras-activated
c-fos (B) and NF-IL6-activated c-fms
transcription (C). The columns showing c-fos and
c-fms activity in HSF1 co-transfection experiments
(lanes 2-6 in B and C) are plotted
below the figures in A representing the structure
of the HSF1 constructs transfected in each incubation. Relative
effectiveness of the transfectants can be observed by comparison with
lane 1, which shows promoter activity in the absence of
co-transfected HSF1. The same mutant constructs were also tested for
effects on the trans-activation of hsp genes
using the hsp70B reporter construct in D.
Transfection conditions and transfection efficiency controls were
performed as under "Experimental Procedures" and Fig. 1. All
experiments were performed three times in triplicate with reproducible
results.
and
c-fms genes by HSF1 suggest a mechanism of HSF1 repression involving direct binding of HSF1 to the bZIP region of NF-IL6, indicating a specific molecular target for repression. We have further
investigated the potential role of HSF1/NF-IL6 binding in repression
(Fig. 4). We examined the in
vitro binding of HSF1 deletion mutants to a glutathione
S-transferase fusion protein containing the bZIP region of
NF-IL6, which we have shown previously to bind HSF1 (20, 21). We
initially used HSF1-(1-279) as the starting template because the
C-terminal amino acid of this fragment appears to be close to the
C-terminal boundary of the repression domain (Fig. 1). As can be seen,
both wild-type HSF1 and HSF1-(1-279), produced by in vitro
transcription/translation in rabbit reticulocyte lysate, bind avidly to
the NF-IL6 construct GST/NF-IL6bZIP in the pull-down experiments (Fig.
4, A and B). In addition, we observed avid
binding to GST/NF-IL6bZIP of the in vitro translated
deletion mutants HSF1-(1-264), -(1-249), -(1-229), and -(1-205).
However, NF-IL6 binding activity was not detected after further
deletion to amino acid 179 (Fig. 4B, lane 18), even though
this polypeptide is synthesized efficiently in vitro and can
be expressed stably in cells in vivo (Fig. 1E).
This experiment suggests that amino acids between 179 and 205, comprising most of leucine zipper 2, are essential for HSF1 interaction
with NF-IL6. HSF1 deletion to amino acid 179 also inhibited the
capacity to bind to HSE in the EMSA assay, although the protein was
still expressed abundantly in cells (Fig. 1, E and
F). This region in HSF1 may thus be required for both
formation of DNA-binding trimers and for interaction with NF-IL6.
Association with NF-IL6 was not observed in HSF1 mutants 272-529,
205-529, and 146-529 (data not shown). However, weak binding with
NF-IL6 was observed with the HSF1-(106-279) mutant (Fig.
4C). The relative avidities of the HSF1 constructs that bind
to GST/NF-IL6bZIP are shown in Fig. 4D. These experiments suggest that HSF1 binds to the bZIP region of NF-IL6 through a region
roughly delineated to amino acid residues 106-205 that we have
designated as the NF domain. Although these studies were carried out
in vitro, our previous studies show that HSF1 and NF-IL6
interact in vivo and can be co-immunoprecipitated with anti-HSF1 and anti-NF-IL6 antibodies (20). This interaction may
partially explain the requirement for sequences in the amino acids
106-279 region of HSF1 for gene repression. Clearly, however, NF-IL6
binding by HSF1 is not sufficient in itself for repression as the
residues in the REP domain (between 205 and 279), which are essential
for repression (Fig. 1), are not required for NF-IL6 binding (Fig. 4).
The REP domain appears to play a role in gene repression that is
independent of NF-IL6 binding.
View larger version (37K):
[in a new window]
Fig. 4.
HSF1 binds to NF-IL6 through a region
containing amino acid residues 179-205. A, the NF-IL6
fragment NF-IL6bZIP was fused to GST and GST/NF-IL6bZIP used as bait to
detect the binding of wild-type, full-length 35S-labeled
HSF1. In vitro translated HSF1 was incubated with either
GST/NF-IL6bZIP or, as a control, with wild-type GST. Samples eluting
from the GST or GST fusion protein were then separated by 10%
SDS-PAGE, and the relative migration of the 35S-labeled
proteins was detected by x-ray film autoradiography. Lane 1 shows the labeled proteins in the in vitro translation
input; lane 2 is the eluate from wild-type GST control
protein, and lane 3 shows the proteins eluted from
GST/NF-IL6bZIP. The upper band corresponds to full-length
HSF1; the lower bands are alternatively translated HSF1
products. B, we show the binding of the deletion mutants
HSF1-(1-279), HSF1-(1-264), HSF1-(1-249), HSF1-(1-229),
HSF1-(1-205), or HSF1-(106-279) to GST/NF-IL6bZIP using similar
conditions to A. For each deletion mutant there are 3 lanes
on the gel, with the 1st lane showing the labeled proteins
in the in vitro translation input, the 2nd lane
showing the eluate from wild-type GST, and the 3rd lane
showing the proteins eluted from GST/NF-IL6bZIP. Thus lanes
1, 4, 7, 10, 13, and
16 show the input controls; lanes 2,
5, 8, 11, 14, and 17 are GST controls; and lanes 3, 6,
9, 12, 15, and 18 are proteins
eluted from GST/NF-IL6bZIP. C, a similar experiment is shown
using the HSF1 mutant HSF1-(106-279). As in A, lane
1 shows the labeled proteins in the in vitro
translation input; lane 2 is the eluate from wild-type GST
control protein, and lane 3 shows the proteins eluted from
GST/NF-IL6bZIP. D, we have compared the relative binding of
HSF1 and deletion mutants to GST/NF-IL6bZIP by densitometric analysis
of the x-ray film autoradiographs. As many of the HSF1 bands were
doublets, for each HSF1 isoform, we quantitated the upper band on the
autoradiograph, corresponding to the full-length in vitro
translated HS1 construct. In these experiments, the GST/NF-IL6bZIP
bound, respectively, 5.1, 5.7, 5.7, 7.1, 6.5, 4.5, and 2.6% of the
35S-labeled constructs 1-279, HSF1-(1-264),
HSF1-(1-249), HSF1-(1-229), HSF1-(1-205), wild-type HSF1, and
HSF1-(106-279). All experiments were performed three times with
reproducible results.
or c-fms promoters (Fig. 5,
C and D). Both promoters were repressed by HSF1
as shown previously, and co-transfection with HSF1-(1-205) prevented
the repression in a dose-dependent manner (Fig. 5,
C and D). In the case of c-fms,
increasing amounts of HSF1-(1-205) not only alleviated HSF1-induced
repression but also increased c-fms activity to 3-fold higher than in cells that had not been exposed to wild-type HSF1 (Fig.
5C). The mechanism behind this finding is not apparent, although it is possible that endogenous HSF1 may exert a background repressive effect on c-fms that is reversed by the
competitive inhibitor HSF1-(1-205). This HSF1 truncation mutant,
HSF1-(1-205) which binds avidly to NF-IL6 (Fig. 4), would be expected
to compete for NF-IL6 with HSF1 within the cell, and this could be at
least part of the mechanism for the c-fms superinduction at
high levels of co-transfected HSF1-(1-205) (Fig. 5C).
Similar results were obtained with IL-1
, which is also
NF-IL6-dependent (Fig. 5D). HSF1-(1-205) caused an even more pronounced super-induction which may
also be due to the reversal of HSF1 repression by endogenous HSF1 (Fig.
5D). It is notable that this effect was not seen in the
c-fos experiments (Fig. 5B). Another possibility
for some of these effects could be direct binding of HSF1-(1-205) to
wild-type, endogenous HSF1. This explanation, however, seems less
likely in light of our in vitro findings that association of
HSF1 with truncation mutants HSF1 205, 229 and 279 is very
inefficient.5 Overall,
however, these experiments using HSF1-(1-205) suggest a broadly
similar common mechanism for the repression of diverse target promoters
by HSF1.
View larger version (22K):
[in a new window]
Fig. 5.
HSF1-(147-279) and HSF1-(1-205) act as
dominant inhibitors of HSF1 repression of c-fos,
c-fms, and il1b. HSF1 mutants,
HSF1-(146-279) and HSF1-(1-205), were tested in transient
transfection assays for their inhibitory effects on repression by HSF1.
Cells were co-transfected with c-fos (A and
B), c-fms (C), or IL-1
(D) reporter genes, alone (1st lane), with the
addition of transcriptional activators for each promoter (2nd
lane), and with increasing amounts (indicated in the figure) of
HSF1-(146-279) (A) or HSF1-(1-205) plasmid DNA
(B-D). A and B, the luciferase
activity of the c-fos promoter-reporter in cells
co-transfected with H-ras expression vector, in the absence of HSF1
co-transfection, was set to 100. C and D, the
luciferase activities of the c-fms and IL-1
reporter genes in cells co-transfected with empty expression vector
were used as controls and set to 100, and the activating effect of
NF-IL6 co-transfection is shown in the 2nd lane. The
3rd lane shows the repressive effect of HSF1 demonstrated by
co-transfecting wild-type HSF1 and NF-IL6. The 4th lane
shows the effect of transfection of HSF1-(1-205) alone into cells
transfected with NF-IL6. The 5th through 8th
lanes show the inhibitory effects of increasing doses of 1-205 on
HSF1-mediated repression of the c-fms and IL-1
reporter genes. As before, luciferase values were normalized to
transfection efficiency; the experiment was carried out in triplicate,
and mean relative luciferase activity is plotted ± 1 S.D.
Experiments were performed three times, with reproducible
results.
involving binding to NF-IL6, we have further pursued this mechanism
here (20, 21). Our current experiments indicate that NF-IL6 binding maps to the N terminus of HSF1 and that residues in the REP domain are
not required (Fig. 4). Instead, residues in the linker and leucine
zipper domains are required for NF-IL6 binding (Fig. 4). The N-terminal
boundary of the NF-IL6 binding region is close to amino acid 106 as
HSF1-(106-279) binds, albeit weakly, to NF-IL6, whereas
HSF1-(146-279) loses all binding ability (Fig. 4 and data not shown).
The C-terminal boundary of NF-IL6 binding is between amino acids 179 and 205 (Fig. 4). A roughly defined NF-IL6 binding region between amino
acids 106 and 205 is thus suggested. Our previous experiments (20, 21)
indicated a mechanism for IL-1
and c-fms
repression by HSF1 involving HSF1 binding to NF-IL6 and quenching the
cooperative interactions between proteins (NF-IL6 and
PU.1/Spi-1) that lead to transcriptional activation of these genes. The NF-IL6 binding "NF" domain may thus constitute a
functionally independent repression domain. Indeed, the N-terminal
boundary of the NF-IL6 binding region (amino acid 106) corresponded
well with N-terminal boundary for gene repression (Figs. 3 and 4). The
HSF1-(106-279) construct bound to NF-IL6 only weakly, and this
fragment, when expressed in cells, also repressed c-fos and c-fms weakly (Figs. 3 and 4). However, the C-terminal
boundary of NF-IL6 binding (amino acid 205) did not correspond to the
C-terminal cut-off for repression (amino acid 279). We have thus
characterized two repression domains in HSF1, including the REP domain
(amino acids 229-279) and the NF domain (amino acids 106-206). Both
regions are necessary for repression but not sufficient in themselves. The NF domain contains the leucine zipper 1 trimerization domain, an
extended region of hydrophobic heptad repeats (amino acids 137-181)
required for stress-induced trimer formation (37, 43). The
trimerization domain is therefore essential for both gene activation
and repression. However, trimerization alone is clearly not indicated
because the N-terminal region of HSF2, which contains an effective
trimerization domain, does not substitute for the HSF1 N terminus (Fig.
2). The NF domain also contains the second leucine zipper region (amino
acids 183-199) that is not required for trimerization but is involved
in the trans-activation step of HSF1 induction (44). We have
found that serine 195 located within this region is required for
effective gene repression by HSF1 (41, 42), and alanine mutation of
this site inhibits repression.4 Leucine zipper 2 may thus
play a direct role in gene repression or may be part of the generic
uncoiling mechanism required for de-repression of HSF1 (44). The amino
acid 106-205 region also contains most of the linker region of HSF1
recently defined by Liu and Thiele (42) as important in regulating the
monomer-trimer transition. Their studies indicate that the linker
region contains sequences not conserved with HSF2 that may interact
with residues within the DNA binding domain (41, 42). These residues in the linker region appear to be required in gene repression and NF-IL6
binding by HSF1 (Figs. 3 and 4). Although binding of the NF domain to
NF-IL6 plays a role in IL-1
and c-fms
repression, its role in the repression of other promoters such as
c-fos, uPA, and TNF
is less clear.
However, NF-IL6 belongs to a larger family of transcription factors
(bZIP family) including AP1-binding proteins, the C/EBP family,
activating transcription factor/cAMP-response element-binding
protein, and others (25). The role of HSF1 binding to DNA during gene
repression is less clear and appears to vary between target genes.
Previous studies (15, 17, 18) show that both the IL-1
and
TNF
genes contain functional HSE that are involved in
repression by HSF1. The c-fos gene, however, does not
contain HSE in the proximal promoter, does not bind avidly to HSF1, and
is repressed by a point-mutated HSF1 (HSF1 Leu-22) that fails to
bind to HSE sequences (16). It is therefore possible that HSF1 can be
recruited to target promoters either through DNA binding,
protein-protein association, or both processes (15, 16, 18).
, we showed that HSF1 binds to NF-IL6 in vivo only after NF-IL6 is activated by the
proinflammatory lipopolysaccharide and phorbol ester treatment and when
HSF1 is activated to a nuclear DNA binding form by heat shock (20). We
suggest a mechanism therefore involving the activation of transcription factors during inflammation or mitogenesis, which migrate to the promoters of genes such as IL-1
, c-fms, or
c-fos (Fig. 6). In the cells
that subsequently encounter stresses such as febrile heat, HSF1 is
activated to a DNA-binding form and migrates to the nucleus as a trimer
(37). Activated HSF1 is then recruited to the promoters of these
induced genes by bZIP factors that bind to the NF domain (20). In
support of this, we have found that NF-IL6 binds to HSF1 and
competitively inhibits HSF1 binding to heat shock promoters in
vitro and represses the activity of heat shock genes in
vivo, whereas HSF1 activation leads to inhibition of
NF-IL6-dependent genes (20, 21). HSF1 may thus become
tethered to the target promoter through binding to DNA-bound b-ZIP
factors and/or by contacting the HSE in genes such as
IL-1
and TNF
(15, 18). HSF1 subsequently
represses IL-1
at least partially by quenching essential
cooperative interactions between NF-IL6 and Spi.1 (Fig. 6) (20, 24). We
have no direct evidence that such a mechanism is involved in the
repression of the other promoters. However, the fact that the HSF1
construct 1-205, which contains the NF domain but not the REP domain,
acts as a dominant negative inhibitor of repression of
IL-1
, c-fms, and c-fos is
suggestive of such a general mechanism (Figs. 1 and 5). However, the
molecular mechanisms involved in repression through the REP domain are
not clear. The REP domain occupies most of the transcriptional
regulatory domain, a region that responds to heat shock and influences
the trans-activation domains to induce heat shock promoters
(47-49). One possible mechanism for repression by this domain is that
the REP domain regulates association of HSF1 with co-repressor
molecules (Fig. 6). Recruitment of co-repressor complexes, many of
which encode histone deacetylases, has been implicated in the
repression of target genes by other factors, most notably the nuclear
receptor family (38, 50, 51). Further experiments will explore this possibility.
View larger version (14K):
[in a new window]
Fig. 6.
Two interdependent domains in HSF1 are
essential for gene repression. A, regions essential for
repression include first the NF domain that binds to NF-IL6 and is
essential for functional repression of each gene. There is a second
region (REP) not involved in NF-IL6 binding but also
essential for repression of target genes. The NF domain overlaps with
the linker region between the DNA binding domain (DB) and
the leucine zipper regions of the trimerization domain (TD).
The REP domain occupies sequences within the regulatory domain
(RD), a temperature-sensitive domain that regulates the
activation domains (AD1 and AD2). However, AD1
and AD2 are not required for gene repression. B, proposed
model for gene repression by HSF1. HSF1 undergoes trimerization after
activation by stress and is competent to bind HSE in target genes. The
activated HSF1 is also enabled to bind essential transcription factors
(TF1) such as NF-IL6 (in the case of the IL-1
and c-fms promoters) on repressed promoters through the NF
domain. We propose that this interaction could recruit HSF1 to
promoters repressed by HSF1, and we have shown that such reactions
participate in repressing the promoter by quenching interactions of
transcription factor 1 with other transcription factors on the enhancer
region of the promoter (transcription factor 2) or general
transcription factors/co-activators. The second domain (REP)
is also essential for gene repression. The function of REP is not
known, although we speculate that it could be involved in binding
co-repressors, common mediators of gene repression. Gene repression
requires both NF and REP domains, and neither has been shown to
function as independent, portable repression domains in experiments
carried out so far.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Carl Wu and Philip Auron for valuable materials and the faculty at the Harvard Joint Center for Radiation Therapy for support and encouragement in the early part of the studies. We are especially grateful to Phil Auron for many valuable discussions over the years in which this project has developed and to Catherine Cahill who helped to initiate the studies.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA47407, CA31303, and CA50642.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Center for the Molecular Stress Response, Boston University School of Medicine, 88 E. Newton St., Boston, MA 02118. Tel.: 617-414-1700; Fax: 617-414-1699; E-mail: stuart_calderwood@medicine.bu.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210189200
2 M. A. Stevenson and S. K. Calderwood, manuscript in preparation.
3 Y. Xie and S. K. Calderwood, unpublished data.
4 Y. Xie and S. K. Calderwood, manuscript in preparation.
5 H. He and S. K. Calderwood, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HSF1, heat shock
factor 1;
IL-1, interleukin 1
;
TNF
, tumor necrosis factor
;
uPA, urokinase plasminogen activator;
bZIP, basic zipper;
HSE, heat
shock element;
EMSA, electrophoretic mobility shift assay;
GST, glutathione S-transferase;
CMV, cytomegalovirus;
CHO, Chinese hamster ovary.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sorger, P. K., and Pelham, H. R. B. (1988) Cell 54, 855-864[Medline] [Order article via Infotrieve] |
2. | Voellmy, R. (1994) Crit. Rev. Eukaryotic Gene Expr. 4, 357-401[Medline] [Order article via Infotrieve] |
3. | Wu, C. (1984) Nature 311, 81-84[Medline] [Order article via Infotrieve] |
4. | Wu, B., Hunt, C., and Morimoto, R. I. (1985) Mol. Cell. Biol. 5, 330-341[Medline] [Order article via Infotrieve] |
5. | Wu, C. (1995) Annu. Rev. Cell Dev. Biol. 11, 441-469[CrossRef][Medline] [Order article via Infotrieve] |
6. | Sarge, K. D., Zimarino, V., Holm, K., Wu, C., and Morimoto, R. I. (1991) Genes Dev. 5, 1902-1911[Abstract] |
7. |
McMillan, D. R.,
Xiao, X.,
Shao, L.,
Graves, K.,
and Benjamin, I. J.
(1998)
J. Biol. Chem.
273,
7523-7528 |
8. | Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986) Science 232, 522-524[Medline] [Order article via Infotrieve] |
9. | Prip-Buus, C., Westerman, B., Schmitt, M., Langer, T., Neupert, W., and Schwarz, E. (1996) FEBS Lett. 380, 142-146[CrossRef][Medline] [Order article via Infotrieve] |
10. | Perisic, O., Xiao, H., and Lis, J. T. (1989) Cell 59, 797-806[Medline] [Order article via Infotrieve] |
11. | Sorger, P. K., and Nelson, H. C. M. (1989) Cell 59, 807-813[Medline] [Order article via Infotrieve] |
12. | Peteranderl, R., Rabenstein, M., Shin, Y. K., Liu, C. W., Wemmer, D. E., King, D. S., and Nelson, H. C. (1999) Biochemistry 38, 3559-3569[CrossRef][Medline] [Order article via Infotrieve] |
13. | Price, B. D., and Calderwood, S. K. (1991) Mol. Cell. Biol. 11, 3365-3368[Medline] [Order article via Infotrieve] |
14. |
Chu, B.,
Soncin, F.,
Price, B. D.,
Stevenson, M. A.,
and Calderwood, S. K.
(1996)
J. Biol. Chem.
271,
30847-30857 |
15. |
Cahill, C. M.,
Waterman, W. R.,
Auron, P. E.,
and Calderwood, S. K.
(1996)
J. Biol. Chem.
271,
24874-24879 |
16. |
Chen, C.,
Xie, Y.,
Stevenson, M. A.,
Auron, P. E.,
and Calderwood, S. K.
(1997)
J. Biol. Chem.
272,
26803-26806 |
17. |
Singh, I. S.,
Calderwood, S. K.,
Kalvokalanu, I.,
Viscardi, R. M.,
and Hasday, J. D.
(2000)
J. Biol. Chem.
275,
9841-9848 |
18. |
Singh, I. S., He, J. R.,
Calderwood, S.,
and Hasday, J. D.
(2002)
J. Biol. Chem.
277,
4981-4988 |
19. | Xie, Y., and Calderwood, S. K. (2001) Curr. Top. Biochem. Res. 4, 81-88 |
20. |
Xie, Y.,
Chen, C.,
Stevenson, M. A.,
Auron, P. E.,
and Calderwood, S. K.
(2002)
J. Biol. Chem.
277,
11802-11810 |
21. | Xie, Y., Chen, C., Stevenson, M. A., Hume, D. A., Auron, P. E., and Calderwood, S. K. (2002) Biochem. Biophys. Res. Commun. 291, 1071-1080[CrossRef][Medline] [Order article via Infotrieve] |
22. | Cahill, C. M., Lin, H. S., Price, B. D., Bruce, J. L., and Calderwood, S. K. (1997) Adv. Exp. Med. Biol. 400, 625-630 |
23. | Westwood, T. J., Clos, J., and Wu, C. (1991) Nature 353, 822-827[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Yang, Z.,
Wara-Aswapati, N.,
Chen, C.,
Tsukada, J.,
and Auron, P. E.
(2000)
J. Biol. Chem.
275,
21272-21277 |
25. | Johnson, P. F., and McKnight, S. L. (1989) Annu. Rev. Biochem. 58, 799-839[CrossRef][Medline] [Order article via Infotrieve] |
26. | Mink, S., Kerber, U., and Klempnauer, K.-H. (1996) Mol. Cell. Biol. 16, 1316-1325[Abstract] |
27. | Stein, B., Cogswell, P. C., and Baldwin, A. J. (1993) Mol. Cell. Biol. 13, 3964-3974[Abstract] |
28. | Mink, S., Haenig, B., and Klempnauer, K. H. (1997) Mol. Cell. Biol. 17, 6609-6617[Abstract] |
29. | Asea, A., Kraeft, S. K., Kurt-Jones, E. A., Stevenson, M. A., Chen, L. B., Finberg, R. W., Koo, G. C., and Calderwood, S. K. (2000) Nat. Med. 6, 435-442[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Asea, A.,
Rehli, M.,
Kabingu, E.,
Boch, J. A.,
Bare, O.,
Auron, P. E.,
Stevenson, M. A.,
and Calderwood, S. K.
(2002)
J. Biol. Chem.
277,
15028-15034 |
31. | McKnight, S. L. (1992) in Transcriptional Regulation (McKnight, S. L. , and Yamamoto, K. R., eds), Vol. 2 , pp. 771-796, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
32. | Tsukada, J., Saito, K., Waterman, W. R., Webb, A. C., and Auron, P. E. (1994) Mol. Cell. Biol. 14, 7285-7297[Abstract] |
33. | Stevenson, M. A., and Calderwood, S. K. (1990) Mol. Cell. Biol. 10, 1234-1238[Medline] [Order article via Infotrieve] |
34. | Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Greenberg, M. E.,
Greene, L. A.,
and Ziff, E. B.
(1985)
J. Biol. Chem.
260,
14101-14110 |
36. | Treisman, R. (1992) Trends Biochem. Sci. 17, 423-426[CrossRef][Medline] [Order article via Infotrieve] |
37. | Rabindran, S. K., Haroun, R. I., Clos, J., Wisniewski, J., and Wu, C. (1993) Science 259, 230-234[Medline] [Order article via Infotrieve] |
38. | Hanna-Rose, W., and Hansen, U. (1996) Trends Genet. 12, 229-234[CrossRef][Medline] [Order article via Infotrieve] |
39. | Rabindran, S. K., Gioorgi, G., Clos, J., and Wu, C. (1991) Procs. Natl. Acad. Sci. U. S. A. 88, 6906-6910 |
40. | Zuo, J., Baler, R., Dahl, G., and Voellmy, R. (1994) Mol. Cell. Biol. 14, 7557-7568[Abstract] |
41. |
Liu, X. D.,
Liu, P. C.,
Santoro, N.,
and Thiele, D. J.
(1997)
EMBO J.
16,
6466-6477 |
42. |
Liu, P. C.,
and Thiele, D. J.
(1999)
J. Biol. Chem.
274,
17219-17225 |
43. | Westwood, T., and Wu, C. (1993) Mol. Cell. Biol. 13, 3481-3486[Abstract] |
44. | Zuo, J., Rungger, D., and Voellmy, R. (1995) Mol. Cell. Biol. 15, 4319-4330[Abstract] |
45. |
Shi, Y.,
Mosser, R. D.,
and Morimoto, R. I.
(1998)
Genes Dev.
12,
654-666 |
46. | He, H., Chen, C., Xie, Y., Asea, A., and Calderwood, S. K. (2000) Cell Stress Chaperones 5, 406-411[Medline] [Order article via Infotrieve] |
47. | Green, M. T., Schuetz, T. J., Sullivan, E. K., and Kingston, R. E. (1995) Mol. Cell. Biol. 15, 3354-3362[Abstract] |
48. | Newton, E. M., Knauf, U., Green, M., and Kingston, R. E. (1996) Mol. Cell. Biol. 16, 839-846[Abstract] |
49. | Knauf, U., Newton, E. M., Kyriakis, J., and Kingston, R. E. (1996) Genes Dev. 10, 2782-2793[Abstract] |
50. |
Burke, L. J.,
and Baniahmad, A.
(2000)
FASEB J.
14,
1876-1888 |
51. | Stein, B., and Yang, M. X. (1995) Mol. Cell. Biol. 15, 4971-4979[Abstract] |
52. | Bruce, J. L., Chen, C., Xie, Y., Zhong, R., Wang, Y., Stevenson, M. A., and Calderwood, S. K. (1999) Cell Stress Chaperones 4, 36-45[CrossRef][Medline] [Order article via Infotrieve] |