From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606
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ABSTRACT |
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Heat shock transcription factors (HSFs) are
stress-responsive proteins that activate the expression of heat shock
genes and are highly conserved from bakers' yeast to humans. Under
basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a
homotrimer that binds to its cognate DNA binding site with high affinity. To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have
used functional complementation in bakers' yeast as a facile assay
system. Whereas wild-type human HSF1 is restrained as an inactive
monomer in yeast that is unable to substitute for the essential yeast
HSF protein, mutations in the linker region between the DNA binding
domain and the first coiled-coil allow HSF1 to homotrimerize and rescue
the viability defect of a hsf The response of all eukaryotic organisms to thermal,
environmental, and physiological stress involves the rapid production of a group of proteins called heat shock proteins
(Hsps)1 (1). At the level of
transcription, this response is orchestrated by the heat shock
transcription factor (HSF). HSF binds as a homotrimer to a conserved
regulatory site, the heat shock element (HSE), composed of inverted
repeats of the 5-base pair sequence 5'-nGAAn-3' located in the
promoters of heat-inducible Hsp genes (2, 3). The architecture of HSF
is modular, conserved among all characterized HSFs, and composed of a
winged helix-turn-helix DNA binding domain, an adjacent coiled-coil
trimerization domain, a central regulatory domain, and a
transcriptional activation domain at the carboxyl terminus (4).
Additionally, many metazoan HSF molecules harbor a second hydrophobic
repeat abutting the activation domain that is believed to play an
important role in suppressing the activity of HSF under normal growth
conditions (5, 6). In yeast and Drosophila, HSF is encoded
by a single gene, but in mammals and higher plants, multiple HSF genes
have been cloned (4, 7). Mammalian HSF1 is activated in response to
many stresses, but the regulation and contribution of other HSF
isoforms to stress responses and normal physiology remain largely unknown.
The activation of HSF1 occurs via a multistep process. Under non-stress
conditions, mammalian HSF1 and Drosophila HSF (dHSF) appear
as monomeric forms that exhibit little DNA binding activity (8-11).
These latent HSF molecules are negatively regulated through intramolecular interactions between the amino-terminal and
carboxyl-terminal hydrophobic domains. In response to stress, HSF
converts from a monomer to a trimer (Fig. 1A). This level of
regulation can be abrogated in the absence of stress by creating
deletions or mutations within either hydrophobic domain that are
predicted to disrupt the integrity of the hydrophobic heptad repeat,
resulting in the formation of constitutively trimeric HSF molecules
with a concomitant loss of monomeric species (5, 6, 12-15). Several deletion mutations of the amino, carboxyl, and internal regions of dHSF
also result in constitutive trimerization, suggesting that the
coiled-coil interactions that restrain the inactive protein may be
stabilized by additional regions within the HSF monomer (5, 13, 16).
Recently, interactions between HSF1 and the molecular chaperone Hsp90
have been detected, suggesting a role for additional factors in its
regulation (17, 18). Upon trimerization, HSF undergoes a significant
conformational change with the amino-terminal hydrophobic domains from
individual monomeric units, forming a stable Significant structural, functional, and regulatory aspects of metazoan
and yeast HSF proteins are conserved. An intriguing difference is that
the single HSF gene in both budding and fission yeast is essential for
viability even under non-stress conditions, whereas dHSF is dispensable
for cell growth (27-30). Flies lacking HSF, like mouse embryonic stem
cell knock-outs of HSF1, are sensitive to heat shock, indicating a
primary role for HSF1 in protection against thermal stress (30, 31).
Consistent with their requirement under all growth conditions, HSFs
from the yeasts Saccharomyces cerevisiae and
Klyveromyces lactis are found as trimers and exhibit constitutive DNA binding activity at all temperatures and conditions tested (20, 32, 33). The occupancy of specific HSEs in the S. cerevisiae HSP82 promoter increases upon heat shock, suggesting that some yeast HSEs are inducibly bound, as observed in higher eukaryotes (34). We previously established an assay to examine the
functional relatedness between the human HSF (hHSF) isoforms and
S. cerevisiae HSF (yHSF) by testing for the ability of hHSF1 and hHSF2 to support the growth of hsf In this investigation, we have used the yeast assay system to identify
novel hHSF1 amino acid residues that regulate the monomer-to-trimer transition. We demonstrate that sequences between the DNA binding domain and first trimerization domain, known as the flexible linker region, strongly modulate the equilibrium between the hHSF1 monomer and
trimer in yeast and mammalian cells. In yeast, linker mutants of hHSF1
trimerize constitutively and support the growth of hsf Yeast Strains, Growth Conditions, and Plasmids--
Yeast
expression plasmids p424GPDHSF1 and p413GPDHSF2 harboring the complete
hHSF1 and hHSF2 cDNAs, respectively, under the control of
constitutive yeast promoters were described previously (35). All linker
mutations were constructed by oligonucleotide-directed mutagenesis and
sequenced to confirm that only desired mutations were introduced. A
complete listing of the mutants generated and used in these studies is
shown in Fig. 2A. The recipient strain for all experiments
was the W303 derivative PS145 (MAT a ade2-1 trp1 can 1-100 leu2,3-112 his3-11,15
ura3 hsf::LEU2 YCpGAL1-yHSF; a gift
from Dr. Hillary Nelson (27)). Plasmids harboring hHSFs were
transformed according to the polyethylene glycol-lithium acetate
procedure and tested for the ability to complement hsf Yeast Cell Biological and Biochemical Techniques--
Yeast cell
extracts were prepared by glass bead disruption in HEGN containing 1%
Triton X-100, 0.5 mM dithiothreitol, and protease
inhibitors, and immunoblotting was performed as described previously
(35) using antibodies specific to HSF1 (a gift from Dr. Carl Wu) or
HSF2 (a gift from Dr. Richard Morimoto). Procedures for ethylene glycol
bis(succinimidyl succinate) cross-linking, immunoblotting, RNase
protection assays, and fluorescence microscopy have been described
previously (35).
Mammalian Cell Culture, Plasmids, and Transfections--
The
mammalian expression vector pBC19 containing wild-type hHSF1 cDNA
under the control of the human cytomegalovirus promoter was a gift from
Dr. Stuart Calderwood. All linker derivatives were directly subcloned
into this vector. Human embryonic kidney 293 cells obtained from
American Type Culture Collection were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum and
supplemented with penicillin and streptomycin. Culture plates
containing approximately 4 × 105 cells were
transfected by calcium phosphate precipitation of DNA consisting of 0.5 µg of expression plasmid and 19.5 µg of carrier
(pKS Mammalian Cell Extracts and Biochemical Techniques--
Whole
cell extracts were prepared by thawing frozen cell pellets in 20 mM Hepes, pH 7.9, 0.5 mM EDTA, 10% glycerol
(HEG) containing 0.42 M NaCl, 1.5 mM
MgCl2, and protease inhibitors (35), dispersing the cells
by repeated pipetting and incubating tubes on ice for 15 min. The
extracts were clarified in a microcentrifuge at 14,000 rpm for 15 min
at 4 °C, and an aliquot of the supernatant (2 µg of protein) was
used in DNA binding reactions in buffer containing 20 mM
Hepes, pH 7.9, 0.5 mM EDTA, 75 mM NaCl, 10%
glycerol, 0.5 µg of poly(dI-dC), and approximately 0.2 ng of
32P-labeled DNA probe containing a consensus HSE with four
inverted repeats of the recognition element 5'-nGAAn-3' (11).
Protein-DNA complexes were resolved on a 3.5% native polyacrylamide
gel in 0.5× Tris borate-EDTA buffer.
To identify regions of the hHSF1 molecule that could influence the
monomer-to-trimer conversion (Fig.
1A), we used a yeast assay
based on the ability of heterologous HSFs to support the growth of
hsf strain. Fine mapping by
functional analysis of HSF1-HSF2 chimeras and point mutagenesis
revealed that a small region in the amino-terminal portion of the HSF1
linker is required for maintenance of HSF1 in the monomeric state in
both yeast and in transfected human 293 cells. Although linker regions
in transcription factors are known to modulate DNA binding specificity,
our studies suggest that the human HSF1 linker plays no role in
determining HSF1 binding preferences in vivo but is a
critical determinant in regulating the HSF1 monomer-trimer equilibrium.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure
consistent with a triple-stranded coiled-coil (10, 19). Trimerization
leads to high affinity DNA binding to the HSE (10, 20); however,
transcriptional activation appears to be distinctly regulated from DNA
binding because these events can be uncoupled by some nonsteroidal
anti-inflammatory drugs (21). Transcriptional activity requires the
unmasking of the carboxyl-terminal activation domain and correlates
with hyperphosphorylation of HSF1 in many, but not all, instances (11,
22, 23). Attenuation of the heat shock response occurs through feedback
regulation of active HSF trimers by interactions with Hsp70 and heat
shock factor-binding protein 1 (24-26).
yeast cells (35).
We found that hHSF2, but not hHSF1, was capable of complementing the
viability defect of hsf
yeast. Human HSF1 expressed in
S. cerevisiae was found as an inactive monomer under both
control and heat shock conditions. A derivative of hHSF1 with point
mutations in the carboxyl-terminal hydrophobic heptad repeat
(hHSF1-LZ4) or chimeric molecules between hHSF1 and hHSF2 joined within
the amino-terminal trimerization domain could both trimerize and
support hsf
cell growth. These observations indicate that
the defect in hHSF1 function in yeast is related to the regulation of
the monomer-to-trimer transition of hHSF1, rather than an inability of
hHSF1 to activate appropriate target genes.
cells. Importantly, when expressed in human cells, these same hHSF1
derivatives are constitutively trimerized and bound to HSEs. The linker
sequences do not alter the preferences for target gene activation by
hHSF1 and hHSF2, suggesting that the hHSF1 linker domain plays a
selective role in modulating oligomerization. These results illustrate
the complexity in the regulation of the hHSF1 monomer-to-trimer
transition and demonstrate the utility of the yeast model for analysis
of the regulation and function of individual mammalian HSF proteins.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells as described previously (35). Cells were routinely grown at
30 °C in selective synthetic complete medium and heat shocked for 15 min by submersing tubes into a shaking water bath equilibrated to
40 °C.
). After a 4-h incubation at 37 °C, the
precipitate was aspirated, and the cells were washed with
phosphate-buffered saline and fed with complete medium. To limit the
level of overexpression, transfections were performed with a low amount
of expression DNA, and the time of expression was limited to no more
than 14 h. Cells were heat shocked for 20 min by submersing sealed
plates into a water bath equilibrated to 42 °C. Cells were harvested
by washing twice with ice-cold phosphate-buffered saline and scraped in
phosphate-buffered saline, and cell pellets were frozen in liquid nitrogen.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (35). We observed previously that sequences amino-terminal to the first trimerization domain of hHSF1 appeared to
modulate trimerization in yeast because replacing the hHSF1 DNA binding
domain and linker with homologous sequences from hHSF2 resulted in a
constitutively trimerized chimeric HSF molecule. To examine the role of
this region in detail, we exchanged sequences encompassing the linker
from hHSF2 into hHSF1 (Fig. 1B). In these studies, we define
the linker as sequences between the end of
-sheet 4 in the HSF1 DNA
binding domain (36, 37) and the first hydrophobic amino acid of LZ1
(amino acids 102-136 for hHSF1; amino acids 94-125 for hHSF2).
Replacement of the complete hHSF1 linker by hHSF2 sequences resulted in
a molecule (M1+M2) that could complement hsf
cells for
growth (Fig. 1C). Subsequently, we interchanged smaller
segments of the linker and identified that amino-terminal sequences
(M1), but not the carboxyl-terminal region (M2), supported growth of
hsf
cells, although both proteins were expressed to
similar levels in yeast cells (Fig. 1D). Comparison of the
hHSF1 and hHSF2 sequences encompassed by M1 revealed only five amino
acid differences. Therefore, to determine the precise residues
responsible for complementation by hHSF1, we constructed two additional
linker mutations encompassing amino acids 103-106 (M3) and amino acids
109-110 (M4). Only M4 conferred viability, although yeast cells
expressing this allele grew more slowly than other hHSF1 alleles that
complemented growth, whereas the M3 allele did not support any growth.
The M4 allele, harboring the double mutation (E109D, Q110D), was the
minimal swap that could confer viability in yeast because a single
amino acid (Q110D) substitution did not allow growth, nor did a double
alanine substitution for EQ (data not shown). Deletion mutants within
the hHSF1 linker,
ST and
TSV, which attempt to mimic the gap in
the hHSF2 linker, also did not complement the viability defect of
hsf
cells (Fig. 1C; data not shown); however,
these proteins were consistently expressed at lower levels than other
hHSF1 linker derivatives, suggesting that they may be unstable in yeast
(Fig. 1D). These data demonstrate that whereas the M4
mutation is the minimal substitution necessary for allowing hHSF1 to
function in yeast, complementation to the extent observed for the LZ4
mutant was obtained by substitution of all five amino acids in the
amino-terminal portion of the hHSF1 linker that differ from hHSF2. To
determine whether these hHSF1 linker sequences could act autonomously
to suppress hHSF2 function, we constructed the reciprocal molecules by
substituting hHSF1 linker sequences into hHSF2. All of the hHSF2
derivatives supported the growth of hsf
cells as well as
wild-type hHSF2 (Fig. 1C), suggesting that the hHSF1 linker
acts through a specific interaction within hHSF1 that is not conserved
in hHSF2.
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Fig. 1.
Complementation of the yeast
hsf allele by hHSF1 and hHSF2 linker
mutants. A, model for the conversion of latent,
monomeric hHSF1 into trimeric HSF that can bind to a HSE with high
affinity (6, 10). B, comparison of primary amino acid
sequences between the hHSF1 and hHSF2 linker regions. DB,
DNA binding domain; L, linker; T, trimerization
or leucine zippers 1-3; LZ4, leucine zipper 4;
AD, transcriptional activation domain. The
numbers refer to the hHSF1 amino acid sequence.
M1 through M4 refer to mutants constructed by
directly swapping the indicated sequences from one HSF isoform to the
other.
ST and
TSV refer to deletion
mutations made in the hHSF1 background. C, glucose shut-off
assays reveal a region of the linker critical for restraining hHSF1 in
an inactive state in yeast. Sequences from hHSF1 are represented in
white, and sequences from hHSF2 are represented in
gray. m refers to a mutation in the LZ4 domain.
PS145 cells harboring the indicated hHSF isoforms were grown in
galactose medium to permit the expression of a plasmid-borne copy of
yHSF under the control of the GAL1 promoter. Three 10-fold
serial dilutions (from left to right) of these
cells were plated to glucose medium that represses
GAL1-driven yHSF expression. Plates were photographed after
a 3-day incubation at 30 °C for hHSF1-expressing cells or a 2-day
incubation for hHSF2-expressing cells. D, levels of hHSF1
and hHSF2 were detected by immunoblotting with specific antibodies
against each protein. Levels of yeast phosphoglycerate kinase
(PGK) were used to normalize sample loading.
We previously observed a strict correlation between the ability of
hHSFs to form trimers in yeast under normal growth temperatures and
their ability to confer viability to hsf cells (35). To examine whether the linker derivatives described in Fig. 1 also show a
similar correlation, we performed ethylene bis(succinimidyl succinate)
cross-linking on extracts from yeast cells expressing both hHSFs and
yHSF to permit the assay of all hHSF1 derivatives, including those that
failed to support viability. In agreement with our previous
observations (35), wild-type hHSF1 was found primarily as a monomeric
species in yeast, whereas the LZ4 mutant was found almost
quantitatively in higher oligomers (Fig.
2). Linker mutants that complemented the
growth defect of hsf
cells (M1+M2, M1, and M4) all formed
some trimers, whereas mutants that failed to support the growth of
hsf
cells (M3; data not shown) also failed to trimerize.
We considered the possibility that complementation by the hHSF1-M1+M2
derivative could have resulted from enhanced nuclear localization
because a hHSF2 bipartite nuclear localization signal is contained
within the linker sequences (38). We have previously shown that green
fluorescent protein-tagged hHSF2 (hHSF2-GFP) was concentrated in the
yeast nucleus, like yHSF-GFP, whereas both hHSF1-GFP and LZ4-GFP
fusions showed diffuse fluorescence throughout the cell (35). The
GFP-tagged linker mutant M1+M2 appeared indistinguishable from LZ4-GFP,
indicating that the hHSF2 linker sequences did not visibly affect the
intracellular distribution of the hHSF1 linker mutant (data not shown).
Thus, only those hHSF1 derivatives capable of constitutive
trimerization, through mutations in either the coiled-coil domain or
the amino-terminal portion of the linker, can support the growth of
yeast cells lacking yHSF.
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To test whether the role of the linker in modulating hHSF1
trimerization in yeast could be recapitulated in human cells, we transfected human embryonic kidney 293 cells with two representative linker mutants that complemented the growth of hsf cells
(M1+M2 and M1) and one that did not (M2). Consistent with previous
reports (5), significant overexpression of hHSF1 either by transfection with large quantities of DNA (>1 µg/4 × 105 cells)
or by allowing transfected cells to express exogenous genes for long
periods (>14 h) resulted in constitutively trimerized and DNA binding
competent wild-type hHSF1 (data not shown). Therefore, we limited the
expression of transfected hHSF1 cDNAs so that we could observe the
heat-inducible activation of ectopically expressed hHSF1. Under these
conditions, hHSF1 linker mutants (M1+M2 and M1) that supported the
growth of hsf
cells and were constitutively trimerized in
yeast also showed significant trimerization under control temperatures
in human 293 cells (Fig. 3). In contrast, the M2 linker mutant showed only partial trimerization under control temperatures, like wild-type hHSF1 (WT), and was extensively
converted to trimers upon exposure to heat shock temperatures.
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Because trimerization of HSF1 is obligatory for high affinity binding
to HSEs (20, 39), we performed DNA binding assays using extracts from
control and heat shocked cells to independently assess the relative
level of oligomerization by each hHSF1 derivative. Consistent with the
trimerization data, 293 cells transfected with wild-type hHSF1
(WT) exhibited significant heat-inducible DNA binding
(9-fold) to the HSE probe, although basal levels of DNA binding were
higher than in vector-transfected (V) cells (Fig. 4A). The hHSF1 leucine zipper
mutant LZ4 was significantly activated at control temperatures, with
little stimulation in DNA binding upon heat shock. The linker mutants
M1+M2 and M1 appear to be quantitatively similar to the LZ4 mutant for
constitutive DNA binding under non-stress conditions. The M2 mutant,
which fails to complement hsf cells, showed a 5-fold
increase in heat-inducible binding to the HSE probe, suggesting that
its DNA binding activity is not deregulated at control temperatures.
Immunoblot analysis revealed that similar levels of protein were
expressed from each transfected plasmid (Fig. 4B). Moreover,
heat shocked extracts show the expected reduced electrophoretic
mobility of hHSF1 that is associated with heat-induced
hyperphosphorylation (9, 11). These results demonstrate that the hHSF1
linker modulates the monomer-to-trimer equilibrium in human cells as
well as in yeast.
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A linker domain connecting DNA binding and oligomerization domains of
the fungal Zn2Cys6 family of transcription
factors has been demonstrated to enforce DNA binding specificity among
the related GAL4, PPR1, and PUT3 proteins to their cognate recognition sequences (40). Similarly, alterations in the length or composition of
the yHSF linker significantly decreased the affinity of HSF trimers to
bind to a HSE (41). Because mammalian HSF1 and HSF2 isoforms exhibit
distinct preferences for binding to different HSEs in vitro
(42) as well as for differential target gene activation in
vivo using our yeast assay system (35), we tested the possibility that the hHSF linker might also influence this specificity. Our previous work showed that hHSF1 preferentially activated transcription of a SSA3 (Hsp70)-lacZ reporter that contains
five tandem repeats of the HSE pentamer (Fig.
5A) and that hHSF2 more
potently activated a CUP1 (metallothionein)-lacZ
fusion that has a shorter, noncanonical gapped HSE (35). To examine the
potential role of the linker in modulating target gene specificity, we
transformed CUP1-lacZ or SSA3-lacZ reporter
plasmids into hsf cells expressing the full-length linker
mutations (M1+M2), wild-type controls, or chimeras fused at a conserved
SphI site (42) that maintains homogeneous DNA binding and
linker domains (Fig. 5B). The levels of lacZ
mRNA were detected by RNase protection assays in control and heat
shocked cells. As observed previously, the CUP1 promoter
reproducibly yielded higher basal levels of lacZ transcripts
than the SSA3 promoter (35); thus, we focused on the degree
of heat-inducible expression. All molecules that harbored the hHSF1 DNA
binding domain (Fig. 5C, HSF1-DBD) displayed
stronger heat-inducible activation of the SSA3-lacZ
(>40-fold) reporter compared with the CUP1-lacZ reporter
(<2-fold). In contrast, all molecules containing the hHSF2 DNA binding
domain (HSF2-DBD) potently activated CUP1-lacZ in
response to heat shock (7-10-fold) but minimally activated the
SSA3-lacZ reporter. For all HSF constructs, heat-inducible expression of the reporter genes was abolished when the HSEs were mutated (data not shown). The similarity in response by all molecules with the same DNA binding domain, regardless of linker or trimerization sequences, supports a model in which DNA binding preferences, and
therefore target gene specificity, are determined by the hHSF isoform
DNA binding domain and are not significantly influenced by the linker
domain.
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DISCUSSION |
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Regulation of mammalian HSF1 activation is tightly controlled to
prevent inappropriate activation of the heat shock response. Under
non-stress conditions, HSF1 is repressed as an inactive monomer.
Activation of hHSF1 occurs through discrete steps involving trimerization, nuclear accumulation, high affinity binding to HSEs, and
transcriptional activation. Here we have used a dual approach,
combining analysis of the structure-function of human HSF1 expressed in
yeast with transfections in a human cell line to identify a region of
the linker that plays an important role in the maintenance of the
monomeric state. We have mapped the critical region to the
amino-terminal end of the linker that abuts the fourth -sheet of the
DNA binding domain. The M4 construct containing the double mutation
(E109D, Q110D) was the minimal swap that supported the growth of
hsf
cells, but a complete swap of the amino-terminal
portion of the linker, construct M1 (amino acids 103-110), gave a more
robust complementation of yeast cell growth than M4, based on colony
growth rates. Therefore, we suggest that these amino acids may form a
surface that interacts specifically with another, as yet unidentified,
region of hHSF1 to stabilize the monomer. A chimera composed of the
hHSF1 DNA binding and linker domains fused to the remainder of hHSF2
(construct hHSF1/2AflII (35)) was sequestered as an inactive
monomer that failed to complement hsf
yeast, suggesting a
potential site of interaction for the hHSF1 linker within the DNA
binding domain. The inability of the hHSF1 linker alone to suppress
hHSF2 trimerization further supports the idea that it is working
through an intramolecular mechanism. Interestingly, analysis of the
solution structure of the dHSF DNA binding domain revealed that two
residues located within the linker, Leu-142 and Ile-145 (corresponding
to amino acids Leu-112 and Ile-115 of hHSF1), interact with the
hydrophobic core of the DNA binding domain (37). These conserved
residues lie directly adjacent to the site that we have mapped as being the critical region for modulating trimerization of hHSF1.
The idea that multiple regions of HSF may contribute to monomer
regulation has been postulated from deletion analyses of HSF (5, 12,
13). Consistent with the results presented here, truncation of the dHSF
DNA binding domain to amino acid 136 did not lead to spontaneous
trimerization; however, removing an additional 17 amino acids that
impinged upon the dHSF linker and align with the human linker sequence
did result in significant trimerization at non-heat shock temperatures
(13). The current analysis extends this finding to hHSF1 and clearly
demonstrates that the substitution of five critical amino acids at the
amino-terminal region of the hHSF1 linker, in the context of the intact
molecule, is sufficient to disrupt the monomeric state. Although no
direct demonstration of an intramolecular interaction within the hHSF1
monomer has yet been reported, there is abundant biochemical and
genetic evidence to support a model (Fig.
6) in which the inactive HSF1 monomer is
restrained by contacts between the coiled-coil domains (5, 6, 10).
Point mutations within either hydrophobic domain that disrupt the
arrangement of heptad repeats lead to constitutive and nearly
quantitative trimerization of hHSF1 when expressed in either yeast
(Fig. 2), human cells (Fig. 3 (5)), or Xenopus oocytes (6).
Furthermore, purified mouse HSF1 or dHSF lacking the last leucine
zipper domain result in constitutively trimerized complexes in
vitro (14, 15). These observations that any number of
perturbations to HSF1 structure, including mutations in the linker,
lead to a significant shift in the monomer-to-trimer equilibrium suggest that the stability of the inactive monomer depends upon multiple interdependent contacts. Intriguingly, purified dHSF can be
reversibly trimerized in vitro in response to heat,
decreased pH, and some chemical inducers of the heat shock response,
indicating that the monomeric HSF protein can directly sense some
environmental changes (15, 43). In vivo, the stabilization
of HSF1 monomers may involve interactions with other cellular factors
such as molecular chaperones (17, 18); however, the precise role of
these proteins in HSF regulation and the sites of interaction between
these proteins remain to be determined.
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The presence of structured linkers between DNA binding and oligomerization domains is a common motif among several classes of fungal and mammalian transcription factors. Among the best characterized is the role of the linker in discriminating between similar binding sites within the yeast Zn2Cys6 family that includes GAL4, PUT3, and PPR1. Analysis of the chimeras between these proteins, together with three-dimensional structural determinations, revealed that the unique linker, rather than the highly similar DNA binding domains, was the determinant of DNA binding specificity (44-47). In our study, we found that interchanging the hHSF1 and hHSF2 linkers did not significantly alter the preferential activation of SSA3-lacZ and CUP1-lacZ by all molecules harboring the hHSF1 and hHSF2 DNA binding domains, respectively. We note, however, that the DNA binding specificity is not absolute, and both HSF1 and HSF2 can bind a variety of HSEs (42). Nevertheless, the SSA3 and CUP1 promoters represent naturally occurring HSEs that show preferential heat-inducible activation by the hHSF isoforms. Thus the role of the HSF1 linker appears to be distinct from that of the Zn2Cys6 family: the HSF1 linker influences trimerization rather than discriminating between different types of HSEs. An implication from the current results is that differential specificity for distinct HSEs and for distinct footprint patterns exhibited by mammalian HSF1 and HSF2 (42, 48) is determined primarily by the highly similar, although not identical, DNA binding domains.2 This prediction is fully consistent with the previous observation that a single amino acid substitution within the DNA binding domain of yHSF dramatically increased the binding affinity for the CUP1 promoter and decreased binding to the SSA3 promoter (49).
Previous studies on the yHSF linker suggested an important role in
aligning the DNA binding domains of HSF to the HSE (41). Because of the
constraints imposed on a trimeric protein by the necessity to bind
three consecutive HSEs on a linear DNA molecule, it is believed that
the HSF linker must provide sufficient flexibility for the individual
DNA binding domains to contact each cognate binding sequence (20). The
minimal yHSF linker required for DNA binding and functional
complementation in yeast was mapped to a 21-amino acid sequence that
aligns in length and, in part, in composition to the mammalian and
Drosophila HSF linkers, whereas a 52-amino acid extension
unique to the yHSF linker was dispensable for either DNA binding or
complementation (41). Thus, the linker appears to play multiple roles:
providing flexibility to all HSF trimers, closing off the DNA binding
domain through hydrophobic interactions, and modulating trimerization
for a subset of HSFs including hHSF1 and dHSF. We propose that within
certain HSF contexts, such as in the hHSF1 monomer, the linker may
adopt a specific conformation that would allow it to make contacts with
another surface of HSF, despite the lack of evidence for a defined
conformation in solution. By analogy, it is interesting that the
solution structures determined by NMR of the GAL4 and PUT3 DNA binding
domains also revealed flexibility in the flanking linker sequences (47,
50, 51), but co-crystal structures of these proteins bound to cognate DNA firmly established that each linker adopted a distinct conformation (45, 52). The precise mechanism by which hHSF1 converts from a monomer
to trimer upon sensing stress remains incompletely understood, and our
results underscore the complexity of this regulation. How the linker
and other regions of HSF1 act to modulate trimerization must await the
complete structural determination of the protein.
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ACKNOWLEDGEMENTS |
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We thank Vincenzo Zimarino for critical reading of the manuscript and helpful suggestions with mammalian transfections. We thank Xiao-Dong Liu, Nicholas Santoro, and Kevin Morano for critical readings, advice, and yeast plasmid constructs; members of the Thiele laboratory for suggestions; and Chen Kuang for excellent technical assistance. The gifts of reagents from Hillary Nelson, Richard Morimoto, Carl Wu, Robert Kingston, and Stuart Calderwood are gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by a Taisho Excellence in Research Program Award from Taisho Pharmaceuticals, Co. Ltd and a grant from the National Institutes of Health.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.
Supported by a National Research Service Award (GM18858)
Postdoctoral Fellowship.
§ A Burroughs Wellcome Toxicology Scholar.
¶ To whom correspondence should be addressed. Tel.: 734-763-5717; Fax: 734-763-4581; E-mail: dthiele{at}umich.edu.
2 P. C. C. Liu, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: Hsp, heat shock protein; HSF, heat shock transcription factor; hHSF, human HSF; dHSF, Drosophila HSF; yHSF, Saccharomyces cerevisiae HSF; HSE, heat shock element; LZ, leucine zipper; GFP, green fluorescent protein; HEGN, 20 mM Hepes, pH 7.9, 0.5 mM EDTA, 10% glycerol, and 100 mM NaCl.
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REFERENCES |
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