Heat shock factor
Mammalian cells express two distinct HSF1 mRNA isoforms that arise via alternative
splicing of the HSF1 pre-mRNA. The two HSF1 mRNA isoforms differ by a single 66 bp exon of the
HSF1 gene, which is spliced into the HSF1-alpha mRNA isoform but skipped in the HSF1-beta mRNA
isoform. This 66 bp exon encodes a 22 amino acid sequence, whose molecular weight (2.3 kDa)
matches the difference in size between the HSF1-beta and HSF1-alpha protein isoforms (69 and 71
kDa). Further analysis reveals that this extra 22 amino acid sequence, whose insertion site in the
HSF1-alpha isoform is located immediately adjacent to a C-terminal leucine zipper motif (leucine zipper
4) previously shown to be involved in maintenance of HSF1 in the non-DNA-binding control form,
contains an additional, previously unidentified leucine zipper motif (leucine zipper 5). The levels of the two HSF1 isoforms are regulated in a tissue dependent manner, with testis
expressing higher levels of the HSF1-beta isoform while heart and brain express higher levels of the
HSF1-alpha isoform. These results demonstrate a new mechanism by which HSF1 expression is
regulated in mammalian cells and suggest a potential role for the HSF1 isoforms in mediating
tissue-dependent regulation of the cellular stress response (Goodson, 1995b).
Heat shock and other proteotoxic stresses cause accumulation of nonnative proteins that trigger activation of heat
shock protein (Hsp) genes. A chaperone/Hsp functioning as a repressor of heat shock transcription factor (HSF) could
make activation of Hsp genes dependent on protein unfolding. In a novel in vitro system, in which human HSF1 can
be activated by nonnative protein, heat, and geldanamycin, the addition of Hsp90 inhibits activation. Reduction of the
level of Hsp90 but not of Hsp/c70, Hop, Hip, p23, CyP40, or Hsp40 dramatically activates HSF1. In vivo,
geldanamycin activates HSF1 under conditions in which it is an Hsp90-specific reagent. Hsp90-containing HSF1
complex is present in the unstressed cell and dissociates during stress. It is concluded that Hsp90, by itself and/or
associated with multichaperone complexes, is a major repressor of HSF1 (Zou, 1998).
Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers as well ashyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including
suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling
cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation
pathway are not known. HSP90, a cellular chaperone known to regulate several signal
transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90
heterocomplexes was demonstrated by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear
extracts: the recognition of HSF1-HSE complexes in vitro was shown by using HSP90 antibodies (Abs), and the recognition of HSF1 in vivo
by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed
using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that
specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delay the disassembly of HSF1 trimers
during recovery from heat shock and specifically inhibit heat-induced transcription from a chloramphenicol
acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activate HSE binding in the absence
of heat shock, an effect that can be reversed by subsequent injection of purified HSP90. GA does not activate HSE binding
under nonshock conditions but increases the quantity of HSE binding induced by heat shock. On the basis of these findings
and the known properties of HSP90, a new regulatory model is proposed in which HSP90 participates in modulating HSF1
at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric
conformations as well as transcriptional activation. HSP90 is proposed to link HSF1 to cellular
signaling molecules coordinating the stress response (Ali, 1998).
HSF1 is the mediator of stress-induced heat
shock gene transcription in humans. HSF1 displays stress-induced DNA-binding activity, oligomerization, and
nuclear localization, while HSF2 does not. HSF1 undergoes phosphorylation in cells exposed to
heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic
acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and
HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity,
oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in
the absence of physiological stress and also provide support for a model of regulation of HSF1 and
HSF2 activity by a titratable negative regulatory factor (Sarge, 1993).
Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. At serine residues distal to the transcriptional activation domain, constitutive phosphorylation of HSF1 functions to repress HSF1's transactivation function. A region of constitutive phosphorylation encompasses serine residues 303-307. Whereas HSF1 is constitutively repressed for transactivation and derepressed by heat shock, mutations of serines 303 and 307 to alanine result in derepression to a high level of constitutive activity. The relationship between MAP kinase phosphorylation of HSF1 and the constitutive activation of HSF1 reported in this paper is unclear (Kline, 1997).
Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of
heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated
by a separate regulatory domain, which confers repression at control temperature and heat
inducibility. A high level of phosphorylation at the low (control) temperature seems to result in HSF1 inactivation. Two specific proline-directed serine motifs are important for
function of the regulatory domain in the following ways: mutation of these serines to alanine results in derepressed HSF1 activity
at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in
normal repression at control temperature, with normal heat shock inducibility. Tryptic mapping
shows that these serines are the major phosphorylation sites of HSF1 at control temperature in
vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation
of these major sites. The regulatory domain is an excellent substrate in vitro for the
mitogen-activated MAPK/ERK. Phosphorylation of the regulatory domain of
HSF1 appears to decrease the activity of HSF1 at control temperature, and suggests a mechanism for
modification of HSF1 activity by growth control signals (Knauf, 1996).
Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions (see Drosophila SUMO). Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2 were used. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression (Anckar, 2006)
The interaction of recombinant mouse heat shock transcription factors 1 and 2 (mHSF1
and mHSF2) with their binding site, the heat shock element (HSE) has been characterized using
human HSP70 HSE. It consists of three perfect 5'-nGAAn-3' sites (1, 3, and 4) and two imperfect
sites (2 and 5) arranged as tandem inverted repeats. Recombinant mHSF1 and mHSF2, which exist as
trimers in solution, both bind specifically to this HSE and stimulate transcription of a human
HSP70-CAT construct in vitro. Footprinting analyses reveal differential binding of mHSF1 and
mHSF2 to the HSP70 HSE. Specifically, mHSF1 binds all five pentameric sites, whereas mHSF2
fails to interact with the first site of the HSE but binds to sites 2 to 5. Missing-nucleoside analysis
demonstrates that the third and fourth nGAAn sites are essential for mHSF1 and mHSF2 binding.
The binding of the initial mHSF1 trimer to the HSE exhibits preference for sites 3, 4, and 5; then
binding of a second trimer occurs at sites 1 and 2. These results suggest that HSF may recognize its
binding site through the dyad symmetry of sites 3 and 4 but requires an adjacent site for stable
interaction. Thus mHSF1 and mHSF2 bind specifically to the HSE through major
groove interactions. Footprinting reveals structural differences in the first and
third repeats of the HSE, suggesting that the DNA is distorted in this region. The possibility that the
HSE region is naturally distorted may assist in understanding how a trimer of HSF can bind to what is
essentially an inverted repeat binding site (Kroeger, 1993).
Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response
in eukaryotes. In addition to its role as a transcription factor, HSF1
stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of
two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory
component, also known as the Ku protein. HSF1 binds specifically to each of the
two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku
protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and
the active HSF1 trimer. The binding is blocked by antibodies against HSF1. These results show that
HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are
dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not
sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both
components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to
heat and other forms of stress (Huang, 1997).
In response to stress, heat shock factor 1 (HSF1) acquires rapid DNA binding and transient
transcriptional activity while undergoing conformational transition from an inert non-DNA-binding
monomer to active functional trimers. Attenuation of the inducible transcriptional response occurs
during heat shock or upon recovery at non-stress conditions and involves dissociation of the HSF1
trimer and loss of activity. The hydrophobic repeats of the HSF1 trimerization domain were used in
the yeast two-hybrid protein interaction assay to identify heat shock factor binding protein 1 (HSBP1),
a novel, conserved, 76-amino-acid protein that contains two extended arrays of hydrophobic repeats
that interact with the HSF1 heptad repeats. HSBP1 is nuclear-localized and interacts in vivo with the
active trimeric state of HSF1 that appears during heat shock. During attenuation of HSF1 to the inert
monomer, HSBP1 associates with Hsp70. HSBP1 negatively affects HSF1 DNA-binding activity, and
overexpression of HSBP1 in mammalian cells represses the transactivation activity of HSF1. To
establish a biological role for HSBP1, the homologous Caenorhabditis elegans protein was
overexpressed in body wall muscle cells and was shown to block activation of the heat shock response
from a heat shock promoter-reporter construct. Alteration in the level of HSBP1 expression in C.
elegans has severe effects on survival of the animals after thermal and chemical stress, consistent with
a role for HSBP1 as a negative regulator of the heat shock response (Satyal, 1998).
The spontaneous expression of heat shock genes during development is well
documented in many animal species, but the mechanisms responsible for this
developmental regulation are only poorly understood. In vertebrates, additional heat
shock transcription factors, distinct from heat shock factor 1 (HSF1) involved in
the stress response, are thought to be involved in this developmental control. In
particular, the mouse HSF2 has been found to be active in testis and during
preimplantation development. In mice, HSF2 is
regulated during the
postimplantation phase of development. It is expressed at least until 15.5 days of embryogenesis. It
becomes restricted to the central nervous system during the second half of gestation.
It is expressed in the ventricular layer of the neural tube, which contains mitotically
active cells, but not in postmitotic neurons. Parallel results are obtained for mRNA,
protein, and activity levels, demonstrating that the main level of control is
transcriptional. The detailed analysis of the activity of a luciferase reporter gene under
the control of the hsp70.1 promoter, as well as the description of the protein
expression patterns of the major heat shock proteins in the central nervous system,
show that HSF2 and heat shock protein expression domains do not coincide. This
result suggests that HFS2 might be involved in other regulatory developmental
pathways and paves the way to new functional approaches (Rallu, 1997).
The inducible DNA-binding
activities of HSF1 and HSF2 are regulated through distinct pathways. HSF1 is activated by heat shock and
other forms of stress, whereas HSF2 is activated during hemin-induced differentiation of human K562
erythroleukemia cells, suggesting a role for HSF2 in regulating heat shock gene expression under
nonstress conditions, such as differentiation and development. The inactive, non-DNA-binding forms of HSF2 and HSF1 exist primarily
in the cytoplasm of untreated K562 cells as a dimer and monomer, respectively. This difference in the
control oligomeric states suggests that distinct mechanisms are used to control the DNA-binding activities of
HSF2 and HSF1. Upon activation, both factors acquire DNA-binding activity, oligomerize
to a trimeric state, and translocate into the nucleus. Simultaneous activation
of both HSF2 and HSF1 in K562 cells subjected to hemin treatment followed by heat shock results in
the synergistic induction of hsp70 gene transcription, suggesting a novel level of complex regulation of
heat shock gene expression (Sistonen, 1994).
Acquisition of heat shock factor 2 (HSF2) DNA binding activity is accompanied by induced transcription of heat shock genes in
hemin-treated K562 cells undergoing erythroid differentiation. HSF2 consists of two alternatively spliced
isoforms, HSF2-alpha and HSF2-beta, whose relative abundance is developmentally regulated and varies between different tissues. To
investigate whether the molar ratio of HSF2-alpha and HSF2-beta isoforms is crucial for the activation of HSF2 and whether the HSF2
isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, cell clones were generated expressing
different levels of HSF2-alpha and HSF2-beta. In parental K562 cells, the HSF2-alpha isoform is predominantly
expressed and HSF2 can be activated upon hemin treatment. In contrast, when HSF2-beta is expressed at levels exceeding those of
endogenous HSF2-alpha, the hemin-induced DNA binding activity and transcription of heat shock genes are repressed, whereas
overexpression of HSF2-alpha results in an enhanced hemin response. Furthermore, the hemin-induced accumulation of globin, known
as a marker of erythroid differentiation, is decreased in cells overexpressing HSF2-beta. It is suggested that HSF2-beta acts as a negative
regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells (Leppa, 1997).
HSF2 alternative splicing is developmentally regulated; a switch in expression occurs during
testis postnatal development from the HSF2-beta mRNA isoform to the HSF2-alpha isoform. Transfection analysis shows that the HSF2-alpha protein, the
predominant isoform expressed in testis cells, is a more potent transcriptional activator than the
HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in
mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional
activity in a tissue-dependent manner (Goodson, 1995c).
Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, HSF2 expression was examined at specific stages of the rat seminiferous epithelial cycle. Expression of the alternatively spliced HSF2-alpha and HSF2-beta isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrate that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I to IV and in the nuclei of round spermatids at stages V to VIIab. In contrast, a strong HSF2 immunoreactivity is detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis reveals that these structures are mainly cytoplasmic bridges between germ cells. These results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells (Alastalo, 1998).
Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions
to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and
activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has
not yet been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of
HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. A stress signal
for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced
upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell
line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) when shifted to the nonpermissive
temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The
downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are
induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory
mechanism to respond to changes in the protein-degradative machinery (Mathew, 1998).
In vertebrates, the presence of multiple heat shock transcription factors (HSFs) indicates that these
factors may be regulated by distinct stress signals. HSF3 is specifically activated in unstressed
proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). These factors
formed a complex through their DNA binding domains that stimulates the nuclear entry and formation
of the transcriptionally active trimer of HSF3. Because c-Myb participates in cellular proliferation, this
regulatory pathway may provide a link between cellular proliferation and the stress response (Kanei-Ishii, 1997).
Human HSF4 (hHSF4) is a novel member of the hHSF family that shares properties
with other members of the HSF family yet appears to be functionally distinct. hHSF4 lacks the
carboxyl-terminal hydrophobic repeat that is shared among all vertebrate HSFs and is thought to be involved in the negative regulation of DNA binding activity. hHSF4 is preferentially
expressed in the human heart, brain, skeletal muscle, and pancreas. Transient transfection of hHSF4 in
HeLa cells, which do not express hHSF4, results in a constitutively active DNA binding trimer that (unlike other members of the HSF family) lacks the properties of a transcriptional activator. Constitutive
overexpression of hHSF4 in HeLa cells results in reduced expression of the endogenous hsp70,
hsp90, and hsp27 genes. hHSF4 represents a novel hHSF that exhibits tissue-specific expression and
functions to repress the expression of genes encoding heat shock proteins and molecular chaperones (Nakai, 1997).
Heat shock factor 1 (HSF1) is the master regulator of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. This study reports that eliminating HSF1 protects mice from tumors induced by mutations of the RAS oncogene or a hot spot mutation in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by orchestrating a network of core cellular functions including proliferation, survival, protein synthesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initiation; human cancer lines of diverse origins show much greater dependence on HSF1 function to maintain proliferation and survival than their nontransformed counterparts. While it enhances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer (Dai, 2007).
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