From the
The anti-inflammatory drug sodium salicylate modulates the
activity of specific transcription factors in humans. Here we show that
salicylate and sorbate, another organic acid, stimulate DNA binding by
yeast heat shock transcription factor (HSF) in vivo.
Surprisingly, salicylate inhibits heat shock gene transcription even in
cells induced by a prior heat shock. This inhibition of transcription
occurs at a step after HSF and transcription factor IID binding but
before promoter melting by RNA polymerase. Salicylate appears to
generate a tight binding but activation-impotent HSF by cytoplasmic
acidification, since inhibiting proton efflux from cells triggers this
same DNA binding and inhibition of heat shock gene expression.
Salicylates are widely prescribed anti-inflammatory drugs, yet
their mechanism of action is not well understood. It has recently been
found that salicylates affect the activity of two human transcription
factors: heat shock factor (HSF)
Here we show that sodium
salicylate can inhibit heat shock gene transcription in yeast. The
inhibition of heat shock gene transcription appears to be specific and
is probably mediated through the formation of an activation-impotent,
tight DNA-binding HSF. By comparison with other chemical agents with
similar effects, we provide evidence that salicylate affects HSF and
heat shock gene transcription by lowering the cellular pH.
We recently demonstrated that, similar to human cells, heat
shock stimulates the DNA binding activity of yeast HSF in vivo(16) .
To test if
cytoplasmic acidification is the condition that activates the binding
of HSF to DNA, we examined HSF binding to the HSP82 promoter
in yeast cells treated with DES. DES-treated cells were indeed found to
have hyperactivated HSF binding as seen by DMS footprinting
(Fig. 4 B). Therefore, lowering cytoplasmic pH by a means
that does not require addition of organic acids leads to the same
activation of HSF binding as seen in salicylate-treated cells.
Interestingly, human monomeric HSF in in vitro extracts from
uninduced cells can also be activated to trimerize and thereby acquire
tight DNA binding activity by simply lowering the pH to between 5.8 and
6.4
(15) . We speculate that a low pH favors formation of the
DNA-binding trimer, but this trimer is locked in a conformation that
cannot activate transcription. Although this inactive trimer does not
respond to heat shock under the conditions tested here, i.e. growth in rich medium with glucose, we have obtained preliminary
evidence that under glucose-derepressing conditions (growth in
galactose), heat-induced transcription can occur in the presence of
salicylate (not shown). Therefore, under glucose-derepressing
conditions, yeast appear to respond to salicylate more like human cells
(1) .
Although our analysis here is limited to the effect of
salicylate on yeast HSF and heat shock gene transcription, it is
possible that salicylate may inhibit NF-
We thank T. O'Brien, L. Shopland, and H. Shi for
critically reading this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
and nuclear
factor-
B (NF-
B)
(1, 2) . During inflammation,
an increased tissue temperature and other forms of physiological stress
induce transcription of the heat shock genes
(1, 3, 4) . The ability of salicylate to
activate the DNA binding activity of HSF could therefore be
contributing to its pharmacological effectiveness. Salicylate has also
been shown to inhibit the activation of NF-
B
(2) .
NF-
B is responsible for activating a spectrum of genes involved in
the inflammatory response, including various cytokines and cell
adhesion molecules
(5, 6, 7) . In addition,
HIV-1 transcription requires NF-
B, and salicylate's
inhibition of HIV-1 replication may be a result of its ability to
inhibit NF-
B activation
(5, 8, 9, 10) . Salicylate also plays an
important role in plant systemic acquired resistance, where it serves
to activate the pathogenesis-related genes
(11, 12, 13) .
Yeast Growth and in Vivo DMS Footprinting
The
yeast strain YPH102 was grown in YEPD medium to an optical density of
1.5 and treated as described in the text. For heat-shocked
samples, yeast were transferred to a flask prewarmed to 39 °C for
15 min. 10 ml of culture was pelleted and resuspended in 150 µl of
fresh medium (either at room temperature or at 39 °C for the heat
shock samples). This suspension is treated with 1 µl of DMS for 1
min. 50 µl was then withdrawn, and the reaction was terminated by
dispersing in 550 µl of sorbitol stop buffer (0.9 M
sorbitol, 0.1 M Tris-HCl (pH 8.0), 0.1 M EDTA, 40
mM
-mercaptoethanol). Spheroplast preparation and DNA
isolation was performed as described
(14) . The sites of DMS
modification were cleaved by treating with 1 M piperidine for
30 min at 88 °C. After piperidine treatment, DNA was dried,
resuspended in 50 ml of 0.3 M sodium acetate,
ethanol-precipitated, and washed with 70% ethanol. Usually, about
one-fourth of this DNA was analyzed by ligation-mediated polymerase
chain reaction as described
(14) . Primers to view DMS
reactivity were: primer 1, GGTTGGTATTAAGATGAGAATTAACC; and primer 2,
TAACCGCTCATAAAACCATGCGCGT.
RNA Preparation and Primer Extension
A 5-ml
culture of yeast was treated as described in the text. Cells were
pelleted, washed with water, and resuspended in 400 µl of 10
mM Tris-Cl, pH 7.5, 10 mM EDTA, 0.5% SDS. This
suspension was extracted with water-equilibrated phenol at 65 °C
over the course of 1 h. Samples were then chilled on ice and
centrifuged for 5 min. The aqueous phase was extracted once more with
phenol and then twice with chloroform. RNA was then
ethanol-precipitated and resuspended in water. 30 µg of RNA was
mixed with
0.2 pmol of end-labeled primer, ethanol-precipitated,
washed, resuspended in 11 µl of water, and heated to 75 °C for
5 min. Samples were cooled on ice, and 9 µl of reaction mixture was
added to give the final concentrations: 1
first strand buffer
(Life Technologies, Inc.); 10 mM dithiothreitol; 2 mM
dNTPs; 1 unit/µl RNasin (Promega); 5 units/µl SuperScript II
reverse transcriptase (Life Technologies, Inc.). This reaction was
incubated for 1 h at 45 °C. DNA was then ethanol-precipitated and
run on a 5% polyacrylamide-urea sequencing gel. The primers were: for
HSP and HSC82, CCAATTGCTTTGGATCAGACAAAG; for
ACT1, GCTGATGTAGTAGAAGATCCTATTC; for GAL1,
TTTGCGCTAGAATTGAACTCAGGTAC.
KMnO
Cultures
were treated as described in the text. 5 ml of culture was pelleted and
resuspended in 50 µl of fresh media either at room temperature
( Footprinting
24 °C) or at 39 °C when heat shocked. This suspension was
treated with 3.5 µl of 0.35 M KMnO
for 1 min.
Reaction was stopped by adding 550 µl of sorbitol stop buffer. DNA
was prepared and treated like the DMS-treated samples above. KMnO
modification of the promoter was observed using ligation-mediated
polymerase chain reaction and the following primers: primer 1,
CCAATTGCTTTGGATCAGACAAAG; and primer 2, CAAAGATTTGTATCTAATTTTATCCAACGC.
DES Treatment
DES (Sigma) was dissolved to 10
mM in methanol and added to the medium dropwise with constant
agitation. For control cells, methanol was added to give a final
concentration equal to that used for the most concentrated DES
treatment. After a 45-min incubation at room temperature, DMS
footprinting was performed.
(
)
We were curious as to
whether sodium salicylate could also stimulate the DNA binding activity
of yeast HSF, as it can in human cells, since yeast would provide a
very tractable system to investigate salicylate's effect on
transcription factor activity. Fig. 1shows a DMS footprint over
the HSP82 heat shock elements (HSEs) in cells treated with 0
to 30 mM sodium salicylate. At 0 mM salicylate, under
non-heat shock conditions (the NHSlane of
Fig. 1
), HSF binding to the strong HSE of this promoter, HSE1, is
revealed by the protection of G residues at -161, -162, and
-171 (compare the NHSlane to the DNAlane). Sodium salicylate increases HSF binding to the
promoter as revealed by a slight increase in protection of the G
residues in HSE1, and, more dramatically, by the formation of a
hyper-reactive site at -210 in a weaker HSE of this promoter,
HSE3 (this hyper-reactive site is also formed when DMS footprinting is
performed in vitro with high concentrations of purified
HSF).
The HSF binding induced by sodium salicylate is
actually greater than that found in heat-shocked cells (Fig. 1).
The salicylate concentration required to stimulate yeast HSF binding is
similar to that needed to affect HSF and NF-
B in human cells,
which is also similar to that required for its anti-inflammatory
activity
(1, 2, 17, 18) .
Figure 1:
Sodium salicylate stimulates HSF
binding to the HSP82 promoter in yeast. Yeast were treated
with 0, 3, 10, and 30 mM sodium salicylate for 10 min before
performing DMS footprinting, as indicated. Also shown is the DMS
modification pattern on purified DNA ( DNA) and DNA in cells
undergoing a 39 °C heat shock ( HS). The locations of the
strong HSE1 and weak HSE2 and HSE3 are indicated, and the numbering is
relative to the transcriptional start site. A long and a short exposure
of the same gel is shown to display clearly all G
residues.
While both
heat shock and salicylate stimulate HSF DNA binding, we found that,
unlike heat shock, salicylate is a potent inhibitor of heat-activated
HSP82 transcription in yeast. Fig. 2 A shows the
levels of HSP82 RNA (and RNA from its cognate gene,
HSC82) quantified by primer extension. Sodium salicylate
strongly inhibits the heat induction of both these genes. This is
unlike the situation in humans where salicylate is non-inhibitory and
possibly potentiates the stress response
(1) . The inhibition of
yeast heat-activated transcription is stable, lasting for at least 2 h
(Fig. 2 B). This effect on transcription is remarkably
specific, however, since salicylate does not interfere with galactose
induction of the GAL1 gene nor does it affect the steady-state
levels of ACT1 RNA (Fig. 2 A) or dramatically
alter cell growth rates (not shown). As would be expected for an
inhibitor of heat shock gene transcription, salicylate inhibited the
acquisition of thermotolerance to a 52 °C heat shock conferred by a
39 °C heat shock pretreatment (not shown)
(19) . It is not
clear how the hyperactivation of HSF's DNA binding activity
inhibits heat-induced transcription in yeast. Perhaps the salicylate
stimulation of HSF DNA binding interferes with another HSF modification
step, such as phosphorylation
(1, 20) , leaving the
promoter occupied only by an inactive form of this transcription
factor.
Figure 2:
A,
the effect of salicylate on HSP82, HSC82, GAL1 and ACT1 gene transcription. In lanes 1-4,
RNA was prepared from yeast heat shocked for 30 min at 39 °C
( HSlanes) and non-heat-shocked yeast ( NHSlanes). In the sodium salicylate ( NaSa) +
lanes, cultures contained 30 mM sodium salicylate. In
lanes 5-8, yeast were grown with raffinose as the carbon
source after which cells were transferred to fresh raffinose ( RAFlanes) or fresh galactose media ( GALlanes). RNA was quantified by primer extension using
primers for HSC82, HSP82, ACT1 and GAL1.B, salicylate's inhibition of heat shock gene
transcription is stable. RNA was prepared from control yeast ( lane1), yeast grown at room temperature in the presence of 30
mM sodium salicylate ( lanes 2-4), or yeast
grown at 39 °C in the presence ( lanes 5-7) or
absence ( lanes 8-10) of 30 mM sodium
salicylate. In lanes5-10, RNA was prepared 30,
60, or 120 min after initiation of heat shock, as indicated, with
salicylate treatment in lanes 5-7 initiated 10 min
before heat shock. Lanes 2-4 are the same as lanes5-7except the cells were maintained at
room temperature for the times shown. Primer extension of isolated RNA
was performed as above.
To gain some insight into which step in the transcription
cycle is blocked by salicylate, we examined the KMnO modification pattern of the HSP82 promoter. KMnO
reactivity with DNA increases upon DNA melting, so this reagent
is useful for detecting RNA polymerase open complexes
(14) .
In addition, a reduction in the TATA
box's reactivity with KMnO
is diagnostic of
transcription factor IID binding
(21) . Salicylate was found to
inhibit promoter melting (note the hyper-reactive sites at -55
and those close to the transcription start site are not detectable in
the presence of salicylate) but not TATA protection (Fig. 3).
These findings suggest that salicylate is inhibiting transcription at a
step after transcription factor IID binding but before polymerase open
complex formation. Salicylate even inhibits promoter melting when added
10 min after heat shock, a time sufficient to fully activate
transcription of heat shock genes (Fig. 3)
(22) .
Figure 3:
Salicylate inhibits the heat shock-induced
melting of the HSP82 promoter, but not TBP binding. In
lanes 2-5, yeast were heat shocked ( HS lanes)
or maintained at room temperature ( NHS lanes) in the presence
of 30 mM sodium salicylate ( NaSa) where indicated. In
these lanes, salicylate treatment was initiated 10 min prior
to heat shocking. Lane 1 shows the KMnO modification reaction on purified DNA. After a 10-min heat shock,
KMnO
footprinting was performed. Lanes 6-9 are the same as lanes 2-5 except salicylate
treatment was initiated 10 min after heat shock. 7.5 min after addition
of salicylate to the indicated samples, KMnO
footprinting
was performed. The arrow indicates the position of the
transcriptional start site, and the numbering is relative to
this start site. The TATA box is labeled.
The effect of salicylate on heat shock gene transcription
observed here is similar to that recently found for other weak organic
acids
(23) . For example, sorbate, a commonly used food
preservative, also blocks the activation of heat shock gene
transcription in yeast. This inhibition is most potent when the
treatment is performed in low pH medium
(23) . Low pH increases
the concentration of protonated acid in the medium, which is probably
the form that most efficiently crosses the membrane. We found that at
pH 4.5 or 5.5, sorbate also stimulated the DNA binding activity of HSF
(Fig. 4 A). The pH of the medium had a similar effect on
the stimulation of HSF DNA binding by salicylate
(Fig. 4 A). The effects of sorbate and salicylate on
yeast heat shock gene transcription and yeast HSF are similar,
indicating that these acids may be working through a common mechanism.
Figure 4:
A, sodium sorbate and sodium
salicylate both stimulate HSF DNA binding in yeast most efficiently in
low pH media. Cells were incubated in YEPD media with pH of 4.5, 5.5,
or 6.5 plus or minus 10 mM sodium sorbate or sodium
salicylate, as indicated. After 10 min, DMS footprinting was performed
as in Fig. 1. In all other experiments the media pH was 5.6 at the
time of DMS treatment. B, DES stimulates the DNA binding
activity of yeast HSF. Yeast were incubated for 45 min at room
temperature with 0, 25, 50, or 100 µM DES, as indicated.
DMS footprinting was then performed as in Fig.
1.
One well characterized activity of acetylsalicylate (aspirin) is its
ability to inhibit prostaglandin synthesis
(18, 24) .
However, sodium salicylate is a poor inhibitor of prostaglandin
synthesis so it is unlikely that this is the mechanism by which it
stimulates the DNA binding of yeast HSF
(18) . Since sodium
salicylate affects yeast heat shock gene transcription and HSF DNA
binding in a manner similar to that of the structurally unrelated
organic acid, sodium sorbate, it could be influencing transcription by
acidifying the cellular cytoplasm
(23) . Organic acids diffuse
through the membrane in their protonated form and dissociate in the
higher pH of the cytoplasm
(25) . Evidence that organic acids
inhibit heat shock gene transcription by cytoplasmic acidification
comes from the finding that a number of other chemical agents and
mutations that decrease cytoplasmic pH also inhibit heat shock gene
transcription. For example, inhibition of the plasma membrane proton
pump, either by diethylstilbestrol (DES) or by mutation, also inhibits
the heat shock gene expression
(23, 26) .
B activation in humans by
reducing cellular pH
(2) . In addition, this activity of
salicylate could be part of its signaling activity in plants. In this
regard, establishing yeast as a model system should facilitate the
study of salicylate's effects on eukaryotic cell physiology and
gene regulation.
B, nuclear factor-
B; HIV-1, human immunodeficiency
virus, type 1; DMS, dimethyl sulfate; HSE, heat shock element; NHS,
nonheat shock; DES, diethylstilbestrol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.