Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 356-362
doi:10.1111/j.1745-7270.2006.00170.x
Effects of Heat Stress on
Yeast Heat Shock Factor–Promoter Binding In Vivo
Ning LI1,
Le-min ZHANG1*,
Ke-Qin ZHANG1, Jing-shi DENG1, Ralf PR?NDL2&, and Fritz SCH?FFL2
1
Laboratory for Conservation and Utilization of
Bio-resources, Yunnan University, Kunming 650091, China;
2
Zentrum f?r Molekularbiologie der Pflanzen – Allgemeine
Genetik, Eberhard-Karls-Universit?t T?bingen, 72076 T?bingen, Germany
Received: January
9, 2006
Accepted: March 22,
2006
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30560012), the Department of Science and Technology of Yunnan Province
(2003C0012R, 2005NG05), the project sponsored by SRF for ROCS, SEM; and the grants
from Deutsche Forschungsgemeinschaft (PR511/1-1; SFB446-A2)
& Present address:
SerCon GmbH, Heinrich-von-Brentano-Sre. 2, D-55130 Mainz, Germany
*Corresponding
author: Tel, 86-871-5031094; Fax, 86-871-5034838; E-mail, [email protected]
Abstract Heat shock factor–DNA
interaction is critical for understanding the regulatory mechanisms of
stress-induced gene expression in eukaryotes. In this study, we analyzed the in
vivo binding of yeast heat shock factor (HSF) to the promoters of target
genes ScSSA1, ScSSA4, HSP30 and HSP104, using
chromatin immunoprecipitation. Previous work suggested that yeast HSF is constitutively
bound to DNA at all temperatures. Expression of HSF target genes is regulated
at the post-transcriptional level. However, our results indicated that HSF does
not bind to the promoters of ScSSA4 and HSP30 at normal
temperature (23 ?C). Binding to these promoters is rapidly induced by heat
stress at 39 ?C. HSF binds to ScSSA1 and HSP104 promoters under
non-stress conditions, but at a low level. Heat stress rapidly leads to a
notable increase in the binding of HSF to these two genes. The kinetics of the
level of HSF-promoter binding correlate well with the expression of target
genes, suggesting that the expression of HSF target genes is at least partially
the result of HSF-promoter binding stability and subsequent transcription
stimulation.
Key words chromatin immunoprecipitation; heat shock factor; heat
shock gene; yeast heat shock factor-promoter binding
Cells respond to elevated temperature and other physiological stresses
by dramatically increasing the expression of heat shock proteins (HSPs), a set
of proteins functioning as molecular chaperones, which are involved in the
folding, trafficking, maturation and degradation of proteins. An increased
accumulation of HSPs is essential for the survival of cells exposed to various
stresses [1,2]. In eukaryotes, the expression of heat shock genes which encode
HSPs is regulated by the binding of heat shock factors (HSFs) to heat shock
gene promoter elements. heat
shock element (HSE) is composed of tandem inverted repeats of a short consensus
sequence 5‘-nGAAn-3‘ [1]. The HSF-HSE interaction is conserved
from yeast to human, but there is wide variability in the number of HSF genes
in nature. Plants and mammals harbor multiple genes encoding HSF isoforms, with
Arabidopsis thaliana possessing 21 distinct HSF genes and
mammals possessing three genes [3,4]. The existence of multiple HSF isoforms
might have a specialized function and regulate different target genes. Yeast,
however, harbors only a single HSF which is thought to play multiple roles that
are shared with the isoforms in higher eukaryotes [5,6]. Recent reports
demonstrated yeast HSF is essential for heat-inducible transcription of not
only HSPs but also genes encoding proteins involved in diverse cellular
processes including growth, development, disease, aging, and in the complex
metabolic reprogramming that occurs in response to stresses [7,8].Considering the important role of HSF in the cellular homeostatic
control, it is important to elucidate the regulatory mechanisms of HSF in
cellular stress response. In plant, animal and mammalian cells, HSF is present
in a latent, monomeric state under normal conditions; the HSF DNA-binding
activity depends on the trimerization of monomeric HSF subunits upon heat
stress, and the event of binding triggers the transcriptional activation of
target genes [4,9,10]. In yeast, early studies using in vitro binding
assay and genetic methods suggested that the stress inducing expression of HSF
target genes is regulated at the post-transcriptional level, for instance, the
heat-inducible phosphorylation of the HSE-bound HSF was suggested to be
responsible for the activation of heat shock gene expression, whereas HSF
binding to HSEs is strictly constitutive [5,11,12]. However, in vivo,
protein-DNA binding is determined by additional factors which stimulate or
inhibit binding. Later, in vivo footprinting was used to study protein-DNA
interaction on the yeast HSP82 gene, and it was proposed that HSF binds
to strong HSEs (three consensuses) constitutively, but binding to weak HSEs
(poor matches to consensus) is dependent on heat stress [13,14]. However,
footprinting does not identify the interacting protein.In this report, we analyzed
the in vivo binding of HSF to target genes using cross-linking chromatin
immunoprecipitation (X-ChIP) which is thought to allow both the identification
of the protein and the interacting DNA sequence [15]. Our results showed that
HSF binds to different targets in a different manner. HSF binding to ScSSA4
and HSP30 is heat stress dependent. HSF binds to ScSSA1 and HSP104
under non-stress conditions, but at a low level; heat stress apparently increases
the binding level. The kinetics of binding correlate well with target gene
expression, suggesting that the binding plays a role in the transcriptional
stimulation of target genes
Materials and methods
Construction of expression
vectors
Plasmid pQE30 was isolated
from Escherichia coli TG1 (Qiagen, Carlsbad, USA) using a Nucleospin
Multi-8 plasmid kit (Machery-Nagel, D?ren, Germany). The yeast HSF
entire coding region DNA fragment (yHSF) was inserted, containing an adaptor of
the PstI restriction site located after the translation stop
codon, and an adaptor of the SacI restriction site located before
the translation start codon. They were produced by polymerase chain reaction
(PCR) using primer pair QE30-hsf/u (5‘-GCACTGCAGCTATTTCTTAACTCGTTTGG-3‘)
and QE30-hsf/d (5‘-GCAGAGCTCATGAATAATGCTGCAAATACA-3‘) at
annealing temperature 53 ?C.
Plasmid pQE30 and the PCR
product of yHSF were digested with SacI, followed by PstI. After
ligation by T4 DNA ligase, the ligated construct pQE30/His-yHSF was transformed
to TG1 by electroporation. Positive colonies were identified by hybridization
with the HSF gene (coding sequence) probe which was synthesized using
the PCR product of yHSF as template. The constructs were verified by
sequencing. The colony with the verified construct was chosen for expressing
the recombinant peptides.
Preparation of
affinity-purified antibodies
Yeast His6-HSF
was expressed from TG1(pQE30/yHSF) and purified using denaturing-renaturing
purification according to the QIAexpressionist (Qiagen), verified by Western
blot analysis and used for the generation of antiserum against yeast HSF in
rabbit. For the generation of monospecific antibodies, His6-HSF
peptide was coupled to AminoLink Plus Coupling Gel (Pierce, Rockford, USA) and
the affinity purification of the antibodies was carried out using ImmunoPure
IgG Elution Buffer (Pierce).
Chromatin immunoprecipitation
Chromatin immunoprecipitation
Yeast cells (Saccharomyces
cerevisiae, strain Y190) were grown in 50 ml SD-Leu medium (26.7 g/L
minimal synthetic defined base, 0.69 g/L Leu drop out supplement, ph 5.8) (BD Biosciences Clontech, Palo
Alto, USA) to an optical density (OD600)
of 2 at 23 ?C, then heated at 39 ?C for 0, 10, 30, and 60 min. Cell cultures
were cross-linked with 1% formaldehyde and chromatin was isolated according to
the method described by Strahl-Bolsinger et al. [16]. Four hundred
microliters of the resulting chromatin from each sample was sonicated for 10?20 s (resulting in fragment
sizes 0.3–0.5 kb) using a Branson
Digital Sonifier (S-250D; Branson Ultrasonic, Danbury, USA) at 40% output
(keeping the sample on ice during sonication). The chromatin extracts were
centrifuged at 10,000 rpm at 4 ?C for 20 min, and the supernatants were
filtered through a 0.22 mm filter.
Ten micrograms of
affinity-purified anti-HSF antibodies was added to 400 ml
chromatin extract samples and incubated at 4 ?C for 3 h, then 60 ml protein A-agarose preincubated with 1%
BSA was added to block non-specific binding, and the samples were incubated at
4 ?C for 30 min. Subsequently, the resin was washed and the immunoprecipitated
material was eluted according to the method described by Strahl-Bolsinger et
al. [16]. Eighty microliters of the resulting elution material was digested
by adding 80 ml of proteinase K (1 mg/ml) in
proteinase K buffer (50 mM Tris, 10 mM EDTA and 0.3% sodium dodecylsulfate),
incubated at 60 ?C for 16 h (from this point, 80 ml
chromatin extract was processed in parallel to obtain total genomic DNA),
extracted twice with phenol/CHCl3, and once with CHCl3.
DNAs were precipitated by ethanol and dissolved in 20 ml
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
PCR analysis of
immunoprecipitated DNA
Immunoprecipitated DNA was
analyzed by PCR. One microlitre of immunoprecipitated material was added to 20 ml PCR reaction mixture (50 mM KCl, 20 mM
Tris-HCl, pH 8.3, 0.05% Tween-20, 0.01 mg/ml gelatin, 2.5 mM MgCl2,
2.5 mM each dNTP, 1 U Taq polymerase). PCR reaction was carried out for
30 cycles (unless elsewhere indicated) of 20 s at 94 ?C, 20 s at 49–56 ?C and 10 s at 72 ?C. The PCR products
were separated on a 10% polyacrylamide gel and visualized by staining with
ethidium bromide. The following primer pairs were used to detect target genes:
ScSSA1–307u, 5‘-TCAACTAAAATCTGGAGAAAA-3‘;
ScSSA1–314d, 5‘-CGGAACGTTTAGAAGCTGTCATT-3‘,
53 ?C; ScSSA1+1893d, 5‘-GGTGCTCCTCCAGCTCCA-3‘; ScSSA1+1899u, 5‘-TCAACGGTTGGACCTTCA-3‘,
54 ?C; ScSSA1+1193d, 5‘-TGTCGCTCCATTATCCTT-3‘; ScSSA1+1199u, 5‘-CACCACCAGCAGTTTCAA-3‘,
54 ?C; ScSSA4–301u, 5‘-AACTCACCGGGCAAAAGA-3‘;
ScSSA4–308d, 5‘-AATGTAATAGGTTTCAAAG-3‘,
49 ?C; ScSSA4–364u, 5‘-GAGAGTACATACCGGAATG-3‘;
ScSSA4–429d, 5‘-ACTAAATTACGTTCATAGGG-3‘,
54 ?C; ScSSA4+1807d, 5‘-GTTAGATGCTTCGCAAGC-3‘; ScSSA4+1814u, 5‘-TCCTTGTATTCCTCGGTG-3‘,
54 ?C; HSP30–503u, 5‘-AAGCACGCTTTCGATGCG-3‘;
HSP30–514d, 5‘-CGTAGGAGGATTCTCTCA-3‘,
52 ?C; HSP104–131u, 5‘-TGAGGCAAGATTACAATGC-3‘;
HSP104–143d, 5‘-CTTATGCAACCTGCCAGA-3‘,
52 ?C; YPL231W–1170u, 5‘-AGTGACTTGCTTGCTCCTC-3‘;
YPL231W–1206d, 5‘-GATTCTTTGCATAAGAGGCTA-3‘,
55?C. Primers were named according to the gene and the distance of the 3‘
nucleotide relative to the ATG translation start codon. The annealing
temperatures are given.
Results
HSF binding site detected at
high resolution by cross-linking chromatin immunoprecipitation and PCR analysis
To detect HSF binding to
target in vivo, we employed the X-ChIP technique and PCR analysis for
immunoprecipitated DNA. We successfully detected the binding of yeast HSF to
the ScSSA4 gene in vivo and examined the binding site at high
resolution. Yeast cells were heat stressed at 39 ?C for 10 min, followed by
formaldehyde cross-linking and immunoprecipitation using affinity-purified
yeast HSF antibodies. The immunoprecipitated DNAs were analyzed by PCR using
different primer pairs. The genes and distribution of primers are shown in Fig.
1. For positive controls, total genomic DNAs were used as the
templates for PCR amplification. For negative controls, we used
immunoprecipitates without prior formaldehyde cross-linking or without HSF
antibodies. The results are shown in Fig. 2. Two primer pairs, ScSSA4–364u/–429d
and ScSSA4–301u/–308d,
were used, which were located in the promoter region of ScSSA4. The
space between primers in ScSSA4–364u/–429d
and ScSSA4–301u/–308d
was 64 and 6 nucleotides, respectively. Both primer pairs properly produced
specific PCR products and gave similar results. Heat stressed samples gave
strong signals after 40 cycles of amplification, but there were weak
background signals for negative controls and weak signals for the sample
without heat stress. However, the signals of the heat stressed samples
were significantly stronger than those of other samples, indicating heat stress
induced HSF binding to the ScSSA4 gene. The weak bands of the
sample without heat stress were comparable with the bands for the negative
controls. With the PCR cycle reduced to 30 cycles, the heat stressed samples
still gave clear signals, whereas the signals were abolished for both
the negative controls and the sample without heat stress, indicating HSF did
not bind to ScSSA4 promoter under non-stress conditions, but the binding
occurred after heat stress.
To determine the spatial
specificity of HSF binding, we amplified immunoprecipitates using primer pair
YPL231W–1206d/–1170u
located in a randomly chosen YPL231W promoter. The results showed that
no signal was detected and HSF did not bind to the non-target promoter. To
further define the binding site, we analyzed whether HSF binds to the space
adjacent to the ScSSA4 promoter by using primer pair ScSSA4+1807d/+1814u
located in the coding region of the ScSSA4 gene, approximately 1800 bp
away from the start codon. No PCR signal was detected with this primer pair,
demonstrating the X-ChIP technique could detect the HSF binding site at high
resolution.
Effects of heat stress on
binding of HSF to target genes in vivo
Early experiments suggested that yeast HSF was localized in the
nucleus and bound to the target gene promoter, even in the absence of heat
stress. However, the above results showed that HSF binding to the ScSSA4
promoter is dependent on heat stress. This contradiction prompted us to further
investigate the effects of heat stress on HSF binding to different target
promoters.Based on multiple independent
chromatin immunoprecipitation experiments, we analyzed the dynamic association
of HSF with the promoters of ScSSA1, ScSSA4, HSP30, and HSP104
genes in vivo. Yeast cells were heat shocked at 39 ?C for 0, 10, 30 and
60 min, followed by formaldehyde cross-linking and immunoprecipitation. The PCR
amplification of immunoprecipitated DNA using primer pairs located in different
promoter regions are shown in Fig. 3. The results showed that HSF bound
to ScSSA1 and HSP104 promoters under non-stress conditions,
whereas no binding occurred to ScSSA4 and HSP30 without heat
stress. Heat stress rapidly increased HSF binding to ScSSA1 and HSP104
promoters and induced HSF binding to ScSSA4 and HSP30 promoters.
The level of HSF binding to these four promoters reached a peak after 10 min of
heat stress and declined with continuous exposure to heat stress for up to 1 h,
however, it was slightly higher than basal level in the cases of ScSSA1
and HSP104. The negative controls without formaldehyde cross-linking or
without antibodies did not give PCR signals. Therefore we excluded the effect
of the background signal. Two primer pairs located in the ScSSA1 coding
regions and one primer pair situated in the YPL231W promoter region did
not have PCR signals, indicating HSF did not bind to non-target DNA. These
results suggested that HSF binds to different targets in a different manner,
constitutively or heat stress-dependently, and heat stress apparently increases
binding affinity. The comparison of HSF binding
levels in the time-course experiment with the profiling of target gene
expression within published data indicates that HSF binding correlates well
with target gene expression, suggesting that induced or increased HSF binding
plays an important role in the regulation of yeast target gene activation.
Discussion
It was initially thought that
yeast HSF trimers constitutively bind to HSEs under both normal and heat stress
conditions. Later, based on in vivo footprinting experiments, Erkine et
al. [13] and Giardina and Lis [14] proposed that HSF binding to strong HSEs
of HSP82 is constitutive, but binding to weak HSEs is induced by heat
stress. However, our results indicate that heat stress significantly affects
binding behavior, and that HSF binds to different promoters in a different
manner. Binding to HSP30 and ScSSA4 genes is heat
stress-dependent. HSF does not bind to these two genes under non-stress
conditions, even the ScSSA4 promoter that contains strong HSE. HSF binds
to ScSSA1 and HSP104 promoters under non-stress conditions, but
at a low level. Heat stress rapidly leads to a notable increase in the binding
of HSF to these two genes. Interestingly, the HSF binding level is attenuated
by prolonged exposure to heat stress. A similar phenomenon was previously found
in higher eukaryotes and it was proposed that the binding stability is
negatively regulated by the association of HSF with other proteins, including
HSP70 and HSP90 [17,18].
However, our experiments
suggested that HSE is not the sole determinant of HSF binding. For instance,
previously published work proposed that HSP30 is not under the control
of HSF because of the lack of a typical HSE in the HSP30 promoter [19].
Our results showed that HSF binds to HSP30 in a heat-induced manner. We
also verified the lack of HSF binding to the YPL231W promoter, which contains
a consensus HSE. Although HSE is known to be important for HSF binding, the
lack of HSF binding to the YPL231W promoter and the heat-induced binding
to HSP30 suggest that its presence is clearly not the sole determinant
of whether a promoter is bound and activated by HSF. It appears that HSF
binding behavior in yeast is more complex than previously thought.
In order to analyze the
putative effects of heat stress-induced or increased HSF binding on
transcriptional activities of target genes, we examined the gene expression in S.
cerevisiae within published data. The ScSSA4 mRNA level rapidly
increased after 15 min and 30 min of heat stress at 39 ?C and declined after 60
min of heat stress [11]. HSP30 mRNA was not detectable under normal
conditions; however, the expression was induced by heat stress and other
stresses [19]. The work by Causton et al. [20] showed that the
expression level of ScSSA4, HSP104 and ScSSA1 was
increased by 95-, 7- and 3-fold over basal levels, respectively, after 15 min
of heat stress, and subsequently declined during continuous heat stress [20].
The timing of induction and the decline of the mRNA correlate well with HSF
binding to the target genes in our time-course experiment, suggesting that
expression of the HSF target genes is, at least in part, the result of the HSF-promoter
binding stability and the consequent transcription stimulation, similar to that
of higher eukaryotes [10]. This information, along with the findings on the
role of heat-inducible phosphorylation of HSF in the expression of target
genes, would promote our understanding of the complexity of yeast HSF
regulatory mechanisms.
In our experiments, the X-ChIP
technique was excellent for detecting HSF target DNA. However, it must be noted
that immunoprecipitated DNA is relative enrichment of targets. To exclude
background signals, it is essential to use negative controls in each experiment
and design the appropriate number of PCR cycles. Only the signals produced by
immuno-enriched targets, shown to be significantly stronger than the background
signals produced by negative controls, can be considered. Furthermore, by
carefully reducing the number of cycles, background signals were abolished,
whereas the signals of the heat shock samples still existed. Therefore we could
clearly determine the true binding signal. Our experiments also demonstrated
that PCR analysis of target DNA is accurate in detecting binding sites. Only
the primer pair located in the promoter region gave signals, whereas the primer
pair located in the adjacent coding region of the same gene did not. To
investigate the binding site and non-binding site at high resolution, we
designed primers that were closely spaced in each primer pair and resulted in a
PCR product with a short fragment (41–175 bp).
Because the PCR reactions were carried out in a short phase of synthesis (10 s
at 72 ?C), the primer pairs listed in this report produced proper PCR products,
and the specificity of the PCR products was further verified by sequencing in
the analysis of a randomly chosen sample. However, we also found that some
primer pairs did not work well in the amplification of such a short fragment,
so it is necessary to test multiple primer pairs and choose the most suitable.
However, with the digestion of formaldehyde-fixed chromatin with nonspecific
proteinases, such as proteinase K, it is difficult to yield a completely
peptide-free DNA [21]. Other cross-linking agents, such as ultraviolet light,
also cause significant DNA damage [22]. These DNA modifications hamper the PCR
analysis as PCR techniques require the DNA integrity for primer extension. The
short space between primers would minimize the effect of DNA damage on PCR.
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