Biochem. J. (2008) 409, 779–788 (Printed in Great Britain)
779
doi:10.1042/BJ20070578
Gcn5- and Elp3-induced histone H3 acetylation regulates hsp70 gene
transcription in yeast
Qiuju HAN*†, Jun LU*, Jizhou DUAN*, Dongmei SU*, Xiaozhe HOU*, Fen LI1 , Xiuli WANG* and Baiqu HUANG*2
*Key Laboratory of Molecular Epigenetics of Ministry of Education (MOE), Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China, and
†School of Environmental and Biological Engineering, Liaoning University of Petroleum and Chemical Technology, Fushun 113001, China
The purpose of this study was to elucidate the mechanisms
by which histone acetylation participates in transcriptional
regulation of hsp70 (heat-shock protein 70) genes SSA3 and
SSA4 in yeast. Our results indicated that histone acetylation was
required for the transcriptional activation of SSA3 and SSA4. The
HATs (histone acetyltransferases) Gcn5 (general control nonderepressible 5) and Elp3 (elongation protein 3) modulated hsp70
gene transcription by affecting the acetylation status of histone
H3. Although the two HATs possessed overlapping function
regarding the acetylation of histone H3, they affected hsp70 gene
transcription in different ways. The recruitment of Gcn5 was
Swi/Snf-dependent and was required for HSF (heat-shock factor)
binding and affected RNAPII (RNA polymerase II) recruitment,
whereas Elp3 exerted its roles mainly through affecting RNAPII
elongation. These results provide insights into the effects of
Gcn5 and Elp3 in hsp70 gene transcription and underscore the
importance of histone acetylation for transcriptional initiation and
elongation in hsp genes.
INTRODUCTION
suggesting that growth defects in gcn5∆elp3∆ cells occur due to the
loss of the HAT activities of both SAGA and Elongator complexes
[8]. It has been reported that a significantly lower level of histone
acetylation in the coding region of several genes correlated with
reduced transcription of the gene in gcn5∆elp3∆ yeast cells [9].
Together, these studies argue that histone acetylation catalysed
by Gcn5 and Elp3 may play an important role in transcription
in vivo, yet the mechanisms underlying this role are unclear.
The HSP (heat-shock protein) 70 family is the most highly conserved of the HSP families, and its members can be found in organisms ranging from bacteria to plants and animals [14]. Intracellular
Hsp70 has several identified functions, including stabilization
of protein structure and prevention of protein aggregation by
binding to unfolded proteins, and regulation of protein activity,
availability and/or transport [15–17]. Hsp70 can also protect cells
from apoptotic stimuli, including DNA damage, UV irradiation,
serum withdrawal and exposure to chemotherapeutic agents
[18–20]. The hsp70 heat-shock-response gene is one of the
best studied examples of eukaryotic transcription elongation
regulation. Dramatic domain-wide nucleosomal disassembly has
been shown to be associated with heat-shock-inducible genes,
including hsp82, hsp12, hsp26 and SSA4 in yeast [21]. This
raised the question of whether histone acetylation is required for
the activation of hsp genes, and if so, how histone acetylation
regulates the transcription of these hsp genes. To address
these questions, we chose SSA3 and SSA4 as target genes for
biochemical and molecular studies. Both SSA3 and SSA4 are
inducible hsp70 genes in yeast. The promoter of SSA3 is weakly
bound by HSF (heat-shock factor), whereas the promoter of SSA4
is tightly bound by HSF. Moreover, it has been reported that the
domain-wide displacement of histones is activated by HSF [21].
The purpose of this study was to investigate the roles of histone
acetylation in transcriptional regulation of these hsp70 genes and
The eukaryotic genome is packaged into chromatin, which acts
as a constant barrier to transcription and other cellular processes
that require access to DNA. Therefore the structure of chromatin
has to be modified before active transcription can proceed.
The first group of chromatin-remodelling factors identified is
composed of enzymes that covalently modify the N-terminus of
histones, such as HATs (histone acetyltransferases) and histone
methyltransferases. Modification of chromatin by HATs and
HDACs (histone deacetylases) plays a key role in transcriptional
regulation [1–4]. Changes to histone acetylation, both at the
gene promoter and coding regions, correlates with transcriptional
activation and repression. A study has suggested that transcript
elongation is required to form an unfolded structure of
transcribing nucleosomes, and histone acetylation is needed to
maintain this unfolded structure [5].
Gcn5 (general control non-derepressible 5) is the catalytic
subunit of SAGA (Spt-Ada-Gcn5-acetyltransferase), a major
transcription-related HAT complex in yeast [6], and Elp3
(elongation protein 3) is the catalytic subunit of Elongator, a HAT
complex originally isolated with hyperphosphorylated, elongating
RNAPII (RNA polymerase II) [7]. Histone H3 is the primary
target for both of these complexes, and the complexes display
significant functional redundancy [6,8–11]. Previous genetic
studies demonstrated that individual mutations in GCN5 and
ELP3 resulted only in fairly mild phenotypic changes [12,13],
whereas deletion of both genes conferred a number of severe
growth defects. These defects include slow growth, temperature
sensitivity and an inability to ferment galactose and sucrose
[8]. Similar phenotypes are conferred by point mutations which
debilitate the HAT activities of these enzymes without affecting
their incorporation into the SAGA and Elongator complexes,
Key words: chromatin remodelling, heat-shock protein (hsp),
histone acetyltransferase (HAT), histone modification, SSA3,
SSA4.
Abbreviations used: Ac-H3; acetyl-histone H3, ChIP, chromatin immunoprecipitation; Elp, elongation protein; Gcn, general control non-derepressible;
HAT, histone acetyltransferase; HSE, heat-shock element; HSF, heat-shock factor; HSP, heat-shock protein; ORF, open reading frame; RNAP, RNA
polymerase; RT – PCR, reverse transcription – PCR; SAGA, Spt-Ada-Gcn5-acetyltransferase; SC, synthetic complete; UTR, untranslated region; YPD, yeast
extract, peptone, dextrose.
1
Present address: College of Life Science, Henan Normal University, Xinxiang, 453007 China.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
780
Table 1
Q. Han and others
Yeast strains used in this study
Strain
Genotype
Reference/source
W303-1B
JSY131
JSY142
JSY144
Z1
Z4 (Y260)
LFY1
MATα ade2-1 ura3-1 his3-11,15 , leu2-3 , 112 trp1-1 can1-100
W303-1B, except elp3∆::LEU2
W303-1B, except gcn5∆::HIS3
W303-1B, except elp3∆::LEU2 gcn5∆::HIS3
MAT* ura3-52 SUC2 GAL +
MATa ura3-52 rpb1-1 gal MATa ade2 can1 his3 leu2 lys2 trp1 ura3 elp3 ::ADE2 hht1-hhf1D ::LEU2
hht2-hhf2D ::kanMX3 +; pRS316:HHT1-HHF1(URA3)
MATa ade2 can1 his3 leu2 lys2 trp1 ura3 elp3 ::ADE2 hht1-hhf1D ::LEU2
hht2-hhf2D ::kanMX3+; pRS313:HHT1-hhf1 (K8R) (HIS3)
MATa ade2 can1 his3 leu2 lys2 trp1 ura3 elp3 ::ADE2 hht1-hhf1D ::LEU2
hht2-hhf2D ::kanMX3+; pRS313:hht1 (K14R)-HHF1 (HIS3)
MATa ade2 can1 his3 leu2 lys2 trp1 ura3 elp3 ::ADE2 hht1-hhf1D ::LEU2
hht2-hhf2D ::kanMX3+; pRS313:hht1 (K14R)-hhf1(K8R) (HIS3)
Matα his3-1 leu2-0 trp1-1 lys2-0 ura3-0
BY4742 background
BY4742 background
[13]
[13]
[13]
[13]
[22]
[22]
[22]
LFY8
LFY14
LFY814
BY4742
snf2∆
snf5∆
to explore the molecular mechanisms underlying this process. We
used yeast strains lacking GCN5 and ELP3, which encode two
transcription-related HATs, as a model to investigate the effects
of histone acetylation on induced transcription of hsp70 genes. We
discovered that histone acetylation indeed plays a key role in both
transcriptional activation and elongation of the SSA3 and SSA4
genes. Our results shed light on to the mechanisms underlying
regulation of hsp70 gene transcription by Gcn5 and Elp3, and
highlight the importance of histone acetylation in transcriptional
initiation and elongation of hsp genes.
Table 2
Yeast strains and media
All Saccharomyces cerevisiae strains used are listed in Table 1.
Cells were grown at 26 ◦C to mid-log phase in rich YPD (yeast
extract, peptone, dextrose) medium. Yeast cells transformed with
pRS313, pRS314 and pRS316 plasmids were grown under similar
conditions in SC (synthetic complete) medium (lacking histidine,
tryptophan and uracil respectively). Heat-shock induction was
performed by transferring the culture to a 37 ◦C water bath and
shaking vigourously; once the temperature reached 37 ◦C, the
incubation was allowed to continue for an additional 10 min.
Cells carrying the rpb1-1 mutation and its wild-type counterpart
[22] were grown in YPD medium at 25 ◦C to mid-log phase and
transferred to 37 ◦C for 30 min to induce heat-shock.
[23]
[23]
Gift from Dr Jinqui Zhou
Gift from Dr Jinqui Zhou
Gift from Dr Jinqui Zhou
Primers used in this study
Name
Primer
Sequence
GCN5 (29–874)
Forward (G1)
Reverse (G2)
Forward (G3)
Reverse (G4)
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
5 -GATATCCATGTAGTGCGCTGTATGG-3
5 -GGTAACATTTCTCTCTTATCGAAAGGTCG-3
5 - AGAGAGAAATGTTACCAAGAATACGATATTTG-3
5 -GAGCTCCACATCGTCTCGCCGTA-3
5 -GCGTCTCAGGAAACCAT-3
5 -TCTCCACCTCCCGTAGC-3
5 -ATTGCGTATGGGCTGGAC-3
5 -AGGGACCTTTGGTTAGTTGTTA-3
5 -AGCCGTTTTGTCCTTGTT-3
5 -GCGGTGATTTCCTTTTGC-3
5 -CGTATTTGACTTGATGCCTAT-3
5 -AATCAATACCAACTGCTC-3
5 -CGTATTATCAATGAACCCACTG-3
5 -GTCTCCTGCGGTAGCCTTA-3
5‘-TGCTACGGGAGGTGGAG-3
5 -CGTGCTGCGGAAACAA-3
5 -TTTGCCCGGTGAGTTGTT-3
5 -AGTCCTTTATTGGCTTGAGTTT-3
5 -ATTGCGTATGGGCTGGAC-3
5 -AGGGACCTTTGGTTAGTTGTTA-3
5 -AACGGTTGAAGAGGTTGATTA-3
5 -AAACTCTGGCTTATGACGATG-3
GCN5 (1124–1741)
SSA3 RT
SSA4 RT
ACT1 RT
EXPERIMENTAL
[23]
SSA3 promoter
SSA3 ORF
SSA3 3 UTR
SSA4 promoter
SSA4 ORF
SSA4 3 UTR
was subjected to sequencing analysis before introduction into
yeast. The constructs pRS316-ELP3 and pRS316-yhelp3HAT−
were amplified as described previously [23]. Yeast cells were
transformed according to Hill et al. [24].
Plasmid constructs
The GCN5 coding sequence was amplified from yeast genomic
DNA by PCR using Pfu polymerase and primers G1 and G4
containing EcoRV and XhoI sites respectively. The PCR products
were cloned into the EcoRV and XhoI sites of the pRS314 vector
to create pRS314-GCN5. The gcn5HAT− insert, which lacks
the sequence encoding the catalytic motif II-III-IV of the HAT
domain of GCN5 (amino acid residues 170–253), was amplified
from pRS314-GCN5 using primers G1/G2 and G3/G4 and the
two products were mixed together and amplified using primers
G1 and G4. The PCR products were cloned into the EcoRV
and XhoI sites in pRS314 to create pRS314-gcn5HAT− . The
oligonucleotide primer sequences used are listed in Table 2.
After cloning, the entire coding region of all cloned fragments
c The Authors Journal compilation c 2008 Biochemical Society
RNA extraction, RT–PCR (reverse transcription–PCR), and
real-time PCR analyses
Total RNA was extracted from yeast cells using the method of
Trotter et al. [25]. RT–PCR was performed using the Access RT–
PCR System supplied by Promega. Real-time PCR was performed
using an ABI Prism 7000 Sequence Detection System (Applied
Biosystems), following the manufacturer’s protocol, with SYBR
Green (TaKaRa, Osaka, Japan) used as a double-stranded DNAspecific fluorescent dye. ACT1 was used as an internal reference
for standardizing SSA3 and SSA4 mRNA expression. The PCR
primers used to amplify SSA3, SSA4 and ACT1 are listed in
Table 2. The data were analysed by calculating 2−Ct [26].
Histone acetylation in hsp70 gene transcription
Figure 1
781
Histone H3 acetylation participated in SSA3 and SSA4 gene transcription
(A, B) Wild-type (W303-1B) yeast cells were heat-shocked for 10 min at 37 ◦C (HS), or left at 26 ◦C (NHS) as a control. ChIP analyses of the relative amounts of histone H3 (A) and acetylation level
of histone H3 (B) in the promoters, ORF and 3 UTR regions of SSA3 and SSA4 were performed. Precipitated DNA was analysed by real-time PCR using primers amplifying the indicated regions of
the SSA3 and SSA4 genes. (C) Wild-type (WT; LFY1), histone H4 K8R (H4K8R; LFY8), histone H3 K14R (H3K14Rl; LFY14) and histone H4 K8R/histone H3 K14R double mutant (H4K8R/H3K14R;
LFY814) yeast cells were heat-shocked for 10 min at 37 ◦C (HS) or left at 26 ◦C (NHS) as a control. Real-time RT–PCR was performed using either SSA3 primers or SSA4 primers. Results are
means +
− S.D., n = 3.
ChIP (chromatin immunoprecipitation)
After the appropriate heat-shock treatment, cells were crosslinked with 1 % (v/v) formaldehyde for 10 min at 37 ◦C, and then
lysed in 700 µl of lysis buffer [50 mM Hepes/KOH (pH 7.5),
140 mM NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100 and 0.1 %
(v/v) sodium deoxycholate] with protease inhibitors. The sonicated lysates were processed using a ChIP assay kit (Upstate Biotechnology), essentially as described by the manufacturer. Antibodies against RNAPII (05-623) and Ac-H3 (acetyl-histone H3;
06-599) were purchased from Upstate Biotechnology; anti-Gcn5
antibody (sc-9078) was purchased from Santa Cruz Biotechnology. The antibody against the C-terminus of histone H3 was a gift
from Dr Alain Verreault (Institute for Research in Immunology
and Cancer, University of Montreal, Montreal, Canada), the
antibody against Elp3 was a gift from Dr Jesper Q. Svejstrup
(Cancer Research UK London Research Institute, Clare Hall
Laboratories, South Mimms, Hertfordshire, U.K.) and the antibody
against HSF was a gift from Dr Gross (Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South
Dakota, Vermillion, SD, U.S.A.). Input and immunoprecipitated
DNA were analysed by real-time PCR using the ABI Prism
7000 sequence detector. To calculate the relative abundance of
a given gene promoter or coding sequence present in an immunoprecipitate, the following formula was used: Qgene = IPgene /
inputgene to calibrate the variation between different samples and
primer pairs. The constitutively expressed ACT1 gene was used
as an internal control for normalization of all RNAPII, histone
H3 and Ac-H3 ChIP results. The data from ChIP assays with
Ac-H3 antibodies were normalized to the total level of H3 present
at the corresponding time point. The primers used are shown in
Table 2.
RESULTS
Histone H3 acetylation affects SSA3 and SSA4 gene transcription
Previous research suggested the removal of nucleosomes as a
mechanism to achieve high levels of transcription [21]. This
theory raised the question of whether histone acetylation is
required for the activation of genes that have lost the histone–
DNA contact. In the present study, we first tested the histone H3
levels at SSA3 and SSA4 genes in response to heat-shock induction.
Cells were subjected to heat-shock treatment for 10 min at 37 ◦C,
and the abundance of histone H3 associated with the promoter and
coding regions of SSA3 and SSA4 was assessed by ChIP using an
antibody recognizing the C-terminus of histone H3. The results
showed that histone H3 associated with SSA4 was displaced,
whereas that associated with SSA3 was not (Figure 1A). In order
to evaluate the roles of histone acetylation in transcription of the
two hsp70 genes, we analysed the histone H3 acetylation state at
c The Authors Journal compilation c 2008 Biochemical Society
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Q. Han and others
SSA3 and SSA4 during heat induction. Because of the observed
loss of histone–DNA contacts (Figure 1A), the data from ChIP
assays using acetyl-histone H3 antibodies were normalized to
the total level of H3 present at the corresponding time point.
As can be seen from Figure 1(B), on induction of transcription,
the level of acetylated histone H3 was increased at the promoter
and coding regions of both SSA3 and SSA4 genes. To further
determine whether the modification of histone H3 and histone H4
is important for hsp70 gene transcription, we used yeast strains
carrying point mutations in sequences encoding histone H3 at
Lys14 (K14R mutant; LFY14), histone H4 at Lys8 (K8R mutant; LFY8) or both histone H3 K14R and histone H4 K8R
(LFY814), and tested the mRNA levels of SSA3 and SSA4 on
induction of transcription. We found that expression of both genes
was decreased in the histone mutant yeast strains (Figure 1C).
These results indicated that the lysine residues in the N-termini
of histone H3 and histone H4 were important for transcription,
and that transcription-associated histone acetylation occurred in
the coding regions of these genes.
Gcn5 and Elp3 regulated SSA3 and SSA4 transcription and the
function was dependent on their HAT activities
It has been reported that cells which lack Gcn5 and Elp3 have
widespread and severe histone H3 hypoacetylation in chromatin, and hypoacetylation of the coding region correlates with
inhibition of transcription [9]. To determine the specific HAT(s)
responsible for H3 acetylation in the regulation of SSA3 and
SSA4, we used strains which were null mutants for the genes
encoding the two transcription-related HATs GCN5 and ELP3.
First, we performed temperature-sensitive experiments and the
results indicated that the gcn5∆elp3∆ yeast strain and the strains
transformed with only one of the HATs exhibited growth defects at
39 ◦C (Figure 2A). When Gcn5 and Elp3 were deleted, the level of
SSA3 and SSA4 mRNA was decreased (Figure 2B). This indicated
that Gcn5 and Elp3 play a role in SSA3 and SSA4 transcription. To
determine whether the function of Gcn5 and Elp3 is dependent on
their HAT activities, functional compensation experiments were
performed using plasmids containing mutated forms of the GCN5
and ELP3 genes pRS314-gcn5HAT− and pRS316-elp3HAT− , and
the consequences for growth were tested by examination of
temperature sensitivity. The results showed that the HAT
activities of Gcn5 and Elp3 were important for compensating
for the temperature sensitive phenotype (Figure 2A), and were
apparently important for efficient transcription of SSA3 and SSA4
(Figure 2B). These results indicated that the HAT activities of
Gcn5 and Elp3 were required for their function.
To investigate whether the effect of Gcn5 and Elp3 on SSA3
and SSA4 transcription was a direct effect, we used ChIP to detect
if Gcn5 and Elp3 could interact with the genes, and the results
showed that Gcn5 and Elp3 do interact with SSA3 and SSA4
(Figures 2C and 2D). Additionally, Gcn5 was found to be recruited
mainly at the promoter regions of the genes (Figure 2C), whereas
Elp3 was located in the coding region (Figure 2D). When the genes
were heat-shock-induced, the recruitment of these two HATs was
enhanced (Figures 2C and 2D). These experiments suggested that
the expression phenotypes associated with the GCN5- and ELP3deletion mutations could be attributed to a direct role for Gcn5
and Elp3 in regulating SSA3 and SSA4 transcription.
Gcn5 and Elp3 affected histone H3 acetylation in SSA3
and SSA4 genes
It has suggested that transcription-dependent histone acetylation
occurred at SSA3 and SSA4, and that both Gcn5 and Elp3 were
c The Authors Journal compilation c 2008 Biochemical Society
the key HATs involved in SSA3 and SSA4 gene transcription.
It is therefore possible to speculate that Gcn5 and Elp3 may
affect SSA3 and SSA4 transcription by acetylating histones
associated with these genes on induction. To experimentally test
this assumption, we investigated the effect of Gcn5 and Elp3 on the
displacement of histones and on histone H3 acetylation at SSA3
and SSA4. We first tested whether Gcn5 and Elp3 could affect the
displacement of histones. ChIP analysis of the histone H3 levels
in gcn5∆ (JSY142), elp3∆ (JSY131) and gcn5∆elp3∆ (JSY144)
yeast indicated that Gcn5 and Elp3 had a more severe effect on
SSA3 than on SSA4 (Figure 3A). The nucleosome density in the
coding region of SSA3 decreased to 50 % on heat-shock induction
in gcn5∆elp3∆ cells, whereas for SSA4, the loss of nucleosomes
occurred to a similar extent both in wild-type and mutant yeast
strains (Figure 3A). We then examined the level of acetylation
of histone H3 at SSA3 and SSA4 in response to heat induction.
The elp3gcn5∆ strain was shown to be deficient in the ability
to acetylate histone H3 associated with the promoter and coding
regions of the two genes (Figure 3B). This result implied that
Gcn5 and Elp3 regulated SSA3 and SSA4 expression by acetylating
histone H3 associated with these genes.
RNAPII is required for the recruitment of Elp3
To address whether the recruitment of Gcn5 and Elp3 to SSA3
and SSA4 was transcription-dependent, we used a yeast strain
expressing the temperature-sensitive allele of the largest RNAPII
subunit, Rpb1 (rpb1-1; Z4). Under the restrictive temperature,
the rpb1-1 expressing strain rapidly ceases transcription due
to the loss of RNAPII polymerization activity [22], as well
as loss of recruitment of the polymerase to promoter regions
[27]. The rpb1-1 strain and its wild-type counterpart were
induced at 25 ◦C and 37 ◦C respectively, and the amount
of Elp3 and Gcn5 recruitment was monitored by ChIP. As
shown in Figure 4(B), when wild-type yeast cells were heatshocked, the binding of Elp3 to SSA3 and SSA4 increased, whereas
in rpb1-1 cells, recruitment of Elp3 was decreased at 37 ◦C. The
recruitment of Elp3 was apparently dependent on active RNAPII
transcription. However, Gcn5 recruitment to SSA3 and SSA4 was
RNAPII-independent, because similar amounts of Gcn5 were
found to be present in SSA3 and SSA4 promoters in both wildtype and rpb1-1 yeast strains grown at 37 ◦C (Figure 4A). These
results suggested that although the two HATs had overlapping
roles in transcription regulation, they might exert their functions
via distinct mechanisms.
Gcn5 affected the recruitment of RNAPII and Elp3 affected
RNAPII-mediated transcription elongation
We then intended to investigate the mechanisms by which Gcn5and Elp3-catalysed histone acetylation affects the RNAPII-based
transcription of SSA3 and SSA4. As members of the HSP70 family,
SSA3 and SSA4 genes may be expressed both at a low (basal) level
under normal growth conditions, and at a high (induced) level
after heat shock. Under non-heat shock conditions, the promoter
sequences of hsp70 genes are occupied by transcription factors,
such as GAF (GAGA factor), TBP (TATA-box-binding protein)
and RNAPII. On heat-shock treatment, the inducible expression
of the hsp70 genes is mediated by the interaction of HSFs with
HSEs (heat-shock elements). To determine whether Gcn5 and
Elp3 influenced the recruitment of RNAPII, ChIP assays were
performed using an antibody against RNAPII. As shown in
Figure 5(A), induction of SSA3 and SSA4 in the elp3∆ yeast strain
resulted in nearly the same levels of RNAPII recruitment to the
Histone acetylation in hsp70 gene transcription
Figure 2
783
Gcn5 and Elp3 regulated SSA 3 and SSA4 gene transcription and this function was dependent on their HAT activities
(A) Examination of the temperature sensitivity of gcn5∆elp3∆ strains. The plasmids pRS314 (V1), pRS316 (V2), pRS314-GCN5 (GCN5), pRS314-gcn5 HAT− (GHAT− ), pRS316-ELP3 (ELP3),
pRS316-yhelp3 HAT− (EHAT− ) were transformed into gcn5∆elp3∆ cells. pRS314 and pRS316 were also transformed into wild-type (WT; W303-1B) cells as control, and the transformants were
plated in a dilution series on to SC minus uracil and tryptophan medium and grown at 30 ◦C for 2 days. The transformants were tested for temperature sensitivity after incubation for 4 days at 39 ◦C.
(B) The plasmids pRS316, pRS316-ELP3 (ELP3) and pRS316-yhelp3 HAT− (EHAT− ) were transformed into elp3∆ cells, and pRS314, pRS314-GCN5 (GCN5) and pRS314-gcn5 HAT− (GHAT− ) were
transformed into gcn5∆ cells. Wild-type yeast (WT) and gcn5∆elp3∆ cells were also cultured. Cells were heat-shocked for 10 min at 37 ◦C (HS) or left at 26 ◦C (NHS) as a control. Real-time PCR
◦
◦
was performed using either SSA3 primers or SSA4 primers. Results are means +
− S.D., n = 3. (C, D) Wild-type (W303-1B) yeast cells were heat-shocked for 10 min at 37 C (HS) or left at 26 C
(NHS) as a control. ChIP analyses of the relative amounts of Gcn5 (C) and Elp3 (D) in the promoter, ORF and 3 UTR regions of SSA3 and SSA4 were performed. Precipitated DNA was analysed by
real-time PCR. Results are means +
− S.D., n = 3.
promoter regions of the genes as demonstrated for the wild-type
strain; this implied that Elp3 did not affect RNAPII recruitment.
However, the amount of RNAPII associated with the promoter
regions of SSA3 and SSA4 in gcn5∆ and elp3gcn5∆ yeast strains
was decreased, and this was more pronounced in elp3gcn5∆
cells, where RNAPII was decreased to 20–30 % (Figure 5A).
c The Authors Journal compilation c 2008 Biochemical Society
784
Figure 3
Q. Han and others
Gcn5 and Elp3 affected histone H3 acetylation in SSA3 and SSA4 genes
(A, B) Wild-type (WT; W303-1B), elp3∆ (JSY131), gcn5∆ (JSY142) and gcn5∆elp3∆ (; JSY144) cells were heat-shocked for 10 min at 37 ◦C (HS) or left at 26 ◦C (NHS) as a control. ChIP
analyses were performed of the relative amount of histone H3 (A) in the promoters (pr.), ORF and 3 UTR (3’) regions of SSA3 and SSA4 . The intensity in all the four non-heat-shocked samples was
defined as 1. (B) ChIP analyses of the acetylation level of histone H3 associated with the promoter, ORF and 3 UTR regions of SSA3 and SSA4 . Precipitated DNA was analysed by real-time PCR.
Results are means +
− S.D., n = 3.
Figure 4
RNAPII was required for the recruitment of Elp3
Cells were heat-shocked for 10 min at 37 ◦C (HS) or left at 26 ◦C (NHS) as a control. ChIP analyses of the presence of (A) Gcn5 in the promoter regions of SSA3 and SSA4 , and (B) Elp3 in the
promoter, ORF and 3 UTR regions of SSA3 and SSA4 in wild-type (WT; Z1) and rpb1-1 (Z4) yeast cells. Precipitated DNA was analysed by real-time PCR. Results are means +
− S.D. n = 3.
This result indicated that the recruitment of RNAPII required
Gcn5.
To further determine whether Gcn5 and Elp3 influenced the
RNAPII-mediated transcription elongation along the hsp70 genes,
we measured the density of RNAPII at the promoters, ORFs
(open reading frames) and 3 UTRs (untranslated region) of
hsp70 genes by using ChIP assays with an antibody against
RNAPII. Previous work has reported that a significant proportion
c The Authors Journal compilation c 2008 Biochemical Society
of RNAPII molecules initiating transcription never progress to the
end of the gene [28]. This experiment was based on the assumption
that if the HATs are involved in transcriptional elongation, the
density of RNAPII would be expected to be lower (in relative
terms) at the 3 end of the gene than at the promoter of the gene
after depletion of Gcn5 and Elp3. Remarkably, we found that
RNAPII density was progressively decreased across the SSA3
and SSA4 genes in elp3∆ and elp3gcn5∆ cells (Figure 5B),
Histone acetylation in hsp70 gene transcription
Figure 5
785
Gcn5 affected the recruitment of RNAPII and Elp3 affected RNAPII-mediated transcription elongation
(A) Cells were heat-shocked for 10 min at 37 ◦C (HS) or left at 26 ◦C (NHS) as a control. ChIP analyses of the relative amounts of RNAPII in the promoters of SSA3 and SSA4 in wild-type (WT;
W303-1B), elp3∆, gcn5∆ and gcn5∆elp3∆ () yeast cells. (B, C) Cells were heat-shocked for 10 min at 37 ◦C (HS) (C) or left at 26 ◦C (NHS) as a control. (B). ChIP analyses of the relative
amount of RNAPII in the promoter, ORF and 3 UTR regions of SSA3 and SSA4 genes in wild-type (WT; W303-1B), elp3∆, gcn5∆ and gcn5∆elp3∆ () cells. Precipitated DNA was analysed by
real-time PCR. Results are means +
− S.D., n = 3.
with an RNAPII density similar to untreated cells observed at
the promoter, but less than 50–60 % density in elp3∆ cells and
10–20 % density in elp3gcn5∆ cells was observed at the 3
end of the genes (Figure 5B). Moreover, when the hsp70 genes
were induced by heat-shock, this decrease in RNAPII density
was more striking (Figure 5C). However, the RNAPII density did
not change noticeably from the promoter regions to the 3 UTR
of SSA3 and SSA4 in gcn5∆ cells (Figures 5B and 5C). The
double mutant had a more severe effect on RNAPII transcription
(Figures 5B and 5C).We propose that Gcn5 affects the recruitment
of RNAPII, whereas Elp3 influences RNAPII elongation along the
genes.
Gcn5 is Swi/Snf-dependent and is required for HSF recruitment
Histone acetylation has been shown to facilitate HSF-induced
transcription of an in vitro-reconstituted heat-shock gene [29].
HSF-mediated transactivation of target genes in response to stress
is thought to occur principally through the SAGA pathway [30].
We were interested in determining whether Gcn5 affected the
binding of HSF. ChIP assays were performed in elp3, gcn5∆
and elp3gcn5∆ cells, using an antibody against HSF. The results
showed that the recruitment of HSF to gene promoters in gcn5∆
and elp3gcn5∆ yeast were decreased, and recruitment was
unchanged in elp3∆ cells (Figure 6A). This implied that Gcn5,
but not Elp3, affected the binding of HSF.
We next sought to test the contribution of the chromatin
remodelling complex Swi/Snf in SSA3 and SSA4 expression,
because of its critical roles in the transcriptional activation of
inducible yeast genes such as HO, SUC2, INO1 and PHO8 [31],
as well as its known roles in transcriptional regulation, particularly
in elongation, of the mammalian hsp70 genes [32,33]. As shown
in Figure 6(B), heat-shock-induced SSA3 and SSA4 expression
was reduced by 2-fold in cells which were either SNF2 or SNF5
null strains. This required further investigation into whether the
Swi/Snf complex interacted with Gcn5 and/or Elp3. As shown in
Figures 6(C) and 6(D), the amount of Elp3 associated with SSA3
and SSA4 did not change, whereas the amount of Gcn5 was sharply
reduced in snf2∆ and snf5∆ cells. This indicated that the Swi/Snf
complex had significant effects on the recruitment of Gcn5, but
not on Elp3. These results suggested that the recruitment of Gcn5
and its roles in hsp70 transcription was dependent on the presence
of the Swi/Snf complex.
DISCUSSION
The information arising from this study provides a further insight
into understanding the connection between histone acetylation
and hsp70 gene transcription regulation. First, histone acetylation was required for both SSA3 and SSA4 transcription, regardless of the occurrence of nucleosome disassembly. Secondly,
the HATs Gcn5 and Elp3 modulated SSA3 and SSA4 gene transcription through altering histone H3 acetylation. Although both
HATs studied were able to acetylate histone H3, they regulated
hsp70 gene transcription through different mechanisms. The
interaction of Gcn5 was Swi/Snf-dependent and was required for
c The Authors Journal compilation c 2008 Biochemical Society
786
Figure 6
Q. Han and others
Gcn5 was Swi/Snf-dependent and required for HSF recruitment
Cells were heat-shocked for 10 min at 37 ◦C (HS) or left at 26 ◦C (NHS) as a control. (A) ChIP analyses of the relative level of HSF in the promoter regions of SSA3 and SSA4 in wild-type (WT;
W303-1B), elp3∆, gcn5∆ and gcn5∆elp3∆ () cells. Precipitated DNA was analysed by real-time PCR. Results are means +
− S.D., n = 3. (B). Real-time RT-PCR was performed in wild-type
(WT; BY4742), snf2∆ and snf5∆ cells using either SSA3 primers or SSA4 primers. Results are means +
− S.D., n = 3. (C, D) ChIP analyses of the relative amounts of Elp3 (C) and Gcn5 (D) in the
promoter regions of SSA3 and SSA4 in wild-type (WT; BY4742), snf2, and snf5∆ cells. Precipitated DNA was analysed by real-time PCR. Results are means +
− S.D., n = 3.
HSF recruitment and affected RNAPII recruitment, whereas Elp3
exerted its roles mainly through affecting RNAPII elongation.
Nucleosome displacement and transcription-associated
histone acetylation
In an attempt to understand the potential diversity in chromatinbased mechanisms of transcription regulation, we investigated
transcription-dependent nucleosome density and acetylation level
in the SSA3 and SSA4 genes. Our ChIP results indicated that
histones lost contact with DNA in both the promoter and
coding regions of the SSA4 gene (Figure 1A, right-hand panel).
Interestingly, an apparent contrast between SSA4 and SSA3
seemed to exist, as in the former gene, histones lost contact with
DNA, whereas in the latter, no net loss of histone–DNA contact
occurred (Figure 1A, left-hand panel). These results suggested
the existence of distinct mechanisms for enabling transcription
through the SSA3 and SSA4 coding regions. However, our results
showed that histone H3 associated with both SSA3 and SSA4 was
c The Authors Journal compilation c 2008 Biochemical Society
acetylated in a transcription-dependent manner (Figure 1B). This
implied that histone acetylation was important for transcription
regardless of whether nucleosome disassembly occurred. Our
ChIP results for the SSA4 gene were in accordance with previously
published work [28], where the loss of nucleosomes also occurred
to a similar extent in both the wild-type and the mutant yeast
strains (Figure 3A, right-hand panel). This indicated that loss
of the histone–DNA contacts detected in this region may have
occurred in an acetylation-independent manner. However, the result from the ChIP assays for SSA3 was surprisingly. The amount
of histone H3 associated with SSA3 in elp3gcn5∆ cells was
decreased to 50 % of that detected in wild-type cells (Figure 3A, left-hand panel). This indicated that either nucleosome
eviction or histone acetylation was required for transcription
through chromatin. Previously published results suggested that
Gcn5 had no effect on nucleosome loss [21]. Presumably, the
absence of a detectable role for Gcn5 may be the result of its
functional redundancy with other proteins (such as Elp3). The
evidence presented here suggested that RNAPII transcription
Histone acetylation in hsp70 gene transcription
along SSA3 might proceed through two distinct mechanisms:
(i) a histone acetylation-dependent mechanism, little associated
with the net loss of nucleosomes, and (ii) a pathway based on
loss of the histone–DNA contact. Furthermore, we demonstrated
that mutation of Lys8 (K8R) in histone H4 also influenced
SSA3 and SSA4 expression (Figure 1C). The levels of SSA3
and SSA4 transcription in histone H3 K14R and histone H4
K8R mutant strains were similar because there was functional
redundancy between different lysine residues, and so individual
lysine residues may not be essential. However, the results in
Figure 1(C) support the theory that the two lysine residues
tested in this experiment, histone H3 Lys14 and histone H4 Lys8 ,
are important because when these sites are mutated to arginine
residues, the transcription of both SSA3 and SSA4 is significantly
reduced as a result of heat-shock induction compared with the
wild-type control. It should be noted that although we have
focused on histone H3, there is evidence that mutations in Gcn5
and Elp3 also affected the acetylation of histone H4 [9,11,34],
and this might contribute to some of the effects described in this
present study.
Functional redundancy of Gcn5 and Elp3: similarities and
differences
Experiments that determined the acetylation level of histone H3
in cells lacking the transcription-related HATs Gcn5 and/or Elp3
demonstrated the functional redundancy of these two HATs in the
regulation of hsp70 gene expression. Histone acetylation at
the promoter and coding regions of hsp70 genes was reduced
by deletion of Elp3 or Gcn5, and the acetylation in elp3gcn5∆
double mutant strain was much lower than that observed in the
individual single mutant yeast strains (Figure 3B). Moreover, the
elp3gcn5∆ double mutant had a more severe effect on hsp70
expression, suggesting that Gcn5 and Elp3 were at least partly
functionally redundant in hsp70 transcription regulation, as the
absence of one of the proteins was compensated for by the
activity of the other protein. This demonstration of the functional redundancy of the two HATs is consistent with previously
published work [8,9].
In this present study, it has been implied that Gcn5 and Elp3
might play distinct roles in SSA3 and SSA4 regulation. Our studies
with the elp3∆ and gcn5∆ deletion mutants revealed that Gcn5
was the major HAT responsible for promoter acetylation, and
Elp3 was the major HAT responsible for coding region acetylation (Figures 2C and 3B). Specifically, our results support the
view that the Elp3-containing Elongator complex may function
via its interaction with the elongating RNAPII, as proposed by
Wittschieben et al. [13]. This is in apparent contradiction to
the model suggested by Pokholok et al. [35], which described
a cytoplasm-based function for the Elongator complex. However,
we showed that Gcn5 exerted its role via affecting the recruitment
of RNAPII, and probably other transacting factors, in the promoter
regions of SSA3 and SSA4 (Figures 5 and 6).
Relationship between Swi/Snf, Gcn5 and HSF
The abundance of Swi/Snf complex is positively correlated with
that of HSF, affects the rate of RNAPII elongation at HSP82 [21],
and mediates the release of paused RNAPII within the 5 UTR
of the hsp70 gene [32]. These reports prompted us to investigate
the roles of Swi/Snf complex in regulating SSA3 and SSA4. Our
results showed that Swi/Snf was required for the recruitment of
Gcn5 to SSA3 and SSA4 (Figure 6D). However, other studies have
suggested that Gcn5 and Swi/Snf were recruited by HSF to wildtype HSP82, but were not recruited to the 2 bp HSE1 mutant
787
hsp82-P2 [21]. In this present study, we showed that Gcn5 was
important for HSF binding to the promoter regions of SSA3 and
SSA4 (Figure 6A). Based on these results, we propose a model in
which a certain degree of chromatin acetylation is necessary for
the recruitment of HSF to the promoter region of a gene, and this
in turn triggers the recruitment of chromatin remodelling factors,
such as Swi/Snf, to the chromatin to facilitate the association of
co-activators to the promoter.
We thank Dr Jesper Q. Svejstrup (Cancer Research UK London Research Institute, Clare
Hall Laboratories, South Mimms, Hertfordshire, U.K.), Dr Richard Young (Whitehead
Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, U.S.A.) and
Dr Jinqiu Zhou (Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China)
for their gifts of yeast strains. We also thank Dr Jesper Q. Svejstrup, Dr Gross (Division
of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota,
Vermillion, SD, U.S.A.) and Dr Alain Verreault (Institute for Research in Immunology
and Cancer, University of Montreal, Montreal, Canada) for their gifts of antibodies. This
work was supported by the grants from the National Natural Science Foundation of
China (30370316), the National Basic Research Program of China (2005CB522404,
2006CB910506), and the Program for Changjiang Scholars and Innovative Research
Team (PCSIRT) in Universities (IRT0519).
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