Trichostatin A Selectively Suppresses the Cold-Induced Transcription of the ZmDREB1 Gene in Maize
et al. (2011) Trichostatin A Selectively Suppresses the Cold-Induced Transcription of the ZmDREB1 Gene in Maize. PLoS
ONE 6(7): e22132. doi:10.1371/journal.pone.0022132
Trichostatin A Selectively Suppresses the Cold-Induced Transcription of the ZmDREB1 Gene in Maize
Yong Hu 0
Lu Zhang 0
Lin Zhao 0
Jun Li 0
Shibin He 0
Kun Zhou 0
Fei Yang 0
Min Huang 0
Li Jiang 0
Lijia Li 0
Michael Freitag, Oregon State University, United States of America
0 State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University , Wuhan , People's Republic of China
Post-translational modifications of histone proteins play a crucial role in responding to environmental stresses. Histone deacetylases (HDACs) catalyze the removal of an acetyl group from histones and are generally believed to be a transcriptional repressor. In this paper, we report that cold treatment highly induces the up-regulation of HDACs, leading to global deacetylation of histones H3 and H4. Treatment of maize with the HDAC inhibitor trichostatin A (TSA) under cold stress conditions strongly inhibits induction of the maize cold-responsive genes ZmDREB1 and ZmCOR413. However, upregulation of the ZmICE1 gene in response to cold stress is less affected. The expression of drought and salt induced genes, ZmDBF1 and rab17, is almost unaffected by TSA treatment. Thus, these observations show that HDACs may selectively activate transcription. The time course of TSA effects on the expression of ZmDREB1 and ZmCOR413 genes indicates that HDACs appear to directly activate the ZmDREB1 gene, which in turn modulates ZmCOR413 expression. After cold treatment, histone hyperacetylation and DNA demethylation occurs in the ICE1 binding region, accompanied by an increase in accessibility to micrococcal nuclease (MNase). The two regions adjacent to the ICE1 binding site remain hypoacetylated and methylated. However, during cold acclimation, TSA treatment increases the acetylation status and accessibility of MNase and decreases DNA methylation at these two regions. However, TSA treatment does not affect histone hyperacetylation and DNA methylation levels at the ICE1 binding regions of the ZmDREB1 gene. Altogether, our findings indicate that HDACs positively regulate the expression of the cold-induced ZmDREB1 gene through histone modification and chromatin conformational changes and that this activation is both gene and site selective.
Funding: This work was supported by the NSFC (nos. 30870261 and 30771204), the National Genetically Modified Organism Breeding Major Project
(no. 200908010-008B), and the Research Fund for the Doctoral Program of Higher Education (no. 20090141110031). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Plants regulate the expression of some genes when they
encounter diverse environmental stresses, such as low temperature,
drought or high salinity. Cold stress induces gene expression
largely through an abscisic acid (ABA)-independent signaling
pathway. In this cold responsive signaling pathway, genes
encoding for transcription factors are particularly pivotal because
they regulate the expression of the target genes mediating the
adaptive response to cold stress. Among these genes, DREB1
(dehydration responsive element binding 1) proteins have been
demonstrated to play an important role in the response of plants to
cold stress . In Arabidopsis, three closely related DREB1/CBF
genes, DREB1B/CBF1, DREB1A/CBF3 and DREB1C/CBF2, are
well studied and located in a tandem repeat on chromosome 4 .
In maize, the ZmDREB1 gene, characterized by high homology
with the DREB1 gene of Arabidopsis, is present as a single locus in
the genome, and it is mapped on chromosome 6 [3,4]. The
DREB1 transcription factor activates the transcription of target
genes, such as COR (cold-regulated), through binding the CRT/
DRE cis-acting element in the promoter regions of these target
genes [5,6]. ICE1 (inducer of CBF/DREB expression 1) is first
activated by cold stress and is an upstream transcription factor
controlling the expression of the DREB1 gene . More recently,
Arabidopsis studies have shown that histone
acetylation/deacetylation is involved in the stress response in plants. For example,
genetic analysis indicated that Arabidopsis HOS15 confers
tolerance to cold through histone deacetylation . Transgenic
Arabidopsis plants over-expressing AtHD2C showed greater
tolerance to salt and drought stresses than wild-type plants .
Similarly, it has been demonstrated that HDA19 may play a key
role in responses to pathogens . Targeted recruitment of
histone acetyltransferases (HATs) to promoters often leads to
localized histone acetylation at the promoter regions and is
generally considered to be associated with transcriptionally active
genes [11,12]. Indeed, HATs such as GCN5 may be recruited
through the CBF1 transcription factor to induce the expression of
cold-regulated genes . Mutations in GCN5 and ADA2 decrease
the expression of the cold-responsive genes and, therefore,
Arabidopsis cold tolerance . Conversely, the histone
deacetylase (HDAC) catalyzes the removal of acetyl groups from histones,
and it is classically believed to repress gene expression [15,16]. For
example, the Arabidopsis ERF7 transcriptional repressor interacts
with the HDA19 histone deacetylase to repress the transcription of
some stress response genes . However, the impact of HDACs
on cold-inducible gene expression has not been intensively studied,
and much less is understood regarding its functions and
mechanisms in this regulation process.
Small-molecule chemical inhibitors rapidly inhibit enzyme
activity in cells, which allows analysis of the time course of change
and immediate assessment of the drug effects . The small
molecule HDAC inhibitor trichostatin A (TSA) represses histone
deacetylation, resulting in histone hyperacetylation [18,19].
Although it is generally accepted that HDACs are negative
regulators of gene expression, it has been reported that HDAC
inhibitors decrease gene expression in yeast, human and animal
culture cells [18,20]. In the present study, we investigate the
function of HDACs during cold acclimation in maize. Our results
demonstrate that TSA selectively suppresses the induction of the
cold-responsive transcription factor gene ZmDREB1 through
histone modification in the defined promoter region, resulting in
reduced transcription of its target gene, ZmCOR413.
Histones H3 and H4 are globally deacetylated after
treatment with cold (4uC)
Recent studies have demonstrated that histone acetylation of
chromatin is involved in plant responses to environmental stress
. To investigate dynamic changes in histone acetylation under
cold stress conditions, we performed western blot detection on the
levels of H3K9Ac, H4K5Ac and H44Ac in both normal and
coldtreated maize seedlings prepared at various time points. As shown
in Figure 1, there were significantly decreased levels of acetylated
histones in cold-treated plants (Figure 1A) compared with plants
grown at normal temperatures (Figure 1B), indicating that cold
stress induced a global decrease in the acetylation levels of histones
H3 and H4. The overall acetylation levels were rapidly recovered
when cold stress was removed (Figure 1A). Plants grown under
normal temperatures showed that acetylation levels were not
significantly altered during growth (Figure 1B), suggesting that the
deacetylation was solely due to the effects of cold stress. Further in
situ chromatin immunostaining of interphase nuclei showed that
the signals for strongly acetylated H3 and H4 histones were evenly
dispersed in the nucleus at interphase, and nucleoli were barely
acetylated (Figure 1C). In comparison, acetylation signal intensity
was reduced after cold treatment, indicating that deacetylation of
histones H3 and H4 occurred during cold acclimation (Figure 1C).
Quantification of the signal intensity by measuring mean gray
values showed that histone acetylation was decreased by
approximately 50% after treatment with cold (Figure 1D). The
chromatin reverted to the normal acetylation state when seedlings
were returned to 25uC (Figure 1D). As a control, immunostaining
using only a secondary antibody showed no specific labeling of
nuclei (data not shown).
Chromatin condensation has been shown to be associated with
histone deacetylation. Because PI (propidium iodide) fluorescence
intensity is directly proportional to the level of accessible DNA and
reflects the condensation state of chromatin , a FACS flow
cytometer was used to examine chromatin conformational changes
during cold acclimation. Flow cytometric analysis showed specific
cold-induced changes in PI fluorescence intensity. After cold
treatment, the nuclei reproducibly showed a slight reduction in
fluorescence intensity and a left shift in the flow peak (Figure 2B)
compared with the control (Figure 2A). Mixing analysis of control
and cold-treated nuclei showed that each population still retained
its position in the FACS histogram, confirming that the shift is
indeed an inherent feature rather than an experimental error
(Figure 2C). When cold-treated plants were returned to culturing
in warm temperatures for 24 h, flow peaks returned to the control
positions. These results revealed that alterations in overall
chromatin conformation may occur after cold treatment, thus
leading to changes in the accessibility of DNA to PI.
HDAC expression levels are increased during cold
Histone acetylation is a reversible modification that is regulated
by two opposing groups of enzymes: HATs and HDACs .
Therefore, reduced histone acetylation should be associated with
the alteration in HDAC expression. We decided to measure and
quantify HDAC messenger RNA levels in control and cold-treated
seedlings by real-time quantitative PCR after reverse transcription
of RNA. Fifteen HDACs have been indentified in maize. They are
encoded by ten maize RPD3/HDA1 genes, one SIR2 gene and four
HD2-like genes [24,25]. RPD3/HDA1 genes and HD2-like genes
are sensitive to the HDAC inhibitor TSA. HDA101, HDA102 and
HDA108 belong to the RPD3/HDA1 families, and HDA103 and
HDA106 belong to the HD2-like gene family . RPD3-type
HDAC expression is required for plant development , and
HD2 genes were found to be associated with plant resistance to
biotic and abiotic stress in barley . Samples were taken from
maize seedlings grown on MS medium and treated with cold (4uC)
at the indicated time points, and maize seedlings grown under
normal conditions (25uC) were used as a control. After cold
treatment, plants were again transferred to normal conditions
(25uC) and sampled at the indicated time points. We found that
HDAC expression levels were rapidly increased immediately after
seedlings were transferred to cold stress conditions (Figure 3AD).
These data indicated that cold stress enhanced HDAC expression
and further led to global deacetylation of H3 and H4 histones.
The HDAC inhibitor selectively interferes with
coldinduced gene transcription
The above results demonstrated that HDAC expression levels
are strongly enhanced by cold stress conditions, implicating
HDACs in the cold stress response in plants. Many genes, such
as ICE1, DREB1 and COR413, are known to rapidly accumulate in
plants during cold acclimation [26,27]. Next, we decided to
examine the presence of a relationship between HDACs and these
cold-responsive genes at the epigenetic level. We first examined
the expression levels of the ZmICE1 and ZmDREB1 transcription
factor genes. As shown in Figure 4A, rapid up-regulation of the
ZmICE1 gene was observed after cold application, whereas basal
levels of ZmICE1 transcripts could be detected in all control plants.
ZmDREB1 was also highly induced by cold stress; however, it was
not induced by ABA application (Figure 4B). Figure 4C shows that
the expression of the ZmDREB1 target gene, ZmCOR413, was also
strongly induced by cold treatment. TSA is an inhibitor of
HDACs, and it has been widely used to characterize the function
of HDACs in yeasts, mammalian cells and plants [18,28,29].
Interestingly, the cold-induced expression of ZmDREB1 was
inhibited after TSA treatment (Figure 4B). However, expression
of ZmICE1 was barely affected by TSA treatment (Figure 4A). As
expected, ZmCOR413 was completely inhibited by TSA
(Figure 4C). Substantial evidence shows that cold, drought and
high salt stress signals and ABA have some overlapping elements
in the signaling network . Drought and salt increase ABA
levels and affect gene expression through an ABA-dependent
signaling pathway. Therefore, we tested whether HDACs affect
ABA-responsive gene expression. ZmDBF1 is induced by ABA,
high salt and drought stress but not by cold, and the rab17 gene is
regulated by ZmDBF1. Seedlings were planted on MS medium
containing various concentrations of ABA, NaCl and mannitol. As
shown in Figure 4D and E, ZmDBF1 and rab17 were strongly
induced by mannitol, NaCl and ABA treatment. However, cold
treatment had a very weak or even no effect on induction of
ZmDBF1, consistent with the previous results . However,
ABAinduced expression of ZmDBF1 and rab17 was not affected by TSA
treatment (Figure 4D and E). Therefore, these findings indicate
that HDACs play an important role in cold-responsive gene
expression independently of the ABA pathway, and it positively
regulates the expression of these genes.
HDACs indirectly regulate the expression of the
DREB1 binds the CRT/DRE element in the promoter region
of the COR gene to activate gene expression [5,6], and the above
results indicated that both ZmDREB1 and ZmCOR413 are
positively regulated by HDACs. Therefore, we further address
whether the decreased expression of ZmDREB1 and ZmCOR413 is
due to direct TSA-mediated suppression. The TSA-treated time
course profiles show that the expression of ZmDREB1 is repressed
within 15 min and down-regulated to an average of 1-fold at
60 min. However, the expression of ZmCOR413 is not significantly
changed during the 60-min time course. These results suggest that
the expression of ZmDREB1 is directly regulated by HDACs,
whereas the effect of HDACs on ZmCOR413 is secondary
TSA-treated plants are hypersensitive to freezing
Because TSA blocks cold-responsive gene transcription, we
examined whether the TSA has an effect on the freezing tolerance
of plants. Therefore, an electrolyte leakage test was performed. As
shown in Figure 5, incubation in 0.5 mM TSA for 24 h decreased
cold hardiness in maize seedlings. Normal plants had an LT50
value of approximately 29.52uC, whereas TSA-treated plants had
an LT50 value of approximately 20.98uC. Ion leakage at
temperature 220uC represents approximately 100% leakage.
Cold and TSA give rise to the different histone
acetylation statuses across the ZmDREB1 promoter
HATs/HDACs are always recruited to target genes and they
cause histone acetylation changes. Thus, we investigated the
acetylation state of histones H3 at Lys 9, H4 at Lys 5 and H4 at
Lys 5, 8, 12 and 16 across the ZmDREB1 promoter region by
ChIP. Primers encompassing the ZmDREB1 gene were designed to
detect dynamic changes in histone acetylation across the
transcriptional regulatory sequences (Table 1). Compared with
the histone acetylation levels in control plants, hyperacetylation in
cold-acclimated plants occurred in the ICE1 binding region (Sets
A, D and E) and a region of the first exon of the gene (Set F)
(Figure 6). However, regions adjacent to the ICE1 binding site (Set
Figure 2. Genome-wide chromatin condensation revealed under cold stress. Nuclei were prepared from seedlings grown under normal (A)
and cold stress conditions (B) and stained with PI. C. Flow cytometric analysis of a mixture of both types of nuclei. Note that the PI fluorescence peak
displayed a slight leftward shift in cold-acclimated plants compared with a control group.
B and C) remain hypoacetylated after cold treatment (Figure 6).
Interestingly, if cold-acclimated plants were treated with TSA, all
of these examined regions (Sets AF) were hyperacetylated
(Figure 6). Under normal conditions, the control region of the
ZmDREB1 gene did not undergo significant changes in acetylation
levels after TSA treatment (Figure 6). Additionally, histone
Figure 3. The transcriptional profile of HDACs in response to the low temperature (46C). Plants grown at 25uC (x-axis 0) were subjected to
cold acclimation (4uC) for 5 days (x-axis 15) and then returned to warm temperature (25uC) for 1 day (x-axis 6). Transcript levels were determined by
quantitative real-time PCR, and beta actin was used as an internal control. The data are the mean 6 standard error for triplicate quantitative PCR
reactions for each time point from three independent experiments.
TSA treatment. TSA treatment rapidly represses ZmDREB1 and slowly represses ZmCOR413. Samples were taken from 7-day-old seedlings grown
under 25uC (x-axis 0), and from plants treated with different reagents or stresses at the indicated time points. The data are the mean 6 standard error
for triplicate quantitative PCR reactions for each time point from three independent experiments.
acetylation of the ZmICE1 and ZmCOR413 promoter regions had
no significant changes after TSA treatment (Figure 7), which is in
agreement with the previous results demonstrating that TSA has
no direct effects on these two genes.
TSA treatment increases the ZmDREB1 promoter
chromatin accessibility to micrococcal nuclease
Histone acetylation/deacetylation usually alters chromatin
conformation to regulate gene expression. Therefore, we next examined
the effect of TSA on local region chromatin accessibility to
micrococcal nuclease (MNase) during cold acclimation. The primer
sets were the same as those used in the ChIP assays (Table 1).
Decondensed chromatin regions are more accessible to MNase
cleavage, and thus MNase can be used to monitor the
conformational change of chromatin across a specific region . Increased
cleavage occurring across the chromatin region resulted in decreased
amplification. Thus, if the treatment of the seedlings induces changes
in the chromatin accessibility of MNase, more or fewer PCR
products will be generated due to increased or decreased digestion
with MNase. As shown in Figure 8A, in untreated seedlings, all
measured regions were inaccessible to MNase. In plants cold
acclimated for 12 h, regions A, D, E and F were accessible to MNase
(Figure 8B), but the other two regions (Sets B and C) remained
inaccessible to MNase. In seedlings treated by cold-TSA for 12 h,
fewer PCR products were gained due to MNase digestion of all of the
regions (Sets AF) (Figure 8C). Thus, chromatin decondensation
may be occurring across the ZmDREB1 promoter region (B and C)
during cold acclimation upon TSA treatment.
The methylation pattern of the ZmDREB1 gene 59 region
in TSA and/or cold-treated seedlings
We further investigated the DNA methylation state of the
ZmDREB1 gene by sodium bisulfite methylation sequencing. Our
results indicate that the region of the ZmDREB1 gene crossing primer
sets A through F contains 59 CpG and 44 CpNpG motifs. The mean
value of the methylated percentages is shown in Figure 9. Compared
with the DNA methylation state in control seedlings, cold stress
caused DNA hypomethylation in the ICE1 binding region (Set A, D
and E) and the first exon (Set F). Two other promoter regions (Set B
and C) were still hypermethylated during cold acclimation.
However, in cold-TSA treated seedlings, all examined regions (Set
AF) were hypomethylated (Figure 9). The DNA methylation state
was not significantly changed in TSA-treated plants.
We have shown that in maize seedlings, HDACs are highly
inducible upon cold treatment (4uC). The increased HDAC expression
results in the global deacetylation of histones H3 and H4. Moreover, a
histone deacetylase inhibitor selectively suppresses the induction of the
cold-responsive transcription factor gene ZmDREB1 through altering
the epigenetic modifications in defined sites of the promoter region.
Furthermore, TSA treatment blocks ZmCOR413, which is a
downstream target, but it does not modulate the expression of several
ABA-induced genes. These findings identify HDACs as a signaling
component that lies between the ZmICE1 and ZmDREB1 transcription
factors in the ICE/DREB1 cold signaling cascade, and it positively
regulates cold signal transduction at the chromatin level independently
of an ABA signaling pathway. This indicates that this activation
function of HDACs is gene and site selective.
HDACs as a positive signaling component selectively
mediated cold-responsive gene expression
A cold-signaling network has been proposed where the cold
signal first induces the expression of the ICE gene that activates the
expression of downstream transcription factor gene CBF/DREB.
Figure 5. TSA-treated plants are more sensitive to freezing. A. Plants were freeze-treated at the indicated temperatures for 15 minutes and
then the survival of plants was quantified. B. Leakage of electrolytes from normal and TSA-treated plants was estimated by determining relative
electro-conductivity of the bathing solution of the leaf tissue. Bars represent standard errors.
TTCAAACTACGCACATCCATAATTT ZmDREB1A Set D
ACAAGTTAATGAGCACACCATCACT ZmDREB1A Set E
ACAGTACAAGGGGCCGCCTAGCAAC ZmDREB1A Set F
TGCTCTGCCACCACCACCTCGTCGT ZmICE1 promoter TTTTGCTCTTCAGGCACCTT
This, in turn, regulates the transcription of downstream target
genes [32,33]. Our results in Figure 4 demonstrate that HDACs
might lie between the ZmICE1 transcription factor and the
expression of ZmDREB1. TSA treatment causes rapid
downregulation of ZmDREB1, suggesting that the HDAC functions as a
direct transcriptional activator regulating the ZmDREB1 gene.
However, these results cannot exclude the possibility that HDACs
may also directly regulate ZmCOR413 gene expression.
FurtherFigure 6. Comparison of H3K9Ac, H4K5Ac and H44Ac levels in the ZmDREB1 gene region. AC. Cold stress caused hyperacetylation in the
ICE1 binding region (Set A, D and E) and the first exon (Set F). Two other promoter regions (Set B and C) were still hypoacetylated during cold
acclimation. When cold-acclimated plants were treated with TSA, all regions were hyperacetylated, and the transcription of ZmDREB1 was repressed.
D. Schematic representation of the ZmDREB1 gene from 23074 to +222 bp. Primer sets A, D and E contain a MYC recognition element that can be
specifically combined with ICE1. Bars represent standard errors.
Figure 7. Comparison of H3K9Ac, H4K5Ac and H44Ac levels in the ZmICE1 and ZmCOR413 gene regions. Cold stress caused
hyperacetylation in the ZmICE1 and ZmCOR413 promoter regions and the first exon. Additionally, these two regions were still hyperacetylated after
cold-acclimated plants were treated with TSA. No significant changes in histone acetylation were detected in TSA-treated normal plants. Bars
represent standard errors.
more, treatment with the HDAC inhibitor TSA indicates that the
ZmCOR413 gene is not a direct target of HDACs, instead HDACs
appear to directly activate the ZmDREB1 gene, which then
mediates activation of ZmCOR413. Therefore, HDACs modulate
cold-responsive gene expression as a positive signaling component
that directly regulates ZmDREB1. Additionally, up-regulation of
upstream ZmICE1 in response to cold stress is less affected by TSA
treatment, and HDACs do not regulate ABA-induced gene
expression. Together, this indicates that modulation of gene
expression by HDACs is gene selective. Similarly, suberoylanilide
hydroxamic acid induces p21WAF1 expression by selective
acetylation of histones associated with p21WAF1 .
TSA represses ZmDREB1 gene expression through
altering chromatin modification
HDACs are classically thought to down-regulate or suppress gene
expression. Therefore, HDAC inhibitors can up-regulate genes. For
example, Arabidopsis AtHD2A, AtHD2B and AtHD2C were
shown to repress transcription  when targeted to the promoter
of genes, and Arabidopsis HOS15 repressed RD29A expression
through RD29A promoter-associated histone deacetylation .
Similarly, TSA induces up-regulation of 45 genes during
Arabidopsis seed germination . However, we show here that
ZmDREB1 and ZmCOR413 were rapidly down-regulated when
HDAC activity is perturbed by TSA, indicating that HDACs may
Figure 8. HDACs induced chromatin accessibility at the promoter region of the ZmDREB1 gene. MNase accessibility by CHART-PCR was
performed on nuclei that were extracted from 7-day-old seedlings grown at 25uC (A) and further treated with cold (B) or cold-TSA (C) for 12 h. Primer
sets AF are the same as those used in the ChIP assay. The Ct values generated were converted to PCR product amounts using the standard curve.
The y-axis indicates the amount of PCR products and the x-axis indicates the different DNA region. The data shown are the mean and standard errors
of triplicate PCRs resulting from three independent experiments.
Figure 9. Comparison of the methylation status of CpG motifs in ZmDREB1 gene region during cold acclimation. Cold stress caused
DNA hypomethylation in the ICE1 binding region (Set A, D and E) and the first exon (Set F). Two other promoter regions (Set B and C) were still
hypermethylated during cold acclimation. TSA treatment did not significantly affect the DNA methylation status in normal seedlings. However, in
cold-TSA treated seedlings, all examined regions (Set AF) were hypomethylated. Bars represent standard errors.
also activate the transcription of cold-responsive genes in maize.
This finding is supported by the similar observation that
overexpression of HDA19 in Arabidopsis results in up-regulation
of ERF1 . The study in yeast indicates that HDACs are essential
for efficient induction of some genes. For example, RPD3 has been
shown to activate telomeric genes . In animal culture cells,
HDAC inhibitors decrease the expression of some cytokine genes
Histone acetylation/deacetylation at the promoter regions of
some genes is usually involved in the alteration of the local
chromatin conformation that regulates gene expression, and our
findings agree with this idea. ChIP and CHART-PCR data
indicate that the acetylation level and the chromatin status of the
ICE1 binding regions remain unchanged in cold-acclimated plants
after TSA treatment, implying that TSA has no effect on these
regions. Additionally, ICE1 may remain bound to these regions in
the promoter. However, in cold-acclimated plants, chromatin
decondensation was observed at other two regions in the promoter
after TSA treatment, and this selective chromatin conformational
change appears to facilitate the binding of certain factors. The
results suggest that TSA inhibits the expression of ZmDREB1
through changing chromatin modification in defined sites that
regulate expression of this gene. Therefore, we speculate that
HDACs likely regulate ZmDREB1 gene expression by preventing
binding of certain factors through maintaining a deacetylated state
in these regulatory regions. Identification of the unknown
repressor and related cis-acting elements of the DREB1 gene will
confirm this speculation. A similar situation has been described for
activation of telomeric genes in yeast through deacetylation of
histones by Rpd3p, which then prevented binding of the SIR
repressor proteins . HDACs may also activate transcription via
chromatin remodeling, enabling transcription factor recruitment.
For example, chromatin remodeling by HDACs activates
proinflammatory gene expression through recruitment of transcription
factors . Furthermore, our recent study indicates that a mutual
reinforcing action between histone acetylation, histone
methylation and DNA methylation occurs during maize mitosis .
DNA methylation has been reported to control methylation of
H3K9 and heterochromatin assembly in Arabidopsis [38,39]. In
Neurospora, TSA could cause selective loss of DNA methylation
. Some reports suggested that histone H4 hyperacetylation
could affect DNA methylation levels [41,42]. It was also reported
that cold stress induces DNA demethylation in maize . Thus,
multiple modifications of histones and DNA may co-function in
various combinations in response to environmental stresses.
Our results indicate that HDACs cause global histone
deacetylation in response to cold stress and regulate the expression
of ZmDREB1 and ZmCOR413. TSA suppresses cell cycle
progression by causing hyperacetylation of histones in tobacco
protoplasts,  and it can delay seed germination through a
global deacetylation event . Studies in yeast show that the
overall acetylation/deacetylation processes may function to
control basal transcription . Global acetylation and
deacetylation also allow for rapid restoration of acetylation levels when the
recruited HAT or HDAC is removed [45,46]. Therefore, we
further extend these findings to show that cold induces a
hypoacetylated state through the genome, and global
acetylation/deacetylation may be involved in rapid reversal of
transcription levels after the removal of cold stress.
Materials and Methods
Plant materials and treatments
Maize seeds (Zea mays L. inbred line Huangzao 4) were sterilized
and grown in 1/5 diluted Murashige and Skoog medium (MS)
under continuous light (120 mmolm22s21) for 7 days at 25uC and
70% relative humidity in a growth cabinet . To investigate the
effects of cold stress, ABA, NaCl, mannitol and trichostatin A
(TSA), nine experimental groups were formed. Each experimental
group contained 30 seedlings. Seedlings in the control group
remained in 1/5 diluted MS medium and were kept in a 25uC
incubator for the entire length of the experiment. For cold
treatment, seedlings in the cold group were transferred to a 4uC
incubator and grown in 1/5 diluted MS medium for 4 days. After
cold stress, the seedlings were grown for another 4 days in the
25uC incubator for recovery. For the ABA, salt and drought
treatments, seedlings belonging to the ABA, NaCl and
mannitol groups were transferred to 1/5 diluted MS medium
containing 100 mM ABA, 300 mM NaCl or 250 mM mannitol,
respectively and then grown in a 25uC incubator for 48 hours. For
TSA treatment, seedlings in the TSA and cold-TSA groups
were incubated in 1/5 diluted MS medium containing 0.5 mM
TSA at 25uC and 4uC, respectively. For ABA-TSA,
NaClTSA and mannitol-TSA treatments, 7-day-old seedlings were
transferred to 1/5 diluted MS medium containing 0.5 mM TSA
combined with 100 mM ABA, 300 mM NaCl or 250 mM
mannitol, respectively and cultivated in a 25uC incubator for
48 hours. Light and humidity conditions were kept constant
throughout the experimental periods.
The following antibodies were used for immunostaining and
ChIP. From upstate (Lake Placid, NY, USA): H3K9ac (catalog
number 07-352), H4K5ac (catalog number 06-759), H44ac
(catalog number 06-866), Anti-Histone H3 (catalog number
06755), and fluorescein-conjugated goat anti-rabbit IgG (catalog
number 16-237). From Sigma (St. Louis, MO, USA):
APconjugated goat anti-rabbit IgG (catalog number A4187).
Western blotting assays
Proteins were extracted by grinding samples in liquid nitrogen
and resuspended in extraction buffer (100 mM Tris-HCl pH 7.4,
50 mM NaCl, 5 mM EDTA and 1 mM PMSF). Western blot
detection was carried out as previously described . Actin and
histone H3 were used as equal loading controls. Western blots
were repeated three times for each sample from three independent
Flow cytometric assays
Nuclei were prepared according to the method described by Li
et al. . The cell-cycle profile was determined with a
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA,
USA) equipped with an argon-ion laser, using the 488 nm laser
line for excitation. CellQuest software running on an Apple
Macintosh computer connected to the flow cytometer was used for
Immunostaining of nuclei was performed according to the
method described by Yang et al. . Nuclei were spread on a
slide, incubated with the primary antibody at 4uC overnight,
followed by 2 h incubation at 37uC with the secondary antibody.
In control experiments, slides were incubated with only the
secondary antibody. All slides were counterstained with DAPI
(Sigma, St. Louis, MO, USA), mounted with Vectashield (Vector
labs, Burlingame, CA, USA) and examined with an Olympus
BX60 fluorescence microscope with filter blocks for DAPI and
fluorescein. Images captured with a CCD monochrome camera
Sensys 1401E were pseudo-colored and merged using the software
MetaMorphH 4.6.3 (Universal Imaging Corp., Downingtown, PA,
USA). Microscope settings and camera detector exposure times
were kept constant for each respective channel (fluorescein or
DAPI) but were optimized for individual experiments. More than
500 nuclei were analyzed for each treatment group. For both
control and treated groups, three independent immunostaining
experiments were performed with each antibody. Image J and
MetaMorph measured the mean gray values of the signal intensity,
and data were analyzed by SPSS10.0.
Reverse transcription and real-time PCR
Total RNA was isolated using the RNAprep pure Plant Kit
(Qiagen, Mannheim, Germany) according to the manufacturers
instructions. To remove residual DNA contamination, 1 mg of
total RNA was treated with 50 units of DNase I (Fermentas,
Burlington, ON, Canada) at 37uC for 30 min. The purified RNA
was reverse-transcribed into cDNA with the RevertAid First
Strand cDNA Synthesis Kit (Fermentas, Burlington, ON,
Canada), as recommended by the manufacturer.
Quantitative real-time PCR was performed using SYBRH
Green Real-time PCR Master Mix (Toyobo, Tokyo Japan) on
the Rotor-Gene 2000 Real-Time Cycler (Corbett Research,
Mortlake, NSW, Australia). For all real-time PCR assays, standard
curves were generated for each primer set to determine their
efficiency, and melting curves were generated to detect
nonspecific amplification products and primer-dimers. The PCR
product was also separated on a 2% agarose gel to confirm its size
(,300 bp), and it was also sequenced to verify its identity. In the
preliminary experiment, we tested the expression of several
reference genes, such as actin (GenBank accession number:
J01238), 18S (GenBank accession number: EU975801.1) and
GAPDH (GenBank accession number: X07156.1), in the control
and treatment seedlings, and we observed that actin transcription
(Ct values: 28.32360.074) was the most stable because it was not
significantly regulated or influenced by the experimental
procedure. Therefore, the actin gene was selected as a reference gene in
this study. Triplicate PCR reactions for each of the three
independently-purified RNA samples were carried out.
Template-free and SYBR Green mix-free samples were amplified for
each gene as negative controls. The standard amplification
conditions were: 95uC for 30 sec, followed by 45 amplification
cycles at 95uC for 10 sec, 5860uC for 10 sec, and 72uC for
15 sec. Fluorescence data were acquired at the 72uC step and
during the melting-curve program. The threshold cycle numbers
(Ct) for each PCR product was determined, and the relative
expression levels for all genes were obtained using 22DDCt
calculations by Rotor Gene software version 6.0.19 (Corbett
Research, Mortlake, NSW, Australia). Quantitative PCR primers
were designed using the Primer Premier 5 software. These primer
sequences and the reaction efficiencies (ranging from 96% to
104%) are detailed in Table 1.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was carried out with H3K9Ac, H4K5Ac and
H44Ac antibodies following the procedure described by Haring
et al. . During the ChIP assay, a negative control was
performed using rabbit serum for mock immunoprecipitation.
After ChIP, DNA was extracted with a standard procedure
(phenol/chloroform/isoamyl alcohol) (25:24:1). Precipitated
genomic DNA was subjected to real-time PCR with primer sets A
F (Table 1) encompassing the promoter region (AE) and the first
exon (F) of the DREB1 gene. Quantitative Real-time PCR was
performed according to the above-mentioned procedure.
Chromatin accessibility measured by CHART-PCR
To analyze the chromatin conformational change, a chromatin
accessibility by real-time PCR (CHART-PCR) assay was carried
out essentially according to the method described by Rao et al.
. Seedlings were cold or cold-TSA treated, and nuclei were
prepared as described by Lo et al. . Nuclei were then digested
for 5 min at 37uC using 5 U micrococcal nuclease (MNase).
Subsequently, DNA was prepared using a Plant genomic DNA kit
(Qiagen, Mannheim, Germany) and quantified using the Gene
Quant calculator (Amersham Pharmacia Biotec, Piscataway, NJ,
USA). One hundred nanograms of genomic DNA from cold or
cold-TSA-treated samples was used for SYBR Green real-time
PCR analysis with the same primer sets that were used for the
ChIP assay (Table 1). MNase accessibility is thought to be
inversely proportional to the amount of amplified product.
Assessment of freezing tolerance
Normal and TSA group (0.5 mM TSA treated for 24 h) plants
were used for cold acclimation tests, and electrolyte leakage
analysis was performed following the method described by Ristic
and Ashworth . For survival analysis, 15 plants from each
group were freeze-treated at each indicated temperatures (22, 26,
210, 214 and 220uC) for 15 minutes, and then the mean relative
survival of 15 seedlings was determined. For the electrolyte leakage
test, 25 fully expanded leaves were taken from 15 plants of each
group and placed in glass-stoppered test tubes (1 leaf per tube
containing 4 ml deionized water). After a 30 min equilibration
period at 4uC, the bath temperature was reduced at 2uC/h rate to
220uC (Masterline Model 2095, Forma Scientific, Marietta, OH,
USA). Five tubes from each group were withdrawn at each
indicated temperature (22, 26, 210, 214 and 220uC) and
placed on ice overnight. Subsequently, the electro-conductivity of
the bathing solution was measured using a conductance meter
(YSI model 35, Yellow spring, Ohio, USA). A value set for 100%
leakage was obtained by boiling the sample for 1 h. The
electroconductivity before boiling was calculated as a percentage
compared to the levels after boiling to give relative
electroconductivity. Then, the average relative electro-conductivity of 5
leaves was calculated. The temperature value for 50% electrolyte
leakage, which was defined as the LT50, was determined by a plot
of freezing temperature versus relative electro-conductivity.
The methylation status of CpG and CpNpG motifs in the
ZmDREB1 promoter region was determined using bisulfite
TGTTAATAGTTTTTTTTATGGGTGGAG CCACAAACAACACAAACATAATCTTATAT CACACCATCACTACTCACTA TTCGGCAAACTATACAACAC
sequencing. Approximately 2 mg of genomic DNA was denatured
in 0.3 M NaOH (30 min, 20uC), neutralized with ammonium
acetate, and ethanol precipitated. Nonmethylated cytosines were
deaminated in 1.5 ml of 4 M NaHSO3 and 500 mM
hydroquinone at 55uC for 16 h. Subsequently, DNA was purified by gel
filtration, incubated in 0.3 M NaOH (10 min, 37uC), and ethanol
precipitated. Purified DNA was resuspended in 50 ml of ddH2O.
Finally, DNA was analyzed using primer sets spanning a region
from 23074 to +222 bp of the ZmDREB1 gene. PCR products
were purified using a gel extraction kit (Qiagen, Mannheim,
Germany) and were directly sequenced using an automated DNA
sequencer. Each sample was sequenced 3 times to determine
sitespecific methylation changes in the amplified regions. To ensure
that only bisulfite-reacted DNA was amplified and to avoid biased
amplification of methylated strands, primers spanning these
regions were designed using methprimer software (Table 2).
Conceived and designed the experiments: LL YH. Performed the
experiments: YH L. Zhang JL SH KZ. Analyzed the data: LL YH L.
Zhao FY MH LJ. Wrote the paper: LL YH.
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