Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy
et al. (2014) Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of
Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy. PLoS ONE 9(8): e104135. doi:10.1371/journal.pone.0104135
Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy
Chang Peng 0
Jing Zhu 0
Hui-Chao Sun 0
Xu-Pei Huang 0
Wei-An Zhao 0
Min Zheng 0
Ling-Juan Liu 0
Jie Tian 0
Robert Dettman, Northwestern University, United States of America
0 1 Heart Centre, Children's Hospital of Chongqing Medical University , Chongqing , China , 2 Key Laboratory of Pediatrics in Chongqing , Chongqing , China , 3 Ministry of Education Key Laboratory of Child Development and Disorders , Chongqing , China , 4 Chongqing International Science and Technology Cooperation Center for Child Development and Disorders , Chongqing , China , 5 Department of Biomedical Science, Charlie E. Schmidt College of Medicine, Florida Atlantic University , Boca Raton, Florida , United States of America
Background: Cardiovascular malformations can be caused by abnormalities in Gata4 expression during fetal development. In a previous study, we demonstrated that ethanol exposure could lead to histone hyperacetylation and Gata4 overexpression in fetal mouse hearts. However, the potential mechanisms of histone hyperacetylation and Gata4 overexpression induced by ethanol remain unclear. Methods and Results: Pregnant mice were gavaged with ethanol or saline. Fetal mouse hearts were collected for analysis. The results of ethanol fed groups showed that global HAT activity was unusually high in the hearts of fetal mice while global HDAC activity remained unchanged. Binding of P300, CBP, PCAF, SRC1, but not GCN5, were increased on the Gata4 promoter relative to the saline treated group. Increased acetylation of H3K9 and increased mRNA expression of Gata4, aMHC, cTnT were observed in these hearts. Treatment with the pan-histone acetylase inhibitor, anacardic acid, reduced the binding of P300, PCAF to the Gata4 promoter and reversed H3K9 hyperacetylation in the presence of ethanol. Interestingly, anacardic acid attenuated over-expression of Gata4, a-MHC and cTnT in fetal mouse hearts exposed to ethanol. Conclusions: Our results suggest that P300 and PCAF may be critical regulatory factors that mediate Gata4 over-expression induced by ethanol exposure. Alternatively, P300, PCAF and Gata4 may coordinate over-expression of cardiac downstream genes in mouse hearts exposed to ethanol. Anacardic acid may thus protect against ethanol-induced Gata4, a-MHC, cTnT over-expression by inhibiting the binding of P300 and PCAF to the promoter region of these genes.
Funding: This study was financially supported by research grants from National Natural Science Foundation of China (Grant Number: 81270234) and the special
fund of Chongqing key laboratory, CSTC, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
In children, congenital heart disease (CHD) accounts for nearly
one-third of all major congenital anomalies , a major cause of
death in infants of 1 year of age or below . Cardiac
development is a very complex process, regulated by both genetic
and epigenetic pathways. Alcohol consumption is a common
teratogenetic factor that is thought to affect epigenetic regulation
of embryonic development and contribute to CHD . Alcohol
consumption during pregnancy is associated with multiple
cardiovascular malformations . However, the potential
mechanism of CHD induced by alcohol is poorly understood.
Many studies indicate that the Gata4 transcription factor plays a
critical role during cardiogenesis, from primitive heart tube to
maturation i.e. the four-chambered heart . Both genetics
and epigenetics play a part in regulating the expression of Gata4
and alterations that lead to uncontrolled expression of Gata4 could
affect normal development of the heart. We have demonstrated
that high consumption of alcohol or its metabolites can induce
histone hyperacetylation that leads to over-expression of Gata4
[12,13]. However, it is not clear whether alcohol exposure
increases myocardial histone acetylation or Gata4 over-expression
in developing hearts. Furthermore, it is not understood which
isoforms of HATs and HDACs take part in Gata4 regulation
Here, we examined the impact alcohol exposure has on HAT
and HDAC activity during heart development, and explored the
effects of P300, CBP, PCAF, SRC1 and GCN5 on Gata4
overexpression. We present data on the effects of alcohol on cardiac
downstream genes a-MHC, cTnT, a-actin. Finally, we tested if a
pan-histone acetylase inhibitor, anacardic acid, has a protective
effect on alcohol-induced Gata4 and cardiac downstream gene
Figure 1. Effects of alcohol exposure on activities of HAT and HDAC. To analyze the impact of alcohol on activities of HAT and HDAC in
cardiac tissues, different doses of alcohol were used to choose optimal exposure dose in pregnant mice. The blood-alcohol concentration (A) after
gavaging with different doses of 56% ethanol in mice (n = 6). The alcohol stress (56%) increases HAT activity (B) in E14.5, E16.5 and PND0.5, while it
remained unchanged in PND7 and no any effects observed on HDAC activity (C) in myocardial tissues. *: P,0.05 vs. control group (n = 9). E14.5:
embryo 14.5 day, E16.5: embryo 16.5 day, PND0.5: postnatal day 0.5, PND7: postnatal day 7.
Materials and Methods
Treatment of Mice
Pathogen free male and female, 9 to 11 week old Kunming mice
(2025 g) were purchased from the Experiment Animal Center of
Chongqing Medical University (Chongqing, China). All the trials
on the animals selected for the experiments were approved by the
Animal Care and Use Committee at the Chongqing Medical
University. Mice were housed and allowed food ad libitum, and
maintained in a controlled environment (2261uC, 5565%
humidity) with a 12 h: 12 h light: dark cycle. Mated female mice
were examined for a vaginal plug in the morning. If a vaginal plug
was observed, embryos were considered to be (E) 0.5 day.
Pregnant mice were randomly assigned to be gavaged with 56%
ethanol (control groups received equivalent normal saline), In
some cases, ethanol treated pregnant mice were administered the
pan-histone acetylase inhibitor anacardic acid. Anacardic acid was
dissolved in sterile DMSO at a concentration of 1 mg/ml and
stored at 4uC. Ethanol (56% v/v) was fed daily to pregnant dams
at a volume of 5 ml per kilogram per day from E8.5E16.5 by
gavage. Anacardic acid was administered by intraperitoneal
injection at a dose of 5 mg per kilogram per day during days
E8.5E16.5. On days, E14.5 and E16.5, pregnant mice were
euthanized using carbon dioxide narcosis and embryonic hearts
were isolated from pups. Hearts from 0.5 day old and 7 day old
neonatal mice were collected, as well.
HAT and HDAC Activity Assays
After homogenization of cardiac tissues, nucleoproteins were
extracted using a Nuclear Extract Kit (Merck Millipore, DA,
GER) according to the manufacturers instructions. HAT and
HDAC activities of the nuclear protein extracts were determined
using a colorimetric assay included in the HAT and HDAC assay
kits (GenMed Scientific Inc, Shanghai, China).
Chromatin Immunoprecipitation (ChIP) Assay
After homogenization of cardiac tissues, formaldehyde (1%) was
added to the samples to cross-link protein-DNA complexes. ChIP
trials were conducted by use of a ChIP assay kit (Merck Millipore,
DA, GER). After cross-linking, the DNA was fragmented by
sonication, this consisted of 20 cycles of 30 seconds each time with
an interval of 30 seconds to cool down. Then protein-DNA
complexes were precipitated by monoclonal anti-P300 antibody,
anti-CBP antibody, anti-PCAF antibody, anti-SRC1 antibody
(ChIP grade, Abcam, Cambridge, UK), anti-GCN5 antibody
(ChIP grade, Epigentek, NY, USA) or polyclonal anti-H3AcK9
antibody (ChIP grade, Abcam, Cambridge, UK) respectively, DNA
was extracted using a DNA purification kit (Merck Millipore, DA,
GER). The experiment contained both a positive control group
(precipitated by anti-RNA polymerase II antibody) and a negative
control group (precipitated by normal mouse IgG). Specific primers
were designed for recognizing the promoter of Gata4, cTnT,
aMHC and a-actin in quantitative Real-Time PCR assays, to
ascertain cardiac development-related genes, which may interact
with HAT proteins and be regulated by HATs. The sequences of
specific primers were as follows: Gata4 sense primer:
59TCTTCCACTTCCACACGTACCAA-39 and antisense primer:
59-CAGAGGGAGTTGGGAGACGTAG-39; cTnT sense primer:
59 -TAACAGTGTCTGGAAGCGTCA-39 and antisense primer:
59-CAGAGTGACTGGCACAAGGT-39; a-MHC sense primer:
59-AGGACAGGGGTTGCCTCT-39 and antisense primer:
59AGGTGCTGCTTTGAATGCC-39; a-actin sense primer:
59TGCCTCAGCCCCCTCTAG-39 and antisense primer:
59-GCAGACAACTGGTGGAAGAG-39; RPL13A sense primer:
59GAAAGCCTTGTCGCATCCCT-39 and antisense primer:
59GAAAGCCAAAGCTGGATGACA-39. Annealing temperatures
were as follows: 59uC for Gata4, 57uC for cTnT, a-MHC, a-actin
and RPL13A. PCR products were detected by 2% agarose gel
electrophoresis. The quantity of DNA immunoprecipitation by each
HAT antibody reflected the HAT isoforms that were bound to the
Western Blot Analysis
Mouse hearts were collected, and nucleoproteins were extracted
as above. Nucleoproteins were separated and electrophoresed on
12% sodium dodecyl sulfate (SDS) polyacrylamide gels and then
transferred to polyvinylidene difluoride (PVDF) membrane (Merck
Millipore, DA, GER). After incubation with 5% non-fat milk for
1 h, the blots were probed with rabbit polyclonal antibodies
against acetylated groups of histone H3K9 (Abcam, Cambridge,
UK, 1:1,000 dilution) or rabbit polyclonal antibody against histone
H3 (Beyotime, Shanghai, China, 1:1,000 dilution) in Tris Buffered
Saline with Tween 20 (TBST) plus 5% non-fat milk at 4uC
overnight. HRP conjugated goat anti-rabbit antibody (Santa Cruz
Biotechnology, CA, USA) was used as the secondary antibody.
After scanning, bands were subjected to analysis using Quantity
One Version4.4 software (Bio-Rad, CA, USA). Western blot
experiments were repeated six times to confirm the results.
Total RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from the collected cardiac tissues using
an RNA extraction kit (Bioteke, Beijing, China). First-strand cDNA
Figure 2. Acetylation sites of HATs on the Gata4 promoter. The sequence of 1.0 kb at up-stream of the transcription start site of mouse Gata4
is indicated in Upper and band density is in the bottom. ChIP-PCR results run on agarose gel electrophoresis showed that five HATs (P300, CBP, PCAF,
SRC1 and GCN5) proteins could bind to multiple sites on the promoter of Gata4 for possible acetylation of histone H3K9 on this site. All the five HATs
(P300, CBP, PCAF, SRC1 and GCN5) were amplified with DNA fragments precipitated by anti-P300, anti-CBP, anti-PCAF, anti-SRC1 and anti-GCN5
antibodies. H3K9 and IgG were amplified with DNA fragment precipitated by anti-H3AcK9 antibody during the later with normal mouse IgG. Site 1:
43801992 bp,43802137 bp, Site 2: 43802226 bp,43802328 bp, Site 3: 43802338 bp,43802475 bp, Site 4: 43802469 bp,43802574 bp, Site 5:
43802817 bp,43802972 bp.
was synthesized from 500 ng to 1,000 ng RNA by using oligo
dTAdaptor primers and AMV reverse transcriptase (Takara, Dalian,
Liaoning, China) following the manufacturers instruction. Then
cDNA was amplified with gene-specific primers (Shanghai DNA
biotechnologies, Shanghai, China) and SYBR Green dye kit
(Takara, Dalian, Liaoning, China). The primer sequences of
cardiac-specific gene and control were designed as follows: Gata4
sense primer: 59-TGCCAACTGCCAGACTACCAC-39 and
antisense primer: 59-TCAGGTTCTTGGGCTTCCGT-39; a-MHC
sense primer: 59 -TGAGACGGATGCCATACAGA-39 and
antisense primer: 59-GCAGCCTGTGCTTGGTCTT-39; cTnT sense
primer: 59-GAAGGAAAGGCAGAACCGC-39 and antisense
primer: 59-GCCTCCAGGTTGTGAATACTC-39; a-actin sense
primer: 59-TGCTGTCCCTCTATGCTTCC-39 and antisense
primer: 59-GCTGTGGTCACGAAGGAATAG-39; b-actin sense
primer: 59-CCTTTATCGGTATGGAGTCTGCG-39 and
antisense primer: 59-CCTGACATGACGTTGTTGGCA-39.
Annealing temperatures were as follows: 59uC for Gata4 and b-actin, 56uC
for a-MHC, cTnT and a-actin. The analysis of relative mRNA
expression was carried out using 22DDCt method , and b-actin
was used as an endogenous housekeeping gene to normalize the
The data was presented as means 6 standard deviation while
the statistical evaluations were performed using
independentsamples by applying T-test, continuity correction chi-square test
and one-way ANOVA. A p-value ,0.05 was considered to be
statistically significant for all analyses.
Effects of alcohol exposure on HAT and HDAC activity
To determine the effect of alcohol on histone acetyltransferases
(HAT) and histone deacetylases (HDAC), we first ascertained an
optimal alcohol exposure dose. Pregnant mice were intragastrically
administrated with different volumes of 56% (v/v) ethanol in water
to find the optimal dose. For this purpose, 5 ml/kg for alcohol
exposure dose was selected according to the blood-alcohol
concentration (Figure 1 A). Alcohol related side effects were
observed (Table 1). The maximum blood-alcohol concentration
was 137.1 mg/100 ml after 40 minutes of the intragastric
administration of 5 ml/kg alcohol.
The results of HAT assay showed that alcohol could increase
significantly the total HAT activity in heart tissue from the fetal
mice exposed to alcohol on E14.5, E16.5 and postnatal day 0.5
(PND0.5) compared to control groups (P,0.05) (Figure 1 B).
Figure 3. Hyperacetylation of histone H3K9 induced by alcohol on the Gata4 promoter. ChIP-Q-PCR results (A,B,C,D) showed that alcohol
is linked to histone H3K9 hyperacetylation on the Gata4 promoter at E14.5, E16.5, PND0.5 and PND7, respectively. However, alcohol could not affect
acetylation of H3K9 on the promoter of RPL13A (E). *: P,0.05 vs. control group (n = 3). E14.5: embryo 14.5 day, E16.5: embryo 16.5 day, PND0.5:
postnatal day 0.5, PND7: postnatal day 7.
However, HDAC activity was not altered in heart tissue from the
fetal mice exposed to alcohol compared to the controls on E14.5,
E16.5, PND0.5 and PND7 (Figure 1 C). This data suggested that
the alcohol enhances selectively HAT activity rather than affecting
HDAC activity in cardiac tissues from fetal mice exposed to
HATs were involved in acetylation on different sites of
the Gata4 promoter
We investigated the regulatory relationship between HATs
(P300, CBP, PCAF, SRC1 and GCN5) and histone acetylation
status on the promoter of cardiac nuclear transcription factor
Gata4. To do this, we isolated cardiac tissues from normal fetal
mice on E14.5 to detect the binding of HATs on the promoter
region of Gata4 using PCR assays following ChIP. Sequence
analysis revealed several putative regulatory domains (H3K9
acetylation sites) and multiple HAT binding sites (P300 binding
sites, CBP binding sites, PCAF, SRC1, GCN5, etc.). P300 and
CBP could bind to sites 1, 2, 3, 4 and 5; PCAF could bind to sites
1, 2 and 5; SRC1 could bind to sites 1, 3 and 4; while GCN5 could
bind to sites 1 and 5 only. It is noteworthy that the binding site of
HATs (P300, CBP, PCAF, SRC1 and GCN5) and the site of
histone H3K9 acetylation within the first 150 bp of the
transcription start site were critical for the gene expression
Alcohol exposure induces hyperacetylation of histone
H3K9 in the developing heart
The level of H3K9 acetylation was analyzed using Q-PCR after
ChIP. The results of ChIP-Q-PCR showed that fetal mice exposed
to alcohol on E14.5, E16.5, PND0.5 and PND7 (P,0.05 vs.
control group) exhibited hyperacetylation of histone H3K9 on the
promoter of Gata4 in cardiac tissue (Figure 3). Western blot data
showed that the acetylation of histone H3K9 in the alcohol treated
group was increased significantly compared to that in control
groups at these stages (Figure 4).
Effects of alcohol on binding of HATs to the promoter of
Gata4 in cardiac tissue
ChIP-Q-PCR data indicated that alcohol exposure could
significantly increase the binding of P300, CBP, PCAF and
SRC1 to the Gata4 promoter at E14.5 (P,0.01) vs. control group
(Figure 5 AD). However, the binding of GCN5 on the Gata4
promoter in the same cardiac samples had no significant change
compared to the control group at E14.5 (Figure 5 E), suggested
that P300, CBP, PCAF and SRC1 may have a regulatory function
for the acetylation of this regulatory site (the first 150 bp upstream
of the transcription start site) that can effect Gata4 gene
Anacardic acid reduces hyperacetylation of H3K9
induced by alcohol exposure
To evaluate the inhibition of anacardic acid in vivo, the optimal
exposure dose should first be established. Pregnant mice were
intraperitoneally injected with anacardic acid at different dosages
Figure 4. Histone H3K9 hyperacetylation induced by alcohol. According to the western blot analyses, the level of acetylation of histone H3K9
increased significantly after alcohol exposure with 5 ml/kg on E14.5, E16.5, PND0.5 and PND7. *: P,0.05 vs. control group (n = 6). (A): statistic analysis,
(B): band density. E14.5: embryo 14.5 day, E16.5: embryo 16.5 day, PND0.5: postnatal day 0.5, PND7: postnatal day 7.
Figure 5. Effect of alcohol on binding of P300, CBP, PCAF, SRC1, GCN5 to the Gata4 promoter. According to ChIP-Q-PCR results, alcohol
could increase binding of P300, CBP, PCAF, SRC1 to the Gata4 promoter on E14.5, whereas binding of GCN5 remained unchanged to the promoter
region. *: P,0.01 vs. control group (n = 3). AE was amplified with DNA-fragment precipitated with their respective antibodies i.e. anti-P300, anti-CBP,
anti-PCAF, anti-SRC1 and anti-GCN5 antibody.
Figure 6. Anacardic acid inhibits histone H3K9 hyperacetylation under alcohol treatment. Pregnant mice were exposed to various
portions (0, 1.25, 2.5, 5 and 10 mg/kg) of anacardic acid to ascertain optimal dose. The result showed that the level of H3AcK9 on the Gata4 promoter
after intraperitoneal injection with 5 mg/kg anacardic acid was the lowest compared with another dose. #: P,0.05 vs. blank group (n = 3) (A).
ChIP-QPCR results further confirms that anacardic acid (5 mg/kg) could reduce significantly hyperacetylation of histone H3K9 induced by alcohol on the
Gata4 promoter at E14.5. *: P,0.01 vs. control group (n = 3), **: P,0.01 vs. alcohol group (n = 3) (B). Western blot analysis showed that the anacardic
acid (5 mg/kg) could inhibit significantly hyperacetylation of histone H3K9 in the myocardium. *: P,0.01 vs. control group (n = 6), **: P,0.01 vs.
alcohol group (n = 6). (C): band density, (D): statistic analysis.
(0, 1.25, 2.5, 5, 10 mg/kg) based on reports in the literature .
The optimal dose (5 mg/kg) was ascertained according to the level
of H3AcK9 on the promoter of Gata4 (Figure 6 A). Pregnant
mice were treated by anacardic acid at this dose to confirm the
inhibitory effect of anacardic acid on H3K9 hyperacetylation
induced by alcohol. ChIP-Q-PCR results showed that the
panhistone acetylase inhibitor anacardic acid reduced significantly the
hyperacetylation of H3K9 on the promoter region of Gata4 from
the fetal mouse hearts in alcohol given groups (Figure 6 B).
Western blot data showed that anacardic acid could significantly
decrease hyperacetylation of H3K9 induced by alcohol (P,0.01)
vs. the alcohol alone group (Figure 6 C, D). In addition, we also
observed that the ratios of abortions, stillbirths and intestinal
Alcohol group (n = 30)
Alcohol+anacardic acid group (n = 35)
Figure 7. Anacardic acid inhibits the binding of HATs to the Gata4 promoter. Alcohol could improve binding of P300, PCAF, SRC1 and CBP
to the Gata4 promoter on E14.5, and anacardic acid could repress binding of P300 and PCAF to the Gata4 promoter in cardiac tissues whereas
anacardic acid could not decrease binding of SRC1 and CBP to the Gata4 promoter in the same cardiac tissues (A,B,C,D). *: P,0.01 vs. control group
(n = 3), ##: P,0.05 vs. alcohol group (n = 3). **: P,0.01 vs. alcohol group (n = 3).
Anacardic acid inhibits binding of P300, PCAF to the
promoter of Gata4
Our results indicated that the anacardic acid could reduce
significantly the binding of P300 and PCAF to the promoter
region of Gata4 in fetal hearts exposed to alcohol (Figure 7 A, B).
However, binding of SRC1 and CBP to the promoter region of
Gata4 had no change in the same tested cardiac tissues (Figure 7
C, D). These data suggest that P300 and PCAF may play a key
role in regulating histone H3K9 hyperacetylation of Gata4.
Anacardic acid down-regulates mRNA over-expression of
Gata4 induced by alcohol
Quantitative RT-PCR data (Figure 8) showed that expression
of Gata4 mRNA in the alcohol group was higher than that in the
control group at E14.5 (1.3260.11 vs. 0.7260.10, P,0.01). This
observation was paralleled by a relative increase in the expression
of mRNA and the acetylation of H3K9 to the promoter of Gata4
on E14.5. Interestingly, anacardic acid could reduce the
overexpression of Gata4 mRNA that was caused by alcohol (anacardic
acid + alcohol group vs. alcohol group = 0.6360.11 vs.
1.3260.11, P,0.005). However, the expression of Gata4 mRNA
in DMSO + alcohol group has unaltered significantly compared to
the alcohol group (P.0.05).
Figure 8. Anacardic acid inhibits the over-expression of Gata4 mRNA induced by alcohol. The mRNA expression of Gata4 was analyzed
using qRT-PCR, and it was discovered that alcohol could significantly increase Gata4 mRNA expression during pregnancy in mice, but anacardic acid
could reverse the situation. *: P,0.01 vs. control group (n = 6), **: P,0.01 vs. alcohol group (n = 6).
Anacardic acid attenuates over-expression of cardiac
downstream genes induced by alcohol
ChIP-PCR data (Figure 9) showed that P300 and PCAF could
bind to the promoter of cardiac downstream gene a-MHC, and
Gata4 protein could also bind to the promoter of a-MHC and
cTnT. However, P300, PCAF and Gata4 protein all could not
bind to the promoter of cardiac downstream gene a-actin.
Quantitative RT-PCR data (Figure 10) and western blot data
(Figure 11) showed that alcohol exposure could lead to
overexpression of cardiac downstream genes a-MHC and cTnT at the
transcriptional and translational level in the fetal mouse hearts (P,
0.01). Note worthily, anacardic acid treatment to alcohol exposed
mice could reverse the over-expression of a-MHC and cTnT (P,
0.01). However, the expression of cardiac downstream gene
aactin have no obvious change in the fetal mouse hearts exposed to
Figure 9. Binding of P300, PCAF and Gata4 protein to the promoter of cardiac downstream genes. ChIP-PCR results showed that the
promoter of a-MHC allow P300, PCAF and Gata4 proteins to bind, while the promoter of cTnT only allows the binding of Gata4. However, P300, PCAF
and Gata4 protein could not bind to the promoter of a-actin. The Gata4, P300 and PCAF were amplified with DNA fragments precipitated by their
respective antibodies (anti-Gata4, anti-P300, anti-PCAF). On the other hand, Input: amplified with DNA fragment without antibody precipitation after
sonication and IgG was amplified with DNA fragment precipitated by normal mouse IgG.
Figure 10. Anacardic acid attenuates over-expression of a-MHC and cTnT mRNA induced by alcohol. The qRT-PCR study was done to find
the mRNA-expression in the mice heart, and a significant increase in a-MHC and cTnT was noted under alcohol treatment during pregnancy with no
effect on a-actin mRNA expression. Meanwhile, anacardic acid could reverse mRNA over-expression induced by alcohol of a-MHC and cTnT. *: P,0.01
vs. control group (n = 6), **: P,0.01 vs. alcohol group (n = 6).
Gata4 expression is essential for heart development, and any
abnormality has been found to cause several cardiac defects.
According to some studies, histone acetylases and histone
deacetylases are involved in cardiogenesis and myocardial
hypertrophy . HATs and HDACs govern gene expression
patterns after being recruited to target genes in association with
specific transcription factors [17,18]. However, the roles of HATs
and HDACs play to induce over-expression of Gata4 after alcohol
exposure are still unknown. Here we observed that alcohol can
increase global HAT activities in vivo, but there was no effect on
global HDAC activities. To date, five HATs isoforms have been
identified in mammalian hearts cells: P300, CBP, PCAF, SRC1
and GCN5 . Some studies have found that steroid receptor
coactivators are important transcriptional modulators that regulate
nuclear receptor and transcription factors activity. These
coactivators are associated with numerous pathologies including
cancer, inflammation and metabolic disorders . GCN5 and
CBP are the critical transcriptional activators for the regulation
and expression of many downstream genes , while PCAF is
involved in the pathogenesis of cardiac dysplasia .
We have observed that P300, CBP, PCAF, SRC1 and GCN5
all are involved in mediating histone H3K9 acetylation on the
promoter of Gata4 during cardiogenesis. The up-stream domain
(,1,000 bp) of mouse Gata4 is highly homologous among mouse,
rat and human. We also revealed several putative regulatory
domains (H3K9 acetylation sites) and binding sites (P300, CBP,
PCAF, SRC1, GCN5, etc.) based on our analysis of the sequence.
Although there are multiple binding sites of P300, CBP, PCAF,
SRC1 and GCN5 on the first 1,000 bp at upstream of Gata4, the
43801992 to 43802137 site can specifically be bound by all HATs
(P300, CBP, PCAF, SRC1 and GCN5) in cardiac tissues, and
histone H3K9 acetylation occur at this site. Therefore, we believe
that this site may be a key site of regulating acetylation on the
promoter of Gata4.
Heterozygous mutation in Gata4 are known to cause familial
septal defects . However, there are many CHD cases that are
not related to Gata4 gene mutations. Spatiotemporal disorder of
gene expression pattern caused by genes or environmental factors
during early heart development can lead to cardiac dysplasia, but
the underlying mechanism(s) remain unclear. Epigenetic
mechanisms play a key role in the regulation of embryonic development,
tissue homeostasis and modulate cardiovascular diseases .
Histones can be modified by several post-translational mechanisms
Figure 11. Inhibitory effects of anacardic acid on the over-expression of a-MHC and cTnT protein induced by alcohol. The protein
expression of a-MHC, cTnT and a-actin was analyzed using western blot that showed that alcohol could significantly increase a-MHC and cTnT
expression at the level of protein in the mice heart exposed to alcohol during pregnancy. Meanwhile, anacardic acid could reverse over-expression
induced by alcohol of a-MHC and cTnT protein. However, alcohol could not change the a-actin protein expression in the same samples. *: P,0.05 vs.
control group (n = 6), **: P,0.05 vs. alcohol group (n = 6). (A): band density, (B): statistic analysis.
including acetylation, methylation, phosphorylation,
ubiquitination, sumoylation and ribosylation of distinct amino acids, and as a
consequence either activation or suppression of gene expression
. It is well known that alcohol is a common teratogenic
factor during pregnancy and may cause heart malformations
during embryonic development, but little is known about the
pathogenesis. Histone acetylation represents a central mechanism
that control the gene expression . To further explore the
specific regulatory roles and the underlying mechanism of HAT
(P300, CBP, PCAF, SRC1 and GCN5) mediated cardiac genes
regulation, we applied chromatin immunoprecipitation (ChIP) to
probe the relationship between HATs and Gata4 in pregnant mice
exposed to alcohol.
Gata4 is a key transcription factor which is involved in the heart
development and cardiac hypertrophy . Many studies have
shown that alcohol can enhance the binding of P300, CBP, PCAF
and SRC1 on the Gata4 promoter, and increase histone H3K9
acetylation in association with the Gata4 promoter. However, the
binding of GCN5 on the Gata4 promoter had no effect on fetal
hearts exposed to alcohol. Interestingly, this observation is
paralleled by a relative increase in the expression of Gata4
mRNA and the acetylation of histone H3K9 that binds with the
Gata4 promoter. These results suggested that during alcohol
induced over-expression of Gata4, P300, CBP, PCAF and SRC1
have effects on Gata4 expression together with
acetylationdependent modifications to expression of other heart development
In order to further demonstrate which HAT isoforms (P300,
CBP, PCAF, or SRC1) are key Gata4 regulatory factors, we
cotreated alcohol exposed mice with the pan-acetylase inhibitor
anacardic acid. Our results demonstrated that anacardic acid can
significantly decrease the binding of P300 and PCAF to the Gata4
promoter. However, binding of SRC1 and CBP was unchanged.
Only down-regulation of P300 and PCAF binding reduced
hyperacetylation of histone H3K9 induced by alcohol, and this
down-regulation played a significant role in the over-expression of
Gata4 induced by alcohol. This suggests that P300 and PCAF may
be key regulatory factors that mediate histone H3K9
hyperacetylation and subsequent over-expression of Gata4 induced by alcohol.
We also found that P300 and PCAF could bind to the cardiac
aMHC promoter. In addition, Gata4 protein was
co-immunoprecipitate with a-MHC and cTnT demonstrating that P300, PCAF
and Gata4 all take part in regulating the expression of cardiac
downstream genes. Our study further demonstrated that alcohol
exposure could lead to over-expression of cardiac downstream genes
(a-MHC and cTnT) in the fetal mouse hearts. Importantly,
anacardic acid inhibited over-expression of a-MHC and cTnT
induced by alcohol. From this point of view, our study may offer
guidance for future advancement of epigenetic medications targeted
at preventing and decreasing morbidity and mortality of CHD.
In this regard, reversibility of histone acetylation modification
presents a new opportunity for the prevention and treatment of
CHD. We observed that the ratios of abortions, stillbirths and
intestinal tympanites declined in mice treated with anacardic acid.
But this work is preliminary and will be the subject of future studies.
Anacardic acid is not approved for human use and little is known
regarding its effectiveness and safety in the human body. We believe
that is an urgent need for further research into the effectiveness and
safety of HATs inhibitor (anacardic acid), with special attention
focused on their effects on cardiovascular diseases.
In conclusion, our study indicated that HATs (P300, CBP,
PCAF, SRC1 and GCN5) can bind with different sites on the
Gata4 promoter under physiological conditions. Covalent
modification imbalance of histone H3K9 acetylation mediated by
HATs (P300, PCAF) in the 43801992 to 43802137 region of the
Gata4 promoter may be regarded as a fundamental factor for the
control of expression of Gata4 in the heart of fetal mice exposed to
alcohol. These results have highlighted the regulatory mechanism
of Gata4 from the view of epigenetics. Epigenetic regulation may
provide novel entry points for therapeutic control of CHD.
Conceived and designed the experiments: CP XPH JT. Performed the
experiments: CP HCS WAZ. Analyzed the data: JZ. Contributed
reagents/materials/analysis tools: MZ LJL. Wrote the paper: CP.
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