DNA methylation regulates mouse cardiac myofibril gene expression during heart development
Xu et al. Journal of Biomedical Science
DNA methylation regulates mouse cardiac myofibril gene expression during heart development
Yang Xu 1
Lingjuan Liu 1
Bo Pan 1
Jing Zhu
Changlong Nan 0
Xupei Huang 0
Jie Tian 1
0 Department of Biomedical Science, Charlie E. Schmidt College of Medicine, Florida Atlantic University , 777 Glades Road, Boca Raton, FL 33431 , USA
1 Department of Cardiology, Heart Centre, Children's Hospital of Chongqing Medical University , 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, P.R. of China
Background: It is well known that epigenetic modifications play an important role in controlling the regulation of gene expression during the development. Our previous studies have demonstrated that the expression of fetal troponin I gene (also called slow skeletal troponin I, ssTnI) is predominated in the fetal stage, reduced after birth and disappeared in the adulthood. The mechanism underlying the developmentally related ssTnI gene regulation is not clear. In this study, we have explored the epigenetic role of DNA methylation in the regulation of ssTnI expression in the heart during the development. Results: The DNA methylation levels of CpG island and CpG dinucleotides region were detected using methylation specific PCR (MSP) and bisulfite sequence PCR (BSP) in 2000 bp upstream and 100 bp upstream of ssTnI gene promoter. Real time RT-PCR and Western blot were used to detect ssTnI mRNA and protein expression levels. We found that DNA methylation levels of the CpG dinucleotides region in ssTnI gene promoter were increased with the development, corresponding to a decreased expression of ssTnI gene in mouse heart. However the DNA methylation levels of CpG islands in this gene were not changed during the development. Application of a methylation inhibitor, 5-Azacytidine, in cultured myocardial cells partially prevented the decline of ssTnI expression. Conclusion: Our results indicate that DNA methylation, as an epigenetic intervention, plays a role in the regulation of the fetal TnI gene expression in the heat during the development.
Troponin I; DNA methylation; 5-Azacytidine; Epigenetic regulation
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Background
Sarcomere protein, troponin, is located on the thin
filament of myocardial cells and plays an essential role in
regulating Ca2+-activated tension of striated muscles.
Troponin component contains three isoforms: troponin
T (TnT), binding to tropomyosion forming
troponintropomyosion complex, troponin C (TnC), binding to
Ca2+ to produce a conformational change in TnT, and
troponin I (TnI), an inhibitory subunit binding to
actintropomyosion and regulating muscles contraction [1].
There are at least two developmentally regulated TnI
isoforms in the heart: the slow skeletal TnI (ssTnI) that
is expressed in the fetal heart and the cardiac TnI (cTnI)
that is predominately expressed in adult hearts [1–3].
TnI isoform switching is common in animals and
human, and it is a good model to investigate the regulation
of cardiac proteins in the development [4].
It is clinically important to clarify the regulation of TnI
expression, because many cardiomyopathy and heart
diseases are associated with abnormal TnI protein expression
leading to diastolic dysfunction and heart failure [5–7]. In
our previous studies, we have demonstrated that ssTnI
expression in heart is partially regulated by thyroid hormone
during the heart development [8, 9]. And we have also
cloned mouse ssTnI gene with upstream promoters and
revealed several regions and domains on the promoters
critically to ssTnI gene expression, such as TnI slow
upstream regulatory elements (SURE), Yin Yang 1 factor
(YY1), proximal 300 bp upstream region and the first
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intron of ssTnI gene [10–13]. However, the mechanisms
of ssTnI down-regulation and finally shut down in the
heart after birth is still not clear.
DNA methylation is one of epigenetic modifications
and many studies have showed that DNA methylation
plays an important role in gene expression, genomic
imprinting, X-chromosome inactivation and chromatin
structure changes [14–16]. DNA methylation occurs on
position 5 of cytosine by the covalent modification of a
methyl group, creating 5-methylcytosine, which is
preferentially found in CpG dinucleotides [17]. The “CpG” is
shorthand for “-C-phosphate-G-”, which is cytosine and
guanine separated by only one phosphate. CpG island is
a (...truncated)