Xu Hao, Yi Qin, Yang Chunmei, Wang Yue, Tian Jie, Zhu Jing
Pediatric Research Institute, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, 400014, China.
China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing, 400014, China.
Cell Prolif. 2016 Jun;49(3):315-29. doi: 10.1111/cpr.12253. Epub 2016 Apr 26.
A previous study of ours confirmed that Islet-1 specifically induces differentiation of MSCs into cardiomyocytes, and that one of the mechanisms underlying that process is regulation of histone acetylation. Here, we further explore the mechanism of MSC differentiation into cardiomyocytes from the perspective of interactions between epigenetic modifications.
We used lentiviral vectors to overexpress Islet-1 in MSCs, and ChIP-qPCR, MSP and BSP were performed to detect levels of histone acetylation/methylation and DNA methylation in the GATA4 and Nkx2.5 promoters. To further explore relationships between these epigenetic modifications, we used 5-aza or TSA to interfere with DNA methylation and histone acetylation, respectively, and detected effects on the other two modifications.
Histone acetylation level increased and its methylation level decreased at GATA4 and Nkx2.5 promoters; DNA methylation level was reduced at the GATA4 promoter but did not change at the Nkx2.5 promoter. Furthermore, 5-aza increased histone acetylation level and reduced its methylation level at the GATA4 promoter but had no effect on the Nkx2.5 promoter; TSA reduced histone methylation and DNA methylation levels at the GATA4 promoter, but it only reduced histone methylation level at the Nkx2.5 promoter.
Histone acetylation/methylation and DNA methylation were both involved in regulating GATA4 expression, but Nkx2.5 expression was not regulated by DNA methylation. These three modifications had high correlation with each other during regulation of GATA4 and produced a regulation loop at the GATA4 promoter.
我们之前的一项研究证实，胰岛-1可特异性诱导间充质干细胞分化为心肌细胞，该过程的潜在机制之一是组蛋白乙酰化的调节。在此，我们从表观遗传修饰之间相互作用的角度进一步探讨间充质干细胞分化为心肌细胞的机制。
我们使用慢病毒载体在间充质干细胞中过表达胰岛-1，并进行染色质免疫沉淀定量聚合酶链反应（ChIP-qPCR）、甲基化特异性聚合酶链反应（MSP）和亚硫酸氢盐测序法（BSP），以检测GATA4和Nkx2.5启动子处的组蛋白乙酰化/甲基化水平以及DNA甲基化水平。为了进一步探究这些表观遗传修饰之间的关系，我们分别使用5-氮杂胞苷（5-aza）或曲古抑菌素A（TSA）干扰DNA甲基化和组蛋白乙酰化，并检测对其他两种修饰的影响。
GATA4和Nkx2.5启动子处的组蛋白乙酰化水平升高，甲基化水平降低；GATA4启动子处的DNA甲基化水平降低，但Nkx2.5启动子处的DNA甲基化水平未发生变化。此外，5-aza增加了GATA4启动子处的组蛋白乙酰化水平并降低了其甲基化水平，但对Nkx2.5启动子无影响；TSA降低了GATA4启动子处的组蛋白甲基化和DNA甲基化水平，但仅降低了Nkx2.5启动子处的组蛋白甲基化水平。
组蛋白乙酰化/甲基化和DNA甲基化均参与调节GATA4的表达，但Nkx2.5的表达不受DNA甲基化的调节。这三种修饰在GATA4的调节过程中彼此高度相关，并在GATA4启动子处形成了一个调节环路。
