MSCs向心肌细胞分化中特异基因的DNA甲基化/组蛋白乙酰化交互调控作用

Recent studies have confirmed that mesenchymal stem cells (MSCs) can be induced to differentiate into cardiomyocyte-like cells.The data reported in the literature and from our group have shown that histone acetylation modification plays an important role in the process of specific differentiation of MSCs into cardiomyocyte-like cells. However,the MSC differentiated level is low, which limits its clinical applications.The key problem is that the mechanisms underlying the induced differentiation are not yet fully understood.It is critical to understand the regulatory network in the differentiation process from MSCs to cardiomyocyte-like cells in a three-dimensional space. Based on our previous experimental data and other reported theories, we hypothesize that: mechanism for the timely modification of cardiac specific gene in regulating MSC differention to cardiomyocytes is associated with DNA methylation and its interaction with histone acetylation.The following specific aims have been developed to address our hypothesis.We will use Islet-1 gene that can cause MSC differentiation as an example and overexpress it in mouse embryonic MSCs. The methylation level in cardiac specific gene promoter regions will be measured with MSP and the methylation sites are detected by methylation microarray and bioinformatics analyses. Meanwhile,the state of histone acetylation on these genes will be monitored as well. In consistent with our preliminary data, we predict that the proposed study will figure out the interacting regulatory network between DNA methylation and histone acetylation during the process of MSC differentiation into cardiomyocyte-like cells.The results obtained will help us to understand better the regulatory network of epigenetics and be useful for the clinical application of stem cells for replacing injured myocardial cells and restoring damaged cardiac function.

国内外资料报道和本课题组前期研究表明：间充干细胞（MSCs）可诱导分化为心肌样细胞，组蛋白乙酰化修饰扮演着重要角色, 但低分化问题限制了其临床应用，关键是分化机制尚未完全明了，故探明MSCs特化心肌细胞的调控网络至关重要。由此，在课题组研究和相关理论基础上，我们提出假设：DNA甲基化与组蛋白乙酰化交互作用开启或沉默特异基因表达，促MSCs向心肌细胞特化。本课题拟采用高表达Islet-1慢病毒载体转染、去甲基化诱导剂与去乙酰化酶抑制剂处理MSCs，分别检测不同时间点Islet-1、心肌早期发育基因Nkx2-5、Gata4和Mef2c表达及其启动子区域甲基化、组蛋白乙酰化水平变化，阐明MSCs向心肌样细胞分化过程中特异基因的DNA甲基化和组蛋白乙酰化时序变化、交互调控及其机制。该研究将进一步明确MSCs分化表观调控网络，为MSCs应用于临床替代病变心肌组织、促进心脏功能恢复奠定坚实的实验基础。

目 的：明确在Islet-1诱导MSCs向心肌样细胞分化过程中组蛋白甲基化/乙酰化和DNA甲基化修饰作用，筛选出参与调控的关键酶GCN5、HP1和DNMT-1，并初步探明其在调控心肌早期转录因子过程中的交互作用机制。.方 法：ChIP-qPCR检测GATA4和Nkx2.5启动子区组蛋白乙酰化/甲基化水平，以及三种修饰的关键酶与启动子区域的结合情况；并用MSP检测启动子区的DNA甲基化水平随后用BSP进行验证；分子水平检测MSCs向心肌细胞分化的关键蛋白表达情况。.结 果：过表达Islet-1可以提高心肌早期转录因子NKx2.5，Gata4和Mef2c的水平，并获得类小鼠心肌细胞电生理特征；研究发现Islet-1诱导过程中，GATA4启动子区组蛋白乙酰化水平逐渐升高，组蛋白甲基化和DNA甲基化逐渐降低；Nkx2.5启动子区组蛋白乙酰化/甲基化与GATA4启动子区的趋势一致，但DNA甲基化在分化过程中没有改变；Islet-1诱导同时用5-aza改变DNA甲基化水平，可引起GATA4启动子区第二位点组蛋白乙酰化升高和组蛋白甲基化降低，但无法改变Nkx2.5启动子区的组蛋白乙酰化/甲基化；Islet-1诱导同时用TSA改变组蛋白乙酰化水平，GATA4启动子区第二位点的组蛋白甲基化和DNA甲基化都明显降低，而Nkx2.5启动子区仅表现出组蛋白甲基化降低；发现Islet-1诱导分化过程中三种表观遗传修饰的关键酶主要有Gcn5、HDAC1、G9A、DNMT-1和HP1；与Islet-1组相比抑制Gcn5后GATA4和Nkx2.5的表达水平明显降低，且没有出现cTnT和Cx43的表达，表明抑制Gcn5能够阻止MSCs向心肌样细胞分化；抑制Gcn5后，与Islet-1组相比其他关键酶与GATA4启动子区的结合都明显升高，且该区域的组蛋白乙酰化显著降低，组蛋白甲基化和DNA甲基化明显升高；而HP1-α、HP1-β和DNMT-1与Nkx2.5的结合不受Gcn5抑制的影响，因而DNA甲基化水平与Islet-1诱导组相比没有明显改变。.结 论：Islet-1引导Gcn5与心肌早期转录因子结合，并连同组蛋白乙酰化/甲基化和DNA甲基化在心肌特异转录因子的启动子区形成交互作用，共同调控该转录因子的表达，从而促进MSCs向心肌样细胞分化以更好地应用于临床治疗。