干细胞心肌分化过程中表观遗传修饰参与Isl1表达的机制研究

Isl1, a member of LIM-homeodomain transcription factor families, plays a crucial role in cardiac differentiation. However, the mechanisms governing the gene expression of Isl1 or the biological roles of Isl1 are poorly known. In this project, we aims at exploiting these problems，focusing on the epigenetic regulatory mechanism on Isl1 expression and function. Based on previous reports and our studies, we propose a hypothesis that EZH2, a histone-lysine N-methyltransferase enzyme, mediates the transcriptional repression of Isl1. Upon cardiac induction, EZH2 is degraded by autophagy mechanism, thereby Isl1 is activated in transcriptional level. Meanwhile, Isl1 can directly activate the expression of histone deacetylases HDAC1 and HDAC2, which inhibit the expression of Oct4 and other pluripotent genes. So Isl1 is invovled in the mechanism for the exit of stem cells from the pluripotent state. In this study, we will examine the potential mechanisms for Isl1 expression and function with gene overexpression/knowndown, ChIP, luciferase reporter, EMSA, and other experiments. Additionally, we will establish new modle for cardiac induction based on the postive roles of autophagy in cardiomyogenesis, and screen the potential drugs among traditional Chinese medicines and components. Therefore, this study will help us to understand the relationships of epigenetic regulatory mechanisms and Isl1 during the cardiac differentiation process, further clarifying the expression and function mechanisms of Isl1. Our study also highlights a novel mechanism for the exit of pluripotentstate.

Isl1是心脏发育过程中的重要转录因子，然而关于Isl1上游表达调控机制以及其生物学作用尚未完全阐明。本课题提出Isl1在心肌细胞分化过程中的表观遗传调控机制工作假说：组蛋白甲基化酶EZH2通过启动子组蛋白甲基化作用，抑制Isl1表达；心肌分化启动后EZH2经自噬途径降解，解除对Isl1启动子的封抑，从而诱导Isl1表达。同时，Isl1表达后通过诱导组蛋白去乙酰化酶HDAC1/2，抑制Oct4等干性基因的表达，从而介导干性程序的退出，确保心肌细胞能够成熟。本课题中，我们将以小鼠胚胎干细胞为研究对象，利用基因过表达/敲除、荧光素酶报告、ChIP和EMSA等技术验证该假说。同时本课题将建立基于促进自噬的心肌分化诱导药物筛选平台，筛选潜在有效的中药或其组分。本课题的完成，可为揭示Isl1与表观遗传机制的相互关联、Isl1的诱导表达机制，并对干细胞心肌分化过程中的干性程序退出机制提供新的线索。

Islet-1 (Isl1)是心脏发育过程中的重要转录因子，是第二生心区心肌前体细胞的分子标志，在心肌分化中发挥了重要作用。然而关于Isl1上游表达调控机制以及其生物学作用尚未完全阐明。本课题阐述了Isl1在心肌细胞分化过程中的表观遗传调控机制。我们发现组蛋白去乙酰化酶HDAC1和HDAC2抑制Isl1基因表达，通过HDAC抑制剂可以促进Isl1表达，从而促进早期心肌分化。但是，在晚期心肌分化中存在HDAC1和HDAC2的表达上调，参与对OCT4等干性基因的基因沉默。因此，在晚期心肌分化中，HDAC抑制剂处理会造成OCT4基因沉默被干扰，从而阻碍了成熟心肌的功能性基因表达。我们进一步发现，Isl1过表达会诱导HDAC1和HDAC2表达上调，抑制OCT4等干性基因的表达，从而介导干性程序的退出，确保心肌细胞能够成熟。此外，本研究也发现PRC2复合物成员EZH2抑制Isl1基因表达；自噬能够清除OCT4、NANOG等分子，促进Isl1基因表达，从而促进早期心肌分化。本课题的完成，揭示了Isl1与表观遗传机制的相互关联，Isl1的诱导表达机制，并对干细胞心肌分化过程中的干性程序退出机制提供新的线索。