干细胞心肌程序分化中HDAC1、EZH2的阶段特异性效应及其成因

Cardiac differentiation in vitro is a complex stepwise process. The effect of exogenous inducers is dependent on the cell context and the differentiation stage during cardiomyogenesis, exhibiting remarkable stage-specific characteristics. Differentiating cells in different status could exhibit distinct action in response to the same inducer. Our previous studies showed that the expression of several enzymes associated with epigenetic modification, such as histone deacetylase 1 (HDAC1) and histone methyltransferase EZH2, fluctuates during the stepwise cardiomyogenesis. Meanwhile, specific inhibitors against HDACs and EZH2 exhibited three-phase effects on cardiomyogenesis. These inhibitors can facilitate cardiomyogenesis at the early-differentiation stage, while exert negative effects at the middle stage and promote the expression of cardiac functional genes at the late stage. However, the underlying mechanisms remain unknown. Herein, we propose a hypothesis that HDAC1 and EZH2 cooperate to control the sequential on-off events of pluripotent genes (such as Oct4) and cardiac differentiation genes, thereby displaying stage-specific effects. HDAC1 and EZH2 mediate the epigenetic silence of Oct4 and other pluripotent genes. Importantly, the on-off status of Oct4 can modify the responsiveness of differentiating cells to inhibitors against HDACs and EZH2, repressing or stimulating the expression of cardiac function genes respectively. Additionally, our previous studies found that the expression alteration of HDAC1 during cardiomyogenesis highly synchronizes with the activation of PKA pathway. In this study, we will investigate the stage-specific effect and cause of HDAC1 and EZH2 during cardiac differentiation, and uncover the relationship between epigenetic modification and the expression of stemness and cardiac genes. And we will explore the function of PKA pathway and its downstream CREB transactivator in regulating cardiac differentiation and epigenetic modification. Collectively, this study will enhance the understanding on the stage-specific feature and mechanism of cardiomyogenesis, and optimize the protocol for cardiac induction based on the sequential process of epigenetic modification.

干细胞的心肌诱导分化是一个连续且分段的过程，选择恰当的发育阶段诱导是提高分化效率的重要策略。我们前期研究发现，干细胞向心肌分化过程中组蛋白的表观遗传修饰分子HDAC1和EZH2等表达具有阶段特异性，协同参与干性基因和分化基因的阶段特异性表达；化学抑制后早期促进、中期抑制心肌分化，而在成熟分化阶段则促进心肌功能基因的表达；PKA通路的活化次序与HDAC1的表达高度同步。在本研究中，我们将阐述表观修饰分子HDAC1、EZH2在心肌分化中的阶段特异性作用及相关机制，揭示表观修饰、干性基因沉默与心肌基因表达之间的调控关系，并阐释PKA通路及其下游CREB转录因子对心肌分化和表观修饰分子表达的阶段性调控作用。本研究有助于对心肌阶段性分化的时相划分、机制成因的理解，并基于表观修饰的不同时相优化心肌诱导分化策略。

干细胞的心肌诱导分化是一个连续但分阶段的过程。本课题发现表观修饰酶HDAC1、EZH2协同参与干性基因和分化基因的次序性启闭，具有阶段特异性效应。我们的研究发现，内质网应激抑制剂4-PBA具有组蛋白去乙酰化酶抑制剂活性，能发挥阶段性效应，影响心肌分化。4-PBA与另一个HDAC抑制剂TSA，在早期促进心肌特异性转录因子表达，在心肌前体细胞分化阶段抑制心肌分化，然后促进心肌成熟阶段心肌功能基因表达和细胞搏动。机制研究显示HDAC1阶段性表达及效应，与干性基因OCT4的表达相关。此外，我们发现HDAC5在内皮诱导分化中有阶段性变化和生物学效应。我们还发现组蛋白甲基化酶EZH2在心肌分化中有着标志性的丰度变化，具有“低-高-低-强高-强高-低”的折叠波动，清晰地区分了中胚层、生心中胚层、心肌前体和跳动心肌四个分化阶段。进一步研究发现，组蛋白甲基化酶EZH2在未分化阶段通过与SUZ12、EED组成PRC2复合物，沉默中胚层基因表达；EZH2在中胚层分化阶段受Wnt信号激活，EZH2蛋白稳定性提高，单独介导β-catenin稳定性增高，协同促进中胚层分化。我们的研究还发现表观修饰酶的变化可能与MAPK和AKT等通路活化相关。我们还研究阐释了干细胞内皮分化中HDAC酶的表达变化和定位变化，阐释了HDAC抑制剂的分化阶段性效应。总之，本研究阐述了表观修饰分子HDAC1、EZH2在心肌分化中的阶段特异性作用及相关机制，揭示表观修饰、干性基因沉默与心肌基因表达之间的调控关系。本项研究增进了对心肌阶段性分化的时相划分、机制成因的理解，并基于表观修饰的不同时相优化心肌诱导分化策略。