Role of Spatiotemporal Epigenetic Dynamics in Regulating Endothelial Gene Expressions under Flows

摘要 血管功能的表观遗传调控已被发现在心血管疾病中起着关键作用。 血管内皮细胞(ECs)暴露在不同的流动模式下，调节血管的动态平衡。 不同流动模式引起的不同表观遗传变化，如组蛋白修饰，调节EC基因 表达模式，从而产生功能后果。组蛋白磷酸化、甲基化、 和乙酰化最近被发现通过不同的染色质来调节基因的表达 重塑复合体，这将改变相应的表型结果。然而，有一种稀缺的 组蛋白修饰在血管细胞中的流动调节研究。我们假设它们之间的耦合 表观遗传组蛋白的磷酸化、甲基化和乙酰化可能是一种转导机制 在不同的流动模式下调节EC基因的表达。我们将为以下目标制定定向进化策略 不同颜色FRET生物传感器的系统优化与调谐 组蛋白修饰具有很高的敏感性和特异性。这些生物传感器将用于追踪多个组蛋白 在同一个活细胞中同时进行修饰，并解开组蛋白不断演变的复合体景观 不同流程下的修改。我们将进一步采用核酸内切酶缺陷型Cas9(DCas9)，小指南 RNAs(SgRNAs)和分裂FFP跟踪EC表型特定基因座组蛋白修饰的动态 标记基因。我们的表观遗传操作系统将被用来调节这些表观遗传学 并确定它们对单个活细胞中基因表达和细胞功能的影响 在不同的流动下。识别出的表观遗传图谱随后将在体内被调节，随后的基因 检测其表达和表型结果。提出了四个具体目标：1)开发和优化FRET 生物传感器可视化单细胞中动态的组蛋白修饰，2)解开时空耦合 组蛋白磷酸化-甲基化-乙酰化在不同流程下对EC功能的调节，3)建立 基因座特异性组蛋白修饰在FLOWS调控EC基因表达中的作用，4)阐明 组蛋白修饰对体内基因表达和损伤形成的影响。的同步跟踪 核中组蛋白修饰的时空动力学与细胞增殖和 单个活细胞中的炎症将使我们能够阐明 调控表观遗传调控和内皮细胞暴露于 血流动力学提示。获得的机械洞察力应该使我们能够识别潜在的分子目标。 并促进对病理过程的药物干预的设计。因此，该项目应该 在血管机械生物学领域产生了革命性的影响，特别是与分子相关的 细胞周期和炎症调控在动脉粥样硬化发展中的作用。

Summary Epigenetic regulation of vascular functions has been found to play crucial roles in cardiovascular diseases. Vascular endothelial cells (ECs), which are exposed to different flow patterns, regulate vascular homeostasis. Differential epigenetic changes, e.g. histone modifications, caused by different flow patterns regulate EC gene expression profile and hence functional consequences. The coupling of histone phosphorylation, methylation, and acetylation have recently been identified to regulate gene expressions through the distinct chromatin remodeling complexes, which would alter the consequential phenotypic outcome. However, there is a paucity of study in the flow-regulation of histone modifications in vascular cells. We hypothesize that the coupling among epigenetic histone phosphorylation, methylation, and acetylation may serve as a transducing mechanism to regulate EC gene expressions under different patterns of flows. We will develop a directed evolution strategy for the systematic optimization and tuning of FRET biosensors with distinct colors to simultaneously monitor different histone modifications with high sensitivity and specificity. These biosensors will be used to track multiple histone modifications simultaneously in the same live cell and unravel the evolving multiplex landscape of histone modifications under different flows. We will further employ the endonuclease-deficient Cas9 (dCas9), small guide RNAs (sgRNAs) and split FPs to track the dynamics of histone modifications at the specific loci of EC phenotype marker genes. Our epigenetic manipulation system will then be employed to modulate epigenetics at these specific loci and determine their effects on gene expressions and consequent cellular functions in single live cells under different flows. The identified epigenetic profiles will then be modulated in vivo, and the consequent gene expression and phenotypic outcome examined. Four specific aims are proposed: 1) Develop and optimize FRET biosensors to visualize the dynamic histone modifications in single cells, 2) Unravel the spatiotemporal coupling of histone phosphorylation-methylation-acetylation in regulating EC functions under different flows, 3) Establish the roles of locus-specific histone modifications in regulating EC gene expression under flows, 4) Elucidate the effect of histone modifications on gene expression and lesion formation in vivo. The simultaneous tracking of the spatiotemporal dynamics of histone modifications in the nucleus in conjunction with cell proliferation and inflammation in a single live cell will allow the elucidation of the spatiotemporal transducing mechanism in regulating epigenetic modulations and pathophysiological consequences upon the exposure of ECs to hemodynamic cues. The mechanistic insights obtained should allow us to identify the potential molecular targets and facilitate the design of pharmaceutical interventions for pathologic processes. As such, the project should have transformative impact in the field of vascular mechanobiology, particularly related to the molecular regulations of cell cycle and inflammation in mediating the development of atherosclerosis.