Integration of single-cell imaging and multi-omics sequencing to study EC mechano-pathophysiology

总结 血管功能的表观遗传调控在心血管疾病中起着至关重要的作用。 血管内皮细胞（EC），暴露于不同的流动模式，调节血管稳态。 由不同的流动模式引起的差异表观遗传变化，例如组蛋白修饰，调节EC基因 表达谱和因此的功能性后果。组蛋白磷酸化，甲基化， 和乙酰化最近已被确定通过不同的染色质调节基因表达 重塑复合物，这将改变相应的表型结果。然而，缺乏 研究血管细胞中组蛋白修饰的流动调节。我们假设， 表观遗传组蛋白磷酸化、甲基化和乙酰化可能作为一种转导机制， 调节EC基因表达的不同模式下的流动。我们将开发一种定向进化策略， 系统优化和调整具有不同颜色的FRET生物传感器，以同时监测不同的 具有高灵敏度和特异性的组蛋白修饰。这些生物传感器将被用于追踪多种组蛋白 同时在同一个活细胞中进行修饰，并解开了组蛋白不断演变的多重景观， 不同流量下的修改。我们将进一步使用核酸内切酶缺陷型Cas9（dCas 9）、小向导RNA（siRNA）和RNA干扰技术。 RNA（sgRNA）和分裂FP来跟踪EC表型的特定基因座处的组蛋白修饰的动态 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。 特定的基因座，并确定其对基因表达的影响，从而在单个活细胞中的细胞功能 在不同的流量下。然后，将在体内调节所鉴定的表观遗传谱，并且随后的基因表达将被调节。 检测表达和表型结果。提出了四个具体目标：1）发展和优化FRET 生物传感器，以可视化单细胞中的动态组蛋白修饰，2）解开时空耦合 组蛋白磷酸化-甲基化-乙酰化在不同流量下调控EC功能的研究 基因座特异性组蛋白修饰在流动条件下调控EC基因表达中的作用; 4）阐明了基因座特异性组蛋白修饰在流动条件下调控EC基因表达中的作用。 组蛋白修饰对体内基因表达和损伤形成的影响。同时跟踪的 与细胞增殖相关的细胞核中组蛋白修饰的时空动力学， 在单个活细胞中的炎症将允许阐明在炎症中的时空转导机制。 调节内皮细胞暴露于 血流动力学提示。所获得的机制见解应该使我们能够确定潜在的分子靶点 并促进病理过程的药物干预的设计。因此，该项目应 在血管机械生物学领域具有变革性的影响，特别是与分子生物学相关的影响。 细胞周期和炎症调节介导动脉粥样硬化的发展。

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.